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Ratarstvo i povrtarstvo Field and Vegetable Crops Research

2010: 47 (2)

Radovi se recenziraju, lektorisu i dostupni su u punom tekstu u bazama / Articles are peer-reviewed, proof-read and available in full-text in the following data bases SCIndeks (Srpski citatni indeks / Serbian Citation Index) (http://scindeks.nb.rs) & EBSCO Academic Search Complete (www.ebscohost.com) do nivoa izvoda u bazi / and abstract level at Agris (FAO) (www.fao.org)

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SADRZAJ / CONTENTS

PREGLEDNI RADOVI / REVIEW ARTICLES

Geneticki resursi / Genetic Resources 381-386 Characterization for Multipurpose Exploitations of Genetic Resources from the Germplasm Collection of Pasture Species Owned by the CNR-ISPAAM in Sassari, Italy / Karakterizacija u cilju visenamenskog korisenja genetickih resursa iz zbirke pasnjackih vrsta u institutu CNR-ISPAAM u Sasariju, Italija Achievements in Research on Vavilovia (Vavilovia Formosa (Stev.) Fed.), a Legume Crop Wild Relative / Dostignua u istrazivanju na vaviloviji (Vavilovia Formosa (Stev.) Fed.), samoniklom srodniku gajenih mahunarki Simonetta Bullitta

387-394

Aleksandar Miki, Petr Smýkal, Gregory Kenicer, Nune Sarukhanyan, Janna Akopian, Ivan Gabrielyan, Armen Vanyan, Andrey Sinjushin, Natalia Demidenko, Branko upina, Vojislav Mihailovi, Margarita Vishnyakova, Mike Ambrose

Genetika i oplemenjivanje / Genetics and Breeding 395-402 Alfalfa Breeding Benefits from Genomics of Medicago truncatula / Doprinos genomike Medicago truncatula oplemenjivanju lucerke Genetic Improvement of Field Pea (Pisum sativum L.) in Bulgaria / Geneticko unapreenje stocnog graska (Pisum sativum L.) u Bugarskoj Bernadette Julier, Isabelle Meusnier

403-408

Valentin Kosev, Ivan Pacev

Molekularna genetika / Molecular Genetics 409-416 Application of Genetic Markers in Seed Testing and Plant Breeding / Primena genetickih markera u kontroli kvaliteta semena i oplemenjivanju biljaka Primena genetickih markera u oplemenjivanju visegodisnjih leguminoza / Use of Genetic Markers in Breeding of Perennial Legumes Zorica Nikoli

417-424

Gordana Surlan-Momirovi, Slobodan Kati, Sanja Vasiljevi, Zorica Nikoli, Gordana Brankovi, Irena Cali, Dragan Mili, Aleksandar Miki

Citogenetika / Cytogenetics 425-434 Primena citogenetskih istrazivanja u oplemenjivanju suncokreta i uljane repice / Significance of Cytogenetic Research in Sunflower and Rapeseed Breeding Jovanka Atlagi, Sreten Terzi, Ana MarjanoviJeromela, Radovan Marinkovi

Fitopatologija / Phytopathology 435-438 Durability of Resistance to Rust in Sunflower: Implications for Resistance Breeding / Postojanost otpornosti suncokreta na ru: preduslovi oplemenjivanja na otpornost Resistance of Cool Season Food Legumes to Ascochyta Blight / Otpornost povrtarskih mahunarki umerenih klimata prema parazitima iz roda Ascochyta Mariá José Llamas, Elena Prats, Diego Rubiales

439-442

Diego Rubiales, Sara Fondevilla

Agroklimatologija / Agroclimatology 443-450 Proizvodnja kukuruza u uslovima globalnih klimatskih promena / Maize Production in Terms of Global Climate Changes Goran Bekavac, Bozana Purar, ore Jockovi, Milisav Stojakovi, Mile Ivanovi, Goran Malidza, Ivica alovi

Modeliranje / Modelling 451-456 Herbage Growth Models as Aids for Managing Intensively Grazed Grassland in Western Europe / Modeli proizvodnje krme za negu intenzivno korisenih travnjaka zapadne Evrope A. Scott Laidlaw

Arheobotanika / Archaeobotany 457-460 A Brief Review on the Early Distribution of Pea (Pisum sativum L.) in Europe / Kratki pregled ranog sirenja graska (Pisum sativum L.) u Evropi Marija Ljustina, Aleksandar Miki

Ekonomska analiza / Economic Analysis 461-466 Obelezja proizvodnje povra u Republici Srbiji / Characteristics of Vegetable Production in the Republic of Serbia Branislav Vlahovi, Anton Puskari, Janko Cervenski

ORIGINALNI NAUCNI RADOVI / ORIGINAL REVIEW ARTICLES Genetika i oplemenjivanje / Genetics and Breeding Kukuruz / Maize 467-472 Geneticka varijabilnost i korelaciona analiza za prinos zrna sinteticke populacije kukuruza NSA15 / Genetic Variability and Correlation Analysis for Grain Yield of a Narrow Base Synthetic Maize Population NSA15 Dusan Stanisavljevi, Sanja Treski, Bojan Mitrovi, Aleksandra Nastasi, Goran Bekavac, Mile Ivanov

Krmno bilje / Forage Crops 473-478 479-484 Correlations among Some Characteristics in Field Pea / Korelacije izmeu nekih osobina stocnog graska Study of Variability and Similarities Among Lotus corniculatus L. Accessions / Studija varijabilnosti i slicnosti meu populacijama zutog zvezdana (Lotus corniculatus L.) Colchicine-Induced Variations in Survival Rate and Morphological Characteristics of Water Yam (Dioscorea alata) / Promene stope prezivljavanja i morfoloskih odlika vodenog jama (Dioscorea alata) izazvane kolhicinom Valentin Kosev, Ivan Pacev Daniela Knotová, Jan Pelikán, Tomás Vymyslický, Helena Hutyrová

485-492

Abiola T. Ajayi, Adenubi I. Adesoye, Robert Asiedu, Alieu Sartie

Povre / Vegetables 493-498 Korisenje starih sorti i lokalnih populacija paradajza kao izvora geneticke varijabilnosti u oplemenjivanju / Heirloom Tomato Cultivars and Local Populations as Sources of Genetic Variability for Breeding Adaptability and Stability of Vegetable Common Bean (Phaseolus vulgaris L.) Accessions from the VIR Collection in Crimea / Adaptabilnost i stabilnost populacija boranije (Phaseolus vulgaris L.) iz kolekcije VIR na Krimu Svetlana Glogovac, Adam Takac

499-504

Margarita A. Vishnyakova, Yulia A. Filimonova

Molekularna genetika / Molecular Genetics Strna zita / Small Grains 505-510 Asocijativna analiza izmeu mikrosatelitskih markera i agronomski vaznih svojstava psenice / Association Analysis between Microsatellite Markers and Agronomically Important Traits in Wheat Ljiljana Brbakli, Ankica Kondi-Spika, Dragana Trkulja, Borislav Kobiljski

Krmno bilje / Forage Crops 511-516 Ispitivanje mogunosti primene RAPD markera u detekciji DNK polimorfizma sorti lucerke/ Research on the Possibility of RAPD Markers Application in Detecting DNA Polymorphism of Alfalfa Varieties Nevena Nagl, Ksenija Taski-Ajdukovi, Goran Bara, Dragan Mili, Slobodan Kati

Semenarstvo / Seed Science Uljane culture / Oil Crops 517-522 Uticaj hibrida, nacina cuvanja i zastite semena na nicanje suncokreta / Effect of Hybrid, Storage Conditions and Seed Protection on Sunflower Field Emergence Jelena Mra, Branislav Ostoji, Sinisa Prole, Goran Joki, Daliborka Butas, Karlo ilvesi, Vladimir Miklic

Krmno bilje / Forage Crops 523-528 Uticaj zaslanjenosti na klijanje i parametre porasta ponika stocnog graska (Pisum sativum L.) / The Effect of Salinity on Seed Germination and Growth Parameters of Field Pea (Pisum sativum L.) Kvalitet semena proteinskog graska u zavisnosti od vlaznosti semena u zetvi i sorte / Dry Pea Seed Quality Depending on Seed Moisture at Harvest and Cultivar Soja / Soybean 535-538 Pouzdanost NIR metode u odreivanju sadrzaja ulja i proteina u semenu soje / Reliability of NIR Method in Protein and Oil Content Determination in Soybean Svetlana Balesevi-Tubi, Vuk orevi, Vojin uki, Jegor Miladinovi, Mladen Tati Dusica Jovici, Milka Vujakovi, Mirjana Milosevi, ura Karagi, Ksenija Taski-Ajdukovi, Maja Ignjatov, Aleksandar Miki Branko Milosevi, ura Karagi, Vojislav Mihailovi, Aleksandar Miki, Sanja Vasiljevi, Imre Pataki, Milka Vujakovi

529-534

Gajenje i agrotehnika / Soil and Crop Management Uljane kulture / Oil Crops 539-544 Uticaj prihrane na prinos i komponente kvaliteta semena uljane repice / Influence of Top Dressing on Yield and Seed Quality Components of Oilseed Rape Soja / Soybean 545-548 Uticaj primene NS-Nitragina na prinos i komponente prinosa kod soje / Effect of NS-Nitragin Application on Soybean Yield and Yield Components Variranje sadrzaja proteina u zrnu soje u zavisnosti od sorte i godine / Variation of Protein Content in Soybean Grain Depending on Cultivar and Year Jelena Marinkovi, Nastasija Mrkovacki, Radivoje Aimovi, Vuk orevi Vera Popovi, Milos Vidi, Vojin uki, Svetlana Balesevi-Tubi, Miladin Kosti, Aleksandar Ili, Dragana Valan Milka Vujakovi, Ana Marjanovi-Jeromela, Dusica Jovici, Radovan Marinkovi, Zorica Nikoli, Jovan Crnobarac, Ksenija Taski-Ajdukovi

549-554

Kvalitet / Crop Quality 555-560 Antioksidativna svojstva fenolnih komponenata semena sociva (Lens culinaris L.) /Antioxidant Activity of Phenolic Compounds in Lentil Seeds (Lens culinaris L.) Jelena Dragisi Maksimovi, Branka Zivanovi, Vuk Maksimovi, Vesna Hadzi-Taskovi Sukalovi

Zastita useva / Crop Protection 561-566 Mikopopulacija semena kukuruza (2006-2008) / Mycopopulation of Maize Seeds (2006-2008) Prisustvo i rasprostranjenost virusa paprike u Srbiji / Presence and Distribution of Pepper Viruses in Serbia Efekti primene diferencijatora populacije Puccinia triticina i predlog stvaranja novih / Effects of Differentials and Suggestion for Creating a New Set Dragana Petrovi, Maja Ignjatov, Milka Vujakovi, Ksenija Taski-Ajdukovi, Zorica Nikoli, Mirjana Milosevi, Dusica Jovici Dragana Petrovi, Aleksandra Bulaji, Ivana Stankovi, Maja Ignjatov, Milka Vujakovi, Branka Krsti Zoran Jerkovi, Marina Putnik-Deli

567-576 577-580

Zdruzeni usevi/Intercropping 581-586 Niche Separation and Nitrogen Transfer in BrassicaLegume Intercrops / Nisno razdvajanje i prenos azota u zdruzenim usevima kupusnjaca i mahunarki Felipe Alfonso Cortés-Mora, Guillaume Piva, Marie Jamont, Joëlle Fustec

Alternativna upotreba / Alternative Uses 587-590 Sericea Lespedeza Biomass Composition for Bioenergy in the Southeastern USA / Sastav biomase lespedeze i njeno korisenje za bioenergiju u jugoistocnom delu SAD Jorge A. Mosjidis

Agroekologija / Agroecology Kvalitet i plodnost zemljista / Soil Quality and Fertility 591-598 Sadrzaj organske materije u zemljistima AP Vojvodine i mogunost korisenja zetvenih ostataka kao obnovljivog izvora energije / Organic Matter Content in Vojvodina Soils and the Possibility of Using Harvest Residues as Renewable Source of Energy Petar Sekuli, Jordana Ninkov, Nikola Hristov, Jovica Vasin, Sran Seremesi, Tijana Zeremski-Skori

Mikrobiologija zemljista / Soil Microbiology 599-606 Uticaj nacina primene Azotobacter chroococcum na mikroorganizme u rizosferi i prinos seerne repe / Effect of Azotobacter chroococcum Application Type on Microorganisms in the Rhizosphere and Sugar Beet Yield Nastasija Mrkovacki, Nikola Caci, Janja Kuzevski, Lazar Kovacev, Snezana Mezei, Nevena Nagl, Dragana Bjeli

Ekonomska analiza / Economic Analysis 607-612 Konkurentnost osnovnih ratarskih useva u Vojvodini / Competitiveness of Basic Field Crops in Vojvodina Danica Bosnjak, Vesna Rodi

Arheobotanika / Archaeobotany 613-616 The "Big Bang" in the Early Iron Age: Charred Crop Storages and Agriculture of the Iron Age Settlement Gradina upon Bosut in Syrmia / "Veliki prasak" u starijem gvozdenom dobu: Eksplodirana skladista ratarskih useva i poljoprivreda starijeg gvozdenog doba u naselju Gradina na Bosutu u Sremu Aleksandar Medovi, Aleksandar Miki

PRETHODNA SAOPSTENJA / RESEARCH NOTES Geneticki resursi / Genetic Resources 617-622 Review of Genetic Resources of Grain Legumes in Albania / Pregled genetickih resursa zrnenih mahunarki u Albaniji Sokrat Jani, Agim Canko

PRIKAZI KNJIGA / BOOK REVIEWS 623 624 Janko Cervenski: Gajenje kupusa (Cabbage Growing ­ in Serbian) uro Gvozdenovi, Sandra Cveji: Oplemenjivanje paprike (Pepper Breeding ­ in Serbian) Nada Hladni: Geni i prinos suncokreta / Genes and Sunflower Yield (in Serbian and English) uro Gvozdenovi Jan Boanski

625

Mile Ivanovi

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Genetic Resources of Pasture Species in CNR-ISPAAM, Sassari, Italy

381 Genetic Resources / Geneticki resursi

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 381-386 review article / pregledni rad

Characterization for Multipurpose Exploitations of Genetic Resources from the Germplasm Collection of Pasture Species Owned by the CNR-ISPAAM in Sassari, Italy

Simonetta Bullitta

primljeno / received: 18.04.2010. prihvaeno / accepted: 28.05.2010. © 2010 IFVC

Summary: This paper reports the results of genetic resources characterization of some pasture species from the germplasm collection held at ISPAAM-CNR in Sassari, Sardinia, Italy. According to the peculiarities of each species, some of the uses suggested by the experimental results were phytoremediation, wildfire prevention, biomass production for bioenergy, forage production and multiple uses, bioactive compounds for health care of domestic animals. Key words: bioactive compounds, forages, Mediterranean germplasm, non-conventional uses

Introduction The ISPAAM u.o.s. of Sassari (formerly Center for Studies on Mediterranean Pastures) was established by the Italian National Research Council in 1984. During the last twenty five years, a main research topic has been the identification and evaluation of genetic resources of forage interest for their introduction into cropping systems for production purposes and for multiple uses, including land recovery and environmental conservation. In such a frame, local germplasm of several species was collected and evaluated for biological and morphological characters useful for forage or multiple uses in the Mediterranean areas. The Institute holds a collection of several herbaceous species, belonging to the more traditional botanic families Leguminosae and Gramineae, and also more unusual species belonging to the Compositae and Geraniaceae, all isolated from the local flora and characterized for biological and morphological traits useful for fodder and multipurpose uses in the Mediterranean environments. A brief description of the collection and the results of some experimental trials aimed at the characterization of accessions of a few species are reported here. Attention is focused on the germplasm characterization for phytoremediation, wildfire prevention, biomass

S. Bullitta ) ( ISPAAM-CNR u.o.s. Sassari, Traversa La Crucca 3, Località Baldinca, 07100 Li Punti ­ Sassari, Italy e-mail: Simonetta.Bullitta@cspm[email protected]

production for bioenergy, forage production and multiple uses, bioactive compounds for health care of domestic animals. The Germplasm Collection of Pasture Species Sixty two species, from the botanic families Leguminosae, Gramineae, Compositae, Geraniaceae and Plantaginaceae, are present in the germplasm collection owned by the CNR-ISPAAM u.o.s. of Sassari. The collection includes twenty-five genera, with the genus Trifolium and Medicago being the most represented with 16 and 15 species respectively. All species in the collection are spontaneous in the Mediterranean flora and are interesting for their potential in multipurpose uses. The germplasm collection includes 547 accessions listed in table 1. Almost all accessions were collected in the regional territory of the island of Sardinia. Annual species are more widespread than perennials in Sardinian pasturelands due to their higher diffusion in the regional territory. This is mainly due to the shallow soil layers that hinder the persistence of perennials during summer drought, to the high grazing pressure that does not allow seed formation of perennials and their natural regeneration on pasturelands. Nevertheless, where perennials are present, they are well adapted to the Mediterranean climate, due to their summer dormancy and good winter growth, features that make them valuable resources for breeding new varieties of perennial

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species suitable for Mediterranean environments. All accessions of the collection represent an important genetic reservoir, also considering the environmental issues deriving from the overgrazing, wildfires, human activities impacts on environment and land desertion occurring in marginal Mediterranean areas.

Genetic Resources for Phytoremediation Purposes The project "Development of symbiotic systems between pasture legumes and soil microorganisms to restore soil fertility in arid and polluted areas", selected in the frame of the third

Table 1. Species in the germplasm collection of the CNR-ISPAAM u.o.s. Sassari, Italy with indications of species already characterized for forage (f), environment (e) or bioenergy (b) purposes. Tabela 1. Biljne vrste u kolekciji germplazme CNR-ISPAAM, Sasari, Italija sa indikacijom vrsta cija je upotreba karakterizovana za krmu (F), zivotnu sredinu (e) i bioenergiju (b) Species Vrste Aegilops sp. (e) Anthyllis sp. (e) Astragalus boeticus (e) Astragalus hamosus (e) Biserrula pelecinus (f) Cichorium intybus (f, e) Chrisanthemum coronarium (f) Cynodon dactylon (e) Cynosurus cristatum Dactylis glomerata (f) Dactylis hispanica Erodium moschatum (f, e) Festuca arundinacea (f, e) Hedysarum coronarium (f, e) Hippocrepis multisiliquosa (e) Hymenocarpos sp. Lathyrus sp. Lolium multiflorum (f) Lolium perenne (f) Lolium rigidum (f, e) Lotus cytisoides (f, e) Lotus edulis (f, e) Lotus ornithopodioides (f, e) Medicago aculeata Medicago arabica Medicago ciliaris Medicago litoralis Medicago marina Medicago minima Medicago murex Medicago orbicularis Medicago polymorpha (f, e) N. of accessions Br. populacija 1 1 2 5 4 7 3 1 1 41 1 1 10 15 1 1 3 2 21 77 5 4 8 1 14 1 1 1 7 29 4 58 Species Vrste Medicago praecox Medicago rigidula Medicago rugosa Medicago sativa (f) Medicago scutellata Medicago truncatula Melilotus sp. Melilotus sulcata Melilotus messanensis Ornithopus compressus (f,e) Phalaris aquatica Plantago lanceolata Sylibum marianum (f, b) Scorpiurus muricatus (f,e) Trifolium campestre Trifolium cherleri Trifolium angustifolium Trifolium campestre Trifolium cherleri Trifolium glomeratum Trifolium ligusticum Trifolium nigrescens (f,e) Trifolium pratense (f) Trifolium repens (f) Trifolium resupinatum Trifolium scabrum Trifolium spumosum Trifolium squarrosum Trifolium subterraneum (f,e) Trifolium tomentosum TOTAL N. of accessions Br. populacija 1 2 4 10 1 13 2 1 1 2 25 4 5 3 1 2 1 2 1 1 2 41 7 14 1 3 2 2 60 2 547

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executive program of scientific and technologic cooperation between Italy and Russia, has been developed in cooperation with scientists from ARRIAM (All Russia Research Institute for Agricultural Microbiology). The project is aimed at identifying plant species tolerant to heavy metals (HMs) and efficient plant-bacteria associations to promote plant growth and root nodulation activity under stress conditions, suitable for phytoremediation purposes. The characterization of different rhizobial strains, symbionts of several Mediterranean pasture legumes species, the identification of some plant-microbe interactions to develop successful techniques for phytoremediation, the selection of rhizobacterial strains particularly efficient in heavy metal polluted soils are among the scientific results of the cooperation (Safronova et al. 2004, Belimov et al. 2005). We identified and collected local populations of legume and non legume species that are able to colonize polluted soils of Sulcis mining region (SW-Sardinia) where concentration of Cd, Zn and Pb can respectively reach about 36, 5900, 2800 mg kg-1 in mine waste areas. We performed hydroponics and pot soil experiments to compare tolerance to HMs of several pasture legume populations collected from polluted and non-polluted sites of Sardinia, and then we estimated the response of the selected plants to inoculation with PGPR containing ACC deaminase and with nodule bacteria, in the presence of elevated HM concentrations. The aim of designed experiments was to identify HM tolerant legume-microbe systems for phytoremediation of mine wastes (Safronova et al. 2010a). Significant interspecific differences in the growth, HM relations and response to inoculation with microorganisms were found. Efficient and metal tolerant plant-microbe associations were detected. Synergistic and additive effects on plant growth and nutrition were observed in Lotus spp. grown in HM-polluted soil after combined inoculations with nodule bacteria Mesorhizobium loti and PGPR Variovorax paradoxus. It is important to collect germplasm from contaminated areas and bring it into cultivation, for establishing germplasm collection and facilities useful for phytoremediation purposes, and for research aimed at selecting fast growing and tolerant genotypes able to colonise polluted areas. A detailed review on the benefits and potential problems in the use of legume-microbe symbioses for phytoremediation of heavy metal polluted soils has recently been completed in the frame of the collaboration with the Institute ARRIAM of Saint Petersburg (Safronova et al. 2010b).

Genetic Resources for the Wildfire Prevention According to the experiences from Sardinia by Franca et al. (2003), firebreaks management by the oversowing of well-adapted pasture species and a correct grazing pressure can allow the constitution of a nine-month green covered firebreak, rotationally grazed by animals and during summer a dry and low­risk firebreak, owing to the control of the fuel biomass made by the animals. Such method represents a valid multi-use system that protects landscape and soil, preserves biodiversity and decreases management costs, compared to the traditional management of firebreaks tillage. Experiences on such a topic by Franca et al. (2003) indicated that: 1) low input management (no-tillage plus low seed rate) with sowing of local ecotypes and commercial varieties of annual self-reseeding legumes (Trifolium spp and Medicago polymorpha L.) and grasses (Lolium rigidum Gaudin) in pure stand or mixture were satisfactory. It allowed the establishment of a complete and persistent herbage cover, to be grazed for reducing fuel accumulation; 2) the management of grazing pressure and/or cutting regimes resulted a parameter as determinant as the green cover establishment; 3) the best management strategy seemed to be a combination of herbage grazing and/or residual cutting. To achieve a suitable forage production, soil protection and fire prevention, it seems crucial to choose adequate plant materials. According to Franca et al. (2003), local flora represents a valuable source of ecotypes to be efficiently utilised in low input firebreaks management. Genetic Resources of Non-Conventional Species for Forage and Multiple Uses Encouraging results were obtained by Sulas et al. (2007a, 2007b, 2007c) in the experiences aimed at the characterization for introduction into cropping of local natural populations of Erodium moschatum (L.) L'Herit., Cichorium intybus (L.) and Chrysanthemum coronarium (L.). E. moschatum showed appreciable nutritional characteristics for ruminants, mainly in autumn, and growth habitus and roots development apt to soil preservation on slopes. According to Sulas et al. (2007a), the E. moschatum first cut plus its regrowth yielded about 2 t of DM ha-1. The E. moschatum herbage dry matter percentage was about 10%; crude protein (CP) ranged from 15% to 20% while NDF and ADF contents were moderate. These results indicate that the forage

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could have a good digestibility. Moreover, the total polyphenols content was on average 5.5% DM and it was similar to that of Mediterranean forage legumes. C. intybus, positively assessed for forage production and quality, can be considered as a valid alternative or, anyway, a complementary species to traditional grass and legume forages. According to Sulas (2007b), on the average of the triennium of trial, DMY per year of a local population of C. intybus reached 7.2 t ha-1 and 6.8 t ha-1 under undisturbed and cut plots, respectively. This suggests that a frequent utilization is profitable for the annual Sardinian population of forage chicory, whose yields from contrasting treatments were surprisingly similar. Moreover, C. intybus can also have a role in environment preservation to protect soil and for landscape embellishment. C. coronarium, considered in the past only as a weed, showed remarkable forage productions and good silage quality; for such species it has also been successfully set up the mechanical harvesting of seed, a very important step for future introduction into cropping (Sulas et al. 2007c). Genetic Resources for Bioenergy from Biomass Production A seed collection of the Sardinian populations of milk thistle (Silybum marianum Gaertner) was made and plots of such germplasm were established in 2006 to evaluate the performance of the species in terms of biomass production under low input conditions. Sulas et al. (2008) report the results of such experience in terms of dry matter accumulation, yield and biomass partitioning into its components (leaves, stems and heads), heating value and energy balance of the crop. According to Sulas et al. (2008), although the species is reputed a dangerous weed in cropping areas, the rusticity and the high growth rate of S. marianum make such species very interesting for bioenergy purposes under rainfed Mediterranean conditions, particularly in those areas where traditional cropping surfaces have recently decreased. The same authors report that about 20 t ha-1 of dry biomass were produced in June and remark that a low level of inputs were applied, making the species interesting for biomass production. They indicate that the high heating value (HHV) was on average 14.8 MJ kg-1 of DM, and report a total energy requirement for cultivation of the species of 8.0 GJ ha-1, 55% of which is represented by indirect energy. Considering an average calorific value of the whole dry biomass of 14.8 GJ t-1, the gross energy output was 301.3 GJ ha-1, with a net

energy gain of 293.3 GJ ha-1 (Sulas et al. 2008). The authors remark that such a value, compared to the output to input energy ratios of other crops such as Arundo donax and Sorghum bicolor, indicate that S. marianum has a better efficiency in energy conversion as well as energy productivity (2.5 GJ per each ton of biomass produced). Genetic Resources for Traditional Health Care of Domestic Animals A study about the diffusion of traditional herbal remedies for health care of domestic animals in Sardinia was made by Bullitta et al. (2007) by means of expeditions in the regional territory, interviews with animal breeders and filling questionnaires. The monitoring of knowledge related to plant species utilised in the past for traditional veterinary practices favoured the recovery of ancient local traditions related to veterinary ethnobotany and also the identification of potential plant resources useful for developing new natural products for animal phytotherapy and as feed additives, to improve animal performances and productions for organic animal farming. According to the information collected, 85 remedies of plant origin were identified. The number of species utilised for the preparations were 41, 9 of which cultivated and 32 from the wild flora. It was found that 19 out of the 41 species are still occasionally used for preparing herbal remedies for domestic animals. Thirteen species were also used to prepare herbal remedies for humans. The plant families mostly represented were Liliaceae and Leguminosae with four species each and Rosaceae and Umbelliferae with three species each. Vitis vinifera, Olea europaea L. var. europaea, Pistacia lentiscus L., Quercus suber L., Malva sylvestris and Parietaria officinalis were the species mostly utilized to prepare herbal remedies for domestic animals. The following step in the research was determination of antioxidant properties and correlations between phenolic content and antioxidant properties in 24 plant species of traditional ethnoveterinary use in the Mediterranean area (Piluzza & Bullitta 2010a) Another topic of research related to the animal well-being is the study of phenolic concentration dynamics in some pasture species and the implications for animal husbandry (Bullitta 2005). Although secondary metabolites have often been studied considering their primary role as plant defensive and their effect on consumers detrimental, their effects are usually dose dependent and it is important to consider their content in all phenological phases and in different

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plant organs. In such a frame, a survey was made on the content of total phenolics (TotP), nontannic phenolics (NTP) and proanthocyanidins (PA) in natural populations of pasture species at defined phenological phases and in different plant organs, and also on pathogen infected plants (Piluzza & Bullitta 2010b). The species included in the study were Hedysarum coronarium L., H. spinosissimum, H. glomeratum Dietrich, Onobrychis viciifolia Scop., Lotus edulis L., L. ornithopodioides L., and L. cytisoides L., collected from local natural populations in semi-arid pastureland areas of Sardinia, Italy. Variation in TotP, NTP and PA content was observed in all species, phenological phases and plant organs. Variations in TotP, NTP and PA content were observed either within and between species in all the phenological phases and plant organs that were examined. For instance, at the flower bud appearance phase, we found differences up to 35%, 78% and 31% respectively in TotP, NTP and PA concentrations among the leaf blades of eight natural populations of H. coronarium (Piluzza & Bullitta 2010b). Such finding could affect the nutritional value and the effects of PA on grazing animals. Moreover, it should be considered that the localization of PA in defined plant tissues and changes in PA concentration associated with plant maturity will affect palatability and ultimately the intake of forage consumed by animals. PA concentrations were always in the range considered beneficial for animals, not exceeding 60 mg delphinidin equivalent g-1 DM. Conclusions The few examples reported on the characterization for production and multiple uses of genetic resources from Sardinian natural pasturelands confirm the idea that native species from Mediterranean pasturelands can be suitable materials for the selection of new varieties. Such genetic resources seem promising materials to accomplish the current requirements of multifunctional agriculture for production, energy, environment and public health needs.

References Belimov A A, Hontzeas N, Safronova V I, Dodd I C, Demchinskaya S V, Piluzza G, Bullitta S, Davies W J, Glick B R (2005): Cadmium tolerant root growth-promoting bacteria of the rhizoplane of Indian mustard (Brassica juncea (L.) Czern). Soil Biol. Biochem 37: 241-250 Bullitta S (2005): Bioactive Compounds in Pasture species for phytotherapy and animal welfare. (issued from the Anfit-MiPAF Project). ISBN 88-901771-1-X, Sassari, Italy Bullitta S, Piluzza G, Viegi L (2007): Plant resources used for traditional ethnoveterinary phytotherapy in Sardinia (Italy). Genet Res and Crop Evol 54 (7): 1447-1464 Franca A, Seddaiu G, Caredda S (2003): Two sylvopastoral approaches for the wildfires prevention in Sardinia. In: A. Franca (ed.) The future of the green Mediterranean. EuroMediterranean Conference. Arti Grafiche Pisano Cagliari Piluzza G, Bullitta S (2010a): Correlations between phenolic content and antioxidant properties in twenty four plant species of traditional ethnoveterinary use in the Mediterranean area (submitted to Pharmaceutical Biology) Piluzza G, Bullitta S (2010b): The dynamics of phenolic concentration in some pasture species and implications for animal husbandry. J. Sci. Food Agric. (Published Online: Apr 13 2010 9:14AM DOI: 10.1002/jsfa.3963) Safronova V I, Piluzza G, Bullitta S, Belimov A A (2010): Use of legume-microbe symbioses for phytoremediation of heavy metal polluted soils: advantages and potential problems (in press Nova Publishers, New York) Safronova V I, Piluzza G, Belimov A A, Bullitta S (2004): Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara island. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 85: 115-127 Safronova V I, Piluzza G, Belimov A A, Bullitta S (2010): Bacterial assistance in the growth, nutrition and heavy metal uptake of pasture legumes grown in polluted mine waste (submitted to International Journal of Phytoremediation) Sulas L, Canu S, Piluzza G, Sassu M, Stangoni A P (2007a): Ecological and forage attributes of Erodium moschatum (L.) L'Herit. In: S. Bullitta (ed.), Plant Genetic Resources of Geographical and "other" Islands. (Conservation, evaluation and use for plant breeding), 159-160 Sulas L, Canu S, Muresu R, Piluzza G (2007b): A new annual forage cichory (Cichorium intybus L.) from Sardinia. In: S. Bullitta (ed.), Plant Genetic Resources of Geographical and "other" Islands. (Conservation, evaluation and use for plant breeding), 115-119 Sulas L, Saba P, Cesaroni C (2007c): Seed production and harvesting for the exploitation of Chrysanthemum coronarium L. germplasm. In: S. Bullitta (ed.), Plant Genetic Resources of Geographical and "other" Islands. (Conservation, evaluation and use for plant breeding), 157-158 Sulas L, Ventura A, Murgia L (2008): Phytomass production from Sylibum marianum for bioenergy. Options Méditerranéennes, Series A, 79: 487-490

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Bullitta S

Karakterizacija u cilju visenamenskog korisenja genetickih resursa iz zbirke pasnjackih vrsta u institutu CNR-ISPAAM u Sasariju, Italija

Simoneta Bulita

CNR, Institut za stocarstvo u mediteranskim uslovima (ISPAAM), Li Punti ­ Sasari, Italija Izvod: Rezultati se odnose na karakterizaciju genetickih resursa nekih pasnjackih vrsta zbirke odrzavane u Institutu za stocarstvo u mediteranskim uslovima (ISPAAM-CNR) u Sasariju (Sardinija), Italija. U skladu sa osobinama svake vrste i rezultatima ispitivanja, mogui nacini korisenja ukljucuju fitoremedijaciju, prevenciju pozara u divljini, proizvodnju bioenergije, proizvodnju krme i visestruke namene, kao i bioaktivne sastojke u veterinarske svrhe. Kljucne reci: bioaktivni sastojci, krmne biljke, mediteranska germplazma, nekonvencionalno korisenje.

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www.nsseme.com/journal.html

Achievements in Research on Vavilovia

387 Genetic Resources / Geneticki resursi Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 387-394 review article / pregledni rad

Achievements in Research on Vavilovia (Vavilovia formosa (Stev.) Fed.), a Legume Crop Wild Relative

Aleksandar Miki Petr Smýkal Gregory Kenicer Nune Sarukhanyan Janna Akopian Ivan Gabrielyan Armen Vanyan Andrey Sinjushin Natalia Demidenko Branko upina Vojislav Mihailovi Margarita Vishnyakova Mike Ambrose

received / primljeno: 21.04.2010. revised / preraeno: 17.05.2010. accepted / prihvaeno: 19.05.2010. © 2010 IFVC

Summary: Vavilovia (Vavilovia formosa (Stev.) Fed.) belongs to the tribe Fabeae along with peas, vetchlings, vetches and lentils. It prefers high mountain areas in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia, Syria and Turkey. A true success in the ex situ conservation has recently been achieved, within the display plot Flora and Vegetation of Armenia in the Yerevan Botanic Garden. The hybridization between vavilovia and other Fabeae was done in the N. I. Vavilov Institute of Plant Industry with F1 seeds and F1 plants that did not produce the next generation. The recent molecular research showed that vavilovia belongs to a Lathyrus-Pisum-Vavilovia clade with a clearly distinct status. Key words: conservation, crop wild relatives, Fabeae, genetic resources, molecular taxonomy, Vavilovia formosa

The Tribe Fabeae The tribe Fabeae (syn. Vicieae) is among the richer ones within the family of legumes (Fabaceae Endl). It is estimated that it comprises more than 300 species, with a taxonomic structure that is still rather dynamic. Many of the Fabeae species have extreme economic importance and repreA. Miki ) ( V. Mihailovi Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia e-mail: [email protected] P. Smýkal Agritec Plant Research Ltd., Sumperk, Czech Republic G. Kenicer Royal Botanical Garden Edinburgh, Edinburgh, UK N. Sarukhanyan A. Vanyan Green Lane Agricultural Assistance NGO, Yerevan, Armenia J. Akopian I. Gabrielyan National Academy of Sciences, Institute of Botany, Yerevan, Armenia A. Sinjushin N. Demidenko M. V. Lomonosov Moscow State University, Moscow, Russia B. upina University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia M. Vishnyakova State Scientific Centre N.I. Vavilov All-Russian Research Institute of Plant Industry of Russian Academy of Agricultural Science, St. Petersburg, Russia (VIR) M. Ambrose John Innes Centre, Norwich, UK

sent one of the most significant crops in the world. They also include one of the most ancient cultivated species in the world (Ljustina & Miki 2010). Among them are common pea (Pisum sativum L.), faba bean (Vicia faba L.) and common lentil (Lens culinaris Medik.), as well as grass pea (Lathyrus sativus L.) and other vetchlings and common vetch (Vicia sativa L.) with other vetches. All these species may be regarded as multi-functional crops that are used both for human consumption and in animal feeding and numerous non-food purposes (Miki et al 2006), in the forms such as green forage, forage dry matter, forage meal, pods with undeveloped grains, immature grain, mature grain, straw, green manure and aboveground biomass (Mihailovi et al. 2010). It is widely regarded that the tribe Fabeae consists of five genera. Two of them, vetchling (Lathyrus L.) and vetch (Vicia L.) comprise more than 100 species each. Another two, lentil (Lens Mill.) and pea (Pisum L.), are less numerous, with four species in the former and two species in the latter. So far, these four have had a well-established taxonomical status. Unlike them, the fifth monospecific genus vavilovia (Vavilovia Fed.) has been considered merely a species within nearly all the

Acknowledgements: The authors would like to thank Nigel Maxted for his sincere support and encouragement in the revival of the vavilovia research. This review is dedicated to the memory of all who took part in the vavilovia research, from C. Steven, A. Fedorov to R. H. Makasheva, A. A. Golubev and many others.

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others. Although it was veiled into many uncertainties and despite its extreme rarity, resulting in the nicknames such as Legume Fata Morgana or Legume Nessie, vavilovia has recently been gaining a lot of new interest due to its potential to provide the legume research community with precious information on the past, the present and the future of the entire tribe Fabeae (Miki et al 2009). Status The first written report ever on vavilovia was made nearly two centuries ago (Steven 1812). The newly discovered species was included in the genus Orobus L. and immediately, since its premier appearance before the scientific community, was acknowledged for the beauty of its flowers by being named Orobus formosus Stev. The genus Orobus was subsequently disregarded and its species were included mostly to the genus Lathyrus. The species O. formosus was incorporated within numerous related genera. Most often, it was considered a pea species and was known as Pisum formosum (Stev.) Alef., symbolically `beautiful' or perennial pea (Alefeld 1861, Alefeld 1866), as well as P. aucheri Jaub. et Spach., Aucher's pea, P. formosum Boiss. (Boisser 1872) and P. frigidum Alef. The results of a more contemporary research of certain floral features, such as diverse androecium and pistil characters, classified this species as a pea subgenus (Gunn & Kluve 1976). Vavilovia was also described as a vetch, named V. aucheri Boiss. and V. variegata var. aucheri (Jaub. & Spach) Bornm., as well as a vetchling species, denoted as L. frigidus Schott & Kotschy. More recently, vavilovia was considered a species belonging to a novel genus within the tribe Fabeae. One of such classifications has become the most widely accepted, denoting it as Vavilovia formosa, `beautiful' or simply vavilovia, belonging to the genus with the same name, Vavilovia Fed. (Fedorov 1939). Initially, this classification distinguished two species, V. formosa (Stev.) Fed. and V. aucheri Fed., with one coming from the Greater and one from the Lesser Caucasus. Later, obvious transitional plant forms in the Armenian material were found and these two species merged into one, under the name V. formosa (Fedorov 1952). The genus was named to honour N. I. Vavilov, who was a pioneer of the study of cultivated plants and remained as symbol of the importance of crop wild relatives. Among the remaining classifications dealing with vavilovia, it is worthy to mention that it was placed in another novel genus, Alophotropis (Jaub & Spach) Grossh. as one or two species,

A. aucheri (Jaub. & Spach) Grossh. (Czerepanov 1981) and A. formosa (Stev.) Grossh. (Grossheim 1949, Lamprecht 1972). There are few reports on the existence of certain variability within vavilovia as a species. This led to a suggestion of hypothetic subtaxa, such as Orobus formosus var. microphyllus Ser. or Pisum formosum var. pubescens C. C. Townsend (Townsend 1968). Characterization Vavilovia is a diploid species with 2n = 14 chromosomes. The karyotype of vavilovia consists of three pairs of submetacentric chromosomes without satellites, one pair of submetacentric chromosomes with small-diameter satellites, one pair of submetacentric chromosomes with satellites of a diameter equal to that of a chromosome alone and two pairs of metacentric chromosomes (Abramova 1971).

Figure 1. A plant of vavilovia, with rhizomes and aboveground organs (Gabrielyan 1962) Slika 1. Biljka vavilovije, sa rizomima i nadzemnim organima

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The results of rare anatomical analysis demonstrated a fragmented similarity between vavilovia on one side and Ethiopian pea (P. sativum subsp. abyssinicum Govorov) and grass pea on the other side (Makasheva et al. 1973). As a perennial and herbaceous plant, vavilovia has a dwarf habit in comparison to other related genera such as pea. Its height, denoting a distance between the soil surface and the highest parts of a plant, varies from 5 cm to 15 cm. Vavilovia roots are long. The underground stem forms rhizomes that may be essentially responsible for its ability to survive grazing and stone deposition on easily movable screes. The aboveground stems are slender, sprawling or creeping, without wings and glabrous (Fig. 1). Vavilovia leaves are compound, like in many other legume species. Its stipules are small, semisagittate, foliaceous and free from the petiole. The leaflets of a vavilovia leaf are broad, of a cuneate-obovate to suborbicular shape, thick, with glabrous surface and with entire margins. Certain populations have leaflets with keel-like basis, being a feature used to distinguish the two vavilovia species that were subsequently discarded in favour of only one (Sinjushin et al 2009). There is only one pair of leaflets in a vavilovia compound leaf. The petiole in vavilovia does not end with tendrils, like in many species of its closest genera, such as pea, grass pea or common vetch, but with one mucrolike formation, similar to that in faba bean.

The flowers of vavilovia are axillary, pedunculate and usually solitary. Bracts are small and/or inconspicuous and without bracteoles. Calyx is campanulate, while its teeth are subequal and narrowly triangular. Corolla in vavilovia flowers is pink or purple. Standard petal is usually oblong, with a short and broad claw. Wing petal is falcate or oblong. Keel is shorter than the wing, non-cristate and sometimes white. Stamens are monadelphous or diadelphous, while anthers are glabrous and smooth. Vavilovia is considered a cross-pollinating species, although without accurate data yet. Vavilovia pods are linearly oblong, long from 20 mm to 35 mm and prone to dehiscence in full maturity. One pod bears between three and five seeds. Vavilovia seeds are globose or oval, smooth and often with dark blotches on the surface. The seed protein composition of vavilovia is more similar to that the one of vetchlings in comparison to that of pea, with globulin, legumin and vicine rather partially identical. The largest portion of the vavilovia seed protein is a component not present in pea or vetchlings (Makasheva 1979). Ecogeography and in situ Conservation Vavilovia prefers mountain areas with altitudes from 1,500 m up to 3,500 m. It grows on shale or rocky ground, with loose limestone scree as a typical example. Although the main homeland of

Figure 2. Geographical distribution of vavilovia Slika 2. Geografska rasprostranjenost vavilovije

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Figure 3. A vavilovia population from Dagestan, Russia (photo A. Ivanov) Slika 3. Populacija vavilovije u Dagestanu, Rusija (fotografija A. Ivanova)

vavilovia are Central and Eastern Caucasus (Grossheim 1952, Galushko 1980), its geographical area of distribution is rather disjunctive (Fig. 2). In the Russian Federation, isolated vavilovia populations may be found in Cabardino-Balkaria, Dagestan (Fig. 3), Karachevo-Cherkessia and Northern Ossetia (Dzuybenko & Dzuybenko 2009). Vavilovia is present in the wild floras of other ex-USSR countries, such as Armenia (Fedorov 1952), Azerbaijan (Carjagin 1954, Gadzhiev & Musaev 1994) and Georgia (Kolakovskiy 1958, Arabuli 1981), as well as in the neighbouring regions in Iran (Rechinger 1979), Iraq (Townsend & Guest 1974), Lebanon and Syria (Maxted & Ambrose 2000) and Turkey (Davies 1970). In many of these countries vavilovia is considered an endangered species and is officially protected (Popov 1988, Gabrielyan 1990). The N. I. Vavilov Institute of Plant Industry carried out several expeditions aimed at collecting vavilovia, between 1960 and 1989 and in the Caucasus region, mostly in Dagestan (Vishnyakova et al. 2007). In the early 21st century, several studies on vavilovia and other wild plants were completed in Turkey, such as those related to floras of a cedar forest near Antalya (Deniz & Sümbül 2004) and a wider region of the We-

stern Taurus Mountains (Eren et al 2004). Most recently, three expeditions have been made in July and August 2009 in Armenia, two in the locations of Mount Ughtasar and one in the vicinity of Lake Aknalitch, as a joint effort of the Green Lane NGO and the Institute of Botany towards an advance in vavilovia ecogeography and conservation (Sarukhanyan 2009). In the light of recent observations, the most serious challenges of an in situ conservation of vavilovia are related to grazing by both domestic and wild animals, especially goats and sheep. Along with this, the area of vavilovia may diminish due to both global climate changes and its own physiology, causing a high susceptibility of vavilovia flowers to early frosts in late summer and an obvious inability of some populations to produce seeds each year. Ex situ Conservation There have been several attempts aimed at the ex situ conservation of vavilovia. The first experiences made in the USSR were mostly unsuccessful due to inappropriate management of soil aeration and water flow (Zhukovskyi 1971, Makasheva 1973). Limited success was achieved in the

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Figure 4. An example of the vavilovia ex situ conservation, Yerevan Botanic Garden, Armenia Slika 4. Primer ex situ konzervacije vavilovije, Botanicka basta, Jerevan, Jermenija

Official Seed Testing Station in Edinburgh and at Southampton University, UK (Cooper & Cadger 1990) but these did not provide the production of new seeds or the plant multiplication. During 1974-1981, sound results were obtained in the N. I. Vavilov Institute of Plant Industry by combining growing the plants in the field and in climatic chambers, with some of the plants surviving for some years, blooming and even producing fruits with seeds (Golubev 1990). According to historical data, vavilovia has been cultivation from time to time at the display plot Flora and Vegetation of Armenia in the Yerevan Botanic Garden since 1940 (Akhverdov & Mirzoeva 1949). A true success in the vailovia ex situ conservation has been achieved only recently, where several individuals of vavilovia from Lake Aknalitch and Mount Ughtasar were planted in the rock garden (Fig. 4), confirming that the conditions similar to natural habitat of this species enable its ex situ cultivation and propagation (Akopian & Gabrielyan 2008). Today, the plot Flora and Vegetation of Armenia contains more than 200 species belonging to 130 genera, with wild relatives of cereals, legumes, vegetables, condiments, fruit and nut crops (Akopian 2009). The artificial creation

of the alpine rock habitat suitable for the vavilovia ex situ conservation was based upon a period of long-term research on the biology and ecology of the alpine plants which enables their introduction from their native altitudes of 2,800-3,500 m into a relatively low-altitude location. It is noteworthy that the N. I. Vavilov Institute of Plant Industry and the Royal Botanical Garden Edinburgh keep the vavilovia herbarium material of diverse geographical origin. Interspecies Hybridization The fact that vavilovia shares the same number of chromosomes with its close cultivated relatives, such as pea, common vetch or grass pea, opens a possibility for their hybridization. It has been often speculated a hypothetical gene for perenniality, present in vavilovia, could be introgressed into its cultivated relatives, especially pea, making vavilovia rather interesting for breeders. The only known accounts of the hybridization between vavilovia and other Fabeae comes from the research undertaken by the N. I. Vavilov Institute of Plant Industry in its centre in St. Petersburg and the station in Dagestan (Golubev 1990).

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The hybridization of V. formosa x P. sativum was successful, resulting in several normally developed F1 seeds. However, only one of these was able to produce a true F1 hybrid plant. This plant had several stems, or basal branches, with long internodes and no lateral branches that are typical for vavilovia. Its leaves were compound, with one pair of leaflets and, instead of the mucro-like organ present in vavilovia, a third and smaller leaflet, resembling the trifoliolate leaves of Medicago or Trifolium species. This, one and only ever received F1 plant did not enter the generative stage and eventually withered due to chlorosis (Golubev 1990). A reciprocal combination of P. sativum x V. formosa also resulted in only one F1 hybrid plant. It had much greater height in comparison to both pea and vavilovia and numerous basal and lateral branches. Unlike the F1 plant from another combination, this one produced flowers and five pods. In two of these six, all the F2 seeds aborted, while in the other three the F2 seeds remained immature (Golubev 1990). According to unpublished data based upon personal communications from A. A. Golubev, the hybridization between vavilovia and its closest relatives, such as red-yellow pea (Pisum fulvum Sm.), was possible if vavilovia is used as the male

parent (Maxted & Ambrose 2000). The evidence on the possibility to produce hybrids of vavilovia and pea, along with a susceptibility of vavilovia to pea-specific pathogens, such as Uromyces pisi, Ascochyta pisi and Ascochyta pinodes, has occasionally been used to demonstrate that the status of vavilovia as a distinct genus is doubtful (Yan'kov & Golubev 1999). Molecular Taxonomy Since the very beginning of its existence as its own genus, vavilovia has been considered rather important for the evolution of the whole tribe of Fabeae, being possibly the closest of all modern taxa to an extinct common ancestor. According to one of such early evolutionary schemes, it is the development of tendrils that accompanies the progress (Fedorov 1939). In that way, first a hypothetical primeval complex of oroboids produced the extinct genus Orobus and then it acted as a common ancestor of all modern Fabeae, with vavilovia and its mucro-like petiole tip, considered a kind of initial tendril, as its closest descendant. Further development of this theory retained vavilovia as being closest to the common ancestor (Makasheva 1975) and brought pea as close to it as vetchlings and vetches.

Figure 5. Hypothesised generic relationships in the tribe Fabeae, based on DNA sequence data (Kenicer et al 2009a) Slika 5. Pretpostavljeni odnosi unutar tribusa Fabeae na osnovu sekvenci DNK

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Akopian J A (2009): Crop wild relative conservation on the plot "Flora and vegetation of Armenia" of the Yerevan Botanical Garden. Electron. J. Nat. Sci. 2: 3-7 Akopian J A, Gabrielyan I G (2008) On high-mountain pea Vavilovia formosa (Stev.) Fed. (Fabaceae) in Armenia. Crop Wild Relatives Newsl. 6: 26-27 Alefeld F. (1861): Pisum formosum. Bonplandia 9: 327 Alefeld F. (1866): Landwirtschaftliche Flora. Verlag Wiegand und Hempel, Berlin Arabuli G (1981): Notulae systematicae ac geographicae Instituti Botanici Tbilissiensis 37: 16-18 Boisser E (1872): Pisum formosum. Flora Orientalis 2: 624 Carjagin I P (1954): Flora of Azerbaijan 5. AN, Baku Cooper S R, Cadger C A (1990): Germination of Vavilovia formosa (Stev.) Davis in the laboratory. Pisum Newsl. 22: 5 Czerepanov S K (1981) Plantae Vasculares. Nauka, St. Petersburg Davies P H (1970): Vavilovia A. Fed. In: Davies P H (ed). Flora of Turkey and East Aegean Islands 3, Edinburgh, UK, 44-45 Deniz G, Sümbül H (2004): Flora of Elmali Cedar Research Forest. Turk. J. Bot. 28: 529-555 Dzyubenko N I, Dzyubenko E A (2009): 2003-2009 Project Interactive Agricultural Ecological Atlas of Russia and Neighboring Countries. Economic Plants and their Diseases, Pests and Weeds [Online]. available at http://www.agroatlas. ru (cited 19 April 2010, verified 20 April 2010) Eren Ö, Gökçeolu M, Parolly G (2004): The flora and vegetation of Bakirli Dai (Western Taurus Mts, Turkey), including annotations on critical taxa of the Taurus range. Willdenowia 34: 463-503 Fedorov A A (1939): Wild high-mountain peas of Caucasus. Trans. Biol. Inst. 1: 39-79 Fedorov A A (1952): Supplement. In: Grossheim A A (ed) Flora of the Caucasus 5, Academy of Science of the USSR, Moscow ­ St. Petersburg, 453 Gabrielyan E Ts (1962): Genus Vavilovia Fed. In: Takhtayan A L (ed), Flora of Armenia 4. Academy of Sciences of Armenian SSR, Yerevan, Armenia, 332 Gabrielyan E Ts. (1990): Vavilovia formosa (Stev.) Fed. In: Gabrielyan E Ts (ed), Red Data Book of Armenia: Plants. Ayastan, Yerevan, Armenia, 123 Gadzhiev V D, Musaev S G (1994): Information from BAK Herbarium. Baku Galushko A I (1980): Flora of Northern Caucasus - A field guide 2. Rostov-on-Don Golubev A A (1990): Habitats, collection, cultivation and hybridization of Vavilovia formosa Fed. Bull. Appl. Bot. Genet. Plant Breed. 135: 67-75 Grossheim A A (1949): Identification of Plants of the Caucasus. Sovetskaya Nauka, Moscow Grossheim A A (1952): Flora of the Caucasus 5, AN USSR, Moscow ­ St. Petersburg Gunn C R, Kluve J (1976) Androecium and pistil characters for tribe Vicieae (Fabaceae). Taxon 25: 563-575 Kenicer G J, Smýkal P, Miki A (2008) Phylogenetic study of mysterious Vavilovia formosa (Stev.) Fed., a Pisum relative? Abstracts, IV International Conference on Legume Genomics and Genetics, Puerto Vallarta, 83 Kenicer G, Nieto-Blásques E M, Miki A, Smýkal P (2009a): Lathyrus ­ diversity and phylogeny in the genus. Grain Legum. 54: 16-18 Kenicer G, Smýkal P, Vishyakova M, Miki A (2009b) Vavilovia formosa, an intriguing Pisum relative. Grain Legum. 51: 8 Kolakovskiy A L (1958): Flora of Abhasiya 3. Tbilisi Lamprecht H (1972): Monographie der gattung Pisum. Steiermarkische Landesdruckerei, Graz Lock J M, Maxted N (2005): Tribe Fabeae. In: Lewis G, Schrire B, Mackinder B, Lock M (eds.), Legumes of the World. Royal Botanical Gardens, Kew, UK, 504-509 Ljustina M, Miki A (2010): Archaeological evidence for the domestication of lentil (Lens culinaris) and its distribution in Europe. J. Lentil Res. 4 (in press)

It has been expected that a molecular research on vavilovia could essentially contribute to defining its ultimate relationship to other Fabeae genera, especially Pisum and Lathyrus (Kenicer et al 2009b). One of the first attempts in this direction included herbarium specimens of vavilovia from two different sources and the material of about 30 species of all other genera of the tribe Fabeae. This investigation was aimed at the four phylogenetically informative regions, namely chloroplast maturase K, trn L-F and trn S-G fragments and the internal transcribed spacer (ITS) region of nuclear DNA. The results of both maximum parsimony and Bayesian analysis of combined sequence data have shown that vavilovia belongs to a Lathyrus­Pisum-Vavilovia clade with a clearly distinct status (Kenicer et al. 2008), as well as that there is a monophyly of Pisum and Vavilovia (Fig. 5), that the Pisum ­ Vavilovia clade is closest to most of Lathyrus and that all of them are nested within Vicia (Smýkal et al. 2009). Other most recent molecular research on vavilovia provides more details for defining its true taxonomical position more precisely. There are suggestions that the similarity between vavilovia and pea is sufficient enough to treat vavilovia once again as P. formosum, although more distant from P. sativum than P. fulvum (Sinjushin & Demidenko 2009) and individual enough to be placed within its own monotypic section (Lock & Maxted 2005). Another study of the phylogenetic status of vavilovia, using nrDNA ITS and cpDNA trnL-F and trnS-G regions and revealing that Vavilovia is closely related to Pisum, strongly indicates that vavilovia should be subsumed under Pisum as P. formosum. Integrated Approach Recent achievements in the vavilovia molecular research and its ex situ conservation surely put even stronger emphasis upon more refined and accelerated approaches to a long-term and integrated preservation of this iconic legume species of a particular interest for taxonomists, geneticist and breeders. References

Abramova L (1971): Chromosome number and some karyotype peculiarities of Pisum formosum. Bull. Appl. Bot. Genet. Plant Breed. 45: 240-243 Akhverdov A A, Mirzoeva N V (1949): The experience of wild herbaceous plants collecting, keeping and planting in the Yerevan Botanic Garden of Academy of Sciences of ArmSSR. Bulleten' Botanicheskogo Sada Akademii Nauk Armianskoy SSR 8: 37-45

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Makasheva R Kh (1973): Gorokh. Kolos, St. Petersburg Makasheva R Kh, Drozd A M, Adamova O P, Golubev A A (1973): Perennial pea. Bull. Appl. Bot. Genet. Plant Breed. 51: 44-56 Makasheva R Kh (1975): Kratkaya istoriya proiskhozhdeniya vozdelimaevogo gorokha. In: Hvostova V V (ed), Genetika i selektsiya gorokha, Nauka, Novosibirsk, Russia, 5-15 Makasheva R Kh (1979): Flora of Cultivated Plants IV, Grain Legumes 1, Pea. Kolos, St. Petersburg Maxted N, Ambrose M (2000): Peas (Pisum L.). In: Maxted N, Bennett S J (eds.), Plant Genetic Resources of Legumes in the Mediterranean, Kluwer, Dordrecht, the Netherlands, 181-190 Mihailovi V, Miki A, Vasi M, upina B, uri B, Duc G, Stoddard F L, Hauptvogel P (2010): Neglected legume crops of Serbia ­ Faba bean (Vicia faba). Ratar. Povrt. / Field Veg. Crop Res. 47: 27-32 Miki A, upina B, Kati S, Karagi Ð (2006): Importance of annual forage legumes in supplying plant proteins. Zbornik radova Instituta za ratarstvo i povrtarstvo / A Periodical of Scientific Research on Field and Vegetable Crops 42: 91-103 Miki A, Smýkal P, Kenicer G, Vishnyakova M, Akopian J, Sarukhanyan N, Gabrielyan I, Vanyan A, Toker C, upina B, Ambrose M, Mihailovi V, Ellis N (2009): A revival of the research on beautiful vavilovia (Vavilovia formosa syn. Pisum formosum). Pisum Genet. 41: 34-39 Oskoueiyan R, Osaloo S K, Maassoumi A A, Nejadsattari T, Mozaffarian V (2010): Phylogenetic status of Vavilovia formosa (Fabaceae-Fabeae) based on nrDNA ITS and cpDNA sequences. Biochem. Syst. Ecol. doi: 10.1016/j.bse.2010.01.011 Popov K P (1988): Red Data Book of the Russian Federation. Pedagogika, Moscow

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Rechinger K H (1979): Flora Iranica. Akademische Druck und Verlagsanstalt, Graz Sarukhanyan N G, Akopian J A, Gabrielyan I G, Vanyan A G (2009): Wild pea, Vavilovia formosa (Stev.) Fed.(Fabaceae) in situ investigation in Armenia. Grain Legum. 52: 25-26 Sinjushin A A, Demidenko N V (2009): Taxonomic position of Vavilovia formosa (Stev.) Fed. evidenced from morphological and molecular data. Materials, International Conference devoted to 120th anniversary of E. Synskaya, St. Petersburg, Russia, 212-214 Sinjushin A A, Demidenko N V, Gostimskii S A (2009): Preliminary report on taxonomical position of Vavilovia formosa (Stev.) Fed. evidenced from morphological and molecular data. Pisum Genet. 41: 15-20 Smýkal P, Kenicer G J, Miki A (2009) `Beautiful vavilovia' (Vavilovia formosa) and molecular taxonomy of tribe Fabeae. Abstracts, IV Congress of the Serbian Genetic Society, Tara, 166 Steven C 1812. Orobus formosus Steven. Mémoires de la Société Impériale des Naturalistes de Moscou 4: 50 Townsend C C (1968): Contributions to the Flora of Iraq: V: Notes on the Leguminales. Kew Bull. 21: 435-458 Townsend C C, Guest E (1974): Flora of Iraq 3, Ministry of Agriculture and Agrarian Reform, Baghdad Vishnyakova M, Yan'kov I, Miki A, upina B (2007): Vavilovia formosa or Pisum formosum? In search of new answers to an old question. Abstracts, 6th European Conference on Grain Legumes, Lisbon, Portugal, 122-123 Yan'kov I, Golubev A A (1999): About the taxonomical status of vavilovia formosa based on the results of crossibility and susceptibility to specialized pathogens. CD XVI International Botanical Congress, St. Louis, USA, 5655 Zhukovskyi PM (1971): Cultural plants and their relatives. Kolos, St. Petersburg

Dostignua u istrazivanju na vaviloviji (Vavilovia formosa (Stev.) Fed.), samoniklom srodniku gajenih mahunarki

Aleksandar Miki1, Petr Smikal2, Gregory Keniser3, Nune Saruhanjan4, Jana Akopijan5, Ivan Gabrieljan5, Armen Vanjan4, Andrej Sinjusin6, Natalija Demidenko6, Branko upina7, Vojislav Mihailovi1, Margarita Visnjakova8, Majk Ambroz9

Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija 2 Istrazivanja na biljkama Agritek, s.o.o, Sumperk, Ceska 3 Kraljevska botanicka basta Edinburg, Edinburg, Velika Britanija 4NVO Poljoprivredno savetodavstvo Grin Lejn, Jerevan, Jermenija 5 Nacionalna akademija nauka, Institut za botaniku, Jerevan, Jermenija 6 Moskovski drzavni univerzitet M. V. Lomonosov, Moskva, Rusija 7 Univerzitet u Novom Sadu, Poljoprivredni fakultet, Novi Sad, Srbija 8 Drzavni naucni centar i Sveruski istrazivacki institut biljne proizvodnje N. I. Vavilov Ruske akademije poljoprivrednih nauka (VIR), Petrograd, Rusija 9 Centar Dzon Ines, Noric, Velika Britanija

1

Izvod: Vavilovija (Vavilovia formosa (Stev.) Fed.) pripada tribusu Fabeae, zajedno sa graskovima, grahorima, grahoricama i socivima. Vavilovija raste u visokoplaninskim oblastima Jermenije, Azerbejdzana, Gruzije, Iraka, Irana, Libana, Rusije, Sirije i Turske. Pravi uspeh u ex situ konzervaciji postignut je nedavno u okviru izlozbenog polja "Flora i vegetacija Jermenije" u botanickoj basti u Jerevanu. Hibridizacija izmeu vavilovije i graska izvrsena je u institutu Vavilov, sa F1 semenima i F1 biljkama koje nisu uspele da daju sledee pokoljenje. Skorasnja molekularna istrazivanja pokazala su da vavilovija pripada grupi Lathyrus ­ Pisum - Vavilovia sa jasno izrazenim posebnim statusom. Kljucne reci: Fabeae, geneticki resursi, konzervacija, molekularna taksonomija, samonikli srodnici gajenih biljaka, Vavilovia formosa

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Medicago truncatula Genomics and Alfalfa Breeding

395 Genetics and Breeding / Genetika i oplemenjivanje Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 395-402 review article / pregledni rad

Alfalfa Breeding Benefits from Genomics of Medicago truncatula

Bernadette Julier Isabelle Meusnier

received / primljeno: 22.04.2010. accepted / prihvaeno : 04.05.2010. © 2010 IFVC

Summary: International programs aim at developing knowledge and tools in the model species Medicago truncatula. Genetic resources, DNA sequences, markers, genetic and physical maps are now publicly available. These efforts contribute to improve breeding schemes of crop species such as alfalfa. However, transfer of information from M. truncatula to alfalfa is not straightforward. The article reviews the gain given by the model species to better analyze genetic determinism of breeding traits in alfalfa. It also shows that investments in alfalfa genomics (DNA sequences, SNP development) are needed to benefit from the model species. Key words: breeding, candidate gene, marker, Medicago sativa, Medicago truncatula, model species

Introduction Alfalfa (Medicago sativa L.) is a major forage species over the world. Its genetics is complex (allogamy and autotetraploidy), it is a perennial plant and cultivars are synthetic varieties that contain a high genetic diversity. Its wild relative Medicago truncatula was chosen by the scientific community as a model for legume species (Barker et al. 1990). This plant, close to many important crops (alfalfa, clovers, pea, soybean, bean, etc.) was selected given its relative small genome size (450Mb/1C, twice less than alfalfa), its simple genetics (diploidy and autogamy) that simplify genetic analysis, its easy growing (short generation time, prolific seed production) and its ability to undergo genetic transformation. The international design strategy was to focus research on the model plant and after all to transfer the knowledge to more complex species such as alfalfa. Many tools are being developed in the model species, such as genomic sequences, EST, markers, genetic and physical maps and expression data (www.medicago.org). It is expected that genomic resources developed in the model species will accelerate genetic analysis in related crop species. However, transfer of information from a model to a cultivated species needs investigation. The aim of this article is to review which data or tools issued from

B. Julier ) ( · I. Meusnier Institut National de la Recherche Agronomique (INRA), UR 4, Unité de Recherche Pluridisciplinaire Prairies et Plantes Fourragères, Lusignan, France e-mail: [email protected]

the model species would be useful in a breeding program of alfalfa. After a description of the genetic analyses that were feasible in alfalfa before genomics on M. truncatula, the knowledge and tools that are being developed on M. truncatula genomics is described. The last part shows that these tools are useful to enhance breeding program strategies. Genetic Analyses in Alfalfa before M. truncatula Genomics Alfalfa cultivars are synthetic populations coming from the cross pollination of several parents during three or four generations. This structure is a way to valorise heterosis and minimize inbreeding depression (Gallais 1990). Each parent being an individual plant or a family, the diversity among plants in a variety is high. Traditional breeding is based on different steps: the identification of breeding criteria related to agronomic traits, the evaluation of genetic diversity for these criteria, a recurrent selection program for the breeding criteria and finally the selection of parental plants or families to create a new variety. Breeding traits are basically forage yield, forage quality, disease and pest resistances, lodging resistance and seed yield. They are mostly quantitative traits governed by several genes. In order to improve breeding schemes, quantitative genetic studies were conducted to analyse genetic diversity, trait heritability and genetic correlations. Although, the genetic progress of these breeding programs is low in alfalfa, due to tetraploidy and synthetic structure of the varieties, significant

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progress was noticed (Tabel and Allerit 2005, Veronesi et al. 2006, Nelson and Burns 2006). Before the development of genomic tools in model species, molecular biology was rarely used to analyse genetic diversity or genetic determinism of traits. For example, as the number of markers was insufficient, it was almost impossible to build a complete genetic map (Yu & Pauls 1993, Brouwer et al. 2000). However, few markers were available and used in combination with phenotypic traits, particularly to analyse genetic diversity. With phenotypic traits related to agronomic value, it was shown that variations among and within populations were high (Julier et al. 1995, Julier et al. 1996, Crochemore et al. 1998, Julier et al. 2000, Bolaños-Aguilar et al. 2001). Isoenzymatic, RAPD, AFLP and RFLP markers also showed a large among individual variation (Pupilli et al. 1996, Crochemore et al. 1996, Jenczewski et al. 1999, Muller et al. 2001, Maureira et al. 2004). However, these markers suffered from several limitations: isoenzymes and RFLP markers exhibited a low level of polymorphism, and RAPD and AFLP were dominant and anonymous. At this time, the availability of SSR markers in alfalfa that combine high polymorphism, codominance and locus-specificity was restricted to about a dozen (Diwan et al. 1997, Diwan et al. 2000). The history of domestication and the relationship between wild and cultivated populations were studied using mitochondrial and nuclear polymorphisms. A demographical bottleneck probably occurred during domestication, even if a low level of divergence was detected between the wild and the domesticated populations (Muller et al. 2001, Muller et al. 2003, Muller et al. 2006). In the 2000's, the available high throughput and reproducible markers, i.e. AFLP, were used in a Bulk Segregant Analysis (BSA) to identify markers linked to a disease resistance. Using two populations specially designed to differ only for their resistance to downy mildew (Peronospora trifoliorum), Obert et al. (2000) identified two AFLP markers that were linked to the resistance. BSA is still an efficient method to test a lot of markers with a reasonable input of funds and time, but it is difficult to sequence an AFLP marker, to get genomic sequence in its region and to convert it into a codominant marker. Progress in M. truncatula Genomics and Genetics From 1990's onwards, international programs were launched on the model legume M. truncatula.

Extensive reviews are available (Young & Udvardi 2009, Rispail et al. 2010). Genetic resources of M. truncatula were collected, population structure was analysed (Ronfort et al. 2006) and their genetic diversity for many traits was described, including first nitrogen-fixing symbiosis (Barker et al. 1990), and more recently other traits that may have an agronomic interest in related species (Duc 2004, Moussart et al. 2006, Julier et al. 2007). Sequencing efforts were first devoted to ESTs (Bell et al. 2001) and lately to gene-rich regions (Young et al. 2005). In parallel, F2 (Thoquet et al. 2002, Choi et al. 2004a, Ané et al. 2008) and RILs (Julier et al. 2007, Pierre et al. 2008) mapping populations were developed. Knowledge in genome sequence of M. truncatula allows to develop low cost and easy-to-use markers, mainly Simple Sequence Repeats (SSRs). Sequence data (EST or BAC) was screened for SSR motifs and primers surrounding these motifs were designed and tested. Large sets of ESTSSR markers or SSR from BAC sequences are now available (www.medicago.org). The constant sequencing technique innovation with higher throughput offers the possibility of more ambitious projects. The next generation sequencing technologies (454, Solexa or Solid) (Rothberg & Leamon 2008, Stangier 2009, Rounsley & Last 2010) gave the opportunity to acquire genomic and/or transcriptomic (cDNA, (Wheat 2010)) sequences of a set of lines for a reasonable cost. Such data are being obtained on M. truncatula by sequencing 30 lines (http://www.medicagohapmap.org/) (Branca et al. 2010). These data sets will reveal sequence polymorphisms that will be used to better understand genetic evolution of the species and will be exploited to identify single-nucleotide polymorphism (SNP) markers. Such markers are supposed to cover the entire genome with a low cost (Ganal et al. 2009). Mapping populations are used to analyse genetic architecture of traits of interest. Having identified genetic variation for the target trait, a QTL analysis indicates the zones of the genome that could be involved in trait variation in a population. A more accurate QTL position is obtained by a fine-mapping strategy developed on a large population and the bio-analysis of the genomic sequence of the confidence interval of the QTL gives a list of candidate genes. Within this list, some genes can be selected by making hypotheses on their role on the trait. Such strategy was applied for flowering date. QTL analysis in three M. truncatula mapping populations revealed a major QTL on chromosome 7 (Pierre et

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al. 2008). A pseudo-F2 population of 1640 individual was derived from the self-pollination of a F6 individual that was heterozygous at the QTL. A confidence interval of 2.4 cM was calculated. The bio-analysis of the BACs comprised in this region revealed 577 genes. Among them, six genes were described in other species to be involved in the flowering pathway (Pierre et al. 2010). Finally, genomic and cDNA sequences of these genes were obtained and their expressions were compared between the two parents. Two genes (CONSTANS-like and FT) showed sequence polymorphism but only CONSTANS-like was differentially expressed. Similarly for the resistance to anthracnose (Colletotrichum trifolii), three genes having motifs specific to resistance genes were identified after a QTL analysis followed by a fine mapping step in M. truncatula (Yang et al. 2007, Yang et al. 2008). A susceptible alfalfa clone was transformed by each of the three genes but only one gene, called RCT1, induced resistance to anthracnose. The genes identified in M. truncatula (CONSTANS-like, FT, RCT1 or others) could possibly explain variation in alfalfa, but it has to be proved. Genetic Analyses in Alfalfa Using M. truncatula Genomic Resources To ensure the contribution of the results obtained on M. truncatula to genetic analyses in alfalfa, the very first question to be solved was the synteny between M. truncatula and cultivated species. Both the macro and the micro-synteny were high among legume species (Julier et al. 2003, Choi et al. 2004b): the order of the genes on the chromosomes are well conserved and the sequence of a given gene is also comparable. This statement is crucial: it means that if a marker is identified in a crop species as being involved in a trait, its position can be predicted in M. truncatula genome. However, if the coding sequences are well conserved, the non-coding sequences with less selection pressure show more divergence. Sequences and data available in M. truncatula helped to develop molecular markers in alfalfa, and more specifically SSR markers that were used for the analysis of genetic diversity and for genetic mapping and QTL detection. Furthemore, discovery of genes involved in trait variation in M. truncatula is a source of candidate genes that can be studied in alfalfa. Genomic and transcriptomic sequences of M. truncatula are references for those who want to analyse a gene or a genome portion in alfalfa.

Marker Development Sequence data (EST or BAC) was exploited to acquire low cost and easy-to-use SSR markers. Indeed, many of the SSRs developed on M. truncatula from EST were transferred to alfalfa (Julier et al. 2003, Sledge et al. 2005), due to synteny. The transfer success of SSR from the model plant to cultivated plant depends of the marker location in the genome. In coding regions, the primers designed on M. truncatula often gave an amplification product when tested on alfalfa (Julier et al. 2003). Conversely, SSR developed in non-coding regions were hardly usable. Sequence Polymorphism Sequence polymorphism is not expected to be the same across legume species because point mutations are different. Thus SNPs identified in M. truncatula are not transferrable in alfalfa. The development of SNPs on alfalfa requires sequencing efforts on this specific species. As on M. truncatula and other species, the next generation sequencing technologies give the opportunity to acquire genomic and/or transcriptomic sequences to reveal polymorphisms in alfalfa. These high throughput sequencers are especially useful for heterozygote species such as alfalfa because all the alleles can be sequenced at once without cloning. Even if a reference sequence of alfalfa is not available, the interpretation of short DNA reads obtained by next generation sequencers will gain from the knowledge of M. truncatula sequence, the model species genome would be used as a reference. In fact, sequencing of alfalfa is closer to a re-sequencing effort than to a de novo sequencing (Young & Udvardi 2009). Specific bioinformatics tools will be needed to handle the high level of polymorphism due to heterozygosity and tetraploidy. Analysis of Alfalfa Genetic Resources The genetic diversity of alfalfa was also analysed with codominant SSR markers that have a higher level of polymorphism than isoenzymes or RFLP. With SSR markers developed from M. truncatula, their number is sufficient to cover the alfalfa genome. However, SSR markers in alfalfa suffer from two limitations: (1) it is often difficult to read their allelic dosage, so they are not truly "codominant" markers: an allele that is present can be in one to four doses, (2) they contain a large number of null alleles, probably due to a lack of cross amplification or sequence

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polymorphisms in alfalfa itself. This reduces the information provided by the markers. SSR markers confirmed the large within-population diversity and the high heterozygosity of the individuals (Flajoulot et al. 2005, Herrmann et al. 2008, Bagavathiannan et al. 2010). They proved that alfalfa varieties or landraces are at panmictic equilibrium, as expected from their mode of production with several cross-pollination generations. Population structure was not described more precisely with SSRs than with AFLPs: populations were highly similar on average. But when sp. falcata wild populations were studied, they were easily differentiated from the sp. sativa cultivated populations (Jenczewski et al. 1999, Muller et al. 2001, Maureira et al. 2004). The consequences of this information on alfalfa breeding are clear: breeding for agronomic traits using the current methods does not reduce the neutral genetic diversity. The distinctness of populations or varieties is difficult except if a large range of variation including both sp. falcata and sativa is concerned. However, it would be interesting to revise this evaluation by an analysis of genetic resources with non-neutral markers. These markers related to agronomic traits would reveal another view of the genetic diversity present in this species and perhaps give other prospects to use genetic resources in breeding programs. Development of Genetic Maps and QTL Detection The SSR markers were used for genetic mapping in alfalfa. As they amplify several alleles per locus, they help significantly to find the four homologous chromosomes that could be detected only by repulsion linkage with dominant markers (Hackett et al. 1998). SSRs contributed deeply to build dense genetic maps of cultivated alfalfa (Julier et al. 2003, Sledge et al. 2005). Genetic mapping confirmed the autotetraploidy of the genome, as supposed by cytogenetic studies (Armstrong 1954). Mapping populations were either F1 or backcross as the high inbreeding depression restricts the analysis of F2 populations and prevents to build RIL populations. QTL detection was carried out for some agronomic traits: disease resistance (Mackie et al. 2007), morphogenesis (Julier et al. 2002), forage yield (Robins et al. 2007), water use efficiency (Julier et al. 2008). In most cases, many QTLs, each with a limited effect, were detected. For disease resistance, a lower number of QTLs was found. Usually, for a trait, each parent carried both positive and ne-

gative alleles that explained the presence of transgressive individuals in the progeny. These QTL could be used to select individuals carrying both valuable phenotypic traits and positive alleles at the QTLs in the mapping population and to create an improved population. However, this strategy was never reported to be used on alfalfa. The QTL position is usually imprecise due to the tetraploidy of alfalfa. To get the same precision than on a diploid species it would require much larger mapping populations. It is a typical case in which a cultivated plant would take advantage of the information available on the model plant. As alfalfa and M. truncatula genomes are syntenic, the position of a QTL detected on alfalfa is the same on M. truncatula. Using the sequence database of M. truncatula and its annotation, it is possible to list all the genes described on M. truncatula in this region. These genes could be responsible for the trait variation in the mapping population, especially if they are involved in the pathway described in other species to contribute to the trait. Association Mapping Association genetics is currently often used in plant species to establish a link between a gene or a genomic region and a trait. Compared to mapping populations based on only two parents, association genetics is based on a wide genetic diversity. In allogamous species such as alfalfa, the linkage disequilibrium (LD: the non-random occurrence of alleles at different loci) is expected to be short (Flint-Garcia et al. 2003). Experimental results confirmed this expectation (Auzanneau et al. 2007). In such a situation, an association study relies on a candidate gene approach in which the polymorphism of the gene is linked to the trait polymorphism. This analysis provides a high accuracy on the location of the causal mutation compared to a genome scan analysis that is carried out when the LD is large. LD can be estimated with markers spread at known distances along chromosomes. For example, in a study of Constans-like gene that was partly sequenced in alfalfa, LD decayed shortly and after 1200 bp, no more linkage was observed (Herrmann et al. 2010). This result is in agreement with the theory and means that only a candidate gene approach is valuable in this species. Moreover, for long genes, it is important to analyse the whole sequence of the gene or several short parts evenly spaced along the gene. In the case of alfalfa, the use of anonymous markers such as SSRs or AFLPs for an association

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study would hardly give positive results. Indeed the chance that the markers are located in the proper gene is extremely low. It would require to genotype all the individuals of the studied population with many markers. In the next years, the development of SNPs covering the alfalfa genome and the ability to genotype large populations will allow such association study. However, if populations having a longer LD decay would be developed, especially by polycrossing inbred parents, a genome-scan approach could be implemented to link a genomic region to a trait. In this situation when LD decay is short, it is valuable to have an association study based on a candidate gene approach. Genes taken in the literature or identified in M. truncatula (CONSTANS, FT, RCT1 or others) could possibly explain trait variation in alfalfa. In the case of the CONSTANS-like gene on chromosome 7, two gene portions were sequenced in an alfalfa population of 400 individuals. Compared to a neutral gene, CONSTANS-like gene showed less SNPs (1 SNP every 125 pb compared to 1 SNP every 30 pb for the neutral gene), indicating that CONSTANS-like gene is probably under selection. All individuals were phenotyped for flowering date and stem height, the SNPs explained up to 4% of the variation (Herrmann et al. 2010). It proved that a gene identified in M. truncatula from a QTL analysis is involved in trait variation in alfalfa. The next generation sequencers can be used to test if a candidate gene, identified in M. truncatula, is involved in a trait variation in alfalfa. Currently, these apparatus are mainly employed to sequence or re-sequence organisms as described above. They can also be useful to sequence a large number of sequences coming from a single gene amplified in numerous genotypes. Bulk segregant analysis, in which the individuals of one population are grouped into two bulks according to their phenotype, is a method to establish interesting plant material. With next generation sequencer, all alleles of the candidate gene can be sequenced in all individuals (total number of alleles = number of populations x 2 bulks x number of individuals per bulk x 4 alleles per individual). Once polymorphism is evidenced, it has to be related to the trait: the frequency of mutations must be different in the two contrasted bulks. Such analysis is handled by bioinformatics tools. The following step is to develop markers in order to genotype individual plants and to assess the link between the gene polymorphism and the phenotype. The reliable markers will be exploited in marker-assisted selection.

Conclusions The development of genetic and genomic tools was delayed on alfalfa because of its biology (allogamous species) and genetics (autotetraploidy). However, efforts in sequencing and analysis of the model species M. truncatula give new perspectives for alfalfa breeding. Locus-specific markers such as SSRs are now available and offer the possibility to analyse genetic diversity, build genetic maps and detect QTLs. All these results can be exploited into breeding programs. Research studies in M. truncatula are progressively identifying genes involved in traits of agronomic interest. These genes are candidates to explain trait variation in the cultivated species and can be tested in association genetics. In addition, considering M. truncatula genome sequence as a reference, next generation sequencers give the ability to sequence parts or even all the alfalfa genome and to develop large set of SNP markers. All this knowledge and tools developed on alfalfa will assist breeding programs.

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Flajoulot S, Ronfort J, Baudouin P, Barre P, Huguet T, Huyghe C, Julier B (2005): Genetic diversity among alfalfa (Medicago sativa) cultivars coming from a single breeding program, using SSR markers. Theor. Appl. Genet. 111: 1420-1429 Flint-Garcia S A, Thornsberry J M, Buckler IV E S (2003): Structure of linkage disequilibrium in plants. Annu. Rev. Plant Biol. 54: 357-374 Gallais A (1990): Théorie de la sélection en amélioration des plantes. Masson, Paris Ganal M W, Altmann T, Roder M S (2009): SNP identification in crop plants. Curr. Opin. Plant Biol. 12: 211-217 Hackett C A, Bradshaw J E, Meyer R C, McNicol J W, Milbourne D, Waugh R (1998): Linkage analysis in tetraploid species: a simulation study. Genet. Res. Camb. 71: 143-154 Herrmann D, Barre P, Santoni S, Julier B (2010): Association of a CONSTANS-LIKE gene to flowering and height in autotetraploid alfalfa. Theor. Appl. Genet. (DOI: 10.1007/ s00122-010-1356-z) Herrmann D, Flajoulot S, Barre P, Huyghe C, Ronfort J, Julier B (2008): Comparison of morphological traits and SSR markers to analyze genetic diversity of alfalfa cultivars. In: North American Alfalfa Improvement Conference, Dallas, USA Jenczewski E, Prosperi J M, Ronfort J (1999): Differentiation between natural and cultivated populations of Medicago sativa (Leguminosae) from Spain: analysis with random amplified polymorphic DNA (RAPD) markers and comparison to allozymes. Mol. Ecol. 8: 1317-1330 Julier B, Bernard K, Abdelguerfi-Laouar M, Udupa S, Semiani Y, Sfarni O, Elboutahiri N, Gasmi F, Barre P, Huguet T, Lelièvre F (2008): QTL for water stress resistance and water use efficiency in alfalfa. North American Alfalfa Improvement Conference, Dallas, USA Julier B, Flajoulot S, Barre P, Cardinet G, Santoni S, Huguet T, Huyghe C (2003): Construction of two genetic linkage maps in cultivated tetraploid alfalfa (Medicago sativa) using microsatellite and AFLP markers. BMC Plant Biol. 3: 9 Julier B, Guines F, Hackett CA, Huguet T, Huyghe C (2002): Genetic mapping and identification of QTL for stem morphogenesis in tetraploid alfalfa. North American Alfalfa Improvement Conference, Dallas, USA Julier B, Guy P, Castillo-Acuna C, Caubel G, Ecalle C, Esquibet M, Furstoss V, Huyghe C, Lavaud C, Porcheron A, Pacros P, Raynal G (1996): Genetic variation for disease and nematode resistances and forage quality in perennial diploid and tetraploid lucerne populations (Medicago sativa L.). Euphytica 91: 241-250 Julier B, Huguet T, Chardon F, Ayadi R, Pierre J B, Prosperi J M, Barre P, Huyghe C (2007): Identification of quantitative trait loci influencing aerial morphogenesis in the model legume Medicago truncatula. Theor. Appl. Genet. 114: 1391-1406 Julier B, Huyghe C, Ecalle C (2000): Within- and among-cultivar genetic variation in alfalfa: forage quality, morphology, and yield. Crop Sci. 40: 365-369 Julier B, Porcheron A, Ecalle C, Guy P (1995): Genetic variability for morphology, growth and forage yield among perennial diploid and tetraploid lucerne populations (Medicago sativa L). Agronomie 15: 295-304 Mackie J M, Musial J M, Armour D J, Phan H T T, Ellwood S E, Aitken K S, Irwin J A G (2007): Identification of QTL for reaction to three races of Colletotrichum trifolii and further analysis of inheritance of resistance in autotetraploid lucerne. Theor. Appl. Genet. 114: 1417-1426 Maureira I, Ortega F, Campos H, Osborn T (2004): Population structure and combining ability of diverse Medicago sativa germplasms. Theor. Appl. Genet. 109: 775-782 Moussart A, Onfroy C, Lesne A, Esquibet M, Grenier E, Tivoli B (2006): Host status and reaction of Medicago trunca-

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Veronesi F, Huyghe C, Delgado I (2006): Lucerne breeding in Europe: results and research strategies for future developments. Sociedad Espanola para el Estudio de los Pastos (SEEP), Madrid, Spain, 232-242 Wheat C W (2010): Rapidly developing functional genomics in ecological model systems via 454 transcriptome sequencing. Genetica 138: 433-451 Yang S, Gao M, Deshpande S, Lin S, Roe B A, Zhu H (2007): Genetic and physical localization of an anthracnose resistance gene in Medicago truncatula. Theor. Appl. Genet. 116: 45-52 Yang S, Gao M, Xu C, Gao J, Deshpande S, Lin S, Roe B A, Zhu H (2008): Alfalfa benefits from Medicago truncatula: The RCT1 gene from M. truncatula confers broad-spectrum resistance to anthracnose in alfalfa. Proc Natl Acad Sci USA 105: 12164-12169 Young N D, Cannon S B, Sato S, Kim D, Cook D R, Town C D, Roe B A, Tabata S (2005): Sequencing the genespaces of Medicago truncatula and Lotus japonicus. Plant Physiol. 137: 1174-1181 Young N D, Udvardi M (2009): Translating Medicago truncatula genomics to crop legumes. Curr. Opin. Plant Biol. 12: 193-201 Yu K F, Pauls K P (1993): Segregation of random amplified polymorphic DNA markers and strategies for molecular mapping in tetraploid alfalfa. Genome 36: 844-851

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Doprinos genomike Medicago truncatula oplemenjivanju lucerke

Bernadet Zilije · Izabel Mesnije

Nacionalni institut za poljoprivredna istrazivanja, IJ 4, Multidisciplinarna istrazivacka jedinica za travnjastvo i krmno bilje, Luzinjan, Francuska Izvod: Meunarodni programi usmereni su na sticanje znanja i razvoj genomickih tehnika na model vrsti Medicago truncatula. Geneticki resursi, sekvence DNK, marker, geneticke i fizicke mape sada su javno dostupne. Ovi napori doprinose usavrsavanju metoda oplemenjivanja gajenih biljaka poput lucerke. Meutim, prenos informacija sa M. truncatula na lucerku nije jednostavan. Ovaj rad daje pregled doprinosa ove model vrste za bolju geneticku analizu pozeljnih svojstava lucerke, a takoe prikazuje i da su ulaganja u genomiku lucerke (sekvence DNK, razvoj SNP) neophodne za dobijanje koristi od model vrste. Kljucne reci: geni kadidati, marker, Medicago sativa, Medicago truncatula, model vrsta, oplemenjivanje

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www.nsseme.com/journal.html

Genetic Improvement of Field Pea in Bulgaria

403 Genetics and Breeding / Genetika i oplemenjivanje Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 403-408 review article / pregledni rad

Genetic Improvement of Field Pea (Pisum sativum L.) in Bulgaria

Valentin Kosev Ivan Pachev

received / primljeno: 14.04.2010. accepted / prihvaeno: 03.05.2010 © 2010 IFVC

.

Summary: Field pea attained greater importance as a cultivated plant in Bulgaria at the beginning of the 20th century. Until 1964, only breeding for forage was in use, with developed winter varieties 5 and Pleven 2. Recently, field pea achieved the greatest increase (283.3%) in the sown areas, since the variety structure was updated annually. There are 10 registered varieties in total, with 7 spring and 3 winter ones. The composition of the Bulgarian Pisum collections is highly variable, with accessions of diverse status. The greatest efficiency is obtained in a combination of bulk method in the early generations and certain features of pedigree, single seed method, with possible modifications and inclusion of the mutational variability. Key words: accession, collections, field pea, grain legumes, improvement, Pisum sativum

Introduction Field pea (Pisum sativum L.) is one of the most important grain legumes in the world. Its grain is a major source of plant-based dietary protein for animals. Pea is a legume native to SouthWest Asia and was one of the first cultivated crops. World production of field pea is 5,389 × 106 ha with the most important producing countries being Canada, China, India and the Russian Federation, all representing almost 70% of the total crop area (FAOSTAT 2006). It is consumed as green vegetables (whole pods or immature seed) in Asian countries and as dry seed in Europe, Australia, America and Mediterranean regions. It ranks third in the world production amongst the food legumes (Ghafoor & Arshad 2008). Field pea can provide protein-rich feed and improve the sustainability of organic systems. The share of agricultural land that is under organic agriculture approaches 4% in EU and 7% or more in Scandinavian countries, Italy, Austria and Greece, and it may reach 25% in EU by 2030 (Annicchiarico & Filippi 2007). The nutritional value of dry pea seed is similar to other grain legumes and contains 18-30% protein, 35-50% starch and 4-7% fiber. Pea protein is deficient in sulfur-containing amino acids, but contains relatively high levels of lysine, making

V. Kosev ) (· I. Pachev Research Institute of Forage Crops, Gen.Vladimir Vazov Street 89, 5800 Pleven, Bulgaria e-mail: [email protected]

it a good dietary complement to cereals. Antinutritional factors, although present in pea, are relatively minor and do not adversely affect crop use (McPhee 2003). History Pea attained greater importance as a cultivated plant in Bulgaria at the beginning of the 20th century. It is supposed that the first true pea varieties were first introduced in Western Europe. In Bulgaria, there are no accurate data for the introduction of pea as a crop. After the establishment of first agricultural schools, pea was grown as a garden plant in southern Bulgaria and as a forage crop in northern Bulgaria (Kalaydzhieva 1980). In Bulgaria in 1939, pea was cultivated at 1,000 ha, with introductions from Romania, while in 1965 it took 49,800 ha including both introduced and Bulgarian varieties. During the years that followed, the areas under pea were small despite the increase of the needs for pea as a protein-rich feed. Until 1964, only breeding for forage was in use. The winter varieties 5 and Pleven 2 were developed during this period, but without wide distribution due to a short generative coefficient. After 1964, the hybridization was introduced in pea breeding, with winter varieties such as Pleven 10. Afterwards, experimental mutagenesis followed (Sachanski 1988).

Acknowledgements: The authors thank the Ministry of Science and Education in Bulgaria for funding the study (contract MSE No.CC 1604/2006).

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The winter forage pea varieties are suitable for arid regions. The utilization of winter forage pea in the conditions with a high water deficit results in higher and more stable aboveground biomass and protein yields, enhancement of the ratio between symbiotic and fertilizer nitrogen in organic farming and the possibilities for more economic use of the agricultural land (Filipov 1997, Filipov 1998). Today The productivity of pea varieties depends to a great extent on climatic conditions and individual characteristics of a variety. The soil-climatic conditions in Bulgaria enable pea to be grown as a cover crop in the spring, summer or autumn. Because of its numerous uses and diverse soil-climatic conditions, there are several breeding directions: both winter and spring varieties for both forage and grain production (Kuzmova 2002a; Kuzmova 2002b).

In Bulgaria, pea is most frequently used in feeding ruminant and monogastric animals, and is regarded as a possible alternative to soybean meal. Among leguminous crops, pea covers the greatest area and has higher yields (4,100 kg ha-1) in comparison to soybean (2,860 kg ha-1), common bean (590 kg ha-1) and lentil (370 kg ha-1). Mainly white-flowered spring varieties are grown with low content of anti-nutritional factors. Its contribution to the biological nitrogen content in the system of organic farming is obvious and expected to increase (Mihov et al. 2002, Mehandzhiev et al. 2006, Krachunov et al. 2007). During the last decade in Bulgaria and Europe, a broad set of spring pea is cultivated for grain, offering a good choice for the Bulgarian farmers. During season 2003-2004, 5,000 ha were sown for grain production in animal feeding. The low yields during this period (1,700 kg ha-1) indicate that the used varieties did not have great potential, while the new varieties were not sufficiently grown.

Table 1. Composition of the Bulgarian grain legume collections in the Institute of Plant Genetic Resources Institute (IPGRI) in Sadovo Tabela 1. Sastav bugarskih zbirki zrnenih mahunarki u Institutu za biljne geneticke resurse u Sadovu Middle-term Long-term Working ex situ Genus Total conservation conservation collection Rod Ukupno Srednjerocno Dugorocno cuvanje Radna ex situ zbirka cuvanje Pisum 2510 610 900 1000 Lupinus 284 70 204 10 Lathyrus 410 270 100 40 Phaseolus 2066 777 1151 138 Cicer 294 234 56 4 Lens 716 385 237 94 Vicia sp. 1808 1150 534 124 Table 2. Bulgarian official catalogue of field pea varieties in 2009 Tabela 2. Zvanicna lista sorti stocnog graska Bugarske 2009. Denomination of the variety Naziv sorte Amitie Bohatyr Drujba Kristal Picardi Pleven 4 Yunak 11 Vesela 23 E Mir

Year of registration Godina priznavanja 2004 1995 2006 2004 2004 2008 1989 1999 2003 2000

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At the present moment, most areas are sown with Pleven 4, registered in 1985, that does not satisfy the increasing requirements of the producers any more. This variety easily lodges and sometimes the grain losses are too great. At the end of the 70s and at the beginning of the 80s, varieties from East Europe were introduced, such as Yunak, Yubileen and Dukat. Relatively recent Dutch varieties, such as Solara, Baccara, Mira and Alex are grown at a limited area (Angelova 2005). The aim of pea breeding is to develop new intensive varieties with increased productivity, better ecological plasticity, improved resistance to biotic and abiotic factors, and desirable biochemical and technological qualities. The new varieties are characterized with high production potential and stable yields. In 2004, the variety Kristal, which has 36.9% higher yield than the former standard Pleven 4, was introduced as another official standard (Mihov et al 2005). In the National Seed Genebank of Bulgaria in Sadovo, each year about 60 characters of its forage pea accessions are analyzed in accordance with the FAO descriptors. Breeding for grain yield prefers spring, early-maturing, white-flowering, short-vining and light-coloured seeds, while breeding for forage yields prefers winter varieties with purple flowers, longer stems, dark seeds and normal leaves (Angelova 1995). The grain legume crops are good preceding crops because of the biological nitrogen fixation by the soil bacterium Rhizobium leguminosarum, which has the capability of fixing atmospheric nitrogen into plant-accesible forms and reduces the need to apply artificial nitrogen fertilizers. However, until 2006 grain legumes were grown on a small area because of the limited needs of animal husbandry, with common bean at 3,676 ha, lentil at 1,918 ha, vegetable pea at 1,086 ha, dry pea at 570 ha, chickpea at 519 ha, soybean at 89 ha and faba bean at 12 ha, making 7,872 ha in total. Despite this, the Institute of Plant Genetic Resources in Sadovo maintained a large grain legume collection (Tab. 1). During the last years, dry pea achieved the greatest increase (283.3%) in the sown areas in 2007 in comparison to 2006, since the variety structure was updated annually (Tab. 2). There are 10 registered varieties in total, with 7 spring and 3 winter ones, namely 11, Vesela ­ 23 E and Mir. All of them are of Bulgarian origin and over 90% of them were included into the List A of the Official Variety List of Bulgaria after 2000 (Stancheva 2007, MZH 2008, Bulgarian Official Catalogue of Varieties of Agriculture and Vegetable Plant Species 2009).

The average grain yields in Bulgaria in the last years were low and varied between 1,220 kg ha-1 and 1,860 kg ha-1. The lasting climatic changes require that the new varieties have good adaptability. The development of the variety Kerpo improves the variety structure of spring pea in Bulgaria and provides a good choice to producers and farmers (Kertikova et al. 2009). The expansion of the areas under pea and the use of various variety groups by farmers are possible only with an efficient and well-organized grain production. Such cultivars are included in both European and Bulgarian Lists (Angelova 2005). The problem for the effectiveness of breeding is heavily present with many different aspects. It is related with a broad inclusion and a rational use of various genetic selection materials, with shortening the deadlines for developing new varieties and increasing the efficiency of the selection in hybrid progenies (Mihov et al. 2006). So far, research has been done regarding the diverse issues related to pea genetics and breeding in Bulgaria. A rich collection is included and a great genetic diversity is developed. An intensive breeding program of common bean and pea has been carried out recently in several centers in Bulgaria, such as Sadovo, Pleven and General Toshevo. All breeding directions are fully developed and new technologies are used. Breeding is mainly targeted towards earliness, winter hardiness and drought resistance. The specific climate conditions and climate changes call for the development of early maturing varieties in all grain legumes. Only early and moderately early cultivars in Bulgaria can express their full yield potential. The composition of the Bulgarian Pisum collections is highly variable (Tab. 3). They include landraces, old cultivars, local populations, all newly developed and cultivated varieties, breeding lines, various forms, wild relatives, etc. Predominant in these pea collections are the accessions with the status of breeding material (Angelova & Stoilova 1998, Angelova 2000, Mihov et al. 2002, Angelova 2008). Selection for productivity is most time-consuming in the process of the pea genetic improvement. Most valuable genotypes for this purpose are those that form two pods at one node. In relation to the global warming, it is demanded that the newly developed cultivars have shorter growing season and avoid the high temperatures in mid June and thus overcome the high temperature stress. The small-seeded varieties, as well as those with wax coating, have more prominent drought resistance and may be successfully used

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Table 3. The classification of the pea collections in the Institute of Plant Genetic Resources (IPGR) in Sadovo and the Research Institute of Forage Crops (RIFC) in Pleven Tabela 3. Klasifikacija zbirki graska u Institutu za biljne geneticke resurse u Sadovu i Naucnom institutu za krmno bilje u Plevenu

IPGRI

Winter hardiness Ozimost 80 ­ 100% 187 samples Pisum arvense Old local varieties, populations, forms. typical fodder varieties and breeding lines used for production of green mass Low sensitivity Niska osetljivost 70 ­ 80% 350 samples Pisum sativum Newly created local and introduced varieties, breeding lines of dry pea High sensitivity Visoka osetljivost 60 ­ 70% 278 samples Pisum sativum hortense Green pea varieties, breeding lines, afila forms, fasciated forms

RIFC 116 samples Pisum arvense Newly introduced typical fodder varieties, afila forms 49 samples Pisum sativum Newly introduced typical fodder varieties, afila forms 1 sample Pisum sativum var. triver 5 samples Pisum sativum var. hiemale 1 sample Pisum sativum var. melanocarpum Fodder populations and forms

as donors of this trait in hybridization. The resistance to insects and pests is a very difficult breeding task due to the limited resources of the knowledge about the genetic control of this resistance and a lack of highly tolerant forms to the main pests. By means of hybridization and the repeated individual selection tolerant forms are being developed with damages by pea weevil (Bruchus pisorum L.) from 2% to 7%. The lines with a partial parchment of pods and a decreased susceptibility to pod dehiscence and seed shattering have also been developed (Mihov et al. 2003). Perspectives The necessary genetic improvement and its use in the development of new genotypes is related to different climatic conditions by using the most appropriate methods of the selection in mutant populations. Hybridization is one of the main methods of combining different characters and quality parameters of parental varieties. However, the method of inter-variety hybridization is closely linked to many difficulties due to a low level of crossing success and a high labour input. The greatest efficiency is obtained in a combination of bulk method in the early generations and

certain features of pedigree single seed method, with possible modifications and inclusion of the mutational variability (Mihov 1988, Kalapchieva 2002). Mutation breeding has been used worldwide for the improvement of grain legumes through an increased genetic variation and the development of novel alleles. Several pea cultivars resulting from mutagenesis have been released with improvements including increased yield, lodging resistance (afila leaf type), larger seed, increased protein content and modified maturity. Induced biochemical mutations can change the content of amino acids by the stimulation of expression of certain genes and thus affect the biosynthesis of various products. Mutant forms with increased content of protein (28.74% to 32.59%) have been developed. The contribution of experimental mutagenesis to the pea genetic improvement is reflected in the fact that there are new line-donors with high productiveness and earliness (15-20 days before the standard), resistance to the economically important diseases (Ascochyta pisi and Erysiphe pisi) and improved standing ability. They can be used in the conventional breeding for the development of varieties with good ecological stability and desirable quality characters (Mihov et al. 2003).

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Filipov H (1998): Polzi ot otglezhdaneto na zimniya furazhen grah pri zasushlivi usloviya, Vliyanie na azotniya rezhim na pochvata. Rastenievadni Nauki 11: 34-39 Ghafoor A, Arshad M (2008): Seed protein profiling of Pisum sativum L., germplasm using sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) for investigation of biodiversity. Pakistan J. Bot. 40: 2315-2321 Kaladzhieva S (1980): Zimen furazhen grah. Zemizdat, Sofia Kalapchieva S (2002): Variabilnost na kolichestveni priznatsi pri sortove i linii gradinski grah. Scientific Reports of the Scientific Session of Jubilee ­ 120 Years of Agriculture Science in Sadovo, IPGR "Konstantin Malkov", Sadovo-Plovdiv, Bulgaria, 21-22 y 2002, 264-267 Kertikova D, Kertikov T, Popov I (2009): Kerpo ­ a new variety of spring forage pea. J. Mountain Agric. Balkans 12: 342-348 Koeva R, Angelova S, Guteva Y (2002): Plant genetic resources and their biological potential integrated to the agricultural and ecological systems. J. Biotech. Biotech Equip. 16: 26-35 Krachunov I, Kirilov A, Ivanov K (2007): J. Mountain Agric. Balkans 10: 22-29 Kuzmova K (2002a): Kolichestvena otsenka na zimni i proletni sortove grah po stepen na ranozrelost. Scientific Reports of the Scientific Session of Jubilee ­ 120 Years of Agriculture Science in Sadovo, IPGR "Konstantin Malkov", SadovoPlovdiv, Bulgaria, 21-22 y 2002, II, 109-112 Kuzmova K (2002b): Svetovni agroklimatichni analozi s Balgariya po usloviyata za otglezhdane na graham. Scientific Reports of the Scientific Session of Jubilee ­ 120 Years of Agriculture Science in Sadovo, IPGR "Konstantin Malkov", Sadovo-Plovdiv, Bulgaria, 21-22 y 2002, II, 113-118 Ministerstvo na zemedelieto i hranite (MZH) (2008): Agrostatistika [online]. Available at www.mzgar.government.bg/ StatPazari/Agrostatistica/agrostatistica.htm (Cited 14 April 2010, verified 14 April 2010). Sofia McPhee K (2003): Dry pea production and breeding - A mini review. Food Agric. Environ. 1: 64-69 Mehandzhiev A, Mihov M, Noveva S, Rodeva R, Kosturkova G (2006): Some results from the investigation on genetic improvement of pea (Pisum sativum L). Field Crop. Studies 3: 397-403 Mihov M (1988): Povishavane produktivnostta na leshtata po selektsionen pat. Selskostopanskata Nauka 3: 27-31 Mihov M, Stoyanova M, Mehandzhiev A (2002): Njakoi rezultati pri selektsiata na proletniya grah za zarno (Pisum sativum L.). Scientific Reports of the Scientific Session of Jubilee ­ 120 Years of Agriculture Science in Sadovo, IPGR "Konstantin Malkov", Sadovo-Plovdiv, Bulgaria, 21-22 y 2002, I, 109-112 Mihov M, Mehandzhiev A, Noveva S (2003): Prinosat na ekperimentalniya mutagenez za genetichnoto podobryavane na graha. Rastenievadni Nauki 4: 325-329 Mihov M, Atanasova D, Stoyanova A, Mehandzhiev A (2005): Kristal ­ nov sort proleten furazhen grah za zarno (Pisum sativum L.). Proc. Yubileyna nauchna konferentsiya Selektsionni i tehnologichni aspekti pri proizvodstvoto i prerabotkata na soya i drugi bobovi kulturi, 276-280 Sachanski S (1988): Sastoyanie i perspektivi pri selektsiyata na furazhniya grah v Instituta po furazhite ­ Pleven, 1, 51-55 Stancheva Y (2007): Analiz na faktorite, indutsirashti neustoychivost v zemedelskoto proizvodstvo na Balgarija. [online]. Available at http://chm.moew.government.bg/SLM/ files/1-Sust_agr.pdf (Cited 14 April 2010, verified 14 April 2010). Sofia

In order to increase the efficiency of breeding process in forage pea it is needed to have a good knowledge on the combining ability of varieties used. Without it, it would be impossible to permanently upgrade and improve the available genetic variability (Koeva et al. 2002, Mehandzhiev et al. 2006). Conclusions Field pea continues to be an important crop worldwide both for food and feed and as a rotational crop with other cultures. The pea breeding programs are based on yield and yield components. Pea grain yield is a quantitative trait which is affected by many genetic and environmental factors. The availability of the genetic diversity of the Pisum germplasm provides breeders with ability to overcome many production constraints. The advanced genetic study and understanding of the pea crop and its genetics as a whole will open the opportunities for its improvement. References

Angelova S (1995): Prouchvane, sahranenie i ispolzivane na rastitelnite resursi ot furazhen grah. Rastenievadni Nauki 6: 105-107 Angelova S. (2000): Grain legume collections in Bulgaria. Report of a Working Group on Grain Legumes, Third Meeting, 41-43 Angelova S (2005): Kakvo novo pri proletniya furazhen grah. Zemedelie Plyus 3: 9-10 Angelova S (2008): Grain legume collection in Bulgaria, Report of a Working Group on Grain Legumes, Fourth Meeting, Lisbon, Portugal, 16-17 November 2007 [online]. Available at www.ecpgr.cgiar.org/workgroups/grain_legumes/Creps/ Bulg_ text.pdf (Cited 14 April 2010, verified 14 April 2010). Bioversity International, Rome Angelova S, Stoikova T (1998): Grain legume collections in Bulgaria. Report of a Working Group on Grain Legumes, Second Meeting, 1-3 October 1998, UK, 41-44 Annicchiarico P, Filippi L (2007): A field pea ideotype for organic systems of northern Italy. J. Crop Improv. 20: 193-203 Bulgarian Official Catalogue of Varieties of Agriculture and Vegetable Plant Species (2009): [online]. Available at www. iasas.government.bg/bg/catalogue-varieties2009.html (Cited 14 April 2010, verified 14 April 2010) Sofia FAOSTAT (2009): FAO Corporate Statistical Database [Online]. [1 p] Available at http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor (Cited 10 May 2010, verified 11 May 2010). FAO Corporate Statistical Database (FAOSTAT), United Nations Food and Agriculture Organization (FAO), Rome Filipov H (1997): Otglezhdane na zimen furazhen grah pri zasushlivi usloviya i poluchavane na visoki dobivi. Rastenievadni Nauki 9-10: 49-54

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Geneticko unapreenje stocnog graska (Pisum sativum) u Bugarskoj

Valentin Kosev · Ivan Pacev

Naucni institut za krmno bilje, Gen.Vladimir Vazov 89, 5800 Pleven, Bugarska Izvod: Grasak je postao znacajan usev u Bugarskoj pocetkom 20. veka. Do 1964. postojalo je jedino oplemenjivanje za krmu, sa stvorenim ozimim sortama 5 i Pleven 2. Tokom poslednjih godina, proteinski grasak je ostvario najvee poveanje setvenih povrsina (283,3%) posto se sortiment osavremenjuje svake godine. Ukupno je priznato 10 sorti stocnog graska, od cega 7 jarih i 3 ozime. Sastav bugarskih zbirki graska je veoma raznovrstan, sa genotipovima razlicitog statusa. Najvea efikasnost oplemenjivanja ostvarena je kombinacijom metoda u smesi tokom ranih generacija i izvesnih odlika pedigre metoda, sa moguim modifikacijama i ukljucivanjem mutantne varijabilnosti.

Kljucne reci: genotip, grasak, kolekcija, Pisum sativum, poboljsanje, zrnene mahunarke

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www.nsseme.com/journal.html

Genetic markers in seed testing and plant breeding

409 Molecular Genetics / Molekularna genetika Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 409-416 review article/ pregledni rad

Application of Genetic Markers in Seed Testing and Plant Breeding

Zorica Nikoli

received / primljeno: 02.03.2010. accepted / prihvaeno: 10.05.2010. © 2010 IFVC

Summary: Genetic markers have been used at Institute of Field and Vegetable Crops in Novi Sad for a number of years, both for seed quality control and for research purposes. The Laboratory for Seed Testing was the first in the former Yugoslavia to use the method of control of hybrid seed genetic purity based on enzymatic polymorphism. This paper presents the application of protein markers, isozymes, seed storage proteins and DNA markers for evaluation of seed and breeding materials of various agricultural crops in Serbia. Key words: DNA markers, isozymes, seed storage proteins

Protein Markers Genetic marker is a gene or a part of DNA having a readily recognizable phenotypic expression, which is used for identification of the entity or cell that contains it or as a probe for nucleus, chromosome or locus marking (King & Stansfield 1990). It is typically defined as a characteristic that marks a part of a genome. As specific gene products, proteins could indicate the genetic specificity of tested plant material, and therefore could be used as markers for characterization of varieties, for seed purity testing, or to resolve taxonomic relationships (Drini-Mladenovi & Konstantinov 2001, Nikoli et al. 2008). Seed Storage Proteins Seed storage proteins in cereals, such as gliadins in wheat, hordeins in barley, zeins in corn and avenins in oats are used for identification of species (Wrigley et al. 1985). Identification of species can also be performed indirectly by comparing the frequencies of protein components obtained by electrophoretic separation. Gliadins are one of the major protein fractions, which are deposited in endosperm protein bodies of grain. Gliadins represent efficient and reliable genetic markers in wheat genetic study.

Z. Nikoli ) ( Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad, Serbia e-mail: [email protected]

Composition of gliadin components for each cultivar was specific (uki et al. 2005). Analysis of 10 Kragujevac wheat cultivars identified only 3 alleles (a, b, k) at the Gli-A1 locus, while 5 alleles were identified in cultivars from Novi Sad (Knezevi 1992). The pronounced polymorphism of the reserve proteins in the cereal species from the family Gramineae may be used for the study of species identity, heterogeneity and origin as well as in the breeding of new cultivars (Nikoli et al. 2007a) (Fig. 1). Composition of high-molecular-weight glutenin subunits (HMW) affects bread making quality. Eighteen bread wheat cultivars developed at Institute of Field and Vegetable Crops in Novi Sad were analysed for two main x-type alleles which code for HMW glutenins 2 and 5, and two main type alleles which code for 10 and 12 HMW glutenin subunits, present at Glu-D1 locus. Among the analysed cultivars, 55.6% expressed the presence of 1Dx5 and 1Dy10 alleles at Glu--D1 locus, which are typically associated with high dough strength and good bread making quality (Vapa et al. 1995, 2003). Together with glutenins, gliadins play an important role for bread making quality. Five gliadin blocks encoded by different alleles at Gli-D1 locus and four gliadin blocks different at Gli-A1 locus were clearly expressed and identified. Variability of determined block components

This research results from project 20081: Stvaranje genotipova uljane repice (Brassica napus L.) za ishranu i industrijsku preradu (20082010) financed by Ministry of Science and Technological Development of the Republic of Serbia

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indicates the existing polymorphisms of gliadins alleles (Knezevi et al. 2007). The cultivars developed in the first 20 years of intensive wheat breeding in Serbia showed high variability at the Glu-B1 locus, similar frequencies of different GSs (N, 1, 2*) at the Glu-A1 locus and the prevalence of GS 2 + 12 over GS 5 + 10 at the Glu-D1 locus. Significant changes occurred in the combinations of HMW GSs as a consequence of 40 years of breeding. The increase in gluten structure stability and appropriate combinations of high molecular weight glutenin subunits have contributed to the improvement of other quality indicators (Hristov et al. 2010).

Seed storage proteins are reliable markers in studying domestication and dispersal of bean cultivars as well as in analysing phylogenetic relationships among species in the genus Phaseolus. Most of the bean cultivars grown in our country have the S type phaseolin, which indicates that Central American germplasm has been used for the development of new cultivars (Nikoli et al. 2007b) (Fig. 2). Considering the similarities in climatic conditions between Serbia and Bulgaria, i.e. high temperatures and irregular rainfall, it seems reasonable to accept the explanation that the cultivars with S type phaseolin are well adapted to the prevailing environmental conditions (Gencev et al. 2002).

Figure 1. Identification of barley cultivars (samples 1-6) based on seed storage protein polymorphism (Nikoli et al. 2007a) Slika 1. Identifikacija sorti jecma (uzorci 1-6) na osnovu polimorfizma rezervnih proteina semena

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2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 2. Different types of phazeolin obtained by SDS PAGE electrophoresis:1 - protein marker (170-11kDa), 2-3 - S type phazeolin control, 4-5 - C type control, 6,7 S type control, 8-11 - C-20, 12-15 - Aster, 16-19 - Ludogorje, 20-23 - Jovandeka (Nikoli et al. 2007b) Slika 2. SDS PAGE elektroforetska analiza fazeolina: 1- proteinski marker (170-11kDa), 2-3 ­S tip fazeolina (kontrola), 4-5 ­C tip fazeolina (kontrola), 6,7 S tip fazeolina (kontrola), 8-11 - C-20, 12-15 - Aster, 16-19 Ludogorje, 20-23 - Jovandeka

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Protein seed analysis confirmed low level of genetic diversity in soybean genotypes. In the plant with a narrow genetic base in their pool, such as soybean, protein markers may not be sufficient for characterization and study of genetic diversity (Nikoli et al. 2005). The utility of embrio salt soluble proteins to characterize maize inbred lines, validate pedigree and show association among inbred lines, was evaluated using a set of inbred lines (Drini et al. 1996). Clustering of hybrids based on embryo salt-soluble protein markers showed good agreement with their pedigree data, because hybrids with similar parental components were joined together in smaller groups. Embryo salt-soluble protein markers are not a marker system of choice for assessing genetic relatedness of maize hybrids because of a relatively low number of protein fractions obtained, low polymorphism and unknown mode of inheritance (Eri et al. 2003). Based on SDS ­ PAGE electrophoresis of different pepper varieties (Capsicum annuum L.) it was found that the seed protein profile could be useful in the study of phylogenetic relationships. The analysis of soluble seed proteins showed that all studied genotypes had a specific protein pattern (Zecevi et al. 2002). Isozymes Izozymes are reliable indicators of genetic purity of various plant species. They are especially suitable for solving problems in seed production and processing, such as inadvertent mixing of seed of hybrids and lines, incomplete pollination in corn, etc. (Geri et al. 1989).

other species, genetic mutations, genetic drift, selection pressure, etc. Corn is the plant species with the largest number of genetic markers on the protein level. Isozymes have been used long in various corn studies: assessment of diversity and variability of breeding material (Zlokolica et al. 1995, Nikoli et al. 1999, Nikoli et al. 2004), identification of combining ability potential in corn lines (Zlokolica et al. 1997), etc. Isozyme polymorphism has been found to be suitable for assessment of genetic uniformity and/or variability of sunflower inbred lines (Zlokolica et al. 1996a) and for identification and registration of lines and hybrids (Carrera & Poverene 1995). Long-term studies of sunflower breeding materials have shown that the following enzymic systems are polymorphic in most lines and hybrids: phosphohexose isomerase (PHI), phosphogluco dehydrogenase (PGD), malate dehydrogenase (MDH) and phosphoglucomutase (PGM) (Fig. 3). The electrophoretic method allows extraction of storage proteins from sunflower seeds and making of helianthin electrophoregrams. The comparative analysis of genetic purity level of the sunflower hybrids have shown that the methods of electrophoresis of isozymes and seed storage proteins were in agreement, but due to low polymorphism levels seed storage proteins and isozymes cannot be used for genetic identification of sunflower hybrids (Nikoli et al. 2008). An analysis of sugar beet haploids obtained by gynogenesis and dihaploid lines obtained by colchicine treatment has indicated that the haploid genome from an unfertilized egg cell is stable. Changes in the loci for phosphoglucomutase

a

b

Figure 3. PHI (a) and PGD (b) polymorphisms detected in the sunflower cultivar Monarch (Nikoli et al. 2008) Slika 3. Polimorfizam PHI (a) i PGD (b) naen u hibridu suncokreta Monarch

In seed production, maintenance of genetic purity requires continual monitoring of seed quality. Here it is important to add that each crop (corn, soybean, sunflower, etc.) has its specific features and requirements with regards to field history, purity standards and production technology. In the course of time, a cultivar may lose its identity due to various reasons, such as mechanical mixing with other seeds, natural crossing with

(Pgm2) and malate dehydrogenase (Mdh1) occurred in response to colchicine application (Zlokolica et al. 1994) or in response to Ti plasmid transformation of sugar beet calli (Zlokolica et al. 1996b). High degrees of isozymic polymorphism have been found during identification and genetic characterization of vegetable crops such as cabbage, cauliflower, cucumber, carrot, tomato

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and pepper (Zlokolica et al. 1996c). Different phenotypic profiles within a cultivar are an indication of seed non-uniformity, i.e. of genetic impurity. An analysis of cabbage breeding material has indicated uniformity in some lines and presence of different phenotypic profiles in others (Nikoli et al. 2007c) (Fig. 4 and Fig. 5).

The marker assisted selection (MAS) permits simultaneous improvement and acceleration of breeding process. Implementation of MAS in conventional breeding programs could allow assessment of the genetic potential of specific genotypes prior to their phenotypic evaluation.

Figure 4. PGM zymogram of the cultivar NS-14 (Nikoli et al. 2007c) Slika 4. PGM zimogram sorte kupusa NS 14

Figure 5. PGM zymogram of the cabbage line (Nikoli et al. 2007c) Slika 5. PGM zimogram linije kupusa

The expression of isozymic loci is codominant and it is generally independent of environmental factors. Most isozymes come to expression in seed or in early stages of germination, which is a major advantage of these markers over field or greenhouse tests which require a considerably longer time. DNA Markers Used in Agricultural Research The numerous shortcomings of the morphological and biochemical markers found in studies of various plant species encouraged the development of molecular markers. Molecular markers have many advantages compared with morphological markers, robustness to environmental change, nearly unlimited number and relative ease and rapidity of data collection (Lombard et al. 2000). Molecular markers are not influenced by environmental factors and are also fast, efficient and more sensitive than field testing for detection of large numbers of distinct differences between genotypes at DNA level (Smith & Smith 1992). They are applicable in the process of identification and approval of cultivars, determination of genomic differences between cultivars and genetic diversity assessment. During registration of cultivars, molecular markers provide additional information which helps distinguish between similar cultivars. Each cultivar or hybrid should have an ID card that would include descriptors of morphological traits and specific combinations of polymorphic molecular markers (Nikoli 2002) (Fig. 6).

The random amplified polymorphic DNA (RAPD) marker system has been used in many different applications involving the detection of DNA sequence polymorphism, but most often in construction of linkage maps and in bulk segregant analysis for identification of markers linked to genes of interest, such as gene(s) for resistance to sugar beet cyst nematode (Heterodera schachtii Schmidt) (Nagl et al. 2007). The study of actual rapeseed breeding material of Southeastern Europe has shown that PCR-based techniques such as RAPD can be successfully used for detecting genetic variability in rapeseed. The simplicity of the technique makes it particularly suitable for breeding programs in which a large number of lines need to be analyzed (Marjanovi-Jeromela et al. 2009). Genetic diversity studies using DNA finger printing techniques have become simple and efficient in detection of sufficient polymorphisms in various crop species including maize (Pejic et al 1998). The main goal of the study conducted by Bauer et al. (2007) was to assess temporal changes in genetic diversity over the past four decades among ZP maize hybrids within the largest planting area in Serbia. Patterns of RAPD markers were unique for each studied genotype, but there was no significant change in genetic variability of hybrids throughout the periods. Genetic distances of ten maize inbred lines of different origin, based on protein and RAPD markers, were similar and in concurrence with the date on the origin of maize inbred lines and also with grain yield heterosis of their crosses (Srdi et al. 2007).

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M 1 2

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Figure 6. RAPD polymorphism in 16 soybean genotypes obtained with primer no.12 (Nikoli 2002) Slika 6. RAPD polimorfizam 16 genotipova soje sa prajmerom broj 12 Drini et al. (2002) reported that simple sequence repeats (SSR) markers provide an effective method for predicting hybrid performance and heterosis. SSR analyses have been used in order to develop genetic fingerprints for their characterization, identification and classification, as well as for estimation of their genetic diversity of local populations from Maize Research Institute gene bank collection. Restriction Fragment Length Polymorphisms (RFLP) and RAPD analysis of some of the local populations for identification of variability and duplicate accessions were performed and the results between different marker techniques were consistent (Ignjatovi-Mici et al. 2003, Ignjatovi-Mici et al. 2008). 1 2 3 4 5 6 7 8 lar markers can be successfully applied to investigate diversity of sunflower inbred lines, wild sunflower species, as well as to identify interspecies hybrids, for some important agronomic traits such as drought tolerance and resistance to downy mildew (Safti-Pankovi 2007). The results on genetic variability between 15 populations of each H. giganteus and H. maximiliani, obtained with SSR markers, confirm clustering pattern between examined populations obtained after analysis of 30 morphological traits (SaftiPankovi et al. 2004). The inheritance of the reaction of sunflower to downy mildew race 730 was investigated by Pankovi et al. (2007). The mapping data indicate that several dominant markers and two CAPS 9 10 11 12 13 14 15 16

Figure 7. SSR polymorphism detected by primer Satt 534 in 16 soybean genotypes (Nikoli et al. 2006) Slika 7. SSR polimorfizam 16 genotipova soje sa prajmerom Satt 534

For genotypes with a relatively narrow genetic basis, such as soybean, DNA markers offer possibilities to differentiate related genotypes as well as to identify those possessing specific germplasm. In the case of incomplete data on origin, molecular markers may provide data on mutual relationships between genotypes (Nikoli et al. 1998, Nikoli et al. 2006) (Fig. 7). In the fields of plant breeding, molecular markers are powerful tools in search for new sources of variation and the analysis of genetic factors that control quantitative traits. Molecu-

markers, developed, completely co-segregate with the Pl6 gene conferring resistance to race 730, the most abundant race in this region of north Serbia. CAPS markers will facilitate efficient marker-assisted selection for sunflower resistance to downy mildew race 730. Correlation between SSR based genetic distance of new sunflower inbred lines and heterosis for six agronomic traits indicated that better results are obtained if hybrid combinations for each tester and each trait are analyzed separately (Gvozdenovi et al. 2009).

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fferent selection cycles obtained by protein markers. Maize Gen. Coop. Newslett. 77: 7-8 Genchev D, Ivanov P, Ivanova I (2002): Phaseolin seed protein variability in Bulgarian dry bean (Phaseolus vulgaris L.) cultivars. Res. Commun. of U.S.B. Branch Dobrich 4: 44-51 Geri I, Zlokolica M, Geri C (1989): Primena izoenzima u kontroli genetske cistoe semena poljoprivrednog bilja. Poljoprivredne aktualnosti 3-4: 555-564 Gvozdenovi S, Safti Pankovi D, Joci S, Radi V (2009): Correlation between heterosis and genetic distance based on SSR markers in sunflower (Helianthus annuus L.). J. Agric. Sci. 54: 1-10 Hristov N, Mladenov N, uri V, Kondi-Spika A, Marjanovi-Jeromela A (2010): Improvement of wheat quality in cultivars released in Serbia during the 20th Century. Cereal. Res. Commun. 38: 111­121 Ignjatovi-Mici D, Mladenovi Drini S, Nikoli A, Lazi-Janci V (2008): SSR analysis for genetic structure and diversity determination of maize local populations from former Yugoslavia territories. Russ. J. Gen. 44: 1317­132 Ignjatovi-Mici D, Cori T, Kovacevi D, Markovi K, LaziJanci V (2003): RFLP and RAPD Analysis of maize (Zea mays L.) local populations for identification of variability and duplicate accessions. Maydica 48: 153­159 King R C, Stansfield W D (1990): A Dictionary of Genetics. 4th Ed. Oxford University Press, New York Knezevi D, Novoselskya-Dragovich A Yu (2007): Polimorphism of Gli-D 1 alleles of Kragujevac's winter wheat cultivars (Triticum aestivum L). Genet. 39: 273-282 Knezevi D (1992): Geneticka varijabilnost rezervnih proteina psenice (Triticum aestivum L.). Doktorska disertacija. Univerzitet u Novom Sadu, Prirodno-matematicki fakultet, Novi Sad Kobiljski B, Denci S, Pilipovi J (2006): Molecular screening of domestic germplasm for allelic variants at the dwarfing gene Rht8 locus in wheat. Genet. 38: 67-74 Kondi-Spika A, Kobiljski B, Denci S, Mladenov N, Hristov N, Kacavenda D, Brbakli Lj (2008): DNA Fingerprinting of wheat (Triticum aestivum L.) varieties using microsatellite markers. Conventional and molecular breeding of field and vegetable crops (Breeding 08), Conference proceedings, Nov. 24-27, Novi Sad, 149-152 Lombard V, Baril CP, Dubreuil P, Blouet F, Zhang D (2000): Genetic relationships and fingerprinting of rapeseed cultivars by AFLP: Consequences for varietal registration. Crop Sci. 40: 1417­1425 Marjanovi-Jeromela A, Kondi-Spika A, Safti-Pankovi D, Marinkovi R, Hristov N (2009): Phenotypic and molecular evaluation of genetic diversity of rapeseed (Brassica napus L.) genotypes. Afr. J. Biotechnol. 8: 4835-4844 Nagl N, Weiland J, Lewellen R (2007): Detection of DNA polymorphism in sugar beet bulks by SRAP and RAPD markers. J. Biotech. 31: 1 Nikoli Z, Zlokolica M (1998): Soybean identification based on isozyme and RAPD markers. Proc of II Balkan Symposium on Field Crops, Novi Sad, Serbia, 403-407 Nikoli Z (1999): Izoenzimska varijabilnost samooplodnih linija kukuruza (Zea mays L.). Magistarski rad, Univerzitet u Novom Sadu, Poljoprivredni fakultet, Novi Sad Nikoli Z (2002): Identifikacija genotipova soje (Glycine max Merrill L.) na osnovu morfoloskih, biohemijskih i molekularnih markera. Doktorska disertacija, Univerzitet u Beogradu, Bioloski fakultet, Beograd Nikoli Z, Zlokolica M, Milosevi M, Vujakovi M (2004): Genetic variability of maize breeding material (Zea mays L.) Proc. Nat. Sci. 106: 21-29 Nikoli A, Srebri M, Drini-Mladenovi S (2005): Genetic similarity of soybean genotypes revealed by seed protein. Sel. Semen. 11: 57-61

Potential uses of microsatellite markers in molecular evaluation of bread making quality were tested in wheat genotypes that were analysed with 3 microsatellites linked to previously mapped QTLs for loaf volume and Hagberg falling number on chromosome 3A. A significant association was found of a specific allele at the GWM674 locus with Hagberg falling number in wheat (Obreht et al. 2006). The microsatellites are very effective molecular markers for the assessment of genetic diversity in wheat. Combined with multiplex PCR, microsatellite markers permit the fast and high-throughput fingerprinting of large number of genotypes (Kondi-Spika et al. 2008). They are easy to use and automate, mapped to specific genomic location and relatively inexpensive. Using STSs and SSR markers, the most important major height reducing genes, Rht-B1b, RhtD1b and Rht8 were evaluated in wheat genetic core collection (Kobiljski et al. 2006, TosoviMari et al. 2008). Conclusions Genetic markers can be used routinely in crop breeding programs, for genetic diversity analysis, cultivar identification, phylogenetic analysis, characterization of genetic resources and association with agronomic traits. Identification of genes and molecular markers underlying these agronomic traits will help accelerate the breeding process and lead to varieties with improved quality, yield, tolerance to unfavourable environmental conditions and resistance to diseases. References

Bauer I, Mladenovi Drini S, Drini G, Ignjatovi Mici D (2007): Assessing temporal changes in genetic diversity of maize hybrids using RAPD markers. Cereal Res. Commun. 5: 1563-1571 Carrera A, Poverene M (1995): Isozyme variation in Helianthus petiolaris and sunflower, H.annuus. Euphytica 81: 251-257 Drini Mladenovi S, Coric T, Konstantinov K (1996): Embryo salt soluble proteins as markers in research on the biological background of heterotic gene expression. Maize Gen. Coop. Newslett. 70: 71-72 Drini Mladenovi S, Konstantinov K (2001): Genetically characterization and simple purity checking of maize seed by protein markers. Sel. Semen. 8: 1-8 Drini Mladenovi S, Trifunovi S, Drini G, Konstantinov K (2002): Genetic divergence and its correlation to heterosis in maize as revealed by SSR-based markers. Maydica 47: 1-8 uki N, Mati G, Konjevi R (2005): Biochemical analysis of gliadins of wheat Triticum durum. Kragujevac J. Sci. 27: 131-138 Eri I, Drini Mladenovi S, Konstantinov K, Stankovi G (2003): Genetic diversity among ZP maize hybrids from di-

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Nikoli Z, Milosevi M, Vujakovi M, Taski-Ajdukovi K, Radovi R (2006): Soybean cultivar identification using microsatellite DNA markers. Biotechnology 2006, University of South Ceske Bujedovica, Faculty of Agriculture, Check Republic, 691-693 Nikoli Z, Milosevi M, Taski-Ajdukovi K, Vujakovi M (2007a): Genetic purity as a component of seeds quality. Proc 42nd Croatian and 2nd International Symposium on Agriculture, Opatija, Croatia, 245-248 Nikoli Z, Vasi M, Milosevi M, Vujakovi M, GvozdanoviVarga J (2007b): Characterization of common bean (Phaseolus vulagris L.) varieties from Serbia. BIC, Annual Report 50: 27-28 Nikoli Z, Milosevi M, Cervenski J, Gvozdenovi (2007c): Izoenzimska analiza genotipova kupusa (Brassica oleracea var. capitata L. ). J. Sci. Agric. Res. 68: 77-83 Nikoli Z, Vujakovi M, Jevti A (2008): Genetic purity of sunflower hybrids determined on the basis of isozymes and seed storage proteins. Helia 31: 47-54 Obreht D, Kobiljski B, Denci S, Djan M, Vapa Lj (2006): Potential uses of microsatellites in marker-assisted selection for improved bread-making quality in wheat. Cereal Res. Commun. 34: 895-902 Pankovi D, Radovanovi N, Joci S, Satovic Z, Skori D (2007): Development of co-dominant amplified polymorphic sequence markers for resistance of sunflower to downy mildew race 730. Plant Breed. 126: 440-444 Pejic I, Ajmon-Marson P, Morgante M, Kozumplick V, Castiglioni P, Tarmino G, Motto M (1998): Comparative analysis of genetic similarity among maize inbred lines detected by RFLPs, RAPDs, SSRs, and AFLPs. Theor. Appl. Genet. 97: 1248-1255 Safti-Pankovi D, Atlagi J, Miljanovi T, Radovanovi N (2004): Morphological and molecular variability of Helianthus giganteus L. and Helianthus maximiliani Sch. species. Genet. 7: 121-130 Safti-Pankovi D (2007): The application of molecular markers in sunflower breeding. Genet. 39: 1-11 Smith J S C, Smith O S (1992): Fingerprinting crop varieties. Adv. Agron. 47: 85-140 Srdi J, Mladenovi Drini S, Paji Z, Filipovi M (2007): Characterization of maize inbred lines based on molecular markers, heterosis and pedigree data. Genet. 39: 355-363 Tosovi-Mari B, Kobiljski B, Obreht D, Vapa Lj (2008): Evaluation of wheat Rht genes using molecular markers. Genet. 40: 31-38 Vapa Lj, Denci S, Soltes-Rak E, Kevresan S (1995): Genetic variation in Glu-1 loci and bread making quality. Cereal Res. Commun. 23: 161-166 Vapa Lj, Obreht D, Kobiljski B, Davidovi M (2003): Molecular characterisation of glutenin alleles at the Glu-D1 locus. Proc. Nat. Sci. 105: 35-41 Zecevi B, Stevanovi D, Mladenovi-Drini S, Konstantinov K, Jakovjevi T (2002): Seed proteins as genetic markers in the study of phylogenetic relations in pepper. Acta Hort. 579: 113-116 Zlokolica, M., S., Mezei, Kovacev, L. (1994): Genetic variability in haploids and induced dihaploids of sugarbeet (Beta vulgaris L.). J. Sci. Agric. Res. 56: 3-9 Zlokolica M, Milosevi M, Geri I (1995): Genetic variability of Yugoslav corn collections. Genet. 27: 63-83 Zlokolica M, Nikoli Z, Turkav S, Milosevi M, Graovac M, Skori D (1996a): Isozymic variability of selfpolinated sunflower (Helianthus annus L.) lines. Helia 24: 113-130 Zlokolica M, Mezei S, Kovacev L, Nikoli Z, Milosevi M, Kupresanin N (1996b): Genetic variability of sugarbeet (Beta vulgaris L.) calli following inoculation with Agrobacterium tumefaciens. Genet. 28: 97-103

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Primena genetickih markera u kontroli kvaliteta semena i oplemenjivanju biljaka

Zorica Nikoli Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija

Izvod: Geneticki markeri se dugi niz godina koriste u Institutu za ratarstvo i povrtarstvo u Novom Sadu u kontroli kvaliteta semena i u istrazivacke svrhe. Laboratorija za ispitivanje semena je prva u bivsoj Jugoslaviji uvela metod kontrole geneticke cistoe semena zasnovan na polimorfizmu enzima. U radu je dat pregled primene proteinskih markera, izoenzima i rezervnih proteina semena, kao i DNK markera u oceni kvaliteta semena i materijala za oplemenjivanje razlicitih biljnih vrsta u Srbiji. Kljucne reci: DNK markeri, izoenzimi, rezervni proteini semena

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 409-416

www.nsseme.com/journal.html

Geneticki markeri i oplemenjivanje visegodisnjih leguminoza

417 Molekularna genetika / Molecular Genetics

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 417-424 pregledni rad / review article

Primena genetickih markera u oplemenjivanju visegodisnjih leguminoza

Gordana Surlan-Momirovi Slobodan Kati Sanja Vasiljevi Zorica Nikoli Gordana Brankovi Irena Cali Dragan Mili Aleksandar Miki

primljeno / received: 14.04.2010. prihvaeno / accepted: 21.05.2010. © 2010 IFVC

Izvod: Oplemenjivanje visegodisnjih leguminoza za mnoge agronomski znacajne osobine kao sto su prinos semena, perzistentnost, dugovecnost, otpornost na bolesti i stetocine, otpornost na limitirajue abioticke uslove i pojavu poliploidije efikasnije je i preciznije ako se u radu primenjuju i geneticki markeri. Polimorfizam genotipova zasnovan na profilima izoenzima moze da potceni ukupan nivo geneticke varijanse jer se odnosi na kodirajue regione DNK koji su bili konzervirani kroz evoluciju da bi se ocuvala funkcija enzima. Kompletna pokrivenost genoma moze se ostvariti samo primenom pokazatelja molekularne varijabilnosti (DNK polimorfizam) tj. molekularnim markerima. Pored toga, molekularni markeri ne zavise od uslova spoljasnje sredine i mogu se detektovati u svim stadijumima fazama razvia biljaka. Glavni aspekti primene genetickih markera u oplemenjivanju visegodisnjh leguminoza se odnose na: karakterizaciju germplazme, mapiranje vezanih gena, QTL analizu, selekciju uz pomo markera (MAS), identifikaciju sorata i zastitu prava oplemenjivaca. Kljucne reci: geneticki markeri, oplemenjivanje, visegodisnje leguminoze

Uvod Korisenje molekularno-genetickih tehnika zajedno sa konvencionalnim oplemenjivanjem povealo je efikasnost i preciznost oplemenjivanja mnogih poljoprivrednih biljaka (Collard & Mackill 2008). Trenutno sve komercijalno dostupne sorte visegodisnjih krmnih leguminoza (lucerka, crvena detelina, bela deteline, zuti zvezdan) jesu sintetici, stvoreni putem meuukrstanja odabranih roditelja tokom cetiri do sest generacija (Woodfield & Brummer 2001). S tim u vezi su metode oplemenjivanja visegodisnjih krmnih leguminoza zasnovane iskljucivo na metodama selekcije u polusrodstvu, ukljucujui intercrossing odabranih roditelja u cilju proizvodnje sintetickih sorti (Hill et al. 1988). Prisustvo heterozisa kod krmnih leguminoza dovelo je do znacajne diskusije o mogunostima razvoja hibridnih sorti. Primena hibridnog sistema oplemenjivanja zahteva poboljsanje najmanje dve nezavisne i komplemenG. Surlan-Momirovi ) ( G. Brankovi I. Cali Poljoprivredni fakultet Univerziteta u Beogradu, Nemanjina 6, 11080 Beograd - Zemun, Srbija e-mail: [email protected] S. Kati S. Vasiljevi Z. Nikoli D. Mili A. Miki Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija

tarne populacije koje u kombinaciji proizvode heterozis (Sengupta-Gopalan et al. 2007, Kati i sar. 2010). Da bi se postigla hibridna snaga tj bujnost krmnih leguminoza kao sto je lucerka neophodne su nove strategije oplemenjivanja u kojima bi bila naglasena karakterizacija i evaluacija genetickih resursa, a posebno identifikovanje heteroticnih grupa, koristei i klasicne i molekularne metode (Woodfield & Brummer 2001). Oplemenjivacki rad na visegodisnjim krmnim leguminozama, kako u svetu tako i u Republici Srbiji, zapoceo je relativno kasno, posle Drugog svetskog rata (Katic et al. 2008) Oplemenjivanje lucerke u Institutu za ratarstvo i povrtarstvo u Novom Sadu, saglasno postavljenim ciljevima vrsilo se u nekoliko ciklusa (Kati i sar. 2002). Za vise od 50 godina rada na oplemenjivanju lucerke u Odeljenju za krmno bilje Instituta za ratarstvo i povrtarstvo u Novom Sadu, stvoreno je 14 sorti lucerke. Aktuelni ciljevi u oplemenjivanju lucerke u Institutu fokusirani su na stvaranje sorti visoke produkcije biomase (zelene krme i sena), namenjenih za razlicite sisteme iskorisavanja sa cetiri i pet otkosa godisnje, kao i proucavanje dugovecnosti ­ trajnosti ovih sorti (Kati i sar. 2007). Ispitivanje parametara kvaliteta posebno hranljive vrednosti novih sorti, kao i njihove svarljivosti, novi je pravac rada na parametrima kvaliteta u Institutu.

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Surlan-Momirovi G i sar.

Intenzivniji rad na kolekcionisanju crvene deteline (Trifolium pretense L.), druge po znacaju visegodisnje krmne leguminoze u Institutu za ratarstvo i povrtarstvo u Novom Sadu zapocet je pre 20 godina, kako bi se obezbedila sto sira geneticka varijabilnost, neophodna za pocetak oplemenjivackog rada na ovoj biljnoj vrsti (Tomi i sar. 2010). Primenom metoda selekcije u polusrodstvu (masovna selekcija, rekurentna fenotipska selekcija) do sada su stvorene tri sorte: Kolubara, Una i Avala. Osnovni cilj u oplemenjivanju crvene deteline je stvaranje dugovecnijih sorti (do tri godine zivota) koje daju visok prinos kvalitetne krme i koje su tolerantne na ekonomski znacajnije bolesti (Vasiljevi et al. 2005). Za uspeh u oplemenjivackom radu osim pravilno odabranih metoda oplemenjivanja (Vasiljevi i sar. 2003) veoma je vazno sakupiti materijal sa sto veom genetickom varijabilnosu (Vasiljevi i sar. 2001). U skorije vreme zapoceto je izucavanje geneticke varijabilnosti kolekcionisanog materijala crvene deteline posredstvom polimorfizma izoenzima (Nikolic et al. 2010), a saglasno utvrenoj metodologiji i stecenom iskustvu stranih autora (Yu et al. 2001, Mosjidis & Klingler 2006). Aspekti upotrebe genetickih markera u oplemenjivanju visegodisnjih leguminoza Karakterizacija germplazme Proteinski i molekularni markeri mogu da pruze brzu procenu genetickog diverziteta (Karp et al. 1996, Powell et al. 1996, Mueller & Wolfenbarger 1999). Dobro analizirani geneticki resursi visegodisnjih leguminoza, na molekularnom nivou, mogu da unaprede oplemenjivacki proces i doprinesu ocuvanju i obnovi biodiverziteta. Polimorfizam genotipova zasnovan na profilima izoenzima moze da potceni ukupan nivo geneticke varijanse, jer se odnosi na kodirajue regione DNK koji su bili konzervirani kroz evoluciju da bi se ocuvala funkcija enzima (Gottlieb et al. 1981). Kompletna pokrivenost genoma moze da se ostvari samo primenom pokazatelja molekularne varijabilnosti (DNK polimorfizam) tj. molekularnim markerima (Galovi et al. 2006). Pored toga, molekularni markeri ne zavise od uslova spoljne sredine i mogu se detektovati u svim stadijumima razvoja biljaka. Tehnike molekularnih markera se ubrzano razvijaju i postoji veliki izbor razlicitih metoda, koje mogu biti apsolutni pokazatelj distanci, stabilnosti i slicnosti razlicitih genotipova ako se pravilno primene (Zlokolica et al. 1999).

Tehnike molekularnih markera (RAPD, AFLP, SSR, RFLP) su korisene za karakterizaciju genetickog diverziteta i genetickih odnosa germplazme gajene lucerke i divljih srodnika u cilju identifikacije najreprezentativnijih populacija odreenog regiona pruzajui mogunost za eliminiciju genotipova duplikata iz kolekcije germplazme (Brummer et al. 1991, Skinner 2000, Yu & Pauls 1993, Jenczewski et al. 1999, Torricelli et al. 2000, Zacardelli et al. 2003, Kölliker et al. 2003). Nasumicno umnozena polimorfna DNK (RAPD) su dominantni markeri koji utvruju polimorfizam DNK primenom kratkih arbitrarnih prajmera. Koristi i jedarnu i citoplazmatsku DNK kao templet za umnozavanje. Korisni su za istrazivanja koja ukljucuju veliki broj polimorfnih lokusa kao sto su mape vezanosti, ka i za pozicioniranje gena (Michelmore et al. 1992). RAPD markeri su koriseni za utvrivanje nivoa geneticke varijabilnosti i geneticke slicnosti 16 elitnih roditelja crvene deteline organizovanih u cetiri podgrupe od po cetiri roditelja (Campos de Quiroz & Ortega Klose 2001), a nize vrednosti polimorfnih lokusa i prosecne heterozigotnosti utvrene su u podgrupi roditelja selekcionisanih za otpornost na nematode stabljike. Grupni uzorci 20 jedinki po sorti Trifolium pratense koriste se kada se zele dobiti RAPD profili sa uklonjenom intrasortnom varijansom (Kongkiatngam et al. 1996), 7 jedinki za Medicago sativa (Yu & Pauls, 1993), 30 jedinki za Lolium perenne (Sweeney & Danneberger 1994). Kongkiatngam et al. (1996) su preporucili da se koriste klijanci crvene deteline umesto listova za DNK ekstrakciju grupnih uzoraka jer je potrebna mala kolicina DNK za test. Utvreno je da manji broj RAPD markera pokazuju kodominantnost i u slucaju da se to zanemari svaka traka markera se oznacava kao razliciti lokus umesto kao razliciti aleli istog lokusa, sto moze dovesti do precenjivanja broja polimorfnih lokusa i potcenjivanja prosecnog broja alela po lokusu (Kongkiatngam et al. 1995). Polimorfizam duzine umnozenih fragmenata (AFLP) su dominantni markeri kao i RAPD ali su tehnicki bolji u sposobnosti da razlikuju homozigote od heterozigota, kao i da detektuju veliki broj lokusa u pojedinacnom testu. Koriste se za svrhe QTL analize na osnovu genetickih mapa vezanih gena i dobre rezultate su pokazali kod crvene deteline za mogunost primene selekcije uz pomo markera (MAS) za perzistentnost (Herrman et al. 2007). Umnozeni jednostavni ponovci (SSR) cine kratke tandemski ponovljene sekvence od 1-6 bp. Polimorfizam je zasnovan na broju

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ponovaka odnosno ukupnoj duzini sekvence. Predstavljaju kodominantne markere i imaju primenu kod visegodisnjih leguminoza za proucavanje genetickog diverziteta, QTL mapiranje i sortnu identifikaciju. Polimorfizam duzine restrikcionih fragmenata (RFLP) su kodominantni markeri koji se detektuju preko elektroforeze i radioaktivnih proba (Southern blotting tehnike). Prikladni su za proucavanje geneticke varijabilnosti hloroplastne DNK (Milligan 1991). Nedostaci vezani za primenu ovih markera su dugotrajnost, poveani troskovi i slaba dostupnost specificnih proba. Kod crvene deteline je utvren visok nivo geneticke varijabilnosti hloroplastne DNK (Milligan 1991) na populacionom nivou primenom RFLP. Ulloa et al. (2003) su proucavali geneticki diverzitet 12 cileanskih populacija crvene deteline i osam sorata poreklom iz Cilea, Argentine, Urugvaja i Svajcarske radi utvrivanja geneticke srodnosti. Herrmann et al. (2005) su sproveli obimno istrazivanje genetickog diverziteta 120 populacija crvene deteline (svajcarskih divljih populacija, svajcarskih ekotipova, "Mattenklee" sorata, "Mattenklee" ekotipova, holandskih divljih populacija i holandskih ekotipova). Odreivanje geneticke divergentnosti, odnosno slicnosti veoma je znacajno, kako zbog ispitivanja potencijala za heterozis roditeljskih parova, tako i zbog mogunosti utvrivanja inbriding depresije u toku selekcije. Identifikacija heteroticnih grupa je, nazalost, bila gotovo bez paznje kod krmnih leguminoza. Kidwell et al. (1994) su pokazali da se M. sativa subsp. falcata i u manjoj meri peruanska germplazma geneticki razlikuju u odnosu na subsp. sativa pul germplazmu. Riday & Brummer (1999) su potvrdili da sativa x falcata interpopulacioni hibridi imaju bolje performanse u odnosu na oba unutar populaciona ukrstanja, dok su Ray et al. (1999) izvestili o postojanju heterozisa izmeu nedormantnih i dormantnih izvora germplazme lucerke. Takoe, M. sativa subsp. falcata je identifikovana kao podvrsta koja pokazuje heterozis u ukrstanjima sa elitnim M. sativa subsp. sativa oplemenjivackim materijalom (Riday & Brummer 2002a, Riday & Brummer 2002b; Riday et al. 2002, Riday et al. 2003). Ispitivanja Kidwell et al. (1999) i Riday et al. (2003) su interesantni primeri korisenja molekularnih markera u proceni geneticke distance izmeu roditelja, sto bi moglo biti vazno pri odabiru adekvatnih roditeljskih genotipova pri stvaranju novih sorti. Kidwell et al. (1999) su stvorili sinteticke populacije korisenjem razlicitog broja roditelja primenjujui RFLP markere u proceni genetickih razlika izmeu roditelja. Meutim,

potomstva iz ukrstanja geneticki razlicitih roditelja nisu dosledno imala visi prinos u odnosu na potomstva iz ukrstanja geneticki slicnih roditelja. Mogui razlozi su ravnoteza vezanih gena u roditeljskoj populaciji ili nemogunost da se pomou korisenih markera identifikuju regioni genoma koji uticu na prinos. Riday et al. (2003) su kod jednostrukih hibrida sativa-sativa, sativa-falcata i falcata-falcata proucavali heterozis za prinos krme. Procena geneticke udaljenosti izmeu genoma M. sativa i M. falcata raena je uz pomo AFLP i SSR markera, a posto nije pronaena znacajna korelacija izmeu geneticke udaljenosti sa heterozisom i specificnih kombinacionih sposobnosti, zakljuceno je da neutralni i random molekularni markeri nisu dobri za predvianje heterozisa kod lucerke. Osim za procenu heterozisa, molekularni markeri su koriseni i u procenama inbridinga. Mada se dobijanje inbred linija kod lucerke smatra zbog velike inbriding depresije, u takvim eksperimentima molekularni markeri mogu biti korisni jer omoguavaju da se direktno proceni progresija homo/heterozigotnosti na genomskom nivou. U tom smislu su Brouwer & Osborn (1997) koristili RFLP markere kod diploidne lucerke, a Scotti et al. (2000) kod tetraploidne lucerke. Mapiranje vezanih gena, QTL analiza i selekcija uz pomo markera (MAS) Meu krmnim leguminozama, mapiranje genoma je najnaprednije kod lucerke (Woodfield & Brummer 2001). Geneticke mape - mape vezanosti gena lucerke dobijene su na osnovu gotovo svih raspolozivih molekularnih markera. Razlicite grupe istrazivaca su se bavile genetickim mapiranjem kod lucerke, a veina genetickih mapa lucerke su konstruisane kod diploida (Kiss et al. 1993, Echt et al. 1994, Tavoletti et al. 1996, Diwan et al. 1997, Diwan et al. 2000, Kaló et al. 2000, Porceddu et al. 2002). Meutim neke istrazivacke grupe su se odlucile da vrse mapiranje tetraploida lucerke (Yu & Pauls 1993, Brouwer & Osborn 1999, Ma et al. 2002, Sledge et al. 2004). M. truncatula je meunarodno priznata model vrsta, ne samo za blisko gajenu M. sativa, ve i za familiju mahunarki, zbog njenih povoljnih osobina kao sto su diploidan broj hromozoma (2n = 2x = 16), mala velicina genoma, autogamija i pogodnost za geneticke transformacije (Veronesi et al. 2003). Geni iz M. truncatula imaju visok stepen identicnosti sekvenci sa genima lucerke tako da je M. truncatula odlican geneticki model za lucerke. Dostupnost M. truncatula mikronizova (Tesfaie et al. 2006) i proteinskih podataka

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(Lei et al. 2005) doprineli su identifikaciji agronomski znacajnih gena. Konsenzus mapu vezanih gena kod crvene deteline sacinili su Isobe et al. (2009), koja je prva takva mapa za stranooplodnu vrstu krmnog bilja. Stvorena je na osnovu sest mapirajuih populacija, ukupne duzine 836,6 cM sa 1804 lokusa markera i prosecnom distancom dva susedna lokusa od 0,46 cM. Za prinos semena po biljci kod crvene deteline, svojstvo koje je inace tesko poboljsati fenotipskom selekcijom, utvrena su 3 QTL na tri razlicite grupe vezanih gena koji su objasnili 33,8% ukupne fenotipske varijanse (Herrmann et al. 2006). Izracunati proseci ocena vigora kroz dve zimske i tri uzgojne sezone su utvreni kao optimalan metod za prikazivanje perzistentnosti i utvren je znacajan QTL za ovo svojstvo, koji je objasnio 12,2% ukupne fenotipske varijanse (Herrmann et al. 2008). Markeri povezani sa utvrenim QTL kod crvene deteline omoguie selekciju uz pomo markera (MAS) za ova dva vazna svojstva. Jedan od najvaznijih segmenata oplemenjivackog rada jeste odabiranje biljaka u generacijama razdvajanja svojstava, kojim se izdvajaju potomstva sa odgovarajuom kombinacijom gena. Selekcija uz pomo markera (MAS - Marker Assisted Selection) moze imati dosta prednosti u odnosu na konvencionalne metode oplemenjivanja. Marker asistirana selekcija (MAS) se zasniva na identifikaciji markera za gene od interesa ili markera koji su blisko vezani sa genima od interesa, a koji se mogu koristiti da se indirektno selektuju osobine cija je procena teska ili skupa (Woodfield & Brummer 2001). Kada se markeri jednom blisko povezu sa genima, odnosno kada se identifikuju QTL markeri za svojstva na koja se vrsi selekcija, umesto poljskog ispitivanja velikog broja biljaka oplemenjivaci mogu koristiti specificne markere kao dijagnosticko sredstvo za odabiranje jedinki koje su nosioci pozeljnih alela (Deleti 2009). MAS selekcija se primenjuje na genotipskom nivou korisenjem DNK markera koji su blisko vezani za gene koji determinisu kvalitativna ili kvantitativna svojstva i koji se nasleuju na isti nacin kao i geni od interesa. Selekcija uz pomo markera je narocito korisna za svojstva koja je dugotrajno i tesko oceniti fenotipski na polju, kao sto su perzistentnost i otpornost na niske temperature. Selekcija uz pomo markera moze da povea efikasnost oplemenjivanja visegodisnjih leguminoza omoguujui preciznije unosenje specificnih gena. Saturisane mape vezanih gena mogu biti veoma korisne u oplemenjivanju visegodisnjih le-

guminoza radi utvrivanja target gena od agronomskog znacaja, za primenu selekcije uz pomo markera i za proucavanje heterozigotnosti i inbriding depresije. Iako se MAS primenjuje kod mnogih kultura (Young 1999), stvarna korist od te tehnologije moze biti najvise uspesno realizovana kod visegodisnjih krmnih kultura, gde svaki ciklus selekcije traje nekoliko godina. Na primer, selekcija na otpornost na niske temperature kod lucerke traje tri do pet godina. MAS pristup moze znacajno da smanji vreme ciklusa, ako bi markeri koji su u vezi sa prezivljavanjem tokom zime mogli biti identifikovani (Woodfield & Brummer 2001). Tako su Brouwer et al. (2000) uspeli da pronau nezavisne regione genoma koji kontrolisu otpornost na niske temperature i dormantnost kod tetraploidne lucerke. Oni su predlozili mogunost korisenja MAS za poboljsanje otpornosti na niske temperature, a bez poveanja dormantnosti. Tu su i drugi pokusaji identifikovanja gena lucerke korisenjem razlicitih markera. Barcaccia et al. (2000) su pomou RAPD, ISSR i AFLP markera identifikovali jednu grupu vezanih gena koji deluju na formiranje 2n jajnih elija kod jednog diploidnog mutanta Medicago falcata oznacenog kao PG-F9. Tavoletti et al. (2000) su proucavali osobinu dzambo polena na diploidnom mejotickom mutanatu koji proizvodi 2n polen i s tim u vezi uspesno su locirali dzp gen koji determinise proizvodnju neredukovanih gameta. Obert et al. (2000) su pronasli dva AFLP markera znacajno povezana sa otpornosu na plamenjacu. Sledge et al. (2002) su proucavali toleranciju na aluminijum (Al) i identifikovali i potvrdili postojanje QTL za to svojstvo kod diploidne M. coerulea. Utvreno je da su dva RFLP markera povezana sa Al tolerancijom kod ukrstanja izmeu tolerantnih i osetljivih biljaka M. coerulea, a predlaze se kao podesno korisenje ta dva RFLP markera za QTL analizu kod tetraploide lucerke. Endre et al. (2002) su primenili mapu molekularnih markera razvijenu na diploidnom nivou za pozicioniranje gena odgovornih za fenotip koji nije u mogunosti da obrazuje kvrzice na korenu tetraploidne lucerke. Identifikacija sorata i zastita prava oplemenjivaca Da bi se novostvorena sorta stavila na nacionalne i internacionalne liste sorata, potrebno je da se testira DUS (Distinctness Uniformity Stability) testovima, kako bi se utvrdilo da li se razlikuje od ve priznatih, sto je osnova zastite intelektualnih prava oplemenjivaca (PBR). Geneticki, a posebno molekularni markeri postaju sastavni deo

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ovih testiranja, pored morfoloskih i biohemijskih metoda, u cilju utvrivanje pouzdanih genetickih profila (fingerprinting). Identifikacija i kvantifikacija geneticke slicnosti varijeteta visegodisnjih leguminoza je teska zbog velike varijabilnosti jedinki u geneticki heterogenim populacijama. Proucavajui proteinski polimorfizam domaih sorti crvene deteline u poreenju sa sortama iz zapadne i severne Evrope koje se koriste kao standardni u procesu registracije i zastite sorti poljoprivrednog bilja prema protokolu UPOV organizacije, pomoi multivarijacione analize posebno se izdvaja podgrupa koja obuhvata NS sorte crvene deteline, sto ukazuje na originalnost i autenticnost domaeg sortimenta, kako u odnosu na morfolosko-bioloske pokazatelje (Vasiljevi i sar. 2006) tako i u odnosu na proteinski polimorfizam (Nikolic et al. 2010). Zakljucak U buduem oplemenjivackom radu na visegodisnjim krmnim leguminozama u nasoj zemlji, saglasno savremenim svetskim trendovima, ocekuje se vea primena genetickih a narocito molekularnih markera, prevashodno kao pomo u oplemenjivanju (marker asistirana selekcija), kao i za analizu geneticke divergentnosti roditelja i tipiziranju varijeteta. Ipak, uprkos mnogim istrazivanjima koja koriste molekularne markere, ni jedna sorta lucerke do sada nije stvorena korisenjem molekularnih markera. DNK sekvence veine gena za agronomski vazne osobine raznih kulturnih biljaka nisu poznate i verovatno e tako ostati neko vreme. Dalja dostupnost obilja informacija o sekvencama olaksae razvoj velikog broja markera koji bi se mogli dovesti u vezu upravo sa onim regionima genoma koji su bogati genima. U meuvremenu, naucnicima preostaje dalje korisenje i usavrsavanje QTL mapa i markera. Ocekuje se da e dalji razvoj genskih mapa visoke rezolucije omoguiti izolaciju stvarnih gena umesto njihovih mapa, a poznavanje DNK sekvenci gena e omoguiti dizajniranje "savrsenih" markera koji bi bili integralni delovi samih gena. Literatura

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Scotti C, Pupilli F, Salvi S, Arcioni S (2000): Variation in vigour and in RFLP-estimated heterozygosity by selfing tetraploid alfalfa: new perspectives for the use of selfing in alfalfa breeding. Theor. Appl. Genet. 101: 120-125 Sengupta-Gopalan C, Bagga S, Potenza C, Ortega J L (2007): Alfalfa. In: Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey). Springer-Verlag Berlin Heidelberg, 321-335 Skinner D Z (2000): Non random chloroplast DNA hypervariability in Medicago sativa. Theor. Appl. Genet. 101: 12421249 Sledge M K, Bouton J H, Dall' Agnoll M, Parrott W A, Kochert G (2002): Identification and confirmation of aluminium tolerance QTL in diploid Medicago sativa subsp. Coerulea. Crop Sci. 42: 1121-1128 Sledge M, Ray I, Rouf Mian M A (2004): EST-SSRs for genetic mapping in alfalfa. In: Hopkins A et al. (eds) Molecular breeding of forage and turf. Springer, Berlin Heidelberg New York, 239-243 Sweeney P M, Danneberger T K (1994): Random amplified polymorphic DNA in perennial ryegras: A comparison of bulk sample vs. individuals. HortSci. 29: 624-626 Tavoletti S, Veronesi F, Osborn T C (1996): RFLP linkage map in an alfalfa diploid mutant based on F1 populations. J Hered. 87: 167-170 Tavoletti S, Pesaresi P, Barcaccia G, Albertini E, Veronesi F (2000): Mapping the jp (jumbo pollen) gene and QTLs involved in multinucleate microspore formation in diploid alfalfa. Theor. Appl. Genet. 10: 372-378 Tesfaye M, Silverstein K A T, Bucciarelli B, Samac D, Vance C P (2006): The affymetrics Medicago genechip array is applicable for transcript analysis of alfalfa (Medicago sativa). Funct. Plant Biol. 33: 783-788 Thorogood D, Laroche S (2001): QTL analysis of chlorophyll retention during leaf senescence in perennial ryegrass. Plant Breeding: Sustaining the Future. Abstr. 16th EUCARPIA Congr., Edinburgh, Scotland, 12 Tomi Z, Sokolovi D, Lugi Z, Radovi J, Vasiljevi S, Mili D, Miki A (2009): Geneticki resursi krmnog bilja u Srbiji. SANU, Naucni skupovi, Knjiga CXXVII, Odeljenje hemijskih i bioloskih nauka 3: 35-46 Torricelli R, Negri V, Silveri D D, Veronesi F (2000): Collection, evaluation and conservation of forage legumes in Abruzzo (Italy). In: Maggioni L, Marum P, Sackville Hamilton NR, Hulden M, Limpan E (comp.): Report of a Working group on Forages. Proc. 7th Meet., Elvas, Portugal, 18-20 Nov. 1999. IPGRI, Rome, Italy, 170-175 Ulloa O, Ortega F, Campos H (2003): Analysis of genetic diversity in red clover (Trifolium pretense L.) breeding populations as revealed by RAPD genetic markers. Genome 46: 529-535

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Vasiljevi S, Surlan-Momirovi G, Luki D, Mihailovi V, Kati S (2001): Iskorisavanje genetickog biodiverziteta kolekcionisanih genotipova crvene deteline u Naucnom institutu za ratarstvo i povrtarstvo ­ Novi Sad. Zbornik radova 1. Meunarodnog simpozijuma »HRANA U 21. VEKU«, 14-17 novembar 2001, Subotica, 167-172 Vasiljevi S, Surlan-Momirovi G, Luki D, Zivanovi T, Kati S, Mihailovi V, Mili D, Miki A (2003): Efikasnost razlicitih metoda selekcije u oplemenjivanju crvene deteline (Trifolium pratense L). Sel. Semen. 9: 77-85 Vasiljevic S, Pataki I, Surlan-Momirovic G, Zivanovic T (2005): Production potential and persistence of red clover varieties. Grassland Sci. Eur. 10: 577-580 Vasiljevi S, Miki A, Mihailovi V, Kati S, Lugi Z, Surlan Momirovi G, Zivanovi T, Mili D, Pataki I (2006): Osobine domaih sorti crvene deteline (Trifolium pratense L) prema protokolu UPOV-a. Treeg simpozijuma Sekcije za oplemenjivanje organizama Drustva geneticara Srbije i Cetvrtog naucno-strucnog simpozijuma iz selekcije i semenarstva Drustva selecionara i semenara Srbije, Zlatibor, Srbija i Crna Gora, 16-20. maj, 2006, 97 Veronesi F, Rosellini D, Albertini E (2003): The use of molecular markers in alfalfa breeding. Czech. J. Genet. Plant Breed. 39: 104-111 Woodfield D, Brummer E C (2001): Integrating molecular techniques to maximise the genetic potential of forage legumes. In: Spangenberg G. (ed.), Molecular Breeding of Forage Crops: Proc. 2nd Int'l Symp., Molecular Breeding of Forage Crops, Lorne and Hamilton, Victoria, Australia, Nov. 19-24, 2000, Dordrecht: Kluwer, 51-65 Young N D (1999): A cautiously optimistic vision for markerassisted breeding. Mol. Breed. 5: 505-510 Yu K F, Pauls K P (1993a): Rapid estimation of genetic relatedness among heterogeneous populations of alafalfa by random amplification of bulked genomic DNA samples. Theor. Appl. Genet. 86: 788-794 Yu K F, Pauls K P (1993b): Segregation of random amplified polymorphic DNA markers and strategies for molecular mapping in tetraploid alfalfa. Genome 36: 844-851 Yu J, Mosjidis J A, Klingler K A, Woods F M (2001): Isozyme diversity in North American cultivated red clover. Crop Sci. 41: 1625-1628 Zaccardelli M, Gnocchi S, Carelli M, Scotti C (2003): Variation among and within Italian alfalfa ecotypes by means of bio-agronomic characters and amplified fragment length polymorphism analyses. Plant Breed. 122: 61-65 Zlokolica M, Milosevi M, Nikoli Z, Balesevi-Tubi S, Galovi V, Vujakovi M (1999): Primena metoda biotehnologije u identifikaciji i genetskoj oceni kvaliteta semena. Zbornik radova Naucnog instituta za ratarstvo i povrtarstvo 31: 369-378

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Use of Genetic Markers in Breeding of Perennial Legumes

Gordana Surlan-Momirovi1 Slobodan Kati2 Sanja Vasiljevi2 Zorica Nikoli2 Gordana Brankovi1 Irena Cali1 Dragan Mili2 Miki Aleksandar2

1

Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Beograd ­ Zemun, Serbia 2 Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia

Summary: Breeding of perennial legumes for many agronomic important traits like grain yield, persistence, longevity, resistance to diseases and pests, resistance to limiting abiotic conditions and polyploidy is more efficient and precise if genetic markers are used. Estimates based on isozyme polymorphism may underestimate overall levels of genetic variation because they are sampling only coding regions of DNA that may be conserved to maintain the function of the enzymes. The complete coverage of a genome can be achieved only by the use of molecular variability indicators (DNA polymorphism), i.e. molecular markers. Molecular markers are independent of environmental influences and can be detected in all plant development stages. The main aspects of genetic markers use in perennial legumes breeding are: germplasm characterisation, genetic linkage mapping, QTL analysis, marker assisted selection (MAS), variety identification and protection of plant breeders' rights. Key words: breeding, genetic markers, perennial legumes

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www.nsseme.com/journal.html

Citogenetska istrazivanja suncokreta i uljane repice

425 Citogenetika / Cytogenetics Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 425-434 pregledni rad / review article

Znacaj citogenetskih istrazivanja u oplemenjivanju suncokreta i uljane repice

Jovanka Atlagi Sreten Terzi Ana Marjanovi-Jeromela Radovan Marinkovi

primljeno / received: 25.03.2010. prihvaeno / accepted: 11.05.2010. © 2010 IFVC

Izvod: Citogenetska istrazivanja suncokreta i uljane repice imaju tradiciju dugu citav vek. Prvo su izucavani broj i morfologija hromozoma kod vrsta iz roda Helianthus i Brassica, a zatim citotaksonomija i filogeneza ovih rodova. Citogenetska istrazivanja su bila primenjivana u transferu gena iz divljih u gajene vrste, u korisenju metoda in vitro gajenja, u izucavanju fenomena citoplazmatske muske sterilnosti (CMS-a), restauracije fertilnosti i dr. U ovom radu su prikazani razliciti aspekti istrazivanja laboratorije za citogenetske analize Instituta za ratarstvo i povrtarstvo u Novom Sadu, ciji su rezultati koriseni u oplemenjivanju suncokreta i uljane repice. Kljucne reci: citogenetska istrazivanja, oplemenjivanje, suncokret, uljana repica

Uvod Citogenetska istrazivanja na suncokretu imaju tradiciju dugu citav vek. Prva istrazivanja su se odnosila na odreivanje broja hromozoma kod gajenog suncokreta. Potom je izraivan kariotip vrsta u rodu Helianthus. Broj i osobine hromozoma su korisene za klasifikaciju vrsta i izucavanje filogenetskih odnosa u rodu Helianthus. Korisenje interspecies hibridizacije u oplemenjivanju gajenog suncokreta, transfer gena iz divljih vrsta u gajeni suncokret iziskivalo je primenu citogenetskih metoda. U stvaranju hibrida suncokreta sedamdesetih godina proslog veka izucavani su fenomeni citoplazmatske muske sterilnosti i restauracije fertilnosti cija je primena bila od izuzetnog znacaja u novim oplemenjivackim programima. Citogenetska istrazivanja su pratila primenu metoda in vitro gajenja, narocito kulturu antera, zatim izucavanje procesa oplodnje u smislu razdvajanja prezigotne i postzigotne inkompatibilnosti u interspecies ukrstanjima, odnosno utvrivanje cross kompatibilnosti u izboru roditeljskih parova kod hibrida suncokreta. Poslednjih godina citogenetska istrazivanja su cesto kombinovana sa primenom metoda molekularnih markera. Citogenetska istrazivanja na uljanoj repici, tacnije u rodu Brassica, zapoceta su pocetkom proslog veka. Prema autorima Prakash i Chopra (1999) odnosila su se na: utvrivanje broja hromozoma i

J. Atlagi ) ( S. Terzi A. Marjanovi-Jeromela R. Marinkovi Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

analizu genoma u rodu Brassica; izucavanje karakteristika somatskih hromozoma; vestacko dobijanje aloploida seksualnim putem ili somatskom hibridizacijom; ispitivanje germplazme; citoplazmatske muske sterilnosti; ugradnju nuklearnih gena hromozomskim manipulacijama u cilju popravljanja agronomskih osobina i restauracije fertilnosti; disekciju bazicnog genoma za dobijanje linija sa hromozomskom adicijom; identifikaciju genskih linkidz grupa i poreenje genskih sinteza izmeu slicnih vrsta; korisenje molekularnih markera za mapiranje hromozoma i analizu odnosa genoma. Svi navedeni aspekti istrazivanja na suncokretu, a samo CMS kod uljane repice, bili su izucavani u laboratoriji za citogenetske analize Instituta za ratarstvo i povrtarstvo u Novom Sadu. Kolekcija divljih vrsta suncokreta Kolekcija divljih vrsta suncokreta u Novom Sadu je nastala kroz 7 ekspedicija izvedenih u periodu od 1980. do 1991. godine, gde je sakupljeno 917 kolekcionih brojeva. Od 49 vrsta suncokreta koje pripadaju rodu Helianthus u kolekciji je bilo 43 vrste. Nazalost, tokom prethodnih godina gajenja izgubljeno je ukupno 15 vrsta. Tako danas kolekcija sadrzi 21 visegodisnju vrstu i 7 jednogodisnjih vrsta. Ukupno se u

Ovo istrazivanje je deo projekta broj TR20081: Stvaranje genotipova uljane repice (Brassica napus L.) za ishranu i industrijsku preradu (01.04.2008-31.12.2010), Ministarstva za nauku i tehnoloski razvoj Republike Srbije / This research results from project TR20081: Development of rapeseed (Brassica napus L.) genotypes for feed and industrial processing (01.04.2008-31.12.2010) financed by the Ministry of Science and Technological Development of the Republic of Serbia

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Tabela 1. Kolekcija jednogodisnjih divljih vrsta suncokreta Table 1. The collection of annual wild sunflower species Broj populacija / No. of accessions Vrsta u gen banci sa rezervom semena Species in gene bank date base with seed reserves H. annuus 108 70 H. petiolaris 33 25 H. neglectus 4 4 H. debilis 21 13 H. praecox 15 14 H. argophyllus 7 7 H. niveus 4 3

Broj semena No. of seeds 10-2539 80-9130 1564-4838 33-5490 335-10710 1616-10396 259-5910

Tabela 2. Kolekcija visegodisnjih divljih vrsta suncokreta Table 2. The collection of perennial wild sunflower species Broj populacija No. of accessions sa rezervom u banci gena semena in gene bank with seed data base reserves 41 16 13 12 7 5 36 32 10 10 8 7 29 9 23 22 20 12 7 7 1 1 16 15 2 2 4 3 1 1 2 2 1 1 1 1 2 2 2 2 1 1 Broj populacija u polju No. of accessions in the field 112 11 6 60 8 7 31 15 20 8 1 19 1 2 1 2 1 1 2 2 1

Vrsta Species H. tuberosus H. rigidus (H.pauciflorus) H. mollis H. maximiliani H. divaricatus H. decapetalus H. grosseserratus H. nuttallii H. strumosus H. laevigatus H. glaucophyllus H. giganteus H. eggertii H. hirsutus H. californicus H. resinosus H. silphioides H. atrorubens H. microcephalus H. smithii H. glaucophyllus

Broj semena No. of seeds 1-650 1-400 3-1162 1-8630 8-680 17-622 2-335 1-399 2-354 7-91 38 1-1800 1-9 3-280 1 125, 4858 13 2 28, 300 13, 90 10

kolekciji nalazi 447 kolekcionih brojeva. Rezerva semena populacija jednogodisnjih vrsta se kree od nekoliko desetina do nekoliko hiljada (Tab. 1), a visegodisnjih od jednog do nekoliko stotina (Tab. 2). Seme se cuva u hladnoj komori (+ 4o C), a populacije visegodisnjih vrsta se odrzavaju i u polju (Atlagi et al. 2006).

Ploidnost vrsta roda Helianthus Osnovni broj hromozoma u rodu Helianthus je n=17, a rod predstavlja poliploidni kompleks koji se sastoji od diploidnih (2n=2x=34), tetraploidnih (2n=4x=68) i heksaploidnih (2n=6x=102) vrsta. Sve jednogodisnje vrste su diploidne, dok

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su visegodisnje diploidne, tetraploidne i heksaploidne. Vrste H. ciliaris i H. strumosus se pojavljuju u tetraploidnoj i heksaploidnoj formi, dok H. decapetalus ima diploidnu i tetraploidnu formu (Schilling & Heiser 1981). U okviru evaluacije vrsta prisutnih u kolekciji bilo je planirano da se odredi kariotip za svaku vrstu, da se pregledaju sve populacije u okviru vrsta u cilju identifikacije vrste, odnosno prirodnih hibrida. Zbog velikog broja kolekcionih brojeva broj hromozoma je odreivan za vrste, odnosno populacije koje su bile ukljucene u program ukrstanja sa linijama gajenog suncokreta. Korisen je Feulgen metod (Georgieva-Todorova 1976) za odreivanje broja hromozoma u somatskim elijama vrha korencia (Sl. 1a). Ovaj metod je uspesno korisen, ali je iziskivao trosenje rezervi semena. Iz tih razloga se preslo na odreivanje broja hromozoma u mejocitama koristei acetokarmin metod za analizu mejoze. U dijakinezi je odreivan broj bivalenata, a time i broj n-hromozoma (Sl. 1b ).

je nekoliko hiljada ukrstanja, a uspesno su preneta pozeljna svojstva i ugraena su u hibride suncokreta iz novosadskog selekcionog programa. Uspesnost ukrstanja je prikazana kroz dva perioda, gde se moze zapaziti da je u prvom periodu izveden vei broj ukrstanja i dobijeno je vise biljaka F1 interspecies hibrida (Tab. 3). Jednogodisnje divlje vrste suncokreta su filogenetski bliske gajenom suncokretu pa ih je mogue koristiti bez velikih teskoa u interspecies programima. Od 11 jednogodisnjih divljih vrsta, 7 vrsta (H. annuus, H. argophyllus, H. petiolaris, H. praecox, H. debilis, H. neglectus, H. niveus) uspesno su ukrstene sa linijama gajenog suncokreta (Tab. 3) (Atlagi 1990, Terzi 2006). Dobijeni interspecies hibridi razlicitih generacija ukrstanja (F1, BC1F1 ­ BC4F1) najcese su koriseni kao izvori CMS-a i Rf gena. Grupa diploidnih visegodisnjih vrsta je vrlo interesantna za oplemenjivace kao izvor otpornosti prema prouzrokovacima bolesti (H. giganteus, H. maximiliani i H. occidentalis), kao izvor visokog sadr-

Slika 1. H. rigidus a) Somatska elija (2n=6x=102); b) Mejocita (51 bivalent) Figure 1. H. rigidus a) Somatic cell (2n=6x=102); b) Meiocyte (51 bivalents) Na taj nacin su pregledane vrste zastupljene u kolekciji, korisene populacije tih vrsta u hibridizaciji sa gajenim suncokretom. Za neke od vrsta je ustanovljeno da se pojavljuju u razlicito ploidnim formama kako je bilo poznato u literaturi, a za neke su ustanovljeni nivoi ploidnosti koji nisu saopsteni ranije. Tako su Atlagi et al. (1992) utvrdili da se diploidna vrsta H. smithii pojavljuje u heksaploidnoj formi, a vrsta H. strumosus pojavljuje se i u diploidnoj formi, osim u tetraploidnoj i heksaploidnoj. Interspecies hibridizacija Mogunost ukrstanja divljih vrsta sa gajenim suncokretom bila je ispitivana prethodnih 28 godina kroz vrlo obiman program hibridizacije uz korisenje klasicnih metoda ukrstanja. Izvedeno zaja ulja u semenu (H. salicifolius), ranostasnost (H. nuttallii) i novog ideotipa (H. mollis). Ove vrste su uspesno ukrstene sa linijama gajenog suncokreta i dobijen je vei broj interspecies hibrida (Tab. 3) (Atlagi 1991, Atlagi et al. 1995). Tetraploidne vrste H. hirsutus, H. decapetalus, H. laevigatus, H. strumosus uspesno su ukrstene sa gajenim suncokretom i korisene su kao izvori otpornosti na bolesti (Tab. 3) (Atlagi 1994, Terzi 2006). Od heksaploidnih vrsta najcese je korisena vrsta H. tuberosus kao izvor otpornosti na razlicite patogene. F1 hibidi su dobijeni sa velikim brojem populacija ove vrste (Atlagi et al. 1993). Dodatne tri heksaploidne vrste H. rigidus, H. resinosus i H. eggertii uspesno su ukrstene sa linijama gajenog suncokreta (Tab. 3) (Atlagi 1996, Terzi 2006).

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Tabela 3. Uspesnost ukrstanja divljih vrsta i gajenog suncokreta Table 3. The success of crossing wild species and cultivated sunflower 1981 - 1991 1992 ­ 2008 Broj Broj populacija Broj populacija F1 gen. Vrste hrom. No. of accessions No. of accessions Species No. of Oprasene Ukrstene chrom. Oprasene Ukrstene Broj bilj. Polinated Crossed No. of plants Polinated Crossed H. annuus H. petiolaris H. argophyllus H. neglectus H. debillis H. praecox H. niveus H. mollis H. salicifolius H. maximiliani H. occidentalis H. nuttallii H. smithii H. decapetalus H. hirsutus H. strumosus H. laevigatus H. tuberosus H. rigidus H. eggertii H. resinosus H. divaricatus H. giganteus H. grosseserratus H. microcephalus 17 17 17 17 17 17 17 17 17 17 17 17 17 17,34 34 34,51 34 51 51 51 51 17 17 17 17 24 22 8 2 12 11 2 4 2 7 3 3 2 5 3 8 5 17 7 1 2 6 6 3 2 17 20 6 2 9 9 1 3 1 3 1 1 2 1 2 1 3 9 3 1 2 1 1 0 0 123 35 24 14 32 24 8 10 7 10 8 9 27 28 66 13 51 90 105 5 89 1 0 0 0 28 23 9 4 13 19 3 7 7 31 0 24 0 10 3 14 3 11 11 1 3 13 12 16 1 11 3 2 1 1 4 0 2 2 5 0 1 0 1 1 5 1 4 4 1 1 2 0 0 0

F1 gen. Broj bilj. No. of plants 169 7 30 12 3 0 0 0 0 0 0 0 0 0 0 24 4 27 0 0 0 3 0 0 0

Citogenetska ispitivanja interspecies hibrida Najcese barijere u primeni interspecies hibridizacije su cross inkompatibilnost (prezigotna i postzigotna - abortivnost embriona), smanjena fertilnost ili potpuna sterilnost F1 i drugih ranih generacija interspecies ukrstanja. Razlike u ploidnosti, filogenetske razlike i razlicita taksonomska pripadnost divljih vrsta u odnosu na gajeni suncokret uzrok su navedenih pojava. Citogenetska ispitivanja divljih vrsta, kao i interspecies hibrida doprinose detektovanju problema i njihovom prevazilazenju. Najcese se koriste citogenetske metode za analizu mejoze ­ mikrosporogeneze i vitalnosti polena. Praenje mejoze podrazumeva posmatranje mejocita u arhesporijalnom tkivu prasnika primenom acetokarmin metode, dok je vitalnost polena odre-

ivana bojenom metodom po Alexandru (Atlagi 1989). Analiza mejotskog ciklusa daje dragocene informacije o homolognosti hromozoma i hromozomskim aberacijama tipa translokacija (konfiguracija u dijakinezi); izmeni genetskog materijala (broj hijazmi); detekcije hromozoma u eliminaciji (univalenti); aberacije tipa neukljucenih hromozoma (izbegli i izostali hromozomi); o hromozomskim aberacijama tipa inverzija (hromozomski mostovi i fragmenti). Ocena vitalnosti polena sluzi za procenu potencijala za oplodnju. Normalan tok mejoze koji je zapazen kod gajenog suncokreta i razlicitih vrsta roda Helianthus prikazan je na slici 2. Kod F1 interspecies hibrida vrlo cesto su detektovane nepravilne faze mejoze (Sl. 3). Dobijeni rezultati su pokazali da ponekad postoje razlike u strukturi hromozoma (pojava multivalenata u dijakinezi biljaka interspecies

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Slika 2. Faze mejoze (pravilne) a) Pahiten; b) Dijakineza; c) Metafaza I; d) Anafaza I; e) Telofaza I; f) Metafaza II; g) Telofaza II; h) Tetrade; i) Jednojedarne mikrospore; j,k) Mikrospore; l) Polenova zrna Figure 2. Phases of meiosis (normal) a) Pachyten; b) Diakinesis; c) Metaphase I; d) Anaphase I; e) Telophase I; f) Metephase II; g) Telophase II; h) Tetrads; i) Mononuclear microspores; j,k) Microspores, l) Pollen grains

Slika 3. Faze mejoze (nepravilne) a) Dijakineza sa kvadri i heksavalentom; b) Metafaza I sa izbeglim hromozomima; c) Anafaza I sa izostalim hromozomima; d) Anafaza I sa hromozomskim mostovima; e) Telofaza II sa izostalim hromozomima; f) Telofaza II sa hromozomskim mostovima Figure 3. Phases of meiosis (irregular) a) Diakinesis with quadrivalent and hexavalent.; b) Metaphase I with fast chromosomes; c) Anaphase I with lagging chromosomes; d) Anaphase I with chromosome bridge; e) Telophase II with lagging chromosomes; f) Telophase II with chromosome bridges

hibrida), iako jednogodisnje vrste imaju isti broj hromozoma kao gajeni suncokret. Genom visegodisnjih diploidnih vrsta se razlikuje od genoma jednogodisnjih ­ gajenog suncokreta, pa je njihovo korisenje praeno velikim brojem teskoa.

Tetraploidne i heksaploidne vrste se razlikuju po broju i strukturi hromozoma od gajenog suncokreta. Kod interspecies hibrida nastalih ukrstanjem ovih vrsta i gajenog suncokreta konstatovan je veliki broj nepravilnosti tipa univalenata

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i multivalenata u dijakinezi, ,,neukljucenih" hromozoma u metafazi i anafazi, kao i hromozomskih mostova u anafazi i telofazi (Sl. 3). Posledica nepravilne mejoze ­ mikrosporogeneze je smanjena vitalnost polena kod interspecies hibrida, narocito F1 i BC1F1 generacije u odnosu na roditeljske vrste, a cesta je bila i pojava musko sterilnih biljaka (Atlagi 2004, Terzi 2006). Interesantno je istai da od svih citogenetskih analiza kod interspecies hibrida najvei broj autora (Georgieva-Todorova 1976, Chandler et al. 1986, Georgieva-Todorova 1990, Jan 1997, Jan & Seiler 2007) izdvaja analizu mejoze - redukcione deobe i vitalnosti polena. Pored mogunosti ukrstanja, karakteristike mejoze, pojave sterilnosti i smanjene fertilnosti kod interspecies hibrida pokazuju mogunost korisenja neke vrste u oplemenjivanju gajenog suncokreta. Citogenetsko-molekularna istrazivanja Pored unosenja pozeljnih gena iz divljih vrsta u gajeni suncokret, u interspecies hibridizaciji unosi se i veliki broj nepozeljnih svojstava (grananje,

mali precnik glave i dr.), zbog cega je potrebno izvesti povratna ukrstanja F1 interspecies hibrida sa gajenim suncokretom. Citogenetske analize BC1F1 hibrida su pokazale visok procenat abnormalnosti u mejozi, pojavu aneuploida, biljaka sa razlicitim brojem hromozoma i smanjenu vitalnost polena (Atlagi & Skori 1999). S druge strane, izvoenjem nekoliko povratnih ukrstanja sa gajenim suncokretom gube se i pozeljni geni, zbog cega je potrebno analizirati prisustvo genoma divlje vrste u odnosu na genom gajenog suncokreta kod interspecies hibrida ne samo na citogenetskom (Sl. 4), ve i na molekularnom nivou (Sl. 5) (Atlagi et al. 2003a). Citoplazmatska muska sterilnost kod suncokreta i uljane repice Stvaranje hibrida suncokreta i uljane repice podrazumeva korisenje postojeih ili stvaranje novih izvora citoplazmatske muske sterilnosti (CMS), kao i njihovo prenosenje u genotipove koji poseduju pozeljne gene za agronomski vazna svojstva. Muska sterilnost je definisana kao pojava da se kod biljaka u prasnicima ne formiraju vitalna polenova zrna. Ekspresija ovog

Slika 4. Citogenetske analize interspecies hibrida a) Dijakineza (bivalenti, univalenti, fragmenti); b) Metafaza I (izbegli hromozomi); c) Telofaza II (hromozomski mostovi) Figure 4. Cytogenetic analysis of interspecific hybrids a) Diakinesis (bivalents, univalents and fragments), b) Metaphase I (fast chromosomes), c) Telophase II (chromosome bridges)

Slika 5. Molekularne analize interspecies hibrida ­ Random Amplification of Polymorphic DNA (RAPD) fragmenti dobijeni amplifikacijom sa prajmerom UBC-39 i sa prajmerom UBC-43 Figure 5. Molecular analysis of interspecific hybrids - Random Amplification of Polymorphic DNA (RAPD) fragments amplified with primers UBC-39 and UBC-43

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svojstva varira od potpunog odsustva prasnika do slabo razvijenih antera, odnosno od odsustva polena do prisustva deformisanih ili normalno razvijenih polenovih zrna u anterama. CMS kod suncokreta i uljane repice je najcese aloplazmatska i nastala je u interspecies ili intergenus ukrstanjima. Svojstvo CMS-a je analizirano pre svega na osnovu morfoloskih razlika u grai cveta, razvijenosti antera i produkciji polena (musko fertilnog i musko sterilnog), procenta vitalnosti polena, kao i na osnovu pojave i pravilnosti toka pojedinih faza mejoze - mikrosporogeneze. U najveem broju slucajeva rezultati analize morfologije musko sterilnog cveta ukazivali su na slabu razvijenost antera, samo ponekad su antere bile bolje razvijene, ali su bile prazne ili su sadrzavale malu kolicinu deformisanih, sterilnih polenovih zrna. Kod musko fertilnih cvetova suncokreta i uljane repice mejoza se odvijala normalno kroz sve faze (pahiten, dijakineza, matafaza I, anafaza I, telofaza II), kao i postmejotskog deljenja do formiranja polenovog zrna (Sl. 7a). Kod CMS biljaka u najveem broju slucajeva mejoza je proticala normalno (jedino je bio manji broj mejocita u deobi nego kod musko fertilnih bi-

ljaka), dok je postmejotsko deljenje izostajalo. Najcesa faza prekida u mikrosporogenezi je bila faza tetrada (Sl. 6a), a samo ponekad i ranije, kasne faze mejoze. Takoe se vrlo retko desavalo da su rezultat mikrosporogeneze bile deformisane mikrospore (Sl. 6b i 7b) ili sterilna polenova zrna. Preko 70 CMS izvora je identifikovano u potomstvima ukrstanja izmeu divljih vrsta i gajenog suncokreta, za 34 su naeni geni za restauraciju fertilnosti, a nacin nasleivanja restauracije za 19 CMS (Seriyes 2002). Citoplazmatsku musku sterilnost dobijenu u interspecies ukrstanju H. petiolaris i H. annuus (Leclercq 1969) ispitivao je Paun (1974), koji je analizirao mejozu kod cetiri sterilne linije, ali i njihovih fertilnih analoga. Kod fertilnih analoga mejoza je proticala normalno, dok je kod dve sterilne linije sporogeno tkivo degenerisalo jos u premejotskom stadijumu, a kod druge dve je doslo do degeneracije posle faze tetrada. Autor pretpostavlja da je degeneracija uslovljena odreenim enzimatskim reakcijama koje su rezultat inaktivacije mehanizama za sazrevanje polena. Horner (1977) je koristio svetlosnu i elektronsku mikroskopiju radi poreenja mikrosporoge-

Slika 6. Mikrosporogeneza u musko sterilnom cvetu suncokreta, a) Tetrade, b) Mikroskore, c) Sterilna polenova zrna, d) Antera sa sterilnim polenom Figure 6. Microsporogenesis in male sterile flower of sunflower a) Tetrads; b) Microspores, c) Sterile pollen grains, d) Anthers with sterile pollen grains

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neze kod fertilne linije HA232 i njenog sterilnog analoga sa izvorom CMS - PET 1. Prvenstveno je posmatrao razvoj antera i sporogenog tkiva, a zatim hromozome. Autor je mikrosporogenezu podelio u 11 faza, gde su faze 1-4 do tetrada, a od 5-11 od tetrada do zrelog polena. Sterilni i fertilni analozi se ne razlikuju u mikrosporogenezi do faze 5. Promene koje se desavaju u tapetumu i tetradama rezultiraju u sterilnosti. Izduzivanje i degeneracija elija tapetuma na kraju 5. faze izaziva degeneraciju mikrospora u tetradama. Stabilnost pet izvora CMS-a (PET-1, PET-2, MAX-1, GIG-1, ANN-6) izucavana je pri unosenju u inbred liniju HA-89, kao i izvora CMS-a PET-1, PET-2, ANN-5, ANN-44, ANN-164 pri unosenju u inbred linije novosadske selekcije (L-1, L-98, L-74 i L-22). Citogenetske analize su pokazale da su antere kod nekih izvora normalno razvijene, dok su kod drugih rudimentirane. Mikrosporogeneza je u najveem broju slucajeva bila prekinuta u fazi tetrada (Sl. 6a). Kod nekih izvora u anterama su bila zapazena polenova zrna, deformisana i sterilna (Sl. 6c, d) (Atlagi et al. 1996). Takoe je utvreno da su izvori CMS-a GIG-1 i PET-2 nestabilni (vitalnost polena je bila 10,42% i 1-63,43%) (Atlagi et al. 1996). Potencijalni izvori CMS-a (interspecies hibridi sa sest populacija H. annuus i jednom populacijom H. petiolaris) bili su citogenetski izucavani. Sve biljke u BC1F1 generaciji su bile musko sterilne. Razlike su postojale u razvijenosti antera i prisustvu pojedinih faza mejoze (Atlagi & Marinkovi 1998). U novosadskom oplemenjivackom programu izucavanje CMS-a uljane repice obuhvatalo je korisenje metoda citogenetskih istrazivanja, pre svega za analizu mejoze i vitalnosti polena kod: 1. potomstva iz samooplodnje biljaka hibrida uljane repice (Atlagi et al. 2003b); 2. inbred linije u sterilnoj formi; 3. potomstva povratnih ukrstanja izmeu izdvojenih steril-

nih biljaka i sorti odrzivaca; 4. razlicitih tipova CMS-a unetih u inbred linije iz novosadskog oplemenjivackog programa (Atlagi et al. 2007).

Slika 7. a) Polen musko-fertilnog cveta; b) Deformisane mikrospore musko-sterilnog cveta Figure 7. a) Pollen grains in male fertile flower; b) Deformed microspores in male sterile flower

Oplodnja Kao kod veine biljnih vrsta, oplodnja kod suncokreta je vrlo kompleksan i osetljiv proces, a izucavana je u smislu razdvajanja prezigotne i postzigotne inkompatibilnosti u interspecies ukrstanjima, kao i u izboru roditeljskih parova kod stvaranja hibrida suncokreta. Podaci o vitalnosti polena dobijeni bojenom metodom bili su indikacija potencijala za oplodnju, sto je relativna ocena, te je trazen metod koji e biti blizi in vivo uslovima. Metod fluorescentne mikroskopije omoguuje praenje klijanja polena na zigu i rasta polenovih tuba kroz stubi i plodnik do embrionove kese (jajne elije), ve nekoliko sati nakon oprasivanja (Sl. 8). Ocena dobijena fluorescentnom mikroskopijom predstavlja sigurnu procenu potencijala za oplodnju.

Slika 8. Oplodnja a) Klijanje polena na zigu (svetlosni mikroskop); b) Klijanje polena na zigu (fluorescentni mikroskop); c) Rast polenove tube kroz stubi (fluorescentni mikroskop) Figure 8. Fertilization a) Pollen grain germination on stigma (light microscope); b) Pollen germination on stigma (fluorescent microscope); c) Pollen tube growth through the style (fluorescent microscope)

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Leclercq P (1969): Une sterilite cytoplasmique chezle tournesol. Ann Amelior Plantes 19: 99-106 Jan C C (1997): Cytology and interspecific hybridization. In: Schneiter A A et al. (ed.), Sunflower technology and production. Agron. Monogr. 35. ASA, CSSA and SSSA, Medison, WI, 497-558 Jan C C, Seiler G J (2007): Sunflower. In: Singh R J (ed.), Genetics resources, chromosome engineering, and crop improvement, Volume 4, Oilseed crops, CRC Press, Taylor and Francis Group, New York, 103-165 Paun L (1974): The cytologic mechanism of male sterility in sunflower. Proc. of 6th Inter. Sunfl. Conf., 22-24 July, Bucharest, Romania, 249-257 Prakash S, Chopra VL (1999): Eighty years of Brassica cytogenetics. Proc. 10th Int. Rapeseed Congres, Canbera, Australia Schilling E E, Heiser Ch B (1981): Infrageneric classification of Helianthus (Compositae). Taxon 30: 393-403 Serieys H (2002): Identification, study and utilization in breeding programs of new cms sources. Progress report 19992001. Technical FAO Meeting and a GRESO Meeting. 7-9 October, Montpellier, France, 1-54 Terzi S (2006): Mogunosti korisenja divljih vrsta roda Helianthus u oplemenjivanju gajenog suncokreta. Magistarski rad. Univerzitet u Novom Sadu, Poljoprivredni fakultet

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Atlagi J (1989): Citogenetika suncokreta, U: Skori D i dr, Suncokret. Nolit, Beograd, 231-258 Atlagi J (1990): Pollen fertility in some Helianthus L. species and their F1 hybrids with the cultivated sunflower. Helia 13: 47-54 Atlagi J (1991): Karakteristike mejoze i fertilnosti biljaka F1 interspecies hibrida suncokreta. Doktorska disertacija, Univerzitet u Novom Sadu, Poljoprivredni fakultet. Atlagi J (1994): Mogunost korisenja tetraploidnih vrsta roda Helianthus L. u oplemenjivanju suncokreta. Zbornik radova Instituta za ratarstvo i povrtarstvo 22: 463-473 Atlagi J (1996): Cytogenetic studies in hexaploid Helianthus species and their F1 hybrids with cultivated sunflower, H. annuus. Plant Breed. 115: 257-260 Atlagi J (2004): Roles of interspecific hybridization and cytogenetic studies in sunflower breeding. Helia 27: 1-24 Atlagi J, Marinkovi R (1998): Cytogenetic study of potential sources of cytoplasmic male sterility in sunflower. Proceedings of 2nd Balkan Symposium on Field Crops, 16-20 June, Novi Sad, Yugoslavia, Vol. 1, 365-368 Atlagi J, Skori D (1999): Cytogenetic study of Helianthus laevigatus and its F1 and BC1F1 hybrids with the cultivated sunflower, H. annuus. Plant Breed. 118: 555-559 Atlagi J, Dozet B, Skori D (1992): Chromosome number ploidy level in some perennial species of the genus Helianthus L. Proc. of the 13th. International Sunflower Conference, 8-10. Septem. 1992., Pisa, Italy, 1349-1356 Atlagi J, Dozet B, Skori D (1993): Meiosis and pollen viability in H. tuberosus L. and its hybrids with cultivated sunflower. Plant Breed. 111: 318-324 Atlagi J, Dozet B, Skori D (1995): Meiosis and pollen grain viability in Helianthus mollis, Helianthus salicifolius, Heliathus maximiliani and their F1 hybrids with cultivated sunflower. Euphytica 81: 259-263 Atlagi J, Skori D, Marinkovi R (1996): Cytogenetic study of different sources of CMS in sunflower. Proc. of 14th Inter. Sunf. Conf., 12-20 June 1996, Beijing/Shenyang, China, 156162 Atlagi J, Pankovi D, Pekanovi A (2003a): Backcrosses in interspecific hybridization in sunflower. Genet. 35: 187199 Atlagi J, Marjanovi-Jeromela A, Marinkovi R, Skori D (2003b): Cytogenetic study of CMS in rapeseed genotypes at the Novi Sad breeding center. Proc. of the 11th International Rapeseed Congress, 6-10 July 2003, Copenhagen, Denmark, 336-339 Atlagi J, Terzi S, Skori D, Marinkovi R, Vasiljevi Lj, Pankovi-Safti D (2006): The wild sunflower collection in Novi Sad. Helia 29: 55-64 Atlagi J, Marjanovi-Jeromela A, Marinkovi R, Terzi S (2007): Cytogenetic studies of cytoplasmatic male sterility in rapeseed. Proceedings of the 12th International Rapeseed Congress, March 26-30, Wuhan, China, I, 66-70 Chandler J M, Jan C C, Beard H (1986): Chromosomal differentation among the annual Helianthus sp. Syst. Bot. 11: 354-371 Georgieva-Todorova J (1976): Interspecies relationships in the genus Helianthus (in Bulgarian). Bulgarian Academy of Sciences, Sofia Georgieva-Todorova J (1990): Genetic and cytogenetic investigation of the genus Helianthus L. (in Bulgarian). Bulgarian Academy of Sciences, Sofia Horner H T (1977): A comparative light- and electron microscopic study of microsporo-genesis in male fertile and cytoplasmic male-sterile sunflower (Helianthus annuus). Am. J. Bot. 64: 745-759

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Significance of Cytogenetic Research in Sunflower and Rapeseed Breeding

Jovanka Atlagi Sreten Terzi Ana Marjanovi-Jeromela Radovan Marinkovi

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia Summary: Cytogenetic research of sunflower and rapeseed has a century long tradition. Chromosome number and morphology were studied at first in species from the Helianthus and Brassica genera, and than their cytotaxonomy and phylogenesis. Cytogenetic research facilitated gene transfer from wild into cultivated species, in vitro plant growing, understanding and usage of cytoplasmic male sterility (CMS), fertility restoration, etc. This paper describes various research aspects used in the cytogenetic laboratory of Institute of Field and Vegetable Crops, Novi Sad, the results of which were used in sunflower and rapeseed breeding. Key words: breeding, cytogenetic research, rapeseed, sunflower

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www.nsseme.com/journal.html

Durability of Resistance to Rust in Sunflower

435 Phytopathology / Fitopatologija Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 435-438 review article / pregledni rad

Durability of Resistance to Rust in Sunflower: Implications for Resistance Breeding

María José Llamas Elena Prats Diego Rubiales

primljeno / received: 18.04.2010. prihvaeno / accepted: 25.05.2010. © 2010 IFVC

Summary: Sunflower rust is caused by Puccinia helianthi Schw. and is an important disease in Argentina, Australia, Canada, Egypt, Israel, Turkey, USA and the former USSR. Sunflower rust is a macrocyclic, heterothallic and autoecious fungus, surviving mainly in plant debris in soil in the form of teliospores. New races of P. helianthi are continuously evolving and overcoming resistance genes, resulting in a continuous threat for the crop. The deployment of hybrids with simple resistances has exacerbated this problem. Combination of the appropriate R-genes could result in a resistance more difficult to overcome. The identification and characterization of new durable sources of sunflower rust resistance is a high priority for both researchers and industry. There is a possibility of breeding for high coumarin content to improve resistance at early stages of fungal development. Key words: breeding, durability, P. helianthi, resistance, rust, sunflower

Introduction Sunflower (Helianthus annuus L.) is one of the four main edible oilseed crops in the world (FAOSTAT 2009). This crop, native from America (Heiser 1978) was likely introduced into Europe from Mexico by the Spanish explorers and later from north eastern Canada and United States by French and British explorers (Putt 1978). Sunflower cultivation started in Eastern Europe in the 18th century with Russia being still at present the first producer followed by Ukraine and Argentina (FAOSTAT 2009). Sunflower rust caused by Puccinia helianthi Schw. is a widespread disease being reported as of importance in Argentina, Australia, Canada, Egypt, Israel, Turkey, USA and the former USSR (Gulya et al. 1997). Severe infections cause premature leaf senescence (Siddiqui & Brown 1977) and a significant reduction in crop yield, seed and oil quality of cultivated sunflower (Markell et al. 2009). Although rust can be controlled by fungicides, their use is limited because of the low unit-value of sunflower (Zimmer & Hoes 1978).

E. Prats D. Rubiales ) ( Institute for Sustainable Agriculture, CSIC, Córdoba, Spain e-mail: [email protected] M. J. Llamas Junta de Andalucía, Consejería de Agricultura y Pesca, Almería, Spain

Sunflower Rust Life Cycle P. helianthi is a macrocyclic, heterothallic and autoecious fungus. It mainly survives in plant debris in soil in the form of teliospores. Teliospores germinate by producing promycelium which gives rise to four haploid basidiospores by meiosis. Under favorable conditions, the basidiospores may establish the infection by direct penetration of cotyledons and leaves. Then, sporidium starts a mycelium resulting in the development of a pycnium which produces pycniospores. Mating between (+) and (-) type pycnia by means of insects or rain water is required to form aecia with binucleate aeciospores. Aeciospores are airborne and penetrate into the leaf mesophyll cells through the stomata establishing a biothrophic interaction in which the fungus uptake nutrients for growing of the colony and production of uredia and uredospores. Annual completion of the life cycle provides the opportunity for sexual recombination of existing virulence combinations and dissemination of new pathotypes (Markell et al. 2009). Uredophase is the most common, conspicuous and economically significant stage of P. helianthi life cycle (Zimmer & Hoes 1978) Uredospores may then reach neighbouring leaves, petioles, stems and bracts. Under high relative humidity and 10-25ºC uredospores germinate and produce a germtube. Once the germtube grows over a stoma, an appressorium is formed. The stoma is penetrated via a

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penetration peg and an H-shaped substomatal vesicle is formed in the substomatal cavity. Approximately twenty-four hours afterwards, two or more infection hyphae grow out from this vesicle. When the infection hyphae contact a mesophyll cell, a haustorium mother cell forms and invaginates a haustorium into the cell. Haustorium is the feeding structure that allows growing of successive hyphae. The hyphae network is called colony. This colony grows and houses new uredial pustules from which uredospores are released 4 to 8 days after first haustorium and can produce a new disease cycle. Sunflower Rust Races The existence of physiologic races of P. helianthi has been known since early in the 20th century (Bailey 1923). Historically, the first change in virulence was detected in 1981, almost 10 years after introduction of R1 rust resistance gene in commercial hybrids (Kochman & Goulter 1984). Since then, more than 78 pathotypes have been identified (Sendall et al. 2006). An initial differential set consisting of three lines of cultivated sunflower enabled the identification of races 1, 2, 3 and 4 (Putt & Sackston 1957). By the 1970s, both wild Helianthus and cultivated sunflower were infected by the race 1 in the north central region of the USA, whereas races 2 and 3 were observed only on wild species (Zimmer & Rehder 1976). Race 1 was also reported in Russia (Pustovoit & Slyusar 1980) and Turkey (Tan 1993). In the late 1980s, race 3 became predominant leading to severe losses (Gulya 1990). Race 3 was also predominant by the 1980s in Canada, followed by races 4 and 1 (Rashid 1991). More than 10 races were described in Argentina (Antonelli 1985). By the early 1990s six races had been identified (Lambrides & Miller 1992) but race 3 was still predominant (31%). Race 4 was the predominant one in Mozambique in the 1990s, followed by a recent rapid development of new races (Zazzerini et al. 2005). Over 39 races were recently identified in USA (Gulya & Markell 2008). Race situation is also well known in Australia, where races 1 and 3 were described in the 1980s (Kochman & Kong 1990), that extended to up to seven races the 1990s and to more than 78 pathotypes today (Sendall et al. 2006). This spectacular virulence development was predictable. We just need to learn from history. Despite the monumental strides made in crop breeding for rust resistance, for many decades (Stakman 1946) breeders and pathologists have been aware that much of their work to introduce resistance in high yielding cultivars has been

inadvertently ephemeral. For decades, selection for resistance was based on highly specific, clearly recognized complete resistance, which is usually controlled by a single gene, and this form of resistance has commonly proved ephemeral due to the evolution of virulent fungal isolates that negated the breeders efforts and lead to spectacular "boom and bust" cycles (Rubiales & Niks 2000). Similarly, new races of P. helianthi are continuously evolving and overcoming resistance genes, resulting in a continuous threat for the crop (Gulya et al. 1997; Sendall et al. 2006). The rapidity with which P. helianthi is able to generate new virulent pathotypes limits the commercial life of hybrids to less than five years. The deployment of hybrids with simple resistances has exacerbated this problem (Lawson et al. 1998). Basis for Sunflower Rust Resistance The first identified resistance genes were designated as R1 and R2 (Putt & Sackston 1963) and controlled races 1, 2 and 3, the main races for many decades in North America. Afterwards, a further number of genes conferring rust resistance were identified. Gene R3 provides resistance against Australian Races 0 and 1 (Goulter 1990). Genes R4 and R5 provides resistance to Race 4 (Miller at al. 1988). At least 21 putative R-genes have been identified at the R4 locus, designated R4a to R4u (Sendall et al. 2006). Genes R6, R7, R8 and R9 confer resistance to North American Races 1, 2, 3 and 4, respectively (Quresh & Jan 1993). Gene Pu6 provides resistance to North American Races 1, 2, 3 and 4 (Yang et al. 1989). Genes Ph1, Ph2, Ph2a and Ph3 confer resistance to Argentine race ARG 340 and North American Race 2 (Antonelli 1985). Gene R10 confers resistance to Race 6 (Lambrides & Miller 1994). New R-genes are continuously identified and sequentially incorporated to commercial hybrids where the resistance gene present has been overcome. The rapid appearance of virulence makes clear that this strategy is not sustainable. The identification and characterisation of new durable sources of sunflower rust resistance is a high priority both for researchers and industry. Gene pyramiding has been proposed as a mean of extending the temporal usefulness of resistance genes that are individually no longer effective. Gene pyramiding is characterized by a combination of different resistance genes to different rust isolates that come from different locations and in this way a specific regional response can be expected and as a result, resistant hybrids for a longer period. There are indications that some virulence genes do not recombine naturally in the sunflower rust

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populations (Sendall et al. 2006). It seems then plausible that the combination of the appropriate R-genes can result in a resistance more difficult to overcome. Genes to be pyramided should be chosen according to what is known about virulence evolution of the pathogen population. In order to facilitate the selection process and to know the presence of a specific gene into the pyramid, molecular marker assisted selection can be used instead of the usual pathogenicity testing. Markers have already been identified for a number of R-genes (Lawson et al. 1998; Sendall et al. 2006; Radwan 2010). How to Increase Durability of Resistance A better understanding of the plant­pathogen interaction is also necessary for a durable control of rust disease. A battery of resistance mechanisms, acting either before or after stomatal penetration, might prevent rust development. There are alternative resistance mechanisms that might prove more durable, for instance those arresting the pathogen before haustorium formation. Prehaustorial resistance has usually been named "partial resistance" and has been identified in a range of crop­rust interactions (Niks & Rubiales 2002). It includes different resistance mechanisms that may act at the cell penetration stage or even earlier, before stomatal penetration. In most cases, prehaustorial resistance is polygenetically inherited and can be selected by recurrent selection or by marker assisted selection. However, there are examples of prehaustorial resistance conferred by single genes, such as Lr34 and Lr46, against P. triticina Eriks. in wheat (Triticum aestivum L.) that are associated with reduced haustorium formation, but not with hypersensitivity (Martínez et al. 2001, Rubiales & Niks 1995). Prestomatal penetration mechanisms are frequent in nonhosts, but have seldom, if ever been reported in hosts against their appropriate rusts. A notable exception is the low appressorium formation by various leaf rusts of cereals in some genotypes of Hordeum chilense Roem. et Schult. and of other wild barleys (up to 10 fold reduction) but unfortunately not in accessions of the cultivated barley (H. vulgare L.) or any cultivated cereal (Rubiales & Niks 1992, Rubiales & Niks 1996). In these, only a little reduction in appressorium formation by P. hordei Otth in some cer-mutants (Rubiales et al. 2001) and in stomatal recognition by P striiformis Westend. in some resistant wheat cultivars (Broers & Lopez-Atilano 1996) were found. Remarkably, strong reduction of P. helianthi germination and appressorium formation was observed in sunflower

AMES 18925 accession (Prats et al. 2007). An excretion of higher amount of the coumarin ayapin to the leaf surface is believed to be, at least partly, responsible for the lack of success in appressorium formation. Ayapin is considered a potent antifungal sunflower coumarin to pathogens such as Sclerotinia spp. and has been shown to inhibit wheat stem rust (P. graminis Pers.) urediospore germination (Prats et al. 2006, Urdangarin et al. 1999). Scopoletin also reduce appressorium formation, though less effectively than ayapin (Prats et al. 2002). Inhibition of P. helianthi urediospore germination was also reported in sunflower treated with the systemic acquired resistance inducer, ASM (Prats et al. 2002). However, this inhibition could not be associated with the excretion of any known coumarins. Excretion of other phenolic compounds was also observed after ASM treatment. These other compounds are also constitutive in sunflower genotypes with Sclerotinia spp. resistance (Prats et al. 2003), and they are currently under further characterization. This highlights the importance of constitutive sunflower coumarins in hampering sunflower rust germ tube growth and appressorium formation and suggest the involvement of other related phenolic compounds inhibiting rust germination. This opens the possibility of breeding for high coumarin content to improve resistance at these early stages of fungal development. References

Antonelli E F (1985): Variabilidad de la población patógena de Puccinia helianthi. Proc. 11th Internationl Sunflower Conference, Mar del Plata, Argentina, 591-596 Bailey D L (1923): Sunflower Rust. Univ. Minnesota Tech. Bull. 16 Broers L H M, Lopez-Atilano R M (1996): Effect of quantitative resistance in wheat on the development of Puccinia striiformis during early stages of infection. Plant Dis. 80: 1265-1268 FAOSTAT (2009): FAO Corporate Statistical Database [online]. [1 p.] Available at http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor (cited 15 April 2010, verified 18 April 2010). FAO Corporate Statistical Database (FAOSTAT), United Nations Food and Agriculture Organization (FAO), Rome Goulter K C (1990): Breeding of a rust differential sunflower line. Proc. 8th Australian Sunflower Association Workshop, Kooralbyn, Queensland, 120-124 Gulya T J (1990): The sunflower rust situation in 1989. Proc. 12th Sunflower Resistance Workshop, Fargo, USA, 106 Gulya T J, Charlet L D, Masirevic S (1997): Compendium of Sunflower Diseases and Insects. APS Press, St. Paul Gulya T, Markell S (2008). Sunflower Rust Status ­ 2008 Race Frequency across the Midwest & Resistance Among Commercial Hybrids. http://www.sunflowernsa.com/uploads/ Gulya_RustStatus_09.pdf (cited 13 May 2010). Heiser C B (1978): Taxonomy of Helianthus and origin of domesticated sunflower. In: Carter J F (ed.), Sunflower Science and Technology. ASA, CSSA, SSSA, Madison, USA, 31-53 Kochman J K, Goulter K C (1984): The occurrence of a second race of rust in sunflower crops in eastern Australia. Australasian Plant Pathol. 13: 3-4 Kochman J K, Kong G A (1990): Investigations of slow rusting and resistance gene pyramiding to control sunflower rust.

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Proc. 8th International Sunflower Conference, Kooralbyn, Queensland, 1301-1304 Lambrides C J, Miller J F (1992): Inheritance of rust resistance in sunflower. Proc. Sunflower Research Workshop, Fargo, USA, 44-49 Lambrides C J, Miller J F (1994): Inheritance of rust resistance in a source of MC29 sunflower germplasm. Crop Sci. 34: 1225-1230 Lawson W R, Goulter K C, Henry R J, Kong G A, Kochman J K (1998): Marker-assisted selection for two rust resistance genes in sunflower. Mol. Breed. 4: 227-234 Markell S, Gulya T, McKay K, Hutter M, Hollingsworth C, Ulstad V, Koch R, Knudsvig A (2009): Widespread occurrence of the aecial stage of sunflower rust caused by Puccinia helianthi in North Dakota and Minnesota in 2008. Plant Dis. 93: 668 Martínez F, Niks R E, Singh R P, Rubiales D (2001): Characterization of Lr46, a gene conferring partial resistance to wheat leaf rust. Hereditas 135: 111-114 Miller J F, Rodriguez R H, Gulya T J (1988): Evaluation of genetic materials for inheritance of resistance to race 4 of rust in sunflower. Proc. 12th International Sunflower Conference, Novi Sad, Serbia, II, 361-365 Niks R E, Rubiales D (2002): Potentially durable resistance mechanisms in plants to specialised fungal pathogens. Euphytica 124: 201-216 Prats E, Rubiales D, Jorrín J (2002): Acibenzolar-S-methylinduced resistance to sunflower rust (Puccinia helianthi) is associated with an enhancement of coumarins on foliar surface. Physiol. Mol. Plant Pathol. 60: 155-162 Prats E, Bazzalo M E, Leon A, Jorrín J (2003): Accumulation of soluble phenolic compounds in sunflower capitula correlates with resistance to Sclerotinia sclerotiorum. Euphytica 132: 321-329 Prats E, Bazzalo M E, Leon A, Jorrín J (2006): Fungitoxic effect of scopolin and related coumarins on Sclerotinia sclerotiorum: A way to overcome sunflower head rot. Euphytica 147: 451-460 Prats E, Llamas M J, Jorrín J, Rubiales D (2007): Constitutive coumarin accumulation on sunflower leaf surface prevents rust germ tube growth and appressorium differentiation. Crop Sci. 47: 1119-1124 Pustovoit G V, Slyusar E L (1980): Inheritance of rust resistance in the sunflower. Sov. Gen. 16: 456-460 Putt E D (1978): History and present world status. In: Carter J F (ed.) Sunflower Science and Technology. ASA, CSSA, SSSA, Madison, USA, 1-30 Putt E D, Sackston W E (1957): Studies on sunflower rust. I. Some sources of rust resistance. Can. J. Plant Sci. 37: 43-54 Putt E D, Sackston W E (1963): Studies on sunflower rust. IV. Two genes, R1 and R2 for resistance in the host. Can. J. Plant Sci. 43: 490-496 Quresh Z, Jan C C (1993): Allelic relationships among genes for resistance to sunflower rust. Crop Sci. 33: 235-238

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Radwan O (2010): Isolation and Expression of an NBS-LRR Protein-encoding Resistance Gene Candidate that Segregates with a Rust Resistance Gene in Sunflower. J. Phytopathol. 158: 433-443 Rashid K Y (1991): Incidence and virulence of Puccinia helianthi on sunflower in western Canada during 1988-1990. Can. J. Plant Pathol. 13: 356-360 Rubiales D, Niks R E (1992): Low appressorium formation by rust fungi on genotypes of Hordeum chilense. Phytopathol. 82: 1007-1012 Rubiales D, Niks R E (1995): Characterization of Lr34, a major gene conferring nonhypersensitive resistance to wheat leaf rust. Plant Dis. 79: 1208-1212 Rubiales D, Niks R E (1996): Avoidance of rust infection by some genotypes of Hordeum chilense due to their relative inability to induce the formation of appressoria. Physiol. Mol. Plant Pathol. 49: 89-101 Rubiales D, Niks R E (2000): Combination of mechanisms of resistance to rust fungi as a strategy to increase durability. Options Méditerraneannes 40: 333-339 Rubiales D, Ramírez M C, Carver T L W, Niks R E (2001): Abnormal germling development by brown rust and powdery mildew on cer barley mutants. Hereditas 135: 271-276 Sendall B C, Kong G A, Gouter K C, Aitken E A B, Thompson S M, Mitchell J H M, Kochman J K, Lawson W, Shatte T, Gulya T J (2006): Diversity in the sunflower: Puccinia helianthi pathosystem in Australia. Australasian Plant Pathol. 35: 657-670 Sidiqqui M Q, Brown J F (1977): Effects of simulated rust epidemics on the growth and yield of sunflower. Austr. J. Agric. Res. 28: 389-393 Stakman E C (1946): Plant pathologist's merry-go-round. J. Heredity 37: 259-265 Tan A M (1993): Identification of rust races of sunflower. Anadolu, J. Aeg. Agric. Res. Inst. 1: 63-72 Urdangarin C, Regente M C, Jorrín J, de la Canal L (1999): Sunflower coumarin phytoalexins inhibit the growth of the virulent pathogen Sclerotinia sclerotiorum. J. Phytopathol. 147: 441­443 Yang S M, Dowler W M, Luciano A (1989): Gene Pu6: A new gene in sunflower for resistance to Puccinia helianthi. Phytopathol. 79: 474-477 Zazzerini A, L. Tosi L, Mondjana A M (2005): Occurrence of Puccinia helianthi Races on Sunflower in Mozambique. J. Phytopathol. 153: 733-735 Zimmer D E, Hoes J A (1978): Sunflower diseases. In J.F. Carter (ed.) Sunflower science and technology. Agron. Monogr. 19. ASA, CSSA, and SSSA, Madison, WI, USA, 225-262. Zimmer D E, Rehder D (1976): Rust resistance of wild Helianthus species of the north central United States. Phytopathol. 66: 208-211

Postojanost otpornosti suncokreta na ru: preduslovi oplemenjivanja za otpornost

Marija Hose Ljamas1 Elena Prac2 Dijego Rubijales2

1

Vlada Andaluzije, Savet za poljoprivredu i ribarstvo, Almerija, Spanija 2 Institut za odrzivu poljoprivredu, Kordova, Spanija

Izvod: Ru suncokreta, koja je znacajna bolest u Argentini, Australiji, Kanadi, Egiptu, Izraelu, Turskoj, SAD i drzavama bivseg SSSR-a, uzrokuje Puccinia helianthi Schw. Ra suncokreta je makrociklicna, heterotalicna i autoecijska gljiva, koja prezivljava uglavnom u biljnim ostacima u zemljistu u obliku teliospora. Do sada je identifikovano vise od 78 patotipova. Novi R geni, koji kontrolisu otpornost na ru, neprestano se ugrauju u komercijalne hibride u kojima je dotadasnji gen otpornosti postao prevazien. Identifikacija i karakterizacija novih postojanih izvora otpornosti prema ri suncokreta od prvorazrednog je znacaja za istrazivanja i industriju. Postoji mogunost oplemenjivanja za visok sadrzaj kumarina kako bi se poboljsala otpornost na rane faze razvoja gljive. Kljucne reci: oplemenjivanje, postojanost, Puccinia helianthi, ra, suncokret

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Resistance of Food Legumes to Ascochyta blight

439 Phytopathology / Fitopatologija Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 439-442 review article / pregledni rad

Resistance of Cool Season Food Legumes to Ascochyta Blight

Diego Rubiales · Sara Fondevilla

received / primljeno: 18.04.2010. accepted / prihvaeno: 03.05.2010. © 2010 IFVC

Summary: Legume cultivation is strongly hampered by the occurrence of Ascochyta blights. Strategies of control have been developed but only marginal successes have been achieved. In the present paper we present and re-evaluate some recent developments in control in the light of recent developments in crop breeding and molecular genetics. The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to speed up the delivery of improved cultivars to the farmer. To date, however, progress in marker development and delivery of useful markers has been slow. The application of knowledge gained from basic genomic research and genetic engineering will contribute to more rapid crop legume improvement for resistance against Ascochyta blight. Key words: Ascochyta spp., biotechnology, crop management, food legumes, resistance

Introduction Cool season food legumes provide an excellent source of high quality plant protein and have a key role in arable crop rotations, reducing the need for fertiliser application and acting as break-crops. However, cool season food legumes are affected by a number of foliar and root diseases that cause wide spread damage and in severe cases cause complete crop loss. The most important foliar diseases worldwide are Ascochyta blights. A number of strategies of Ascochyta blight control have been developed including cultural practices and chemical control. However, only marginal successes have been achieved, most control methods being uneconomical, hard to achieve or resulting in incomplete protection. Breeding for resistance is the most economic and environmentally friendly control method. Breeding for Resistance Sources of incomplete resistance have been identified in all of the cool season food legumes and the currently available resistance is being used in breeding programmes designed to develop cultivars with improved resistance. Since there are some recent reviews on breeding methods, screening procedures, the Ascochyta

D. Rubiales ) ( CSIC, Institute for Sustainable Agriculture, Córdoba, Spain e-mail: [email protected] S. Fondevilla University of Córdoba, Genetic Department, Córdoba, Spain

diseases and biology of the pathogens (Ye et al. 2002, Pande et al. 2005, Bretag et al. 2006, Tivoli et al. 2006, Sillero et al. 2010), in this review we will mainly focus on the recent developments in understanding the genetics of host resistance for each of the major cool season food legumes and point out immediate needs in research that in our opinion will further advance deployment of resistance in managing Ascochyta blight in cool season food legumes. Ascochyta blight of pea is a disease complex caused by three pathogens: Ascochyta pisi which causes well-defined lesions (spots) on leaves, stems and pods; Phoma medicaginis var. pinodella, previously Ascochyta pinodella, which causes lesions on leaves and stems, and foot rot; and Didymella pinodes, previously Mycosphaerella pinodes which causes blight starting with small purple to black spots, enlarging and turning brown to black. Resistance to A. pisi race C is controlled by a mayor gene modified by minor genes or QTLs (Darby et al. 1985, Dirlewanger et al. 1994). Some levels of incomplete resistance against M. pinodes and P. medicaginis have been reported (Ali et al. 1978, Kraft 1998, Wroth 1998, Prioul et al. 2003, Fondevilla et al. 2005). The majority of the genetic studies concerning resistance to M. pinodes in pea have concluded that resistance is a polygenic trait (Fondevilla et al. 2007). Numerous QTLs explaining from low to moderate percentage of the variation of the trait have been identified (Timmerman-Vaughan et al. 2002, Tar'an et

Acknowledgments: Financial support by Spanish project AGL200801239 is acknowledged.

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al. 2003, Prioul et al. 2004, Fondevilla et al. 2008). In addition, candidate genes approached and comparative mapping have revealed the co-localization of QTLs for resistance to M. pinodes and resistance gene analogs, the putative transcription factor PsDof1 and the pea defensin DRR230-b (Timmerman-Vaughan et at. 2002; Prioul-Gervais et al. 2007). Incomplete levels of resistance to the Ascochyta blight of lentil (Ascochyta lentis, teleomorph Didymella lentis) is also available in lentil germplasm. Single genes, either dominant (Ford et al. 1999) or recessive (Chowdhury et al. 2001) have been reported together with molecular markers flanking the resistance gene. In addition, at least five QTL for blight resistance have been mapped that together accounted for 50% of phenotypic variation (Rubeena et al. 2006). Ascochyta blight, caused by Ascochyta rabiei (teleomorph: Didymella rabiei), is responsible for widespread damage to chickpea crops worldwide. Incomplete resistance is available in chickpea germplasm (Reddy & Singh 1984, Singh & Reddy 1994) and is being used in breeding programs. Early inheritance studies concluded that resistance could be controlled by one, two or three genes (Singh & Reddy 1993, Collard et al. 2001, Chen et al. 2004). More recently, two major QTLs have been identified in LG4, conferring resistance to pathotype II. Other QTLs have been identified in LG2, mainly associated with resistance to pathotype I, (Santra et al. 2000, Tekeoglu et al. 2002, Collard et al. 2003, Rakshit et al. 2003, Udupa and Baum 2003, Cho et al. 2004, Iruela et al. 2006). Current efforts are focused in saturating these regions with additional markers. Some genes involved in defence co-localizing with resistance QTLs have been reported (Winter et al. 2000, Huettel et al. 2002, Flandez-Galvez et al. 2003, Cho et al. 2004, Iruela et al. 2009). Ascochyta blight of faba bean is caused by Ascochyta fabae (teleomorph Didymella fabae). Incomplete levels of resistance have been identified in faba bean germplasm (Tivoli et al. 2006, Sillero et al. 2010) that are being used in breeding programmes to develop improved cultivars. The information about the genetic basis of the resistance has been conflicting since both polygenic and major gene inheritance have been suggested. Rashid et al. (1991) proposed a model consisting of 7 major genes controlling the resistance to 5 different isolates of A. fabae. Kophina et al. (2000) identified a major dominant gene as well as a complex of minor genes controlling resistance. More recently, Kharrat et al. (2006) suggested dominant monogenic control

at the level of leaves and, in addition, a recessive gene controlling resistance of stems. Román et al. (2003) reported a polygenic control determined by at least two QTLs. In a subsequent study Avila et al. (2004) detected 6 additional QTLs. Lessons to Learn from Model Legumes Although genomic regions involved in resistance to Ascochyta blight and candidate genes located in these regions have been identified, still very little is known about the mechanisms of response to Ascochyta at the histological, molecular and biochemical level. Utilisation of model systems, together with the modern molecular biology tools will be of great help for understanding the physiological and genetic nature of the Ascochyta-host interaction (Dita et al. 2006, Rispail et al. 2010). Considering the high level of synteny between the genomes of the model plant Medicago truncatula and the economically important grain legume crops, the results obtained in this model legume should be transferred between species and used in breeding programs. In M. truncatula several microarray platforms have been developed including an Affymetrix system that can be used to identify genes involved in M. truncatula defence against Ascochyta. Furthermore, this microarray can also be hybridized to cDNA from other legumes and has been already used to identify genes differentially expressed in response to M. pinodes in pea (Fondevilla et al. 2009). Other tools for this model legume include a library of more than 1000 transcription factors available at the Max-Planck Institute of Molecular Plant Physiology (Potsdam, Germany) that could be used to quantify the level of expression of these transcription factors in the interactions Medicago M. pinodes by real-time quantitative PCR and high resolution genetic maps under construction that could facilitate the identification and cloning of genes and QTLs involved in resistance to Ascochyta by comparative mapping with crop legumes maps. The omic tools developed for M. truncatula will allow us to investigate whether genes expressed in different processes in the model plant M. truncatula, including interaction with pathogens, are involved in the resistance to Ascochyta blight in crop legumes. However, these techniques demand previous sequence information knowledge, being limited to known genes. As a consequence, genes specifically involved in the Ascochyta-crop legumes interaction may not be detected. For this reason

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Collard B C Y, Pang E C K, Ades P K, Taylor P W J (2003): Preliminary investigation of QTL association with seedling resistance to ascochyta blight from Cicer echinospermun, a wild relative of chickpea. Theor. Appl. Gen. 107: 719-729 Darby P, Lewis B G, Matthews P (1985): Inheritance and expression of resistance to Ascochyta pisi. In: Hebblethwaite P D, Heath M C, Dawkins T C K (eds.), The Pea Crop. Butterworths, London, UK, 231-236 Dirlewanger E, Isaac P, Ranade S, Belajouza M, Cousin R, Devienne D (1994): Restriction fragment length polymorphism analysis of loci associated with disease resistance genes and developmental traits in Pisum sativum L. Theor. Appl. Gen. 88: 17-27 Dita M A, Rispail N, Prats E, Rubiales D, Singh K B (2006): Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes. Euphytica 147: 1-24 Flandez-Galvez H, Ades P K, Ford R, Pang E C K, Taylor P W J (2003): QTL analysis for ascochyta blight resistance in an intraespecific population of chickpea to six races of Ascochyta rabiei in the world germplasm collection of chickpea. Crop Sci. 23: 9-10 Fondevilla S, Avila C M, Cubero J I, Rubiales D (2005): Response to Mycosphaerella pinodes in a germplasm collection of Pisum spp. Plant Breed. 124: 313-315 Fondevilla S, Cubero J I, Rubiales D (2007): Inheritance of resistance to Mycosphaerella pinodes in two wild accessions of Pisum. Eur. J. Plant Pathol. 119: 53-58 Fondevilla S, Krajinski F, Küster H, Rubiales D (2009). Identification of genes involved in resistance to Mycospherella pinodes in pea using micro-array technology. 2nd International Ascochyta Symposium, Pullman, USA, 68 Fondevilla S, Rubiales D, Zatovic S, Torres A M (2008): Mapping of quantitative trait loci for resistance to Mycosphaerella pinodes in Pisum sativum subsp. syriacum. Mol. Breed. 21: 439-454 Ford R, Pang E C K, Taylor P W J (1999): Genetics of resistance to Ascochyta blight (Ascochyta lentis) of lentil and the identification of closely linked RAPD markers. Theor. Appl. Gen. 98: 93-98 Huettel B, Santra D, Muehlbauer F J, Kahl G (2002) Resistance gene analogues of chickpea (Cicer arietinum L.): isolation, genetic mapping and association with a fusarium gene cluster. Theor. Appl. Gen. 105: 479-490 Iruela M, Rubio J, Barro F, Cubero J I, Millán T, Gil J (2006): Detection of two quantitative trait loci for resistance to ascochyta blight in an intra-specific cross of chickpea (Cicer arietinum L.): development of SCAR markers associated with resistance. Theor. Appl. Gen. 112: 278-287 Iruela M, Pistón F, Cubero J I, Millán T, Barro F, Gil J (2009): The marker SCK13603 associated with resistance to ascochyta blight in chickpea is located in a region of a putative retrotransposon. Plant Cell Rep. 28: 53-60 Kharrat M, Le Guen J, Tivoli B (2006): Genetics of resistance to 3 isolates of Ascochyta fabae on faba bean (Vicia faba L.) in controlled conditions. Euphytica 151: 49­61 Kohpina S, Knight R, Stoddard F L (2000): Evaluating faba beans for resistance to ascochyta blight using detached organs. Aust. J. Exp. Agric. 40: 707-713 Kraft J M (1998): A search for resistance in peas to Mycosphaerella pinodes. Plant Dis. 82: 251-253 Pande S, Siddique K H M, Kishore G K, Bayaa B, Gaur P M, Gowda C L L, Bretag T W, Crouch J H (2005): Ascochyta blight of chickpea (Cicer arietinum L.): A review of biology, pathogenicity, and disease management. Aust. J. Exp. Agric. 56: 317-332 Pfaff T, Kahl (2003): Mapping of gene specific markers on the genetic map of chickpea (Cicer arietinum L.) Mol. Gen. Genomics 269: 243-251 Prioul S, Frankewitz A, Deniot G, Morin G, Baranger A (2004): Mapping of quantitative trait loci for partial resistance to

these approaches could be complemented with large scale expression techniques such as cDNAAFLPs, Supersage or Suppression Subtractive Hybridisation cDNA libraries that are able to identify novel genes. Conclusions Though limited in efficacy in many cases, the control methods available today represent a major progress when compared to the lack of any means for the control of these plants one or two decades ago. Crops can be protected by resistance, by selective fungicides, by biocontrol agents, and by cultural methods that did not existed before. The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to allow marker ­assisted selection with the hope that this will speed up the delivery of improved cultivars to the farmer. To date, however, progress in marker development and delivery of useful markers has been slow. We are now also facing an accelerated progress in the genomic and biotechnological research, which should soon provide important understanding of some crucial developmental mechanisms in both the pathogen and their host plants. The application of knowledge gained from basic genomic research and genetic engineering will contribute to more rapid legume improvement. References

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Rubiales D, Fondevilla S

Singh K B, Reddy M V (1993): Resistance to 6 races of Ascochyta rabiei in the world germplasm collection of chickpea. Crop Sci. 33: 186-189 Singh K B, Reddy M V (1994): Registration of 8 Ascochyta blight-resistant, early-maturing, large-seeded chickpea germplasms. Crop Sci. 34: 1416-1417 Tar'an B, Warkentin T, Somers D J, Miranda D, Vandenburg A, Blade S, Woods S, Bing D, Xue A, DeKoeyer D, Penner G (2003): Quantitative trait loci for lodging resistance, plant height and partial resistance to Mycosphaerella blight in field pea (Pisum sativum L.). Theor. Appl. Gen. 107: 1482-1491 Tekeoglu M, Rajesh P N, Muehlbauer F J (2002): Integration of sequence tagged microsatellites to the chickpea genetic map. Theor. Appl. Gen. 105: 847-854 Timmerman-Vaughan G M, Frew T J, Russell A C, Khan T, Butler R, Gilpin M, Murray S, Falloon K (2002): QTL mapping of partial resistance to field epidemics of ascochyta blight of pea. Crop Sci. 42: 2100-2111 Tivoli B, Baranger A, Avila C M, Banniza S, Barbetti M, Chen W, Davidson J, Lindeck K, Kharrat M, Rubiales D, Sadiki M, Sillero J C, Sweetingham M, Muehlbauer F J (2006): Screening techniques and sources of resistance to foliar diseases caused by major necrotrophic fungi in grain legumes. Euphytica 147: 223-253 Udupa S M, Baum M (2003): Genetic dissection of pathotypespecific resistance to ascochyta blight disease in chickpea (Cicer arietinum L.) using microsatelite markers. Theor. Appl. Gen. 106: 1196-1202 Winter P, Benko-Iseppon A M, Huttel B, Ratnaparkhe M, Tullu A, Sonnante G, Pfaff T, Tekeoglu M, Santra D, Sant V J, Rajesh P N, Kahl G, Muehlbauer F J (2000): A linkage map of the chickpea (Cicer arietinum L.) genome based on recombinant inbred lines from a C. arietinum × C. reticulatum cross: localization of resistance genes for fusarium wilt races 4 and 5. Theor. Appl. Gen. 101: 1155-1163 Wroth J M (1998): Possible role for wild genotypes of Pisum spp. to enhance ascochyta bligt resistance in pea. Aust. J. Exp. Agric. 38: 469-479 Ye G, McNeil D L, Hill G D (2002): Breeding for resistance to lentil Ascochyta blight. Plant Breed. 121: 185-191

Otpornost povrtarskih mahunarki umerenih klimata prema parazitima iz roda Ascochyta

Dijego Rubijales1 · Sara Fondevilja2

2

CSIC, Institut za odrzivu poljoprivredu, Kordova, Spanija Univerzitet u Kordovi, Odeljenje za genetiku, Kordova, Spanija

1

Izvod: Gajenje mahunarki je u velikoj meri otezano prisustvom bolesti koje prouzrokuju gljive iz roda Ascochyta. Razvijane su strategije u cilju njihovog suzbijanja, ali sa ogranicenim uspehom. Rad prikazuje i ponovo ocenjuje neka od savremenih dostignua mera suzbijanja u svetlu skorasnjih pomaka u oplemenjivanju biljaka i molekularnoj genetici. Primenjeno oplemenjivanje je trenutno usredsreeno na primenu biotehnoloskih metoda u razvoju veeg broja boljih markera koji treba da ubrzaju stvaranje poboljsanih sorti. Meutim, do danas je napredak u razvoju markera uopste i stvaranju korisnih markera bio spor. Primena znanja dobijenog putem osnovnih genomskih istrazivanja i genetickog inzenjeringa doprinee brzem oplemenjivanju gajenih mahunarki na otpornosti prema parazitima iz roda Ascochyta. Kljucne reci: Ascochyta spp., biotehnologija, otpornost, povrtarske mahunarke, zastita useva

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Kukuruz i globalne klimatske promene

443 Agroklimatologija / Agroclimatology Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 443-450 pregledni rad / review article

Proizvodnja kukuruza u uslovima globalnih klimatskih promena

Goran Bekavac Bozana Purar ore Jockovi Milisav Stojakovi Mile Ivanovi Goran Malidza Ivica alovi

primljeno / received: 30.04.2010. prihvaeno / accepted: 25.05.2010. © 2010 IFVC

Izvod: Klimatske promene i ocekivana varijabilnost klimatskih parametara predstavljaju ozbiljan izazov za poljoprivredu 21. veka. Na globalnom nivou, ocekuje se porast temperature vazduha, promene kolicine i rasporeda padavina, poveanje varijabilnosti klimatskih parametara i pojava ekstremnih klimatskih dogaaja. U cilju izbegavanja, ili barem redukovanja negativnih efekata globalnih klimatskih promena, predlozeno je nekoliko adaptacionih strategija. Kao dve najznacajnije mere adaptacije predlazu se prilagoavanje tehnologije proizvodnje i oplemenjivanje biljaka na tolerantnost prema izmenjenim uslovima spoljasnje sredine. Kljucne reci: klima, kukuruz, oplemenjivanje

Uvod Poljoprivredna proizvodnja se duzi niz godina suocava sa brojnim izazovima prouzrokovanim globalnim klimatskim promenama. Meunarodni panel o klimatskim promenama u svom cetvrtom izvestaju o proceni promene klime iz 2007. predvia dalji porast temperature, promene kolicine i rasporeda padavina, poveanje varijabilnosti klimatskih parametara i pojavu ekstremnih klimatskih dogaaja, kao sto su suse, poplave i olujni vetrovi. Ukoliko koncentracija gasova staklene baste nastavi sa rastom, mogao bi se ocekivati jos dramaticniji porast temperature (za 1,1°C do 6,2°C) do kraja ovog veka (IPCC WGI 2007). Iako su promene klime na globalnom nivou primeene jos pre nekoliko desetina godina (National Science Board U.S. 1974), iako su dobro poznati uticaji istih na gajene kulture, stocni fond, hidroloski balans, agrarna ulaganja, proizvodne resurse i druge komponente agroekosistema (Adams et al. 1998), svest o problemima i posledicama klimatskih promena na poljoprivredu je i dalje nedovoljno nerazvijena, ulaganja u resavanje problema nesrazmerna, a sistematska multidisciplinarna istrazivanja prisutna samo u najrazvijenijim zemljama.

G. Bekavac ) ( B. Purar . Jockovi M. Stojakovi M. Ivanovi G. Malidza I. alovi Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

Klima i kukuruz Kukuruz kao C4 biljka ima vei temperaturni optimum od C3 biljaka i bolje podnosi visoke temperature. Ipak, kriticni period u kojem su biljke kukuruza narocito osetljive na visoke temperature i nedostatak vode od velikog je znacaja za formiranje prinosa. Krai periodi ekstremnih temperatura praeni susom u periodu cvetanja imaju daleko pogubniji efekat na prinos od globalnih promena srednjih temperatura u duzem vremenskom intervalu (Sage & Kubien 2003). Visoke temperature i druge vrste abiotickog stresa uticu na smanjenje kolicine vlage u zemljistu, pojavu suse, ubrzano starenje biljke, susenje polena, slabiju oplodnju i smanjenje prinosa (Zaidi 2002). Povisena radijacija stimulise asimilaciju listova i poveava prinos, ali poveava i evapotranspiraciju, sto se u uslovima ogranicene obezbeenosti biljaka vodom moze negativno odraziti na prinos. Promene u rezimu padavina imaju veliki uticaj ne samo na prosecan prinos kukuruza, nego i na stabilnost prinosa, koji se smanjuje shodno opadanju srednjih vrednosti sezonskih padavina i porastu varijabilnosti istih. Osetljivost agroekosistema na promene klime specificna je za lokalitet i zavisi od postojanja jednog ili vise limitirajuih faktora (vodni deficit, visoke temperature, hemijske i fizicke karakteristike zemljista) i ravnoteze, odnosno izbalansiranosti samog sistema (von Braun 2007). Rezultati dosadasnjih istrazivanja pokazuju da se pozitivan

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efekat klimatskih promena na prinos kukuruza moze ocekivati samo u severnijim i hladnijim proizvodnim regionima, sto pruza mogunost sirenja povrsina pogodnih za gajenje kukuruza ka severu, dok bi veina regiona juzne Evrope mogla osetiti manje ili vee gubitke prinosa (Olesen et al. 2007, Zaidi 2002). Prema rezultatima brojnih klimatskih modela, juzni delovi kontinenta bi se mogli suociti sa vodnim deficitom i cestim vremenskim ekstremima. Bez primene odgovarajuih mera adaptacije, moglo bi doi do variranja i znacajnog pada prosecnih prinosa, kao i redukcije povrsina pogodnih za gajenje useva. Na bazi porasta temperature za 5°C i smanjenja kolicine padavina u toku letnjih perioda, moze se ocekivati pad prinosa kukuruza za 10%, cak i kada se uzme u obzir pozitivan efekat duplo vee koncentracije CO2 (Torriani et al. 2007). U svetu se izvodi veliki broj istrazivanja o uticaju klimatskih promena na rast i razvoj kukuruza primenom simulacionih modela poput CERES-Maize, WOFOST, MACROS i EPIC (Travasso et al. 2009, Zalud & Dubrovsky 2002, Jones & Thornton 2003, Meza et al. 2008, Torriani et al. 2007, Tsvetsinskaya et al. 2003, Mera et al. 2006, Abraha & Savage 2006, Erda et al. 2005, Trolove et al. 2008). Modeli obrauju podatke o genotipu, karakteristikama zemljista, agrotehnickim operacijama i vrednostima osmatranih vremenskih parametara (koncetracija CO2 i serija dnevnih vremenskih parametara) koji se prethodno modifikuju u skladu sa jednom ili vise scenarija klimatskih promena (SRES A1, A2, B1, B2, GCM). Simulacioni modeli se izvode za set nelimitiranih uslova (u kojima nivo snabdevenosti vodom i hranivima ne ometa razvoj useva) i set limitiranih uslova (nedovoljna obezbeenost vodom i hranivima) (Zalud & Dubrovsky 2002). Rezultati veeg broja modela ukazuju na uglavnom negativan uticaj klimatskih promena na prinos zbog skraivanja fenoloskih faza razvoja kukuruza u uslovima povisenih temperatura. Travasso et al. (2009) prognoziraju variranje prinosa kukuruza u Argentini od +46% do -17%, u zavisnosti od scenarija klimatskih promena i od toga da li je efekat CO2 uzet u obzir. Prema istim autorima, povisene temperature bi mogle skratiti vegetacioni period kukuruza za 27 dana, sto za posledicu moze imati smanjenje prinosa. Do slicnih zakljucaka dosli su takoe Zalud & Dubrovsky (2002) primenom simulacionog modela u proizvodnim uslovima Ceske, predviajui kretanje prinosa kukuruza od +18% do -29% i skraivanje vegetacionog perioda za jedan mesec. Ispitivanja klimatskih parametara u Srbiji pokazuju da je u poslednjih 50 godina doslo do

progresivnog produzenja susnih perioda, poveanja temperature, smanjenja kolicine padavina i poveanja povrsina semiaridnih oblasti, pre svega u severoistocnim delovima Republike. Osim toga, doslo je do poveanja broja tropskih dana (sa maksimalnom temperaturom iznad 300C) kao i smanjenja broja mraznih dana (sa minimalnom temperaturom ispod 00C). Neki regionalni klimatski modeli predviaju da bi do 2100. maksimalne temperature vazduha mogle porasti za 2,6-2,80C u odnosu na maksimalne temperature vazduha s kraja proslog veka, sto bi moglo imati snazne implikacije na agroekosisteme, kako u nas tako i u okruzenju. Pri primeni sadasnjih agrotehnickih i drugih mera gajenja, prema tri scenarija klimatskih promena prinos kukuruza u Grckoj bi se mogao smanjiti za 4,5-8,5% pri porastu temperature za 2°C, odnosno za 4,3-14% pri porastu temperature za 4°C u zavisnosti od lokacije (Kapetanaki & Rosenzweig 1997). Ne uzimajui u razmatranje mogunost adaptacije, Torriani et al. (2007) utvrdili su ocekivani pad prosecnog prinosa u Svajcarskoj sa 8,5 t ha-1 na 7,3 t ha-1. Simulirani prinos kukuruza u Juznoj Africi raste pri duplo veoj koncentraciji CO2 i pri porastu temperature za 2°C, ali se smanjuje pri porastu temperature za 4°C (Abraha & Savage 2006). Projektovani prosecni prinosi kukuruza u Kini u uslovima suvog ratarenja i navodnjavanja u narednih 20-80 godina mogli bi opasti za 37% zbog skraivanja vegetacionog perioda u uslovima povisenih temperatura, dok bi u Cileu u zavisnosti od scenarija, primenjenih mera adaptacije i hibrida, snizenje prosecnih prinosa moglo iznositi 14% do 28%. Smanjenje prinosa kukuruza u zemljama Latinske Amerike prema podacima Adams et al. (1998) moglo bi se kretati od -61% u Meksiku do -2% u Brazilu. Iako rezultati istrazivanja Jones & Thornton (2003) ukazuju na smanjenje proizvodnje kukuruza u Africi i Latinskoj Americi do 2055. godine za samo 10% u odnosu na 2000. godinu, ovaj procenat je ekvivalentan gubitku od 2 milijarde dolara godisnje. Klima i bolesti Osim uticaja na samu biljku, klimatske promene bi mogle dovesti do ekspanzije ve postojeih prouzrokovaca bolesti i/ili introdukciju novih, kserofilnih vrsta na dva nacina: postepenim promenama klime, menjajui distribuciju vektora biljnih bolesti ili poveavajui vodni i temperaturni stres biljaka i frekventnijim pojavama ekstremnih vremenskih prilika, neuobicajenih za dato podrucje. Blaze zime, vise none tempe-

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rature i vise srednje dnevne temperature bi poveale prezimljavanje patogena, ubrzale zivotni ciklus vektora i patogena, sporulaciju i infekciju folijarnim gljivama. Ceste i ekstremne kolicine padavina koje se predviaju po nekim modelima mogu imati uticaj na periode povoljne za razvoj bolesti, pre svega zbog visoke relativne vlage u biljnom sklopu (Gregory 2009). Efikasnost fungicida i baktericida bi u novonastalim uslovima mogla biti umanjena, jer ceste padavine dovode do ispiranja kontaktnih preparata i shodno tome potrebe za cesim tretmanima (Petzoldt & Seaman 2006). Visoke temperature i susni uslovi mogu uticati na pojavu trulezi klipa kukuruza i stvaranje mikotoksina. Pojava aflatoksina, vrlo opasnog mikotoksina koji produkuje gljiva Aspergillus flavus poveava se u uslovima vodnog stresa. Savremeni hibridi i sorte su manje ili vise tolerantni prema gljivama iz roda Fusarium i Aspergillus, ali vremenske prilike koje favorizuju razvoj ovih gljiva, osteenja od ptica, insekata, mehanicke povrede, kisni period neposredno pre i u toku berbe, mogu poveati pojavu mikotoksikoza na kukuruzu (Anderson et al. 2004). Takoe, odnos vrsta iz roda Fusarium moze biti izmenjen zbog promena temperatura, favorizujui F. graminearum koja produkuje osam razlicitih mikotoksina i koja ima vei temperaturni optimum od ostalih vrsta (Waalwijk et al. 2008). Toksini gljiva Penicillium i Aspergillus, patulin i ohratoksin, produkuju se na nizim temperaturama i sa porastom temperature mogu postati problem u severnim i hladnijim regionima (Paterson & Lima 2009). Promene u plodoredu bi mogle favorizovati pojavu novih sojeva ili porast znacaja ekonomski manje vaznih patogena. Ukoliko zagrevanje severnijih krajeva omogui gajenje silaznog kukuruza, zetveni ostaci mogu predstavljati izvor velike kolicine inokuluma fuzarium vrsta, sto moze znacajno ugroziti proizvodnju psenice i jecma (Maiorano et al. 2008). Klima i insekti Dobro je poznato da senzitivnost poljoprivrednih sistema na klimatske promene zavisi od toga sta je limitirajui faktor: temperatura ili voda i da li ti sistemi funkcionisu blizu bioloskog i proizvodnog optimuma ili ne. Efekti povisenih temperatura na insekte mogu se ispoljiti u vidu promene geografske rasprostranjenosti, reproduktivne sposobnosti, prezimljavanja, rasta populacija, broja generacija, sinhronizacije razvia insekata i biljke domaina, nacina sirenja, migracije i prisutnosti biljaka domaina. Na modelu dinamike ra-

zvoja lisne vasi Rhopalosiphum padi, vektora virusa zute patuljavosti jecma, ciji je domain i kukuruz, vidi se porast populacije vasi u uslovima povisene koncentracije CO2 pri neizmenjenim vrednostima ostalih parametara. Ranija pojava pikova generacija i poveanje brojnosti krilatih formi za 10% moze izazvati sirenje i cesu pojavu virusa zute patuljavosti jecma (Newman 2004). Simulacionim istrazivanjem Diffenbaugh et al. (2008) su predvideli sirenje areala rasprostranjenosti vrsta Diabrotica virgifera virgifera i Ostrinia nubilalis u severnije, vise i hladnije regione kao i kontinentalne delove SAD. D. virgifera virgifera preti da se prosiri na nove regione zbog smanjenog mortaliteta prezimljujue generacije i poveane brojnosti u uslovima visih temperatura. Predvieno sirenje kukuruznog plamenca nije znacajno, ali usled porasta sume minimalnih temperatura, mogue je da ova vrsta razvije i treu generaciju godisnje i time nanese vee stete proizvodnji kukuruza. Onstad et al. (2003) ukazuju na rizik koji nosi sirenje povrsina pod kukuruzom na rejone u kojima D. virgifera virgifera moze da zavrsi zivotni ciklus i dostigne brojnost populacije koja je ekonomski stetna. Do slicnih zakljucaka dosli su i MacLeod et al. (2005), Baker et al. (2000) i Baufeld & Enzian (2001) za povrsine pod kukuruzom u Evropi. Istrazivanje koje su sproveli Zavala et al. (2008) pokazuje da povisena koncentracija CO2 remeti funkciju gena odgovornog za sintezu inhibitora cistein proteinaze, koji igra ulogu u tolerantnosti soje prema kukuruznoj zlatici (Diabrotica virgifera virgifera). Ovo moze predstavljati ozbiljan problem ne samo za soju ve i za kukuruz, zbog poveanja rasprostranjenosti stetocine, neefikasnosti plodoreda i poveane brojnosti populacije usled dodatnog izvora hrane. Klima i korovi Korovi imaju vei geneticki diverzitet od useva, kompetitivniji su i bolje adaptirani na promene uslova spoljasnje sredine u odnosu na gajene biljke. Pojava i sirenje korovskih vrsta van dosadasnjeg areala mogu stvarati nove probleme u proizvodnji kukuruza. Prema autoru McDonald (2009), termofilna korovska vrsta Sorghum halepense mogla bi postati cest i opasan korov u kukuruzu severnijih krajeva SAD-a. Malidza & Vrbnicanin (2006) su na podrucju centralne Backe utvrdili prisustvo Ambrosia trifida, invazivne korovske vrste, koja zbog svoje agresivnosti, velike kompetitivne sposobnosti i alergentnosti moze predstavljati ozbiljan problem ne samo sa agronomskog nego i medicinskog aspekta. Povean rizik od pojave alergija

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zbog enormne produkcije polena ambrozije pri visim koncentracijama CO2 utvrdili su Wayne et al. (2002). Vea koncentracija CO2 moze dovesti do smanjenja efikasnosti glifosata u suzbijanju palamide, iako biohemijska osnova ove pojave nije u potpunosti objasnjena (Ziska et al. 2004). Ukoliko CO2 smanjuje efikasnost herbicida, postoji potreba da se odrede specificnost herbicida, koncentracije i doze primene, kao budue adaptivne mere hemijskog suzbijanja korovskih vrsta (Ziska 2006). Klima i adaptacione mere U cilju izbegavanja ili barem redukovanja negativnih efekata, a korisenja potencijalnih pozitivnih efekata globalnih klimatskih promena, predlozeno je nekoliko adaptacionih strategija koje se mogu svrstati u dve grupe: kratkorocne i dugorocne. Kratkorocne mere podrazumevaju prilagoavanje, odnosno optimizaciju procesa proizvodnje bez veih strukturalnih promena. One su autonomne, primenjuju se na nivou farme odnosno preduzea i podrazumevaju uglavnom promenu sortimenta, datuma setve, kolicine i nacina korisenja mineralnih hraniva, pesticida i sl. Zalud & Dubrovsky (2002) isticu da se smanjenje prosecnog prinosa moze ublaziti ukoliko se kukuruz seje ranije. Do slicnog zakljucka dosli su i domai autori. Primenom linearne regresione analize, Latkovi i sar. (2008) su ustanovili da se u periodu 2003-2006. prinos zrna kukuruza u proseku smanjivao po stopi od 290 kg ha-1 na svakih 14 dana zakasnjenja u setvi. Ukoliko bi umesto optimalnog roka sredinom aprila setvu kukuruza obavili sredinom maja, pad prinosa prouzrokovan kasnjenjem setve bi mogao iznositi 630 kg ha-1 do 790 kg ha-1. Pad prinosa zbog zakasnjenja u setvi je karakteristika svih hibrida, ali je najizrazeniji kod hibrida duze vegetacije (FAO 600 i FAO 700; Latkovi i sar. 2007). U prilog ovoj cinjenici idu i istrazivanja autora Trolove et al. (2008) koji pokazuju da ranija setva moze rezultirati u veoj produkciji biomase kukuruza. Ipak, mora se konstatovati da su ovakve mere adaptacije specificne za svaku zemlju, odnosno region i da u krajnjem slucaju mogu samo umanjiti negativan efekat klimatskih promena (Tsvetsinskaya et al. 2003). Istrazivanja Erde et al. (2005) potvruju ovu cinjenicu navodei da bi i primena navodnjavanja samo ublazila negativan trend smanjenja prinosa. Dogorocne mere adaptacije se odnose na krupne strukturalne promene koje se implementiraju sa ciljem prevazilazenja problema prouzrokova-

nih klimatskim promenama. Ovde se podrazumeva promena nacina korisenja zemljista: npr. zamena kultura koje se karakterisu visokim sezonskim variranjem prinosa (psenica) kulturama koje su manje produktivne, ali imaju stabilnije prinose (pasnjaci); promena nacina korisenja zemljista i primena agrotehnickih mera sa ciljem cuvanja vlage u zemljistu, poveanje efikasnosti navodnjavanja, itd. Kao tipican primer dugorocnih mera adaptacije globalnim klimatskim promenama navodi se zamena stocne repe i strnih zita silaznim kukuruzom u Danskoj (Olesen & Bindi 2002). Ipak, svim ovim merama mogu se samo ublaziti negativni efekti klimatskih promena, jer ukoliko se u sirokoj proizvodnji ne budu gajili hibridi tolerentni prema faktorima spoljasnje sredine, nastavie se trend pada prinosa prouzrokovan klimatskim faktorima. Zbog toga se oplemenjivanje biljaka navodi kao jedna od najznacajnijih mera adaptacije. Oplemenjivanje kao mera adaptacije Jedan od najinteresantnijih primera znacaja genotipa jeste slucaj osetljivosti inbred linija kukuruza prema prouzrokovacu Juzne pegavosti lista (Bipolaris maydis) u Americi pocetkom 70-tih godina proslog veka. Uzrok iznenadne pojave i brzog sirenja bolesti bila je pojava rase-T B. maydis, specificne za cms-T citoplazmu (tzv. Teksaski ili T tip citoplazme) koja je u to vreme bila vrlo zastupljena u semenskoj industriji Amerike. Bolest je zahvatila velike povrsine i nanela ogromne stete semenskim kompanijama u jako kratkom vremenskom periodu. Resenje problema naeno je u korisenju linija sa normalnom citoplazmom. Snazan doprinos genetike i oplemenjivanja kukuruza u stvaranju genotipova tolerantnih prema faktorima spoljasnje sredine vidi se i na primeru hibrida NSSC70. Jedan od najrodnijih hibrida pripadajueg ciklusa selekcije (II ciklus selekcije), koji se gajio vise od decenije imao je znacajan nedostatak u smislu osetljivosti prema trulezi klipa (gljive iz roda Fusarium). U godinama sa veom kolicinom padavina i visokom vlaznosu vazduha u jesenjem periodu, dolazilo je do znacajnog gubitka prinosa (Ivanovi i sar. 2002). Resenje je naeno ve u tzv. IV ciklusu selekcije. Hibrid NS640 je u pravom smislu pokazao znacaj oplemenjivanja u resavanju nekih od goruih problema u proizvodnji kukuruza. U ovom hibridu su na vrlo izbalansiran nacin objedinjeni visok prinos, tolerantnost prema susi, siroka adaptabilnost i izuzetna tolerantnost prema prouzrokovacima trulezi klipa. Ovaj hibrid i danas drzi svoju lidersku poziciju u proizvodnji kukuruza u Srbiji.

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Iako efekti globalnih klimatskih promena i poveane koncentracije CO2 na biljne bolesti mogu biti pozitivni i negativni, u najveem broju slucajeva primeeno je sirenje areala i agresivnosti patogena (Chakraborty et al. 2000). Crvenilo kukuruza, koje je registrovano u Srbiji krajem pedestih godina proslog veka i do sada se periodicno javljalo, nije predstavljalo ekonomski znacajan problem u proizvodnji kukuruza. Ipak, poslednja pojava crvenila veih razmera zabelezena je 2002. i 2003. kada se na nekim poljima stepen crvenila i susenja biljaka kretao u intervalu od 10% do 90%. Tesko je ustanoviti vezu izmeu globalnih klimatskih promena i ekonomski znacajnijeg snizenja prinosa prouzrokovanog crvenilom u poslednjoj dekadi, ali je alarmantno sto su svi komercijalni hibridi u veoj ili manjoj meri pokazali osetljivost prema ovoj pojavi. Iako polemika oko bioticke ili abioticke prirode crvenila kukuruza jos uvek traje, u nekoliko eksperimenata je utvreno da ne postoji jasna veza izmeu ove pojave i corn stunt spiroplazme, te da plodnost zemljista i prisustvo nematoda u zemljistu i biljnom materijalu nemaju uticaja na pojavu crvenila (Purar et al. 2009). Autori navode da je na eksperimentalnim parcelama tretiranim insekticidima utvrena manja pojava crvenila nego na kontroli, sto bi moglo upuivati na vezu izmeu ove pojave i odreenih biotickih faktora. Bez obzira na uzrok, interesantan izvor tolerantnosti prema crvenilu kukuruza otkriven

je u jednoj lokalnoj populaciji. U periodu 20042005. i ogledima postavljanim u epicentru ove pojave, ustanovljena je izuzetna tolerantnost populacije NS 1-257 CRS prema crvenilu (Graf. 1; Bekavac et.al. 2007). Sve S1 familije selekcionisane iz ove populacije bile su potpuno tolerantne prema crvenilu. Ipak, TC potomstva (nastala ukrstanjem S1 familija sa nesrodnim inbred testerom) pokazivala su diskretnu osetljivost, ali neuporedivo nizu nego standardni hibridi (Graf. 1). Ovi rezultati upuuju na zakljucak da rad na oplemenjivanju lokalno razvijenih materijala moze biti vrlo interesantan, ali da treba biti umereni optimista posto je to trenutno jedini izvor tolerantnosti prema crvenilu stvoren u Institutu za ratarstvo i povrtarstvo u Novom Sadu. Kombinovanom familijskom selekcijom pokusavaju se popraviti srednje vrednosti osnovnih agronomskih svojstava i kombinacione sposobnosti ovog materijala uz ocuvanje neizmenjenog nivoa tolerantnosti prema crvenilu (Bekavac i sar. 2010). U sestogodisnjem periodu testiranja (2004-2009) na S1 familijama razvijenim iz populacije NS 1-257 CRS nije determinisana ni jedna biljka sa tipicnim simptomima crvenila. Strategija proizvodnje kukuruza u uslovima globalnih klimatskih promena zasniva se na gajenju hibrida siroke adaptiranosti za podrucja razlicitih agroekoloskih karakteristika i rejonizaciji odnosno izboru hibrida prilagoenih speci-

12,00 10,00

% of CR plants

8,00 6,00 4,00 2,00 0,00 2004 2005 S1 0,00 0,00 TC 0,32 0,30 M St 6,71 5,84 W St 10,80 8,73 2004 2005

Grafikon1. Tolerantnost S1 i TC potomstava populacije NS 1-257 CRS i standardnih hibrida prema crvenilu kukuruza (S1 potomstva; TC potomstva; M St ­ srednja vrednost cetiri standarda; W St ­ srednja vrednost dva najosetljivija standarda) Graph 1. Tolerance of S1 and TC progenies of the population NS 1-257 CRS and check hybrids to corn reddening (S1 progenies; TC progenies; M St ­ mean of four control hybrids; W St ­ mean of two most susceptible control hybrids)

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Bekavac G i sar.

ficnim klimatskim i zemljisnim karakteristikama pojedinacnih rejona. Sve vei znacaj rejonizacije posledica je kako potreba da se shodno proizvodnim uslovima i zahtevima trzista proizvodi sto vise i sto racionalnije, tako i novih uslova koje namee izmenjena klima. U nasoj zemlji rad na rejonizaciji je poceo jos u proslom veku, kada je uraena detaljna studija i definisano deset rejona ratarske proizvodnje na teritoriji Vojvodine (Stojkovi 1972; cit. prema Stojakovi i sar. 2010). Na osnovu prosecnih prinosa u Vojvodini od 1981. do 2000. kao i prirodnih uslova u ostalim delovima Srbije, izdvojeno je pet rejona proizvodnje kukuruza u nas (Stojakovi i sar. 2001, Stojakovi i sar. 2006). Ova istrazivanja su pomogla da se za svaki rejon preporuci najbolje adaptirani sortiment i time povea proizvodnja ili barem stabilizuje na zadovoljavajuem nivou. Izbor hibrida otpornih prema susi i prilagoenih odreenom rejonu gajenja, kao mera adaptacije na klimatske promene, sve vise dobija na znacaju (Reidsma et al. 2009). Pod uticajem faktora spoljne sredine hibridi se razlicito rangiraju bez obzira na njihov geneticki potencijal (Ivanovi i sar. 2007), pa se utvrivanju interakcije genotip x spoljasnja mora pazljivo pristupati. Efekti nepovoljnih klimatskih prilika, pre svega suse mogu se umanjiti gajenjem hibrida vee stabilnosti, siroke adaptabilnosti ili gajenjem hibrida krae vegetacije (FAO grupe 300 do 500), koji u nasoj zemlji kriticne faze razvoja, oplodnju i nalivanje zrna, zavrsavaju pre nastupanja susnog perioda (Stojakovi i sar. 2009). Pojedini hibridi poseduju visok potencijal za prinos zrna i natprosecnu stabilnost prinosa, zbog cega se sa uspehom mogu gajiti u gotovo svim proizvodnim rejonima Srbije. Sa druge strane, razlikujemo hibride visoko adaptirane na specificne uslove gajenja jednog uzeg regiona ili cak lokaliteta. U cilju rejonizacije proizvodnog podrucja, Stojakovi i sar. (2010) su ispitivali agronomska svojstva 15 hibrida kukuruza razlicitih grupa zrenja (FAO300-700) na 30 lokaliteta u Srbiji. Metodom multivarijacione analize izdvojeni su hibridi sa visokim prinosom i interakcijskom vrednosu bliskoj nuli i utvrene specificne veze izmeu lokaliteta i hibrida. Na ovaj nacin je mogue dati preporuku sortimenta od kojeg se u datom lokalitetu i uz primenjenu agrotehniku moze ocekivati maksimum. Zakljucak Veina modela globalnih klimatskih promena predviaju vise temperature, promene u kolicini i distribuciji padavina, kao i veu koncentraciju

atmosferskog CO2. Iako rast temperature moze imati i pozitivan i negativan uticaj na prinos useva, generalno povisene temperature dovode do ubrzanih respiratornih procesa, kraeg perioda nalivanja zrna i slabije produkcije biomase, sto utice na pad prinosa i kvaliteta. Klimatske promene uticu na promenu sastava i sirenje korovskih vrsta, stetocina i bolesti gajenih biljaka. Najznacajnije mere adaptacije podrazumevaju raniju setvu, uvoenje navodnjavanja i izbor tolerantnih hibrida. Korisenje visokoproduktivnih genotipova, tolerantnih prema izmenjenim faktorima spoljasnje sredine imae presudnu ulogu u proizvodnji kukuruza u budunosti. Literatura

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Maize Production in Terms of Global Climate Changes

Goran Bekavac Bozana Purar ore Jockovi Milisav Stojakovi Mile Ivanovi Goran Malidza Ivica alovi

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia Summary: Climate changes and expected variability of climatic parameters represent a serious concern of the 21st century agriculture. At the global level, the further rise in temperature, changed quantity and distribution of precipitation, increased variability of climate parameters and the occurrence of extreme climate events are expected. In order to avoid, or at least reduce the negative effects of global climate change, several adaptation strategies are proposed. Adjustment of production technology and breeding for tolerance to changed environment are proposed as two most important adaptation measures. Key words: breeding, climate, maize

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www.nsseme.com/journal.html

Predicting Herbage Growth with Models

451 Modelling / Modeliranje Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 451-456 review article / pregledni rad

Herbage Growth Models as Aids for Managing Intensively Grazed Grassland in Western Europe

A. Scott Laidlaw

received / primljeno: 23.03.2010. accepted / prihvaeno: 04.05.2010. © 2010 IFVC

Summary: Estimates of variation in herbage growth are presented to demonstrate the problems which unforeseen variation can create for management of intensively grazed grassland in temperate Western Europe. The role of growth models in predicting herbage availability as a component in feed budgeting is described. Herbage growth models share a common structure and produce output in response to weather conditions; others also take account of soil properties. Growth models can be applied as aids to grassland management either as `stand alone' models or can be an integral part of a decision support system and used to predict outcomes from whole farming systems. Although the reliability of models has been well demonstrated in many instances, their value to farmers as an aid to managing grassland intensively needs to be re-enforced. Key words: grasslands, feed budgeting, herbage growth models, Western Europe

Introduction Forage can be considered as a raw material in the `manufacture' of ruminant products. However, unlike most industrial raw materials it is not possible to control with accuracy the amount which will be available. Consequently, any means of predicting growth and quality of herbage for a given set of conditions offers the farmer some control over forage as an input. Problems of variation apply to herbage grown either for forage conservation or grazing. However, it is more critical for management of grazing swards as grass is utilised more closely to the time that it has been produced than for forage grown for conservation and so there is less opportunity to make contingency plans when herbage is either scarce or is more plentiful than required by the grazing animals. Grass growth models, as a means of predicting production for a given set of conditions, can be used to help farmers plan their grazing strategy and as an early warning system of periods of excess or shortage before they arise. Variation in Herbage Growth Seasonal grass growth rate curves vary widely over western and northern Europe reflecting the

A. S. Laidlaw ) ( Agri-Food and Biosciences Institute, Applied Plant Science and Biometrics Division, Crossnacreevy, Belfast, Northern Ireland, UK e-mail: [email protected]

range of climatic conditions (Fig. 1). So, while countries with short growing seasons, such as Finland and Iceland, rely heavily on conserved feed to meet the needs of ruminants, those with longer growing seasons have the opportunity to exploit fully the benefits of grazing. However, even when grassland at the same site is managed consistently, herbage growth can vary widely from year to year, especially in intensively managed grassland systems. This variation is mainly due to year-to-year variation in weather and its interaction with other environmental conditions such as soil. Spring growth is important for grassland farms in which animals are housed over winter as a late spring extends the time the animals remain indoors and have to be fed expensive conserved forage and/or supplements. Grass growth rates for spring production in variety assessment trials of perennial ryegrass in Northern Ireland ranged from 21 to 100 kg DM/ ha/day over 15 years. Feed Budgeting The concept of feed budgeting provides a basis for allocating grassland areas for short term and long term use (Mayne et al. 2000). In rotational grazing where the grassland area is divided into paddocks, a combination of a) knowing the daily feed requirements of the grazing animals, b) the current amount of herbage available and c) the anticipated growth allows the number of paddocks required for

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140 120 100

Iceland Finland Netherlands Ireland

kg DM/ha/day

80 60 40 20 0 March 3 April May 13 June July 23 Aug. Sept. Oct. 33

Figure 1. Daily grass growth rates for a range of sites in western and northern Europe (Corrall 1984) Slika 1. Dnevne kolicine proizvedene krme na veem broju lokaliteta u zapadnoj i severnoj Evropi

grazing in the near future to be determined and so the rest of the paddocks can be set aside to be cut for silage or hay. This ensures that maximum use is made of the herbage available. The excess conserved forage can be fed as a `buffer' when grazing grass becomes scarce or can supplement the pool of winter fodder. The farmer will know approximate intake requirements of animals either from experience or from tables of information. There are techniques available to help farmers estimate the amount of herbage currently in the paddocks. As it is not feasible to expect a farmer to take sample cuts to estimate herbage availability, indirect methods have been developed. A common method used is a `rising plate' in which a disc or plate of specific weight slides on a graduated shaft. The assembly is placed vertically into the sward and the density and height of the sward determines how high the disc is above ground level. Equations are supplied with the equipment relating height above ground to herbage mass. So the estimated herbage mass (availability) can be calculated by the equation for a given sward height. The third criterion for budgeting feed is anticipating growth. Grass growth models have a role to play in reducing uncertainly in intensive grassland management either as stand-alone aids

or as integral components in decision support systems in which they are used in conjunction with whole-farm models which predict intake and animal or milk production. Basis of Growth Models A model is a representation of a real life system. So, a herbage growth model which represents herbage growth for a given set of conditions at a specific time of the year, is able to provide outcomes from a range of scenarios i.e. predictions. Whether complex or a crude representation of reality, most grass growth models have a number of components in common i.e. a conversion of light (solar radiation) to dry matter, distribution of dry matter between plant parts, and loss of herbage due to utilisation or senescence and death (Fig. 2). The amount of solar radiation obviously influences the first component, while temperature, available soil moisture and nutrients (especially the major nutrients and nitrogen in particular) and stage of development influence all of these components. These components interact, such as the amount of leaf lamina produced has an affect on the interception of solar radiation. In

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Figure 2. Schematic representation of an herbage growth model Slika 2. Shematizovani prikaz modela proizvodnje krme

addition some models include herbage quality as an output, especially digestibility and crude protein content (Groot & Lantinga 2004). Probably the most comprehensive grass growth model is that of Thornley (1998) which is based on the Hurley Pasture Model. The model is complex but routines and submodels within it can be isolated and applied to specific aspects of grassland growth and herbage species development. Due to its complexity, the Hurley model is often used more as a research tool. Some advanced models include more than one species e.g. grass and white clover and so the interaction between the species both above and below ground is considered (e.g. Thornley et al. 1995). So the model is extended to take account of soil factors which influence growth. Inclusion of the grazing animal embraces the interaction between different species in the mixture and intake, including selection by the animal (e.g. Parsons et al. 1991). These plant-animal-soil models form the basis of decision support systems in which the next step is to frame the

model to fit specific production systems. This is considered in the next section. Other grass growth models have been developed to take account of different seminatural grassland communities. For example, the model of Jouven et al. (2006) includes differences in grassland species, classified as different functional groups, and their adaptation to different grassland conditions. The most productive functional group contains species which are adapted to fertile sites and frequent defoliation (with high specific leaf area, high digestibility, short leaf lifespan and early reproductive growth). In contrast, the least productive contains those which are adapted to poor sites and infrequent defoliation (with low specific leaf area, low digestibility, very long leaf life span and very late reproductive growth). Application of Herbage Growth Models in Grassland Management A herbage growth model can be used as a management aid either as a stand-alone method

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Figure 3. Actual and predicted a) daily growth rate and b) white clover content in dry harvested dry matter in two series of plots at each of 7 CloverCheck sites in Northern Ireland in 2009 Slika 3. Trenutne i predviene vrednosti a) dnevnih proizvedenih kolicina i b) udela bele dateline u pokosenoj suvoj materiji u dva niza parcela na svakom od sedam CloverCheck lokaliteta u Severnoj Irskoj 2009. godine

to predict growth, and so can be used as an input to grass budgeting calculations, or as an integral component of a decision support system. In the latter, the growth model is linked to a herbage intake model which in turn can predict animal production. Application of `stand-alone' grass growth models In the herbage growth monitoring and forecasting system in Northern Ireland, called GrassCheck, the model originally developed as GrazeGro (Barret & Laidlaw 2005), is used to predict growth while the network of monitoring plots provides information on growth and quality of herbage up to a specific date. The monitored and forecasted growth information is publicised weekly in farming press and on appropriate websites for farmer to use in their grass budgeting calculations. The model is specifically built for intensive grassland management and so inputs to drive the model include N fertiliser application, and forecasted daily air temperature, photosynthetically active radiation and rainfall. In contrast, a grass/white clover monitoring and forecasting system in the Province, CloverCheck, has been developed for farmers who are applying no (e.g. organic dairy and beef farmers) or very low levels of nitrogen fertiliser and so are relying heavily on white clover as a source of nitrogen (Laidlaw et al. 2007). CloverCheck bulletins are published fortnightly and emphasis is on herbage growth and clover content. Plots are in two series at each site, harvested every 4 weeks 2 weeks apart. Actual and predicted herbage growth

and clover content from the 7 monitor sites in 2009 are presented in figure 3. Despite the sites ranging from upland beef farms to lowland organic dairy farms agreement between actual and predicted outputs is reasonably good. Although the data from cut plots are applied to grazed swards, the disadvantage of grazing plots instead of cutting them is that high variation is introduced and it is not practical to manage a network of grazed plots widely distributed. While grazing has a relatively small effect on grass swards compared with cutting, especially those receiving relatively high rates of nitrogen fertiliser, the difference between cut and grazed swards is high for grass/ clover swards. In CloverCheck the data have to be converted when applied to grazed swards. Growth in grazed swards is about 75% of that of plots and clover content relative to that of cut plots declines to 40%, or less, that of plots in summer (Laidlaw 2008). While a model may be capable of predicting growth with sufficient accuracy when the inputs are known, relying on forecasted weather as a set of inputs introduces an additional source of variation in the prediction. So limitations to forecasting weather, especially one to two weeks ahead, may limit the effectiveness of application of a model Use of models in decision support systems for grassland management When a growth model is linked to other models, especially animal intake and production models, and represent complete systems of production, they can function as decision support systems. The

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whole farm model SEPATOU in France (Cros et al. 2003) and Dairy-sim in Ireland (Fitzgerald et al. 2008) each incorporate a grass-growth sub-model which predicts herbage growth and, in the case of SEPATOU, herbage quality. Dairy-sim, having been tested against output data from different climatic areas in Ireland, was used to simulate and predict outcomes on dairy farms on wet heavy soils. Contrasts were drawn between these farms and those on light free draining soils, with the model predicting lower practicable stocking rates and higher housing requirements than on farms on the drier soils. The model developed by Jouven et al. (2006) already discussed, has been built into a full systems model simulating suckler cow and calf production in permanent grassland in France, called SEBIEN (Jouven & Baumont 2008). They have used the model to determine the extent to which acceptable animal production can be achieved using biodiversity-friendly management. The output of the model has shown that, overall, balance between animal production and biodiversity is possible but while some paddocks had a high level of floristic diversity, others were adversely affected, emphasising the need for whole farm simulation rather than simulating events in only one paddock. Conclusions The herbage growth models cited are only a small selection of those either being developed or are currently being applied to grassland farming in Europe. While models can be effective aids to understanding complex interactions between components in the sward-animal-soil system, more effort is required to educate farmers in the value of output of herbage growth models. Formal courses are unlikely to be effective but repeated demonstration of the reliability of prediction of herbage growth models, particularly in periods where weather has resulted in forage shortages, may convince farmers of their value.

References

Barrett P B, Laidlaw A S (2005): Grass growth modelling: to increase understanding and aid decision making on-farm. In: Murphy J J (ed.), Utilisation of Grazed Grass in Temperate Animal Systems. Proc. Satellite Workshop of the 20th International Grassland Congress, Cork, Ireland, 79-88 Corrall A J (1984): Grass growth and seasonal pattern of production under varying climatic conditions. In: Riley H, Skjelvåg A O (eds), Impact of Climate on Grass Production and Quality. Norwegian State Agricultural Research Stations, Ås, Norway, 36-45 Cros M J, Duru M, Garcia F, Martin-Clouaire R. (2003): A biophysical dairy farm model to evaluate rotational grazing management strategies. Agronomie 23: 105-122 Fitzgerald J B, Brereton A J, Holden N M (2008): Simulation of the influence of poor soil drainage on grass-based dairy production systems in Ireland. Grass Forage Sci. 63: 380-389 Groot J C J, Lantinga E A (2004): An object-oriented model of the morphological development and digestibility of perennial ryegrass. Ecol. Modell. 177: 297-312 Jouven M, Carrere P, Baumont R (2006): Model predicting dynamics of biomass, structure and digestibility of herbage in managed permanent pastures. 1. Model description. Grass Forage Sci. 61: 112-124 Jouven M, Baumont R (2008): Simulating grassland utilization in beef suckler systems to investigate the trade-offs between production and floristic diversity. Agric. Syst. 96: 260-272 Laidlaw A S, Moore N, Dale A J (2007): Monitoring and modelling growth in a proposed management support system for grass-white clover swards. In: Holden N M et al. (eds), Making Science Work on the Farm. AGMET, Dublin, Ireland, 83-88 Laidlaw A S (2008): Development of a model of growth of grass/white clover swards for a grassland management support system. Proc. 8th International Rangeland Congress and 21st International grassland Congress, Huhhot, China, 2: 54 Mayne C S, Wright I A, Fisher G E J (2000): Grassland management under grazing and animal response. In: Hopkins A (ed.), Grass ­ Its Production and Utilisation. Blackwell Science, Oxford, UK, 247-291 Parsons A J, Harvey A, Woledge J (1991): Plant-animal interactions in a continuously grazed mixture. 2. The role of differences in the physiology of plant growth and selective grazing on the performance and stability of species in a mixture. J. Appl. Ecol. 28: 635-658 Thornley J H M, Bergelson J, Parsons A J (1995): Complex dynamics in a carbon-nitrogen model of a grass-legume pasture. Ann. Bot. 75: 79-94 Thornley J H M (1998): Grassland Dynamics- An Ecosystem Simulation Model. CAB International, Wallingford

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Modeli proizvodnje krme za negu intenzivno korisenih travnjaka zapadne Evrope

A. Skot Lejdlo

Institut za poljoprivredu, hranu i bioloske nauke, Odsek za biljne nauke i biometriku, Krosnakrivi, Belfast, Severna Irska, Velika Britanija Izvod: Procene variranja proizvodnje krme izlozene su u svrhu ukazivanja na probleme koje mogu da izazovu nepredviena variranja u nezi intenzivno korisenih travnjaka u oblastima zapadne Evrope sa umerenom klimom. Opisana je uloga modela proizvodnje u predvianju raspolozivosti krme kao cinioca krmnog budzeta. Modeli proizvodnje krme poseduju zajednicku strukturu i ostvaruju proizvode u zavisnosti od vremenskih uslova, dok drugi, pak, uzimaju u obzir i zemljisne uslove. Modeli proizvodnje mogu da se primene u nezi travnjaka bilo kao pojedinacni modeli, bilo kao integralni deo sistema odlucivanja i u svrhu predvianja ishoda citavih sistema poljoprivredne proizvodnje. Iako je isplativost modela dokazana na vise nacina, potrebno je potvrditi vrednost koju imaju za farmere prilikom nege intenzivno korisenih travnjaka. Kljucne reci: krmni budzet, modeli proizvodnje krme, travnjaci, zapadna Evropa

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www.nsseme.com/journal.html

Early Distribution of Pea in Europe

457 Archaeobotany / Arheobotanika Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 457-460

review article / pregledni rad

A Brief Review on the Early Distribution of Pea (Pisum sativum L.) in Europe

Marija Ljustina Aleksandar Miki

received / primljeno: 18.04.2010. accepted / prihvaeno: 03.05.2010. © 2010 IFVC Summary: Pea was a part of the everyday diet of the European hunter-gatherers at the end of the last Ice Age. The major criteria to determine the domestication in pea are non-dehiscent pods, larger seed size and smooth seed testa. Pea seeds were found among the earliest findings of cultivated crops at the site of Tell El-Kerkh, Syria, from 10th millennium BP. Along with cereals and lentil, pea has definitely become associated with the start of the `agricultural revolution' in the Old World. Pea entered Europe in its southeast regions and progressed into its interior via Danube. Its distribution was rapid, since the available evidence reveals its presence in remote places at similar periods. The linguistic evidence supports the fact that pea had been present in nearly all regions of Europe. Most of European peoples have their own words denoting pea, meaning that it preceded the diversification of their own proto-languages. Key words: archaeology, crop history, distribution, Old Europe, pea, Pisum sativum L.

Introduction Legumes (Fabaceae Endl.) are one of the richest plant families in the world, extending with hundreds of genera and thousands of species over all continents. Numerous members of this family have economic importance and have been used for diverse purposes, including human consumption, animal feeding and green manure, for millennia (Miki et al. 2006). One of such species is pea (Pisum sativum L.), being one of the most widespread and most cultivated annual legume crops, especially in temperate regions (Miki et al. 2007). The aim of this brief review is to remind the pea research community on a part of its rich history, as well as to encourage further archaeological research and its integration into a holistic legume history of the Old World. Centres of Origin It is widely accepted that many of the traditional European grain legumes, such as chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.) and common vetch (Vicia sativa L.) originate primarily

A. Miki ) ( Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia e-mail: [email protected] M. Ljustina University of Belgrade, Faculty of Philosophy, Cika Ljubina 18-20, 11000 Belgrade, Serbia

from the Near Eastern centre of diversity. Among them is pea, with the Near Eastern centre as the primary, while both Mediterranean and African are considered the secondary centres or diversity (Zeven & Zhukovsky 1975). An abundant presence of the wild taxa of pea, such as red-yellow pea (Pisum fulvum Sm.), as well as of beautiful vavilovia (Vavilovia formosa (Stev.) Fed.), the closest relative of the genera Pisum L. and Lathyrus L. (Kenicer et al. 2009), in the local floras of the Near East region, may be regarded as another testimony that it was the very centre of an eventual distribution of pea in all directions. It is considered that the phenomenon of pod dehiscence, present in pea and other related genera, with an ability of shattering the seeds up to 2 m in diameter, significantly contributed to the spreading of these species. The only wild or semi-wild taxon of pea present in Europe is tall pea (Pisum sativum L. subsp. elatius (Steven ex M. Bieb.) Asch. & Graebn. var. elatius (Steven ex M. Bieb.) Meikle), that still may be found in southern regions of the Balkans and the Black Sea coast, i.e. in Greece, Bulgaria and Serbia, with latter as the northernmost border of its areal (Miki et al 2009a). Process of Domestication Grain legumes, including pea, had been known to humans before they became cultivated. Together with vetches (Vicia spp.) and vetchlings

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(Lathyrus spp.), pea was a part of the everyday diet of the hunter-gatherers at the end of the last Ice Age in Europe, as witnessed by the remains from the site of Santa Maira, Spain, from 12,000­9,000 BP (Aura et al. 2005). In all plant species, the process of domestication led to certain morphological changes that, in many aspects, strongly resemble the methods of selection used in contemporary plant breeding programmes (Ljustina & Miki 2008). In grain legumes, the major criteria to determine the domestication are non-dehiscent pods, larger seed size and smooth seed testa. It seems that the pea gene Dpo, controlling pod dehiscence (Weeden et al 2002), underwent modifications during the domestication and thus became responsible for the development of nondehiscent genotypes. Larger seed size in cultivated forms in comparison to that in wild ones is not typical only for grain legumes, but is often very hard to interpret. A smooth testa, due to the domestication, is the most reliable characteristic in telling wild from cultivated forms (Butler 1998). Apart from these three major indicators, there are several other indicators of the pea domestication, such as absent seed dormancy, dwarf growing habit, less prominent basal branching, neutral photoperiodical reaction and improved grain quality (Weeden 2007). The remains of domesticated grain legumes often occuring at high frequencies during the 10th and 9th millennia (Willcox et al. 2007) may contribute to the possibility that the domestication of grain legumes could predate cereals (Kislev & Bar-Yosef 1988). However, little is known about the early stages of pulse domestication and there is very little evidence of how, when and where they were domesticated, mostly due to a fact that all those changes, being mostly morphological, have not survived to the present day. Among the earliest findings of cultivated grain legumes is the site of Tell El-Kerkh, Syria, from 10th millennium BP, with the seeds of lentil, bitter vetch (Vicia ervilia (L.) Willd.), chickpea, grass pea (Lathyrus sativus L.), faba bean (Vicia faba L.) and pea (Tanno & Willcox 2006). Conquest of Europe It is quite certain that pea was one of the most ancient crops that entered Europe, after it had become more suitable place for living again, following the end of the last Ice Age. Thus pea, along with several cereals and lentil (Ljustina &

Miki 2010), has definitely become associated with the start of the `agricultural revolution' in the Old World (Erskine 1998). Pea entered Europe in its south-east regions and roughly progressed into its interior via Danube. Its distribution was a rapid one, since the available evidence reveals its presence in mutually remote places at similar periods. It is only Iberian Peninsula where Lathyrus crops seem to be most abundant of all grain legumes including pea (Miki et al 2009b). The following selection of archaeological findings offers nice examples that confirm the extreme importance pea had in the primeval European agriculture. 5790-5630 BC, Kovacevo, southeast Bulgaria. The final early Neolithic site in southwest Bulgaria, with lentil, grass pea, chickling vetch (Lathyrus cicera L.) and bitter vetch found along with pea and several cereal species (Marinova & Popova 2008). 5600-4300 BC, south-western Germany. The results of more than 100 archaeobotanical investigations from this region reveals that pea was one of the commonest pulses, with similar curves with high presence values in the early Neolithic, the Bronze and Iron Ages and in the early Medieval period and most frequent in the early Neolithic and in the Bronze Age (Rösch 1997). 5470-5260 BC, Aknashen, Armenia. A still disputable presence of pea, along with lentil and bitter vetch, could be an evidence of the pea distribution to the opposite, easternmost, regions of Europe (Hovsepyan & Willcox 2007). 5000-4800 BC until 600 AD, the modern Paris area, France. Here pea was one of the first introduced crops, along with lentil and cereals (Bakels 1999). 3900 BC, Lake Constance, southern Germany. In a Neolithic village Hornstaad Hiirnle I A, there were found carbonsied seeds and non-carbonised pods (Maier 1999). 3400-3300 BC, Lake Biel, Switzerland. A Late Neolithic site where the small numbers of peas was found, as another confirmation that due to poor preservation it is not possible to reliably indicate the role of peas in this period (Brombacher 1997). 2750-2500 BC, an Early Bronze age site of Arslantepe, Malatya, central Turkey. According to the present evidence, pea was used much less than chickpea and equally to lentil (Sadori et al 2006), with no evidence that it was grown there, but perhaps imported from somewhere else. This could lead to a conclusion that pea in some

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regions, such as this one, just passed through further and was not truly cultivated. 1600-1300 BC, a Bronze Age Vatin culture settlement of Zidovar, Banat, northern Serbia. Archaeobotanical research of this multi-layered site revealed that pea was in use along with other grain legumes, such as lentil or vetch (Medovi 2003). Pea was cultivated in the Serbian section of Danube quite early (Renfrew 1979) and was found in the sites such as the Neolithic Starcevo, the Neolithic - Early Iron Age - La Tène site of Gomolava (Van Zeist 2001/2002), the Bronze and Early Iron Age site Feudvar (Kroll 1998) and the Early Iron Age site of Gradina upon Bosut (Medovi 2010). 700-200 BC, a Celtic, La Tène site in Dürrnberg, Austria. Here, in a salt-mining and metal-working settlement, pea was used as a sole legume with cereals, significantly contributing to the quality of miners diets (Swidrak 1999). 600-400 BC, four late Hallstat and early La Tène sites in Baden-Wurttemberg, Germany. Together with lentil, bitter vetch and faba bean, pea was an important crop in everyday nutrition (Stika 1999). 100-200 AD, a Gallo-Roman cemetery in Moselle, France. Pea seeds were found among the other crops as grave offerings. (Preiss 2005). The linguistic evidence supports the fact that pea had been present in nearly all regions of Europe before the modern European language families were developed. Peoples like IndoEuropeans, Turkic, Caucasians or Basques, each have their own words denoting pea, meaning that it preceded the diversification of their own proto-languages into their contemporary descendants (Miki 2009). Similar is with other most ancient legume crops, such as lentil (Miki 2010). Among the most ancient words denoting pea in European languages are the Proto-IndoEuropean *kek-, the Proto-Turkic *burcak, the Proto-Caucasian *qr' and the Proto-Basque *ilha (Miki et al 2008). Conclusions As one of the most ancient crops in the world, pea played an important role in the introduction of agriculture in post-glacial Europe, often representing the main pulse in the diets of local communities across the continent. The future research on this subject certainly must make a more detailed map of its paths over Europe and, especially, its long-term and essentially important ties with the pea domestication and distribution in Asia Minor, Near East and North Africa.

References

Aura J E, Carrión Y, Estrelles E, Jordà G P (2005): Plant economy of hunter-gatherer groups at the end of the last Ice Age: plant macroremains from the cave of Santa Maira (Alacant, Spain) ca. 12000 ­ 9000 B.P. Veget. Hist. Archaeobot. 14: 542-550 Bakels C (1999): Archaeobotanical investigations in the Aisne valley, northern France, from the Neolithic up to the early Middle Ages. Veget. Hist. Archaeobot. 8: 71-77 Brombacher C (1997): Archaeobotanical investigations of Late Neolithic lakeshore settlements (Lake Biel, Switzerland). Veget. Hist. Archaeobot. 6: 167-186 Butler A (1998): Grain legumes: Evidence of these important ancient food resources from early pre-agrarian and agrarian sites in southwest Asia. In: Damania A B, Valkoun J, Willcox G, Qualset C O (eds.), Origins of Agricultural and Crop Domestication. ICARDA, Aleppo, Syria, 102­117 Erskine W (1998): Use of historical and archaeological information in lentil improvement today. In: Damania A B, Valkoun J, Willcox G, Qualset C O (eds.), Origins of Agricultural and Crop Domestication. ICARDA, Aleppo, Syria, 191-198 Hovsepyan R, Willcox G (2007): The earliest finds of cultivated plants in Armenia: evidence from charred remains and crop processing residues in pisé from the Neolithic settlements of Aratashen and Aknashen. Veget. Hist. Archaeobot. 17: 63-71 Kenicer G, Smýkal P, Visnhakova M, Miki A (2009): Vavilovia formosa ­ an intriguing Pisum relative. Grain Legum. 51: 8 Kislev M E, Bar-Yosef O (1988): The legumes: the earliest domesticated plants in the Near East? Curr. Anthrop. 29: 175­179 Kroll H. (1998): Die Kultur- und Naturlandschaften des Titeler Plateaus im Spiegel der metallzeitlichen Pflanzenreste von Feudvar. In: Hänsel B, Medovi P (eds), Feudvar 1. Das Plateau von Titel und die Sajkaska. Prähistorische Archäologie in Südosteuropa 13, Kiel, Germany, 305-317 Ljustina M, Miki A (2008): Grain legumes technology transfer in Old Europe - archaeological evidence. Abstracts, Second Grain Legumes Technology Transfer Platform Workshop, Novi Sad, Serbia, 42-43 Ljustina M, Miki A (2010): Archaeological evidence for the domestication of lentil (Lens culinaris) and its distribution in Europe. J. Lentil Res. 4 (in press) Maier U (1999): Agricultural activities and land use in a Neolithic village around 3900 B.C.: Hornstaad Hiirnle I A, Lake Constance, Germany. Veget. Hist. Archaeobot. 8: 87-94 Marinova E, Popova Tz (2008): Cicer arietinum (chick pea) in the Neolithic and Chalcolithic of Bulgaria: implications for cultural contacts with the neighbouring regions? Veget. Hist. Archaeobot. 17: 73-80 Medovi A (2003): Archäobotanische Untersuchungen in der metallzeitlichen Siedlung Zidovar, Vojvodina/Jugoslawien. Ein Vorbericht. Starinar 52: 181-190 Medovi A (2010): Biljna privreda Gradine na Bosutu (ili Savi?) u starijem gvozdenom dobu. In: Medovi P, Medovi I (eds.), Gradina na Bosutu. Naselje starijeg gvozdenog doba. Novi Sad, Serbia (in press) Miki A (2009): Words denoting pea (Pisum sativum) in European languages. Pisum Genet. 41: 29-33 Miki A (2010): Words denoting lentil (Lens culinaris) in European languages. J. Lentil Res. 4 (in press) Miki A, upina B, Kati S, Karagi Ð (2006): Importance of annual forage legumes in supplying plant proteins. Zbornik radova Instituta za ratarstvo i povrtarstvo / A Periodical of Scientific Research on Field and Vegetable Crops 42: 91-103 Miki A, Mihailovi V, Duc G, upina B, Étévé G, LejeuneHénaut I, Miki V (2007): Evaluation of winter protein pea

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cultivars in the conditions of Serbia. Zbornik radova Instituta za ratarstvo i povrtarstvo / A Periodical of Scientific Research on Field and Vegetable Crops 44: 107-112 Miki A, upina B, Mihailovi V, Vasi M, orevi V (2008): Diversity of words denoting traditional annual legumes in modern European languages. Proc. 18th EUCARPIA General Congress, Valencia, Spain, 193-197 Miki A, Angelova S, Burstin J, uri B, upina B, Lejeune I, Sabeva M, Vishnyakova M, Duc G (2009a): The pea genetic resources of the Balkans, to represent the first cultivated peas of Europe. Grain Legum. 52: 16-17 Miki A, Ljustina M, Kenicer G, Smýkal P (2009b): A brief historical review on Lathyrus in Europe. Grain Legum. 54: 33 Preiss S, Matterne V, Latron F (2005): An approach to funerary rituals in the Roman provinces: plant remains from a GalloRoman cemetery at Faulquemont (Moselle, France). Veget. Hist. Archaeobot. 14: 362-372 Renfrew J M (1979): The First Farmers in South East Europe. Festschrift Maria Hopf. Archaeo-Physika 8, Köln, Germany, 243-265 Rösch M (1997): The history of crops and crop weeds in south-western Germany from the Neolithic period to modern times, as shown by archaeobotanical evidence. Veget. Hist. Archaeobot. 7: 109-125 Sadori L, Susanna F, Persiani C (2006): Archaeobotanical data and crop storage evidence from an early Bronze Age 2 burnt house at Arslantepe, Malatya, Turkey. Veget. Hist. Archaeobot. 15: 205-215

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Stika H-P (1999): Approaches to reconstruction of early Celtic land-use in the central Neckar region in southwestern Germany. Veget. Hist. Archaeobot. 8: 95-103 Swidrak I (1999): A Celtic, La Tène trade centre in Ramsautal in the Dürrnberg, Austria: macrofossil data towards reconstruction of environment and food plants. Veget. Hist. Archaeobot. 8: 113-116 Tanno K, Willcox G (2006): The origins of cultivation of Cicer arietinum L. and Vicia faba L.: early finds from Tell el-Kerkh, north-west Syria, late 10th millennium B.P. Veget. Hist. Archaeobot. 15: 197-204 Van Zeist W (2001/2002): Plant husbandry and vegetation of Tell Gomolava, Vojvodina, Yugoslavia. Palaeohistoria 43/44: 87-115 Weeden N F (2007): Genetic changes accompanying the domestication of Pisum sativum: is there a common genetic basis to the `domestication syndrome' for legumes? Ann. Bot. 100: 1017-1025 Weeden N F, Brauner S, Przyborowski J A (2002): Genetic analysis of pod dehiscence in pea (Pisum sativum L.). Cell. Mol. Biol. Lett. 7:2B: 657-663 Willcox G, Fornite S, Herveux L (2008): Early Holocene cultivation before domestication in northern Syria. Veget. Hist. Archaeobot. 17: 313-325 Zeven A C, Zhukovsky P M (1975): Dictionary of Cultivated Plants and Their Centres of Diversity. Centre for Agricultural Publishing and Documentation, Wageningen

Kratki pregled ranog sirenja graska (Pisum sativum L.) u Evropi

Marija Ljustina1 Aleksandar Miki2

1

Univerzitet u Beogradu, Filozofski fakultet, Cika Ljubina 18-20, 11000 Beograd, Srbija 2 Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija

Izvod: Grasak je predstavljao deo svakodnevne ishrane evropskih lovaca-sakupljaca krajem poslednjeg ledenog doba. Glavni kriterijumi za odreivanje odomaivanja graska su nepucajue mahune, krupno seme i glatka semenjaca. Semena graska pronaena su meu najstarijim ostacima gajenih biljaka na lokalitetu El-Kerk u Siriji, starosti 10,000 godina. Zajedno sa strninama i socivom, grasak je odigrao kljucnu ulogu u otpocinjanju `poljoprivredne revolucije' u Starom svetu. Grasak je usao u Evropu u njenim jugoistocnim delovima i prodro u unutrasnjost uz Dunav, sirei se brzo, sto je potvreno njegovim prisustvom na udaljenim mestima u slicno vreme. Lingvisticki dokazi takoe govore u prilog da je grasak brzo postao prisutan u skoro svim delovima Evrope. Veina evropskih naroda poseduje svoje sopstvene reci za grasak, sto znaci da je postojao pre razvia njihovih prajezika. Kljucne reci: arheologija, grasak, istorija useva, Pisum sativum L., Stara Evropa, sirenje

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www.nsseme.com/journal.html

Obelezja proizvodnje povra u Srbiji

461 Ekonomska analiza / Economic Analysis Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 461-466 pregledni rad / review article

Obelezja proizvodnje povra u Republici Srbiji

Branislav Vlahovi Anton Puskari Janko Cervenski

primljeno / received: 14.04.2010. prihvaeno / accepted: 24.05.2010. © 2010 IFVC

Izvod: Cilj istrazivanja ovoga rada jeste da se sagledaju osnovni trendovi kretanja proizvodnje povra u Republici Srbiji, koji su ostvareni u vremenskom periodu 2000-2009. Zadatak je, takoe, da se kvantifikuju nastale promene i utvrde faktori koji su determinisali ispoljeno kretanje proizvodnje u zemlji. Autori ukazuju na osnovne probleme i daju predloge neophodnih mera koje e uticati na poveanje obima proizvodnje, odnosno na mogue pravce razvoja ove veoma znacajne grane poljoprivrede za Republiku Srbiju. Ukazano je takoe na znacaj i mogunosti proizvodnje zdravstveno bezbednog povra u sistemu organske poljoprivrede. Uz odgovarajuu kreditno-monetarnu, investicionu i izvoznu podrsku, povrtarska proizvodnja moze postati vazan sektor poljoprivrede Republike Srbije. Kljucne reci: povre, proizvodnja, Srbija

Uvod Srbija je tradicionalno povrtarska zemlja sa izvanrednim klimatskim i zemljisnim potencijalima za ovaj vid proizvodnje. Proizvodnja je raznovrsna i dominantno se odvija na otvorenom polju, a manjim delom u zastienom prostoru. U poslednjim godinama proizvodnja u zastienom prostoru dobija na znacaju, jer se njome postize ranije prispee povra i ostvaruje znacajna profitabilnost proizvodnje. Proizvodnja povra u najveem obimu organizovana je u dolinama veih reka (nizije) i u blizini gradova (trzista). Sela koja gravitiraju na ovim potezima u strukturi svoje poljoprivredne proizvodnje, pored ostalog, imaju ozbiljniju proizvodnju povra. Brdsko-planinsko podrucje redukuje broj povrtarskih vrsta koja mogu uspesno da se gaje, ali s druge strane, mikroklima smanjuje pojavu odreenih bolesti i stetocina koje u niziji predstavljaju veliki problem. Ova vrsta proizvodnje ima znacajnu perspektivu zbog povoljnih prirodnih uslova za uspevanje

B. Vlahovi ) ( Poljoprivredni fakultet Univerziteta u Novom Sadu, Trg Dositeja Obradovia 8, 21000 Novi Sad, Srbija e-mail: [email protected] A. Puskari Institut za ekonomiku poljoprivrede, Volgina 15, 11060 Beograd, Srbija J. Cervenski Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija

svih povrtarskih vrsta i izrazene traznje na domaem i meunarodnom trzistu. Materijal i metod rada Osnovni cilj istrazivanja jeste da se sagledaju osnovna obelezja proizvodnje povra u Republici Srbiji za vremenski period 2000-2009. Istrazivanje je bazirano na raspolozivim podacima, uz primenjeni metod "istrazivanja za stolom" (desk research). Osnovni podaci preuzeti su iz Republickog zavoda za statistiku, Beograd. Najznacajnija obelezja prezentovana su putem tabela i grafikona, a obraena su standardnim matematickostatistickim metodama. Rezultati i diskusija Povrtarska proizvodnja predstavlja znacajnu i tradicionalnu privrednu delatnost u Republici Srbiji. Povrtarstvo omoguava intenzivno korisenje zemljista i sistema za navodnjavanje smenom dve do tri vrste u toku godine na njivi i zastienom prostoru. Specificnost velikog broja povrtarskih kultura omoguuje i proizvodnju hrane u klimatski i edafski nepovoljnim uslovima, korisenjem razlicitih nacina i sistema proizvodnje. Proizvodnja povra predstavlja jednu od najintenzivnijih grana biljne proizvodnje, sto se izrazava velicinom prinosa po jedinici povrsine, ostvarenim dohotkom, neto prihodom i ucesem ljudskog rada. Moze da obezbedi pet do osam puta

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Vlahovi B i sar.

veu vrednost proizvodnje, dok ista proizvodnja u plasteniku ima 190-250 puta veu vrednost u odnosu na psenicu. Povrtarska proizvodnja tesno je povezana sa razvojem preraivackih kapaciteta, odnosno povre cini sirovinu za razlicite oblike prerade (www.pks.rs)

280

(000 ha)

0,2 ha, a njihova proizvodnja nije namenjena trzistu ve pretezno licnoj potrosnji. Proizvoacima je cilj redukovanje troskova, a ne ostvarenje ekstremno visokih prinosa. Za visok i stabilan prinos neophodan je kvalitetan sadni materijal, kao i odgovarajue i pravovremene agrotehnicke

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200 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

originalni podaci linija trenda

Grafikon 1. Kretanje povrsina pod povrem u Republici Srbiji (2000-2009) Graph 1. Trends of areas under vegetables in the Republic of Serbia (2000-2009)

U strukturi ukupne vrednosti poljoprivredne proizvodnje u Republici Srbiji u 2009. povrtarska proizvodnja ucestvuje sa 16,5%. Prosecna povrsina pod povrem u Srbiji iznosi 247.000 ha. U istrazivanom periodu prisutan je trend pada povrsina pod povrem po stopi od 1,45% godisnje (Graf. 1). U poslednjoj godini se povrsina smanjila za oko 30.000 ha u odnosu na pocetnu godinu. Najvei deo povrsina (oko 70%) lociran je na malim poljoprivrednim gazdinstvima, koja nisu usko specijalizovana za ovaj vid proizvodnje. U strukturi ukupnih povrsina najvee ucese ima krompir sa 35%. Od analiziranih deset vrsta povra, prikazanih u tabeli 1, samo povrsina pod krastavcima ima tendenciju rasta, dok povrsina svih ostalih vrsta belezi tendenciju pada. Najintenzivniji pad povrsine ima beli luk (-2,36%). Prinos povra u Republici Srbiji znacajno je manji u odnosu na ostvareni evropski prinos (Vlahovi i sar. 2006). Prinos povra zavisi, pre svega, od sledeih faktora: toplote, svetlosti, vode, kvaliteta zemljista, unosa mineralnih hraniva, nivoa zastite od bolesti i stetocina (odnosno nivoa ulaganja u ovu vaznu agrotehnicku meru) i slicno. Za sadasnji nivo agrotehnike moze se rei da je na dosta skromnom nivou. Osnovni razlog lezi u tome sto se povre kod najveeg broja proizvoaca gaji na malim parcelama i okunicama povrsine ispod

mere. Ohrabruje cinjenica da u analiziranom periodu sve vrste povra imaju trend porasta prinosa. Najintenzivniji rast prinosa ima pasulj, po prosecnoj godisnjoj stopi od preko 5%. Porast prinosa kompenzovao je opadanje povrsine pod povrem, sto je rezultiralo porastom proizvodnje povra u Republici Srbiji. Osim dinja, lubenica i belog luka, sve ostale vrste povra ostvaruju trend porasta proizvodnje (Tab. 1). Najintenzivniji porast proizvodnje imaju pasulj i grasak, sto je uslovljeno, pored ostalog, rastom traznje na trzistu. Najznacajniji okrug u proizvodnji krompira je moravicki, paprike i paradajza macvanski, pasulja branicevski, graska i crnog luka juzno-backi, kupusa podrucje grada Beograda. U cilju poveanja proizvodnje povra, potrebno je preduzeti odreene mere na razlicitim nivoima. Na makroekonomskom nivou potrebno je povoljnim kreditima uticati na ekonomsku motivisanost proizvoaca da se odluce za ovu proizvodnju. Na mikroekonomskom nivou, mora se voditi racuna o svim neophodnim agrotehnickim merama u smislu dobijanja visih prinosa i boljeg kvaliteta proizvoda. Jedan od limitirajuih faktora dalje proizvodnje jeste nesreeno trziste i prisustvo velikog broja nakupaca, koji cesto ,,diktiraju" enormno nisku cenu, zbog cega proizvoaci cesto ostvaruju gubitke u proizvodnji i nisu ekonomski motivisani za prosirenje iste.

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Tabela 1. Proizvodnja povra u Republici Srbiji (2000-2009) (1.000 t) Table 1. Vegetable production in the Republic of Serbia (2000-2009) (1,000 t) Interval varijacije Prosecna Variation interval CV Vrsta vrednost (%) Species Average value min max

Stopa promene Change rate (%)

Krompir 859 621 1.015 15,64 1,18 Potato Paradajz 176 152 199 8,47 0,34 Tomato Grasak 33 23 42 18,07 5,14 Pea Kupus i kelj 304 261 342 8,73 0,26 Cabbage and kale Crni luk 126 94 143 13,49 1,89 Onion Paprika 150 132 177 9,43 2,35 Pepper Pasulj 33 15 55 36,07 10,88 Bean Dinje i lubenice Melon and 252 205 295 11,66 -0,72 Watermelon Mrkva 60 45 70 15,81 3,13 Carrot Krastavac 60 51 67 8,75 2,17 Cucumber Beli luk 25 21 28 11,32 -0,93 Garlic Obracun autora na bazi podataka Republickog zavoda za statistiku, Beograd, 2010. Authors' calculation based on data provided by Statistical Office of the Republic of Serbia, Belgrade, 2010. Kvalitet povra zavisi i od samog svojstva ali i pripreme za prodaju, kao sto su pakovanje, ambalaza i marketing. Svojstva koja najvise doprinose kvalitetu voa su: estetska, organolepticka i hemijsko-bioloska (sadrzaj vitamina, mikroelemenata i enzima i odsustvo rezidua pesticida). Proizvodnja u zastienom prostoru ima velike prednosti. U svetu se primenjuje, kako u urbanim sredinama i u blizini trznica, tako i u seoskim podrucjima, gde obezbeuje radna mesta i dodatne prihode tamosnjem stanovnistvu. Proizvodnja u zastienom prostoru intenzivnija je od drugih oblika proizvodnje jer se, uz neophodno opste i specificno znanje, ostvaruju vei prinosi. Zastieni prostor treba da obezbedi optimalne uslove za gajenje biljaka u jesenje-zimsko-prolenom periodu, koji omoguuju planiranu berbu u vreme manje ponude na trzistu, sto osigurava veu cenu. Ekonomicnost proizvodnje u zastienom prostoru zavisi, pre svega, od vremena pristizanja proizvoda na trziste. Klimatski uslovi i trziste uslovljavaju izbor vrste zastienog prostora, vrstu i sortu povra i vreme proizvodnje. Proizvodnja u zastienom prostoru omoguuje punu kontrolu uslova uspevanja, sto znaci i mogunost da se dobije povre dobrog kvaliteta i zdravstveno bezbedno povre. Proizvodnja u plastenicima i staklenicima treba da se zasniva na ekoloskim principima, uz postovanje mera preventive i predostroznosti. Ekoloski princip obuhvata izbor lokaliteta na neophodnoj udaljenosti od zagaivaca (500-5.000 m) kontrolu kvaliteta zemljista, vode i vazduha. Proizvodnja u zastienom prostoru ekonomicna je za porodicno gazdinstvo, a u celini moze da obezbedi dovoljno kvalitetnog povra i za potrebe domaeg trzista i za izvoz (www.aic.ba). Jedan od osnovnih problema sirenja proizvodnje u zastienom prostoru u nasoj zemlji jesu visoka investiciona ulaganja. Promene klimatskih prilika uticale su da se u svetu proizvodnja povra s otvorenih povrsina premesti u zatvorene prostore, plastenike i staklenike kako bi se proizvodnja sacuvala od

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vremenskih nepogoda. Podizu se plastenici namenjeni hidroponskoj proizvodnji. Proizvodnja se odvija bez prisustva zemlje, na osnovu supstrata, u kockama kamene vune. Kocke kamene vune postavljaju su na blokovima kamene vune veih dimenzija kako bi biljka mogla da pruzi sto vei rod. Prihranjivanje je kompjuterizovano, sto znaci da su biogeni elementi rastvoreni u vodi. Prednost ovakvog nacina proizvodnje jeste u tome sto nema bespotrebne potrosnje vode, nema potrebe za plodoredom, hraniva su dozirana i apliciraju se uz biljku prema njezinim potrebama. Srbija je 2008. na plenarnoj sednici meunarodne organizacije OECD (Organizacija za ekonomski razvoj i saradnju), primljena u clanstvo Seme za sveze voe i povre OECD-a. Odluku o uclanjenju donele su 23 zemlje clanice te meunarodne organizacije, posle boravka Evaluacionog tima OECD - Seme za sveze voe i povre u Srbiji. Na sednici je odluceno da nasa zemlja moze aktivno da ucestvuje u meunarodnoj spoljnotrgovinskoj razmeni svezeg voa i povra. Pored obaveza o uvoenju OECD standarda, koji su gotovo jednaki sa EU standardima, Srbija je dobila pravo izdavanja meunarodnih certifikata koji joj daju pravo ravnopravnog ucesnika u meunarodnoj trgovini. U narednom periodu, zadatak Srbije vezan je za izmenu zakonske regulative u smislu usaglasavanja nacionalnih standarda sa meunarodnim (OECD/UNECE) standardima kvaliteta za sveze povre, cime bi postali konkurentniji na inostranom trzistu. Ulaskom u OECD-semu za sveze voe i povre, zaokruzen je ciklus uclanjenja zemlje u sve cetiri OECD seme koje propisuju standarde za trgovinu semenom, sumskim materijalom, svezim voem i povrem, kao i uslove koje moraju da ispunjavaju masine kojima se obavlja poljoprivredna proizvodnja. Navedeno treba da predstavlja podstrek domaim proizvoacima u cilju poveanja izvoza svezeg i preraenog povra. S obzirom da Srbija poseduje povoljne agroekoloske uslove, postoje realne mogunosti za ostvarivanje znatno visih prinosa i vee proizvodnje. Za unapreenje povrtarske proizvodnje predlaze se sledee (Markovi 2004): · poveanje povrsina pod povrem, pri cemu bi ucese povra u setvenoj strukturi bilo 10% do 15% · posebno poveanje gajenja povra kao drugog useva · poveanje povrsine pod zastienim prostorom (plastenici, staklenici i slicno)

· uvoenje u proizvodnju povra vise vrsta za svezu potrosnju, tokom cele godine, kao i za razlicite oblike prerade · primena odgovarajuih agrotehnickih mera, gajenje kvalitetnih i otpornih sorti, korisenje kvalitetnog semena i sadnog materijala, realizacija ekonomicnog prinosa povra, visokog nutritivnog i zdravstvenog kvaliteta · osavremenjivanje bastenskog nacina proizvodnje (uvoenjem novih prinosnijih sorti, specificne mehanizacije i gajenjem vrsta koje se tesko mogu mehanizovati, gde je potrebno vee angazovanje ljudske radne snage) i kombinovanje sa proizvodnjom u zastienom prostoru · poveanje broja gajenih vrsta u poljoprivrednim preduzeima i zadrugama uz produzenje kontinuiteta proizvodnje · poveanje broja istrazivaca i strucnjaka u proizvodnji i uspostavljanje transfera rezultata nauke u direktnu proizvodnju, sto bi za posledicu imalo poveanje prinosa ovakve proizvodnje povra i ukupne proizvodnje povra · podizanje obrazovnog nivoa poljoprivrednih proizvoaca u zimskim skolama, seminarima i savetovanjima, kao i angazovanje lokalnih medija za emitovanje emisija sa raznim savetima i prenosenjem poruka, narocito prognozne sluzbe · stvaranje novih sorti i proizvodnja kvalitetnog semena povra · obezbeivanje savremenih uslova od berbe do plasmana (transport, pakovanje, ambalaza, distributivni centri, kratkorocno i dugorocno cuvanje) · razvijanje marketinga koji opredeljuje trzisnost proizvedenog povra. U intenzivnoj poljoprivrednoj proizvodnji, kao sto je povrtarska proizvodnja, razvojem povrtarstva podstice se i razvoj industrija, koja cini input za ovu proizvodnju, kao sto je industrija poljoprivrednih masina, industrija mineralnih ubriva, sredstva za zastitu bilja, hemijsku industriju, sto znaci vise povra u ishrani kako na domaem, tako i na stranom trzistu, sa osnovnom karakteristikom visokog ukupnog nutritivnog i zdravstvenog kvaliteta. Mogunost proizvodnje organskog povra u Srbiji U nasoj zemlji postoje dobri preduslovi za proizvodnju organskog povra. Prednost organske proizvodnje sastoji se u odsustvu upotrebe hemijskih sredstava uobicajenih u konvencionalnoj proizvodnji, meu kojima su sinteticka ubriva, pesticidi i herbicidi, koji su posledicno povezani sa mnogim zdravstvenim smetnjama.

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Prema Zakonu o organskoj proizvodnji, uslovi za zasnivanje ove proizvodnje su sledei: · prostorna izolacija zemljisnih parcela i preraivackih pogona od moguih izvora zagaenja · nezagaeno zemljiste ciji je sadrzaj stetnih materija ispod propisanih maksimalno dozvoljenih kolicina · propisani kvalitet vode za navodnjavanje i minimalna zagaenost vazduha na proizvodnom podrucju Osnovne prednosti organske proizvodnje povra su sledee (Moravcevi 2006): · cuva se ekosistem i dobija se zdravstveno bezbedan proizvod kvalitetnijeg sastava · otvara se mogunost prevoenja jedne ekstezivne proizvodnje u intenzivniju i obezbeuje se kontinuiran priliv proizvoda i novca · otvara se veliki potencijal za brendiranje roba i promociju seoskog turizma · u potpunosti se koriste klimatski, zemljisni i ljudski resursi · organsko povre je deficitarna roba na trzistu Evopske unije · stvara se motiv za zajednicki rad (udruzivanje) i proizvoaca koji se nisu usko specijalizovali za pojedini oblik proizvodnje · manja su ulaganja u preskup repromaterijal (mineralno ubrivo, pesticide i slicno) Napred navedeno govori u prilog tvrdnji da se sa veim ili manjim uspehom proizvodnja povra obavlja na teritoriji cele drzave. Uvoenje alternativnih nacina gajenja povra, kao sto je organska proizvodnja, na vee povrsine od sadasnjih (oko 50 ha) doprinelo bi raznovrsnosti ponude i dalo bi pecat ve postojeoj proizvodnji povra. Meutim, postoje i prepreke za sirenje organske proizvodnje povra u Srbiji: · malo pristupacnog znanja i o konvencionalnoj i o organskoj proizvodnji povra, · losa drzavna politika vezana za organsku proizvodnju, · potencijalni potrosaci su neinformisani o prednostima organskog povra, · trziste organskih proizvoda je usko i neorganizovano i dr. Integralna proizvodnja odvija se na osnovu standarda udruzenja za integralnu proizvodnju, a oznacava prelaz ka odrzivoj, organskoj proizvodnji. Osnovna odlika organske (ekoloske, bioloske) proizvodnje jeste izbegavanje primene sintetickih mineralnih ubriva i sredstava za zastitu bilja. Ekoloski prihvatljiva proizvodnja vodi racuna

da proizvodi sadrze sto manje (svakako daleko ispod dozvoljenih kolicina) zagaenja, ne samo iz poljoprivrede ve i iz drugih izvora, a da se troskovi te proizvodnje kompenzuju veom cenom u odnosu na konvencionalnu proizvodnju. U preradi se polazi od primene svih sanitarnih i organskih metoda prerade. Dozvoljeni su mehanicki, fizicki, biofer mentacioni postupci i kombinacija ovih postupaka (Lazi i sar. 2005). Proizvoaci proizvoda organske poljoprivrede u Srbiji treba da imaju odgovarajuu finansijsku, pravnu, ali i marketinsku podrsku za postizanje rezultata i uspesan nastup na domaem i inostranim trzistima (Vlahovi & Strbac 2007). Vlada Republike Srbije je 2009. donela Uredbu o uslovima i nacinu korisenja podsticajnih sredstava za podrsku proizvodnji semena, rasada i sadnog materijala metodama organske proizvodnje. Fizicka lica, nosilac poljoprivrednog gazdinstva, pravno lice, preduzetnik pod propisanim uslovima imaju prava na podsticajna sredstva u iznosu do 50.000 dinara (500 evra) po hektaru za organsku povrtarsku proizvodnju (www.minpolj.gov.rs). Preduslov za uspesniji nastup na trzistu jeste udruzivanje proizvoaca povra. Na taj nacin obezbedile bi se vee kolicine i kontinuirane isporuke. Postoji niz dobrih primera kao sto je begecko udruzenje povrtara i udruzenje povrtara ,,Banatski Brestovac", koje je u saradnji sa Institutom za ekonomiku poljoprivrede Beograd uvelo HACCP koncept za pet povrtarskih kultura: lubenicu, papriku, krastavac, paradajz i kukuruz seerac, a u budunosti se ocekuje uspesan prodor povra ovog udruzenja u domae hiper markete i na probirljivo evropsko trziste (Parausi i sar. 2007). Zakljucak U istrazivanom vremenskom periodu (20002009) ostvareni su znacajni naturalni proizvodni rezultati u proizvodnji povra. Povrsina pod povrem belezi pad po prosecnoj godisnjoj stopi od 1,45%. Prinos svih analiziranih vrsta povra ostvaruje tendenciju umerenog rasta. Proizvodnja svih povrtarskih vrsta belezi tendenciju porasta. Najintenzivniji rast belezi proizvodnja pasulja. Postoje preduslovi za proizvodnju organskog, odnosno zdravstveno bezbednog povra za kojim raste traznja na meunarodnom trzistu. Neophodno je povezivanje proizvoaca u specijalizovana strukovna udruzenja sa ciljem poveanja proizvodnje i izvoza. Njihov cilj jeste rentabilnija i profitabilnija proizvodnja. Povrtarska proizvodnja moze, uz odgovarajuu kreditno-monetarnu, investicionu i izvoznu podrsku, postati veoma bitan sektor poljoprivrede Republike Srbije.

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Vlahovi B, Strbac M (2007): Osnovne karakteristike trzista i marketinga proizvoda organske poljoprivrede. Ekonomika poljoprivrede 2: 131-147. Agrarni informacioni centar (2008): Povre iz plastenika, [Elektronski izvor], dostupno na adresi http://aic.ba/poljoprivreda/povrtlarstvo/56-povrtlarstvo/1610-povre-iz plastenika. html, (citirano 15.09.2008., verifikovano 17.03.2010.), AIC, Banja Luka, BiH Ministarstvo poljoprivrede, sumarstva i vodoprivrede (2009), Uredba o korisenju podsticajnih sredstava za podrsku razvoju sela kroz investiranje u prosirenje i unapreenje ekonomskih aktivnosti seoskog stanovnistva za 2009. godinu [Elektronski izvor], dostupno na adresi http://www.minpolj.gov.rs/postavljen/120/uredbausl. pdf, (citirano 20.03.2009., verifikovano 18.03.2010.), Beograd, Srbija Privredna komora Srbije (2010): Proizvodnja povra [Elektronski izvor], dostupno na adresi http://www.pks.rs/PrivredauSrbiji/Poljoprivreda/Povrtarstvo/tabid/2050/language/ sr-Latn-CS/Default.aspx, Beograd, Srbija Republicki zavod za statistiku (2010): Baza podataka biljne proizvodnje [Elektronski izvor], dostupno na adresi http://webrzs.stat.gov.rs/axd/poljoprivreda/index.php?ind=1, (citirano 20.03.2009., verifikovano 15.03.2010.), Beograd, Srbija

Literatura

Lazi B, Babovi D, Novakovi N (2005): Put ka ekoloskoj poljoprivredi. Institut za ratarstvo i povrtarstvo, Novi Sad Markovi V (2004): Stanje i perspektive povrtarske proizvodnje u Vojvodini. Agrarna saznanja, Novi Sad Moravcevi (2006): Perspektive organske proizvodnje povra u Srbiji. [Elektronski izvor], dostupno na adresi http:// www.bioserbia.info/fileadmin/templates/bioserbia_info/ gallery/2009_02_19_brosura/perspektive%20organske%20 proizvodnje%20povrca%20u% 20srbiji.pdf Parausi V, Cvijanovi D, Subi J (2007): Afirmacija udruzivanja i marketinga u funkciji kreiranja konkurentnosti agrarnog sektora Srbije. Institut za ekonomiku poljoprivrede, Beograd Seavert C, Hinman H R, Stodic L (1995): CPA Version 2.00, Oregon State University, Oregon Vlahovi B, Ilin Z, Umievi B (2002): Stanje i mogunosti za plasman povra na meunarodno trziste. Savremeni povrtar 3-4: 74-79. Vlahovi B, Puskari A (2008): Izvoz povra iz Republike Srbije, XIII savetovanje o biotehnologiji, Cacak Vlahovi B, Stevanovi S, Tomasevi D, Zelenjak M (2006): Agrarna proizvodnja u Republici Srbiji, DAES, Beograd

Characteristics of Vegetable Production in the Republic of Serbia

Branislav Vlahovi1 Anton Puskari2 Janko Cervenski3

1 Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovia 8, 21000 Novi Sad, Serbia 2 Institute of Agricultural Economics, Volgina 15, 11060 Beograd, Serbia 3 Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia

Summary: The main goal of research in this paper was to consider basic trends in vegetable production in the Republic of Serbia for the period 2000-2009. Additionally, the task was to quantify of all changes, as well as to determin the factors that affected expressed production trends in Serbia. Authors point out the basic problems, suggesting both the necessary measures that would affect production increase and possible development of this very important branch of agriculture for Serbia. Also, the importance and possibilities of safe vegetables production in the system of organic agriculture are pointed out. With adequate support regarding credits, investments and export, vegetable production may become a significant sector of Serbian agriculture. Key words: production, Serbia, vegetables

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www.nsseme.com/journal.html

Geneticka varijabilnost prinosa kukuruza

467 Genetika i oplemenjivanje / Genetics and Breeding Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 467-472 originalni naucni rad / original research article

Geneticka varijabilnost i korelaciona analiza za prinos zrna sinteticke populacije kukuruza NSA15

Dusan Stanisavljevi Sanja Treski Bojan Mitrovi Aleksandra Nastasi Goran Bekavac Mile Ivanovi

primljeno / received: 20.04.2010. prihvaeno / accepted: 10.05.2010. © 2010 IFVC

Izvod: S1 potomstva populacije uze geneticke osnove NSA15 nastale ukrstanjem inbred linija NS15 x NS61, testirana su istovremeno sa dva nesrodna inbred-testera: NS732 (ID geneticke osnove) i NS27 (BSSS osnove). Proizvedeno je po 40 parova HS potomstava, tako da svaki par ima zajednicku majcinsku komponentu (S1 liniju). Porodice su posebno randomizirane i zasejane u dva nezavisna ogleda, po modelu nepotpunog blok dizajna, svaki u dva seta, sa dva ponavljanja u okviru seta i 20 HS familija u setu. Ogledi su izvedeni tokom 2008. (cetiri lokaliteta) i 2009. (tri lokaliteta). Primenjena je standardna tehnologija gajenja. Vei prosecan prinos u veini kombinacija S1 familija x tester ostvaren je sa linijom NS27. Oko 50% testiranih familija sa oba testera ostvarilo je vei prinos zrna od proseka odgovarajuih standarda. Heritabilnost za prinos zrna kod HS-1 potomstava nastalih ukrstanjem sa testerom NS732 bila je signifikantna (h2=0,519±0,228), dok je kod HS-2 potomstava nastalih ukrstanjem sa testerom NS27 zabelezena niza i nesignifikantna vrednost (h2=0,337±0,231). Geneticka korelacija izmeu dve grupe HS potomstava za prinos zrna je bila nesignifikantna (rg=0,309±0,253). Visoke vrednosti standardnih greski za heritabilnost i geneticku korelaciju uslovljene su najveim delom driftom nastalim slucajnom koncentracijom rodnijih genotipova u II setu HS-2 ogleda. Na osnovu dobijenih rezultata za prinos zrna, inbred-tester NS27 je pokazao perspektivnije rezultate za budui oplemenjivacki program sa populacijom NSA15. Kljucne reci: geneticke korelacije, half-sib (HS), heritabilnost, kukuruz, tester, Zea mays L.

Uvod Poznavanje korelacione povezanosti izmeu razlicitih svojstava od velikog je znacaja u primenjenim (oplemenjivackim) i bioloskim istrazivanjima. Falconer (1981) navodi tri osnovna razloga za to: geneticku determinaciju korelacija, pre svega plejotropski efekat; uticaj promene jednog na nivo (istovremene) promene drugog karaktera; i najzad, znacaj korelativnih odnosa metrickih svojstava i adaptivne vrednosti sa stanovista prirodne selekcije. U programima oplemenjivanja organizama, korelacionoj analizi je posveena velika paznja primenom razlicitih parametrijskih i neparametrijskih metoda. Od posebnog znacaja su geneticka (pre svega aditivna) i fenotipska korelacija izmeu svojstava. Jacina korelacione veze (obicno izmeu osnovnog i prateih svojstava) i heritabilnost kljucni su parametri pri izboru pocetnog materijala, metoda selekcije i nivoa selekcionog intenziteta.

D. Stanisavljevi ) ( · S. Treski · B. Mitrovi · A. Nastasi · G. Bekavac · M. Ivanovi Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

Kod kukuruza je prinos zrna, zbog ekonomskog znacaja, najcese korelirano svojstvo (u okviru populacije) sa komponentama prinosa, morfoloskim i fizioloskim karakteristikama biljke. Korisene su populacije uze ili sire geneticke osnove, kao i razliciti tipovi potomstava (puni srodnici, FS; polusrodnici, HS; samooplodna potomstva, S1, S2; Hallauer & Miranda 1988). Iako mogu biti od velikog prakticnog i teorijskog znacaja, korelacije za jedno svojstvo (npr. prinos zrna) izmeu razlicitih tipova potomstava, ili odgovarajuih familija u okviru istog potomstva (npr. HS) znatno se ree sreu u literaturi. Cilj ovog rada je bilo proucavanje varijabilnost i geneticke korelacije za prinos zrna izmeu dve grupe polusrodnika (HS), potomaka populacije kukuruza uze geneticke osnove. Materijal i metod rada Za istrazivanja u ovom radu korisena je populacija kukuruza uze geneticke osnove NSA15, standardnog kvaliteta zrna, srednje kasne vegetacije (FAO grupe zrenja 700). Tokom 2005. izvrseno je ukrstanje dve inbred linije NS15 (nastala kombinacijom 25% materijala iz Juzne Amerike i

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75% adaptiranih izvora, nesrodnih sa drugim heteroticnim grupama) i NS61 (Lancaster osnove). Samooplodnjom F1 biljaka u zimskoj generaciji (2005/2006) dobijeno je seme F2 generacije, od koga je proizvedeno oko 300 F2 biljaka u 2006. na oglednom polju Instituta za ratarstvo i povrtarstvo u Novom Sadu. Metodom slucajnog izbora samooploeno je 100 biljaka, a odabrano 50 klipova (biljaka) u berbi, koji su imali dovoljan broj zrna za setvu naredne godine. Tokom 2007. zasejano je svih 50 S1 familija metodom klip na red. Po 15 slucajno odabranih biljaka od svake S1 familije (majcinske komponente) ukrsteno je sa svakim od dva meusobno i u odnosu na populaciju, geneticki nesrodna testera - inbred linije NS 732 i NS 27 (ocinske komponente). Linija NS732 (FAO grupa zrenja 500) pripada ID heteroticnoj grupi, dok je NS 27 (FAO grupa zrenja 500), BSSS osnove. Dve grupe HS potomstava, svaka sa 40 genotipova (familija), sa dovoljnom kolicinom izbalansiranog semena, formirane su tako da svaka S1 familija bude istovremeno zastupljena (ukrstena) sa oba testera. Poljski ogledi po modelu nepotpunog blok dizajna, sa ponavljanjima u okviru seta (Cochran & Cox 1957), posejani su tokom 2008. i 2009. na cetiri lokaliteta: Rimski Sancevi, Srbobran, Sremska Mitrovica i Kikinda. Usled jake suse i neujednacenog nicanja i razvoja biljaka ogledi u Kikindi 2009. nisu koriseni, tako da je analiza obuhvatila 7 lokaliteta (cetiri u 2008. i tri u 2009). Za svaki tip HS potomstava postavljeni su nezavisni ogledi, sa dva seta (u svakom setu po 20 slucajno odabranih potomstava) i dva ponavljanja po setu. U okviru svakog ogleda bila su zastupljena po cetiri hibrida - standarda (dva inbred-testera x linije koje su formirale populaciju): NS732 x NS61, NS27 x NS61, NS732 x NS15 i NS27 x NS15. Standardi su koriseni samo radi poreenja vrednosti proucavanih svojstava u odnosu na odgovarajua testirana potomstva, te nisu ukljuceni u analizu i procenu genetickih parametara. Povrsina elementarne parcele iznosila je 8,1 m2 za svaki genotip (dva reda, svaki duzine 5,4 m), sa rastojanjem od 0,75 m izmeu redova i 0,22 m izmeu biljaka u redu. Gustina useva iznosila je 60.606 biljaka/ha. Setva i berba ogleda (za procenu prinosa zrna) obavljena je masinski. Primenjena je uobicajena tehnologija gajenja, koja je prilagoena agroekoloskim uslovima lokaliteta. Proucavan je vei broj svojstava biljke i klipa, ali su u radu prikazani samo rezultati za prinos zrna (t ha-1 sa 14% vode). Analiza i komponente varijanse, kovarijanse i heritabilnost (u sirem smislu) nekompletnog blok dizajna uraena je na principu modela II (Hallauer

& Miranda 1988). Geneticke korelacije za prinos zrna izmeu dve grupe HS familija izracunate su po standardnoj proceduri, iz odnosa genetickih kovarijansi i varijansi (Falconer 1981). S obzirom da je randomizacija svake HS grupe (2 x 20 familija) bila meusobno nezavisna, analiza iskljucuje mogunost procene komponenti fenotipskih varijansi, jer je njihova fenotipska kovarijansa jednaka genetickoj kovarijansi (Bradshaw 1983). Rezultati i diskusija U tabeli 1. prikazan je prosecan prinos zrna (t ha-1) dve grupe (HS-1 i HS-2) potomstava sa 7 lokaliteta (cetiri u 2008. i tri u 2009). Na osnovu rezultata ANOVA-e (nisu prikazani u ovom radu) utvrena je signifikantna razlika izmeu setova kod HS-2 familija. Zbog toga su rezultati uporeivani za svaki set posebno, kod oba ogleda, izmeu prinosa familija i prosecnog prinosa odgovarajuih standarda. Kod HS-1 potomstava za poreenje je sluzio prosek hibrida NS732 x NS15 i NS732 x NS61, a za drugu grupu ogleda (HS-2) korisen je prosek standarda NS27 x NS15 i NS27 x NS61. Drugim recima, standardi su odgovarali HS potomstvima prema izabranom testeru (NS732 ili NS27, kao majcinske komponente hibrida), dok su ocinske linije u standardnim hibridima (NS15 i NS61) korisene kao komponente pri stvaranju pocetne populacije. Direktno poreenje prosecnih i pojedinacnih prinosa HS-1 i HS-2 grupe genotipova nije pouzdano zbog nezavisne randomizacije, iako se jasno uocavaju vee vrednosti za setove, standarde i veinu familija HS-2 grupe, posebno u drugom setu (Tab. 1). Dobijeni rezultati ukazuju da inbred-tester NS27 ima prednost u odnosu na NS732 za nastavak oplemenjivackog programa sa navedenom populacijom (NSA15). Od ukupno 40 testiranih familija, 22 u HS-1 i, 19 u HS-2 (ili oko 50% po grupi), ostvarile su vei prinos (Tab. 1) u odnosu na odgovarajui prosek standarda. Istovremeno, 11 familija (27,5% od ukupno testiranih) pripada grupi najrodnijih sa oba testera (npr. S1 familije br. 5, 7, 8, itd.). Korisenje elitnih linija kao testera svakako ima prakticni znacaj. Ipak, u hibridnim kombinacijama ove linije mogu izazvati maskirajui efekat (Ivanovi et al. 1987), koji je utoliko vei ukoliko su performanse testera bolje (Smith 1986). Detekcija znacajnog broja ,,zajednickih" (za HS-1 i HS-2 grupe) najrodnijih kombinacija, u konkretnom slucaju se samo delimicno moze objasniti maskirajuim efektom testera, tim pre sto se radi o geneticki nesrodnim linijama, cije F1 potomstvo (NS732 x NS27) predstavlja hibrid sa

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Tabela 1. Prosecan prinos zrna (t ha-1) dve grupe HS potomstava NSA15 populacije kukuruza (S1 x NS732 = HS-1 i S1 x NS27 = HS-2) Table 1. Average grain yield (t ha-1) of two HS progeny groups of NSA15 maize population (S1 x NS732 = HS-1 i S1 x NS27 = HS-2)

Testeri () Setovi Sets S1 familije () S1 families 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Prosek seta Set mean Prosek standarda Mean values of checks 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Prosek seta Set mean Prosek standarda Mean value of checks Testers NS732 (HS-1) NS732 (HS-1) 11,10* 10,90* 10,78* 9,74 10,84* 11,27* 11,58* 10,77* 9,88 10,78* 10,96* 11,29* 11,56* 11,36* 10,40 10,34 10,36 11,48* 11,17* 10,62 10,86 10,62 10,45 11,73* 10,72 10,87* 10,95* 10,71 10,80 11,42* 10,44 10,45 10,94* 11,06* 11,15* 10,34 10,58 10,81 10,76 10,53 10,24 10,89* 10,79 10,84 NS27 (HS-2) NS27 (HS-2) 10,78 11,18 10,38 10,82 11,26* 11,11 11,42* 11,60* 10,78 10,97 10,54 11,22 10,48 10,04 11,26* 10,95 10,70 11,58* 11,36* 10,85 10,96 11,24 11,33* 12,10* 11,32* 11,68* 11,74* 11,11 10,89 11,30 11,33* 11,38* 11,14 11,87* 12,27* 11,39* 11,16 10,73 11,71* 11,51* 11,44* 11,79* 11,46 11,30

I

II

* Prinos vei od

proseka standarda Yiels higher than mean values of checks Checks for HS-1: NS732 x NS15 NS732 x NS61 for HS-2: NS27 x NS15 NS27 x NS61

Standardi za HS-1: NS732 x NS15 NS732 x NS61 za HS-2: NS27 x NS15 NS27 x NS61

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komercijalnim performansama, sto podrazumeva i razlike u frekvenciji pozeljnih alela izmeu roditelja. Verovatno da respektujuu ulogu u objasnjenju navedenih rezultata igra i geneticka struktura pocetne populacije (NSA15). Rekombinanti (S1 familije) izmeu pretezno ,,starijih" Lancaster izvora i juznoamerickih tvrdunaca, donori su pozeljnih alela za prinos zrna, komplementarni inbred linijama novijih ciklusa selekcija.

izmeu ova dva tipa potomstava. Bekavac i sar. (2007) su ispitivali varijabilnost i meuzavisnost veeg broja agronomski znacajnih svojstava kod dve sinteticke populacije kukuruza, koristei S1 i HS (inbred-tester) potomstva. Utvrena je relativno visoka heritabilnost (u sirem smislu) za veinu proucavanih svojstava, ali i niska geneticka korelacija za prinos zrna izmeu S1 i HS potomstava.

Tabela 2. Geneticka korelacija (rg) i heritabilnost (h2) za prinos zrna, HS potomstava NSA15 populacije kukuruza Table 2. Genetic correlation (rg) and heritability (h2) for grain yield of HS progeny groups of NSA15 maize population HS-1 Geneticka varijansa (Vg) Genetic variance (Vg) Heritabilnost (h2) ± Se Heritability (h2) ± Se Koeficijent varijacije (CVg) Coefficient of variation (CVg) Geneticka korelacija HS-1:HS-2 (rg) ± Se Genetic correlation HS-1:HS-2 (rg) ± Se HS-1= S1 x NS732 HS-2= S1 x NS27 Se= standardna greska/standard error Maskirajui efekat dominantnih alela posebno moze biti izrazen kod korelacione analize (Smith 1986). Razlicita geneticka struktura pocetne populacije (uze ili sire geneticke osnove) primenjeni metod selekcije, efektivna velicina populacije, geneticki drift, kao i drugi faktori koji dovode do promene frekvencije alela, mogu drasticno da menjaju geneticke korelacije izmeu svojstava (Bohren et al. 1966, Falconer 1981). Problem se dodatno komplikuje kod korelacija izmeu razlicitih tipova familija (S1, S2, HS, FS). Ivanovi et al. (1987) su utvrdili jace korelacione veze za prinos zrna kukuruza izmeu familija u okviru populacije (S1, S2, FS) u odnosu na HS potomstva (inbred-tester), korelirana sa navedenim tipovima porodica. Ovaj zakljucak odnosi se na ispitivane populacije, uze i sire geneticke osnove. Ovakve korelacione odnose autori objasnjavaju maskirajuim efektom inbredtestera (HS porodica). Korelirajui prinos zrna izmeu S1 i HS potomstava (u okviru populacije), Vancetovi i sar. (2001) konstatuju da korelaciona povezanost zavisi od populacije (varira od 0,359 do 0,663), te predlazu naizmenicnu ciklicnu selekciju (S1, HS), ali samo kod kompozita sa visokom korelacijom za prinos zrna 0,105 0,519±0,228 3,000 0,309±0,253 HS-2 0,060 0,377±0,231 2,183

Dobijeni rezultati u ovom radu za heritabilnost prinosa zrna (h2=0,519 za HS-1 i h2=0,377 za HS-2) i geneticku korelaciju izmeu dve grupe HS potomaka (rg=0,309; Tab. 2) kreu se u granicama vrednosti veine rezultata koje navode Hallauer & Miranda (1988) i Srekov et al. (2007). Signifikantna vrednost heritabilnosti utvrena je samo za HS-1 potomstva, dok je kod HS-2 grupe heritabilnost nepouzdana (0,377<(2x0,231)). Relativno visoke standardne greske za heritabilnost i korelaciju posledica su svakako efekta drifta, koji uglavnom uzrokuju rezultati II seta kod HS-2 potomstava (vea koncentracija rodnijih genotipova u odnosu na I set). Homogenost zivotne sredine je manje verovatan uzrok, obzirom da je bilo 7 lokaliteta u dve godine ispitivanja (4+3), koji u velikoj meri mogu da izbalansiraju efekat sredine. Zakljucak U radu je proucavan prinos zrna dve grupe HS potomstava (S1 x inbred-tester) sinteticke populacije kukuruza uze geneticke osnove NSA15. Od ukupno 40 testiranih familija, 11 pripada grupi genotipova rodnijih od proseka odgovarajuih standarda u ukrstanju sa oba testera. Zbog

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Bohren B B, Hill W G, Robertson A (1966): Some observations on asymmetrical correlation responses. Genet. Res. 7: 44-57 Cochran W G, Cox M G (1957): Experimental designs. 2nd ed. John Wiley and Sons, Inc., New York Falconer D S (1981): Introduction to quantitative genetics. 2nd ed. Longman, New York, USA Ivanovi M, Stojni O, Greder R R (1987): Correlations among grain yields of full-sib, half-sib, and selfed progenies in broad and narrow base populations of maize (Zea mays L.). Genet. 19: 37-45 Hallauer A R, Miranda J B (1988): Quantitative genetics in maize breeding. 2nd ed. Iowa State Univ. Press. Ames Smith O S (1986): Covariance between line per se and testcross performance. Crop Sci. 26: 540-543 Srekov Z, Boanski J, Ivanovi M (2007): Genetic and phenotypic correlations between oil content and morphological traits in high oil maize population NSU. Genet. 39: 103-112 Vancetovi J, Rosulj M, Paji Z (2001): Geneticke korelacije za vaznija agronomska svojstva izmeu S1 i HS familija u dva kompozita kukuruza (Zea mays L.). Sel. Semen. 8: 57-61

neizbalansirane distribucije genotipova izmeu setova, vrednost geneticke korelacije za prinos zrna izmeu dve grupe HS potomstava je bila nesignifikantna, optereena visokom standardnom greskom (rg=0,309±0,253). Dobijeni rezultati u ovim istrazivanjima pokazuju da pocetna populacija (geneticki nesrodna sa drugim heteroticnim grupama) moze biti donor pozeljnih alela, komplementarnih, pre svega ID i BSSS genetickoj osnovi. Literatura

Bekavac G, Purar B, Stojakovi M, Jockovi , Ivanovi M, Nastasi A (2007): Varijabilnost i meuzavisnost svojstava u sintetickim populacijama kukuruza. Zbornik radova Instituta za ratarstvo i povrtarstvo, Novi Sad 43: 103-113 Bradshaw J E (1983): Estimating the predicted response to S1 family selection. Hered. 51: 415-418

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Genetic Variability and Correlation Analysis for Grain Yield of a Narrow Base Synthetic Maize Population NSA15

Dusan Stanisavljevi · Sanja Treski · Bojan Mitrovi · Aleksandra Nastasi · Goran Bekavac · Mile Ivanovi Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia

Summary: S1 progenies of a narrow base maize population NSA15 unrelated to other heterotic sources, derived from a cross of NS15 x NS61 inbred lines, were tested simultaneously with two unrelated inbred-testers: NS732 (ID genetic background) and NS27 (BSSS background). Forty pairs of each HS progenies were made in such a manner that every pair has female parent (S1 line) in common. The families were separately randomised and planted in two trials, using incomplete block design, in two sets each with two replications per set and 20 HS families within a set. In each set, four hybrid checks, which represent a combination of tester lines and component lines of the populations, were planted. The trials were carried out during 2008 (at four locations) and 2009 (at three locations). The standard crop management practices were applied. Higher average yield in most combinations of S1 families x tester were obtained with NS27 line. Approximately 50% of families tested with both testers achieved grain yield higher than average value of the corresponding checks. Heritability for grain yield of HS-1 progeny group (NS732) was significant (h2=0.519±0.228), whereas HS-2 progeny group (NS27) had lower and insignificant heritability (h2=0.337±0.231). At the same time, genetic correlation between two groups of HS progenies for grain yield was also insignificant (rg=0.309±0.253). High values of standard errors for heritability and genetic correlation were mainly caused by drift due to random concentration of more productive genotypes in the set II of the HS-2 trial. The results for grain yield indicated that NS27 inbred-tester is of a greater value for further breeding programme with the population NSA15. Key words: genetic correlations, half-sib (HS), heritability, maize, tester, Zea mays L.

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Correlations among Some Characteristics in Field Pea

473 Genetics and Breeding / Genetika i oplemenjivanje Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 473-478 original research article / originalni naucni rad

Correlations among Some Characteristics in Field Pea

Valentin Kosev Ivan Pachev

received / primljeno: 14.04.2010. accepted / prihvaeno: 14.05.2010 © 2010 IFVC

Summary: Pea grain yield is a quantitative character affected by many genetic and environmental factors. The trial was carried out during the period of 2007-2009 in the Research Institute of Forage Crops, Pleven, Bulgaria, including seven spring field pea lines. The line L-12AB had the greatest average values of both plant height (116.90 cm) and first pod height (56.12 cm). The largest 1000 seed mass was in the line L-470-16 (273.33 g). The winter cultivars had the longest growing season (239 days). The highest seed weight per plant was in the line L-12AC (8.54 g), while the highest seed yield was in the line L-12AB (2970 kg ha-1). Seed weight per plant was in the highest positive correlation with both number of pods per plant (r = 0.908) and number of fertile nodes per plant (r = 0.901). The highest positive correlation were between plant height and growing season (r = 0.990). Key words: correlations, grain yield, grain yield components, pea, Pisum sativum

Introduction Field pea (Pisum sativum L.) is one of the most important legume crops with grain rich in crude protein and mineral elements for the animal feed industry. It is used as an alternative source of crude protein to soybean meal. Its quality chemical contents and wide ecological stability and adaptability are responsible for its wide distribution and make it an irreplaceable source of protein (Acikgoz et al. 2009). Pea breeding is based on yield and yield components. Pea grain yield is a quantitative character affected by many genetic and environmental factors such as temperature, rainfall, soil type and moisture that represent factors important for a proper selection of parental lines (Angelova 1995, Filipov 1997, Filipov 1998, Mehandzhiev et al. 2006, Turk et al. 2008, Aydin et al. 2009). In the contemporary breeding programs, genotypes with wide genetic base and great diversity of the phenological, morphological and economic characters are used. The major achievements of a breeder rely upon the appropriate use of genetic resources (Nisar et al. 2008). The aim of our study was to determine the relationship among grain yield components in

V. Kosev ) ( I. Pachev Research Institute of Forage Crops, Gen.Vladimir Vazov Street 89, 5800 Pleven, Bulgaria e-mail: [email protected]

spring field pea lines using correlation coefficients and variability. Materials and Methods The trial was carried out during the period of 2007-2009 in the Research Institute of Forage Crops, Pleven, Bulgaria, situated in the central part of the Danube hilly plain. The trial included seven spring field pea lines, namely L-470-16, L-470-18, L-470-21, L-470- 22, F-R1, F-R2, F-R3, developed from the institute working collection, as well as two winter field pea lines, L-12AB and L-12AC that served as controls. The lines were sown in October and March, at a row spacing of 70 cm and a distance within the row of 5 cm. The following phenological phases and characters were studied: growing period (day), plant height (cm), first pod height (cm), number of pods per plant, number of seeds per plant, number of fertile nodes per plant, number of seeds per pod, seed weight per plant (g), number of branches per plant, 1000 seed mass (g) and seed yield (kg ha-1). In the technological maturity of each line, 30 plants per line were used for the analysis. Correlation coefficients and variability (VC, %) were determined for all studied traits (Dimova & Marinkov 1999). The variation was considered low, medium or high at the VC values of 10%, 10-20% and over 20%, respectively. All experimental data were processed statistically by the computer software MS Excel (Microsoft Office 2002) for Windows XP.

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Results and Discussion The weather conditions during the trial had a strong impact on the productivity of the pea lines (Acikgoz et al. 2009). The years of 2007 and 2009 years may be considered as dry, while the year of 2008 was favourable for the pea production (Fig. 1). The rainfall distribution during the pea growing season was relatively uniform in 2008 and irregular in other two trial years. Similarly, the years of 2007 and 2009 may be regarded as having warmer growing seasons in comparison with the year of 2008 that was colder. The line L-12AB had the greatest average values of both plant height (116.90 cm) and first pod height (56.12 cm), while the line F-R2 had the smallest average values of both plant height (31.10 cm) and first pod height (18.83 cm) (Tab. 1). The line L-12AB also had the greatest average values of both seed number per plant (60.17) and pod number per plant (15.87). The line F-R1 had the smallest average value of seed number per pod (14.03), while the line F-R3 had the smallest average value of pod number per pod (5.60).

The average values of number of branches per plant ranged between 0.37 in L-470-21 and 0.83 in F-R1, while the average values of seed number per pod varied from 2.78 in L-470-22 to 4.18 in L-470-18 and L-470-21. The line L-12AC had the greatest average value of fertile nodes per plant (10.72). The largest 1000 seed mass was in the line L-470-16 (273.33 g). The winter cultivars had the longest growing season, 239 days in both lines. The highest seed weight per plant was in the line L-12AC (8.54 g), while the highest seed yield was in the line L-12AB (2,970 kg ha-1). The influence of specific year on all examined quantitative characteristics was obvious (Tab. 2). It is also noteworthy that the winter lines had different distribution of the variation of all examined quantitative characteristics in comparison to the spring lines. Basically, nearly all of them had the smallest variability in the third trial year (2009). Among the most variable characteristics were seed number per plant and seed weight per plant. The presence of correlations is important because they are important criterion which could

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100 90

Temperature, C

100 80

80 70 60 50 40 30 20

60

40 20

10 0

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-10

Figure 1. Weather conditions during the trial with field pea lines Slika 1. Vremenske prilike tokom trajanja ogleda sa linijama stocnog graska

September, October November December January February March April May June July August September, October November December January February March April May June July August September, October November December January February March April May June July August September,

2006

2007 Years

2008

2009

rainfall sum, mm

monthly mean air temperature, ºC

humidity, %

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Rainfall sum, mm, Humiditi,%

L-470- 22 Spring Prolee Afila 36.33 a 19.93 a 23.90 a 0.44 ab 6.63 a 2.78 a 4.41 a 4.97 a 210.72 c 87 a 1422 a 0.83 c 8.87 ab 3.15 a 5.39 a 6.80 abc 208.19 bc 89 a 2954 b 14.03 a 19.20 a 18.83 a 21.33 a 0.44 ab 6.07 a 3.26 a 4.04 a 4.73 a 222.45 c 87 a 2042 ab 37.43 a 31.10 a

F-R1 Spring Prolee Normal Obican 36.67 a 22.56 a 18.33 a 0.44 ab 5.60 a 3.28 a 3.77 a 4.20 a 222.97 c 87 a 2151 ab

Correlations among Some Characteristics in Field Pea

F-R2 Spring Prolee Normal Obican

F-R3 Spring Prolee Normal Obican

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 473-478

Table 1. Morphological traits and quantitative characteristics of the forage pea lines in 2007-2009 Tabela 1. Morfoloske odlike i kvantitativne osobine linija krmnog graska 2007-2009. L-12AB L-12AC L-470-16 L-470-18 L-470-21 Sowing season Winter Winter Spring Spring Spring Vreme setve Jesen Jesen Prolee Prolee Prolee Leaf type Normal Normal Afila Afila Afila Tip lista Obican Obican Plant height (cm) 116.90 b 104.07 b 39.57 a 39.97 a 43.30 a Visina biljke First pod height (cm) 56.12 c 44.08 b 20.69 a 24.14 a 26.54 a Visina prve mahune Seed number per plant 60.17 b 60.13 b 22.20 a 24.73 a 14.21 a Broj semena po biljci Number of branches per plant 0.73 bc 0.70 abc 0.43 ab 0.59 abc 0.37 a Broj grana po biljci Pod number per plant 15.87 c 14.57 bc 5.83 a 5.77 a 6.30 a Broj mahuna po biljci Seed number per pod 3.94 a 3.89 a 3.93 a 4.18 a 4.18 a Broj semena po mahuni Fertile nodes per plant 10.00 b 10.72 b 3.66 a 3.43 a 4.19 a Broj rodnih kolenaca po biljci Seed weight per plant (g) 7.96 bc 8.54 c 5.73 abc 5.53 ab 5.93 abc Masa semena po biljci 1000 seeds mass (g) 125.93 a 137.23 ab 273.33 c 220.58 c 227.54 c Masa hiljadu semena Growing season (days) 239 b 239 b 89 a 89 a 88 a Duzina vegetacije (dani) -1 Seed yield (kg ha ) 2970 b 2636 ab 2670 ab 2719 ab 2636 ab Prinos semena Multiple Range test: a, b, c - significant at P < 0.05 Visestruki test intervala: a, b, c ­ znacajno pri P < 0,05

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Table 2. Variability (VC %) of the quantitative characteristics in field pea lines Tabela 2. Varijabilnost (%) kvantitativnih osobina linija stocnog graska First pod height (cm) Visina prve mahune Seeds number per plant Broj semena po biljci Pod number per plant Broj mahuna po biljci Seed number per pod Broj semena po mahuni Seeds weight per plant (g) Masa semena po biljci

Years Godine

Plants height (cm) Visina biljaka

L-12AB

L-12AC

L-470-16

L-470-18

L-470-21

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L-470- 22

F-R1

Kosev V & Pachev I

F-R2

F-R3

2007 2008 2009 2007 2008 2009 2007 2008 2009 2007 2008 2009 2007 2008 2009 2007 2008 2009 2007 2008 2009 2007 2008 2009 2007 2008 2009

25.35 13.63 3.48 24.97 22.03 11.54 18.30 16.04 24.28 23.12 15.59 19.15 10.38 19.06 11.29 14,28 11.68 7.62 11.62 12.83 11.74 19.95 9.38 15.97 17.24 19.80 10.28

12.11 21.21 3.83 12.83 23.50 12.82 37.32 50.74 37.32 25.67 36.60 25.67 10.97 34.06 10.97 10.26 34.11 10.25 11.86 41.74 11.85 14.64 22.38 14.63 13.30 19.52 13.29

86.47 58.60 4.52 40.56 56.08 20.65 22.28 62.70 33.78 60.20 22.24 24.87 30.00 67.66 29.41 28.49 34.21 28.49 64.09 69.14 35.76 51.84 54.81 38.04 78.50 39.38 40.43

73.90 53.52 5.24 37.15 44.18 19.91 31.86 42.80 27.17 32.49 34.09 24.94 22.45 57.36 24.18 35,13 35.72 26.96 36.57 30.79 34.75 42.61 44.72 37.42 50.37 41.05 35.79

28.70 31.42 3.05 23.89 17.44 10.35 19.73 38.25 35.04 33.95 40.19 16.45 17.54 29.80 15.79 25.08 9.71 25.08 58.33 38.27 11.05 46.74 26.82 17.77 63.63 20.00 15.09

Fertile nodes per plant Broj rodnih kolenaca po biljci 60.52 41.23 16.72 32.10 46.35 19.91 35.13 36.00 27.17 37.15 34.31 24.29 21.51 47.53 24.18 29,87 28.05 26.29 32.81 28.83 34.75 45.14 36.44 37.42 40.25 39.83 35.79 76.20 63.70 6.49 55.42 62.85 23.31 14.14 31.22 32.34 68.72 24.99 19.32 30.58 69.19 28.61 18,34 45.81 27.27 49.38 43.12 37.07 48.76 54.57 41.35 73.35 40.85 39.09

Correlations among Some Characteristics in Field Pea

477

make breeding progress more efficient (Sardana et al. 2007, Togay et al. 2008, Kalapchieva & Angelova 2009). All examined quantitative characteristics were in mutually positive correlations (Tab. 3). Seed weight per plant was in the highest positive correlation with both number of pods per plant (r = 0.908) and number of fertile nodes per plant (r = 0.901). The highest positive correlations were between plant height and growing season (r = 0.990), between number of pods per plant and number of fertile nodes per plant (r = 0.986) and between growing season and seed number per plant and growing season and number of fertile nodes per plant (both r = 0.978). In their studies, Abnasan et al. (1987), Balashova (1989) and Raudseping et al. (1996) reported a high positive correlation between seed weight per plant and number of pods and number of seeds per plant.

Conclusions The obtained results may provide a field pea breeder with valuable data for the better understanding of seed yield components, their variabi lity and their mutual relationship. This knowledge may be essential for accelerating the success and increasing the breeding benefit.

Table 3. Correlation coefficients (r) among the quantitative characteristics of the lines with normal leaf type Tabela 3. Koeficijenti korelacije izmeu kvantitativnih osobina linija sa obicnim tipom lista Number of branches per plant Broj grana po biljci Fertile nodes per plant Broj rodnih kolenaca po biljci 0.901 0.978* 0.859 Seed number per pod Broj semena po mahuni Seed number per plant Broj semena po biljci Seeds weight per plant Masa semena po biljci Pod number per plant Broj mahuna po biljci 0.226 0.217 0.967* First pod height Visina prve mahune 0.468 0.888 0.905* 0.455* 0.880* 0.783* 0.928 0.499 0.707 0.022 0.646 0.741 0.562 0.917 0.316*

First pod height Visina prve mahune Number of branches per plant Broj grana po biljci Seed number per plant Broj semena po biljci Pod number per plant Broj mahuna po biljci Seed number per pod Broj semena po mahuni Fertile nodes per plant Broj rodnih kolenaca po biljci Seeds weight per plant Masa semena po biljci Growing season Vegetacioni period *P<0.05

0.551* 0.962 0.964* 0.396* 0.959* 0.862* 0.990

Plants height Visina biljke 0.968

0.930* 0.986*

0.786* 0.908* 0.417* 0.978 0.334

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Kalapchieva S, Angelova S (2009): Morfologichna harakteristika na obraztsi gradinski grah (Pisum sativum L.). Proc. Mezhdunarodna nauchna konferentsiya Rastitelniyat genofond ­ osnova na savremennoto zemezelie, I, 175-178 Mehandzhiev A, Mihov M, Noveva S, Rodeva R, Kosturkova G (2006): Some results from the investigation on genetic improvement of pea (Pisum sativum L). Field Crop. Studies 3: 397-403 Nisar M, Ghafoor A, Ahmad H, Khan M R, Qureshi A S, Ali H, Islam M (2008): Evalution of genetic diversity of pea germplasm through phenotypic trait analysis. Pakistan J. Bot. 40: 2081-2086 Raudseping M, Puhm M (1996): Correlation between yield, morphological and biochemical characteristics of the garden pea and its importance for breeding work. Trans. Estonian Academic Agric. Soc. 1: 18-21 Sardana S, Mahajan R K, Gautam N K, Ram B (2007): Genetic variability in pea (Pisum sativum L.). Sabrao J. Breed. Genet. 39: 31-41 Togay N, Togay Y, Yildirim B, Dogan Y (2008): Relationships yield and some yield components in pea (Pisum sativum ssp. arvense L.) genotypes by correlation and path analysis. African J. Biotechnol. 7: 4285-4287 Turk M, Çelyk N, Bayram G, Budalki E (2008): Relationships between seed yield and yield components in narbon bean (Vicia narbonensis L.) by path analysis. Bangladesh J. Bot. 37: 27-32

References

Abnasan N T, Garin C S, Tandang L L (1987): Varietal evaluation and correlation studies in fifteen lines of garden pea, Philippine J. Crop Sci. 1: 11 Acikgoz E, Ustun A, Gul I, Anlarsal E, Tekeli A S, Nizam I, Avcýoglu R, Geren H, Cakmakci S, Aydinoglu B, Yucel C, Avci M, Acar Z, Ayan I, Uzun A, Bilgili U, Sincik M, Yavuz M (2009): Genotype ×environment interaction and stability analysis for dry matter and seed yield in field pea (Pisum sativum L.). Spanish J. Agric. Res. 7: 96-106 Angelova S (1995): Biologichna i stopanska harakteristika na nashi i introdutsirani sortove grah. Rastenievadni Nauki 1-2: 149-151 Aydin Ü, Cesurol B, Yava (2009): Determining relationships among agronomic characteristics and seed yield in pea (Pisum sativum L.). J. Plant Environ. Sci. 1: 1-4 Balashova N (1989): Selekciâ i semenovodstvo ovonyh bobovyh kul'tur. Stiinca, Kisinev Dimova D, Marinkov E (1999): Opitno delo s biometriya. Zemizdat Filipov H (1997): Otglezhdane na zimen furazhen grah pri zasushlivi usloviya i poluchavane na visoki dobivi. Rastenievadni Nauki 9-10: 49-54 Filipov H (1998): Polzi ot otglezhdaneto na zimniya furazhen grah pri zasushlivi usloviya, Vliyanie na azotniya rezhim na pochvata. Rastenievadni Nauki 11: 34-39

Korelacije izmeu nekih osobina stocnog graska

Valentin Kosev Ivan Pacev

Naucni institut za krmno bilje, Gen.Vladimir Vazov Street 89, 5800 Pleven, Bugarska Izvod: Prinos zrna graska je kvantitativna osobina na koju uticu brojni geneticki cinioci i cinioci spoljasnje sredine. Ogled je izveden tokom perioda 2007-2009. u Naucnom institutu za krmno bilje u Plevenu, Bugarska, ukljucivsi sedam linija jarog i dve linije ozimog stocnog graska. Linija L-12AB imala je najvee prosecne vrednosti visine biljke (116.90 cm) i visine prve mahune (56.12 cm). Najvea prosecna masa hiljadu semena bila je kod linije L-470-16 (273.33 g). Ozime sorte imale su najduzu vegetaciju (239 dana). Najvea masa semena po biljci bila je kod linije L-12AC (8.54 g), dok je najvei prinos semena bio kod linije L-12AB (2970 kg ha-1). Masa semena po biljci bila je u najveoj pozitivnoj korelaciji sa brojem mahuna po biljci (r = 0.908) i brojem rodnih kolenaca po biljci (r = 0.901). Najvea pozitivna korelacija bila je izmeu duzine vegetacije i visine biljke (r = 0.990). Kljucne reci: grasak, komponente prinosa zrna, korelacije, Pisum sativum, prinos zrna

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479 Variability and Similarities Among Lotus corniculatus L. Accessions and Breeding / Genetika i oplemenjivanje Genetics Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 479-484 original research article / originalni naucni rad

Study of Variability and Similarities Among Lotus corniculatus L. Accessions

Daniela Knotová Jan Pelikán Tomás Vymyslický Helena Hutyrová

received / primljeno: 15.04.2010. accepted / prihvaeno: 04.05.2010. © 2010 IFVC

Summary: In an individual planting 25 birdsfoot trefoil (Lotus corniculatus L.) accessions constituting the Czech collection were assessed. Fourteen accessions were varieties from the world collection and eleven accessions were wild forms obtained during the collecting expeditions in the Czech Republic and abroad. Morphological, yield and qualitative traits were measured on ten plants of each accession. A total of 34 traits were evaluated. From these values point and interval estimates of means and variation coefficients were produced for each accession as a measure of variability of each particular trait within and among accessions. Point estimates of mean values of accessions formed a data matrix which was then processed by cluster analysis. Accessions with the highest similarity were determined based on these results. Key words: birdsfoot trefoil, genetic resources, Lotus corniculatus, similarity, variability

Introduction Birdsfoot trefoil (Lotus corniculatus L.) is a species covering a wide area of Europe, western Asia and northern Africa. In other regions its occurrence is not so widespread (Rak 2007). It grows on warm and moist sites such as meadows, pastures, stream banks and grass banks. As a forage crop it is valued for its high protein content. It is most often grown in mixtures, only rarely as monoculture, and it increases the nutritional value of forage and hay (Chrtková 1995). Lotus corniculatus L. is a tetraploid, even though it was sometimes characterized as a diploid (Grant & Small 1996). The surveys show that birdsfoot trefoil is one of the most widespread species occurring in natural pastures all the year. It is well adapted to a wide range of altitudes, pH and precipitation (Sardaro et al. 2008). Steiner and Santos (2001) pointed out that geographical distance is not related to the genetic distance. The similarity of genotypes is associated with the similarity of sites, even though they are geographically very distant. Study of similarities among accessions in genetic resources collections is important for preventing duplicates in the stored materials of the genetic resources in the gene banks. The collecD. Knotová ) ( · J. Pelikán · H. Hutyrová Agricultural Research, Ltd., Zahradní 1, 664 41, Troubsko, Czech Republic e-mail: knotova@vup[email protected] T. Vymyslický Research Institute for Fodder Crops, Ltd., Troubsko, Czech Republic

tions of important agricultural crops are usually quite extensive and there are often duplicates in them (Dotlacil 2005). Therefore, working with these collections can be very difficult for users. Basic studies of plant genetic resources covered assessment of variability of traits and characters among particular populations and genotypes (Brindza 1998). The description and evaluation of morphological and yield traits of wild red clover populations and their comparison with those of registered varieties were made by Drobná (2005). The practical application of statistical methods in diversity studies was shown on chick pea genetic resources by Záková & Benková (2003). Materials and Methods During 2008, a total of 25 accessions of birdsfoot trefoil (Lotus corniculatus) from the Czech collection of this species were assessed in an individual planting under field conditions. Fourteen accessions were varieties of the world collection and eleven accessions were wild forms obtained during the collecting expeditions in the Czech Republic and abroad. A brief characterization of wild forms is shown in table 1. On 10 plants of each accession the following 34

Acknowledgements: This work was conducted in the frame of the project "National Programme on Conservation and Utilization of Plant Genetic Resources and Agro-biodiversity", supported by the Ministry of Agriculture of the Czech Republic and in the frame of institutional research project number MSM 2629608001 financed by the Ministry of Education, Youth and Sports of the Czech Republic.

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traits were assessed: petiole width, stem length, stem thickness, number of internodes, plant height, root length, root collar thickness, number of stems, number of lateral branches, middle internode length, plant green matter yield, plant dry matter yield, number of inflorescences per plant, number of flowers per inflorescence, flower length, flower width, calyx length, pod length, pod width, number of seeds per pod, number of inflorescences per stem, terminal leaflet width, terminal leaflet length, leaf area, terminal leaflet area, seed yield. In the traits, such as nitrogen substances content, fat content, fibre content, ash content, digestible nitrogen substances content and sugar content, fibre digestibility and 1000seed weight only one value from the sample mixed from the biomass from 10 evaluated plants was acquired. The obtained values were used to receive point and interval estimates of the mean value as a measure of variability among accessions and also estimates of variation coefficients as a measure of intra-varietal variability. The matrix of mean values was processed by cluster analysis to determine similarities among accessions. Cluster analysis was performed in the software Statistica for Windows (STATSOFT, INC., 2003) for

all the accessions together. The complete linkage method was used for clustering and Euclidean distance as the measure of distance. Results and Discussion Some morphological parameters of this species in the stand were studied e.g. by Sareen & Dev (2003). Because of a large amount of the obtained data, it is impossible to give basic statistical characteristics of all the accessions. For this reason, table 2 gives only minimum and maximum values for mean values and variation coefficients and the names of the accessions. Still it is possible to draw some conclusions even from this overview. It is generally assumed that wild forms will have lower values for the means than the cultivated varieties and vice versa. The variety Bekesi, however, had the shortest stems and the smallest number of internodes and the Grassland Goldie variety had the narrowest flowers, shortest calyx and narrowest pods. Polom exhibited the smallest number of inflorescences per stem and the smallest number of seeds per pod. On the contrary, the wild accession denoted as TROU 151/96 had the longest and widest pods and also

Table 1. Basic characteristics of wild accessions Tabela 1. Osnovne osobine samoniklih populacija Origin Country of origin Locality Poreklo Zemlja porekla Lokalitet Czech Republic TROU 164/95 Horní Beckov Ceska Czech Republic TROU 151/96 Onsov Ceska Czech Republic ZN 1/00 Lukov Ceska Czech Republic POD 1/98 Podyjí national park Ceska Slovenia SVN 55/00 Zagarji Slovenija Czech Republic Omice TROU 8/01 Ceska Czech Republic TROU 144/95 Podmolí Ceska Czech Republic TROU 6/97 Havraníky Ceska Czech Republic TROU 28/96 Masovice Ceska ROH 63/00 SUM 382/94 Slovakia Slovacka Austria Austrija Roháce - Blatná valley Obertraun

Characteristic of locality Osobina lokaliteta Forest margin Obod sume Forest margin Obod sume Forest path Sumska staza Forest margin Obod sume Brook margin Obala potoka Leafy forest Listopadna suma Meadow Livada Dry heathland Susno vresiste Xerothermic meadow Kserotermna livada Mesophilous meadow with Agrostis Mezofilna livada sa rosuljom Parking lot, forest margin Parking, obod sume

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Table 2. Minimum and maximum values for mean values and variation coefficients for each accession Tabela 2. Najmanje i najvee vrednosti oko srednjih vrednosti i koeficijent varijacije populacija

the highest seed yield per plant. Another accession denoted as ROH 63/00 exhibited the longest root, the highest green matter yield, and the highest number of inflorescences per plant. From the viewpoint of variability, there was a similar situation within the accessions. A number of wild forms showing low variability in some of the traits were found. For example, a wild accession denoted as POD 1/98 showed high uniformity in flower length, number of inflorescences per stem, terminal leaflet width and length, leaf area and terminal leaflet area. On the contrary, high non-uniformity was exhibited by the Víglasský variety in the number of inflorescences per plant, number of flowers per inflorescence, terminal leaflet width, leaf area and terminal leaflet area. The lowest values of variation coefficients were found in the following traits ­ number of flowers per inflorescence, flower length, pod length, terminal leaflet width and terminal leaflet length. On the contrary, the highest variability was found in the traits such as petiole width, stem number and plant green matter yield. These findings are important to birdsfoot trefoil breeders. It was proved that even among wild forms there are interesting materials that could be utilized in plant breeding. It was also revealed that there are traits with low variability in this species which, however, need not be focused on in the selections. Attention should be given to traits with high variability. Cluster analysis of 28 world genotypes based on 18 morphological characteristics was reported e.g. by Steiner & Santos (2001). The matrix of mean values was used as the basis of cluster analysis and the results are shown in figure 1. The highest similarity among the accessions was found in a pair of wild accessions TROU 164/95 and TROU 151/96; high similarity was also exhibited by wild accessions POD 1/98 and SVN 55/01. In neither case were these accessions duplicates because they came from collections at different localities. High similarity was also between Táborák (CZE) and Grassland Goldie (NZL). These were not duplicates, either as they come from different countries. The materials were basically divided into 5 clusters. The first cluster contained wild accessions (TROU 164/95, TROU 151/96 and ZN 1/00) and also the above-mentioned varieties Táborák and Grassland Goldie. These accessions exhibited high values for the following traits - number of lateral branches, middle internode length and nitrogen substances content, fat content, ash content and digestible nitrogen substances content. The second cluster was composed only of wild accessions (POD 1/98, SVN 55/01

Var. coefficient max. Najvei koeficijent varijacije

37.34 (TROU 164/95)

33.42 (TROU 164/95)

45.77 (TROU 164/95)

Var. coefficient min. Najmanji koeficijent varijacije

9.11 (Skrzeszowiecka) 27.87 (ROH 63/00) 15.4 (TROU 144/95)

10.42 (TROU 28/96)

Mean value max. Najvea vrednost oko srednje vrednosti

13.83 (TROU 164/95)

22.1 (Malejovský)

8 (Bekesi, TROU 28/96, TROU 144/95)

Mean value min. Najmanja vrednost oko srednje vrednosti

1.25 (TROU 144/95)

0.31 (TROU 8/01)

Stem - number of internodes Broj clanaka stabla

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Characters Osobina

Petiole - width (mm) Sirina lisne drske Stem - length (cm) Duzina stabla Stem - thickness (mm) Debljina stabla

Plant - height (cm) Visina biljke Root - length (cm) Duzina korena Root collar - thickness (mm) Debljina korenskog vrata

5.85 (TROU 8/01)

5.5 (TROU 8/01)

23.92 (Bekesi)

10.81 (Szabolci)

48.80 (Upstart)

3.26 (Upstart)

1.22 (Puszta)

10.5 (ROH 63/00)

19.48 (Szabolci)

9.18 (Szabolci)

10.10 (Polom)

7.10 (Polom)

40.16 (TROU 6/97)

68.65 (ZN 1/00)

27.85 (ZN 1/00)

33.27 (Bull)

482

14.1 (Skrzeszowiecka) 6 (TROU 144/95) 1.51 (Szabolci) 56.0 (TROU 144/95) 9.0 (TROU 144/95) 234 (TROU 144/95) 3.9 (Víglasský) 10.64 (TROU 28/96) 3.38 (Grassland Goldie) 5.31 (Grassland Goldie) 2.11 (ROH 63/01) 1.91 (Grassland Goldie) 15.1 (Polom) 9.78 (Polom) 1.8 (TROU 151/96) 10.7 (TROU 28/96) 1.16 (TROU 28/96) 0.41 (TROU 28/96) 4.88 (TROU 28/96) 3.54 (Lotar) 1.32 (Lotar) 12.45 (TROU 151/96) 18.7 (Upstart) 10.4 (Lotar) 33.25 (ROH 63/00) 28.4 (Upstart) 3.2 (TROU 151/96) 3.25 (TROU 151/96) 7.76 (Leo) 4.2 (TROU 28/96) 7.14 (TROU 164/95) 6.98 (TROU 151/96) 12.53 (Skrzeszowiecka) 9.67 (POD 1/98) 10.9 (POD 1/98) 5.72 (POD 1/98) 12.2 (POD 1/98) 12.73 (POD 1/98) 10.58 (Bull) 11.58 (Skrzeszowiecka) 3.96 (Mirabel) 13.16 (SVN 55/00) 1.09 (POD 1/98) 5.3 (Upstart) 9.32 (ZN1/00) 635.1 (ROH 63/00) 14.45 (TROU 164/95) 47.0 (Leo) 16.07 (Polom) 274.1 (ROH 63/00) 19.93 (Lotar) 4.29 (Leo) 17.77 (Mirabel) 62.82 (POD 1/98) 65.65 (TROU 8/01) 61.38 (Bekesi) 51.62 (Víglasský) 25.50 (Víglasský) 16.72 (TROU 28/96) 40.32 (Grassland Goldie) 29.83 (Grassland Goldie) 19.16 (Szabolci) 32.14 (SVN 55/01) 38.41 (Szabolci) 69.2 (Táborák) 28.61 (Víglasský) 25.44 (TROU 151/96) 39.86 (Víglasský) 45.29 (Víglasský) 95.44 (Táborák) 14.67 (POD 1/98) 14.18 (TROU 8/01) 57.38 (TROU 151/96)

47.0 (Polom)

19.38 (Upstart)

71.69 (TROU 6/97)

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Stem - number on plant Broj stabala po biljci Stem - number of lateral branches Broj bocnih grana po biljci Stem - length of middle internode (cm) Duzina srednjeg clanka Yield of green matter - plant (g) Prinos zelene krme Yield of dry matter - plant (g) Prinos suve materije krme Number of inflorescence per plant Broj cvasti po biljci Inflorescence - number of flowers Broj cvetova po cvasti Flower - length (mm) Duzina cvasti Flower - width (mm) Sirina cvasti Calyx - length (mm) Sirina casice Pod - length (cm) Duzina mahune Pod width (mm) Sirina mahune Pod - number of seeds Broj semena po mahuni Number of inflorescence per stem Broj cvasti po stablu Leaflet terminal - width (mm) Sirina vrsne liske Leaflet terminal - length (mm) Duzina vrsne liske Leaf - area of compound leaf Povrsina slozenog lista Leaflet terminal - area (cm2) Povrsina vrsne liske Yield of seeds - plant (g) Prinos semena po biljci

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and TROU 8/01). The accessions in this cluster had high values for root length and 1000-seed weight. The third cluster contained the Bekesi (HUN) and the Skrzeszowiecka (POL) varieties and also the wild accession TROU 144/95. The accessions of this cluster excelled at digestibility. Another cluster contained the Víglasský (SVK), Lotar (CZE), Puszta (HUN) and Malejovský (CZE) varieties. These genotypes differed from the other accessions by having high values for such traits as terminal leaflet area, leaf area and stem thickness. The last cluster contained the remaining varieties of the collection and the wild accession SUM 382/94 from Austria. As for varieties, these were the Canadian varieties Mirabel, Upstart, Bull and Leo and also the Szabolci (HUN) and Polom (SVK) varieties. The accessions of this cluster excelled at such traits as green matter yield of plant, number of inflorescences per plant, terminal leaflet length and leaf area. The wild accession ROH 63/00 was quite independent. This accession had the highest values for the traits such as number of internodes, green matter yield of plant, dry matter yield of plant, number of inflorescences per plant and number of inflorescences per stem.

Conclusions Wild populations of crops may be a very valuable source of properties that might be utilized for pest control, improved plant persistence or maybe for delayed pod dehiscence in seed production. Large genetic variation was described in birdsfoot trefoil by Chrtková-Zertová (1973). Steiner & Poklemba (1994) reported that the present varieties were developed from a narrow genetic basis. It was shown that cluster analysis is suitable for evaluating plant genetic resources on the basis of morphological and yield characteristics. The accessions are divided into several clusters on the basis of high similarity. This enables to find duplicates in the accessions and the user can then choose the most suitable cluster, or possibly accessions exhibiting the highest similarity. These results can be further used in plant breeding programs, as it is possible to choose from genetically distant accessions.

Figure 1. Dendrogram of Lotus corniculatus L. accessions Slika 1. Dendrogram populacija zutog zvezdana

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Rak L (2007): Lotus corniculatus [Online]. Available at http:// botany.cz/cs/lotus-corniculatus (cited 1 April 2010, verified 14 April 2010). Sareen S, Dev I (2003): A preliminary study to explore potential of Lotus corniculatus L (Birdsfoot trefoil) in Palampur (Himachal Pradesh, India). Lotus Newsletter 33: 3-5 Sardaro M L S, Atallah M, Tavakol E, Russi L, Porceddu E (2008): Diversity for AFLP and SSR in Natural Populations of Lotus corniculatus L. from Italy. Crop Sci. 48: 10801089 Steiner J J, de los Santos G G (2001): Adaptive ecology of Lotus corniculatus L. genotypes: I. Plant Morphology and RAPD Marker Characterizations. Crop Sci. 41: 552-563 Steiner J J, Poklemba C J (1994): Lotus corniculatus classification by seed globulin polypeptides and relationship to accession pedigrees and geographic origin. Crop Sci. 34: 255-264 Záková M, Benková M (2003): Vyuzitie statistických metód v studiu diverzity kolekcie genetických zdrojov v príklade cícera baraníno. In: Hodnotenie genetických zdrojov rastlín, VÚRV Piesany, Piesany, Slovakia, 104-111

References

Brindza J (1998): Hodnotenie genofondu rastlín z pohadu teorie chaosu. Zborník: Hodnotenie genetických zdrojov rastlín, 16-20 Chrtková A (1995): Lotus L. In: Slavík B (ed.), Kvtena Ceské republiky 4. Academia, Praha, Czech Republic, 492-497 Chrtková-Zertová A (1973): A monographic study of Lotus corniculatus L. I. Central and Northern Europe. Rozpravy Ceskoslovenské Akademie Vd 83: 1-94 Dotlacil L (2005): Cíle a metody pro lepsí management a vyuzití kolekcí. In: Racionalizace managementu a vyuzívání genetických zdroj zemdlských plodin. Agritec Plant Research, s.r.o. Sumperk a VÚRV Praha-Ruzyn. Genetické zdroje 93: 4-12 Drobná J (2005): Hodnotenie divorastúcich populácií ateliny lúcnej (Trifolium pratense L.). In: Hodnotenie genetických zdrojov rastlín, VÚRV Piesany, Piesany, Slovakia, 154155 Grant W F, Small E (1996): The origin of Lotus corniculatus (Fabaceae): A synthesis of diverse evidence. Can. J. Bot. 74: 975-989

Studija varijabilnosti i slicnosti meu populacijama zutog zvezdana (Lotus corniculatus L.)

Daniela Knotova1 Jan Peljikan1 Tomas Vimisljicki2 Helena Hutirova1

2 1 Poljoprivredna istrazivanja, s o. o., Troubsko, Ceska Naucni institut za krmno bilje, s. o. o., Troubsko, Ceska

Izvod: U pojedinacnoj setvi je ispitivano 25 populacija zutog zvezdana (Lotus corniculatus L.) iz zbirke Republike Ceske. Cetrnaest populacija su bile sorte iz svetske zbirke, dok je jedanaest populacija bilo samoniklo i sakupljeno tokom ekspedicija u Republici Ceskoj i inostranstvu. Na deset biljaka svake populacije odreivane su morfoloske osobine, prinos i kvalitet. Ukupno je odreeno 34 osobina za svaku akcesiju, odreene su tacke i razmaci procene sredina i koeficijenti varijacije kao merilo varijabilnosti svake osobine unutar i izmeu populacija. Od tacaka procene srednjih vrednosti sacinjena je matrica podataka, koja je potom obraena klaster analizom. Na osnovu ovih vrednosti odreene su akcesije sa najveom slicnosti. Kljucne reci: geneticki resursi, Lotus corniculatus, slicnost, varijabilnost, zuti zvezdan

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www.nsseme.com/journal.html

Colchicine-Induced Variations in Water Yam

485 Genetics and Breeding / Genetika i oplemenjivanje Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 485-492 original research article / originalni naucni rad

Colchicine-Induced Variations in Survival Rate and Morphological Characteristics of Water Yam (Dioscorea alata)

Abiola T. Ajayi Adenubi I. Adesoye Robert Asiedu Alieu Sartie

received / primljeno : 17.04.2010. revised / preraeno : 21.05.2010. accepted / prihvaeno: 24.05.2010. © 2010 IFVC

Summary: The effects of 0.2% aqueous solution of colchicine on the survival rate and morphological features of five accessions of Dioscorea alata were investigated. Sprouting buds of two month old plants were treated with 0.2% colchicine and their performances were monitored until maturity. Survival of buds was lower in all the treated plants (ranging between 6.25% and 8.75%) compared to controls (between 13.25% and 15.25%). However, colchicine treated vines had higher survival rates when exposed to drought (13.8% - 24%) compared to controls (7.2% - 16.69%). Higher number of leaves, larger leaf width and fewer numbers of stomata were observed among the treated plants. The survival of buds between the treated plants and control plants was significantly different at P 0.05. The treated and non-treated plants were also significantly different at P 0.05 for leaf width in accessions TDa02/00246, TDa98/00116 and TDa99/00240, and for stomata number in accessions TDa02/00151 and TDa02/00246. Our results suggest that colchicine can be used to induce mutagenic changes in yam which may be of agronomic importance in the production of the crop. Key words: colchicine, Dioscorea alata, drought tolerance, morphological variations, polyploidy, tubers

Introduction Water yam (Dioscorea alata L.) is an important tuber crop and is a staple food for millions of people in tropical and sub-tropical countries (Hahn 1995). It was first cultivated in South-East Asia but is now grown in Africa, the Pacific Island, and the West Indies. Although it is not grown to the same extent as the Africa yams (D. rotundata and D. cayensis), it has the widest global distribution than any other cultivated yams (Mignouna 2003). In the United States it has become an invasive species in some southern states. In the Philippines it is known as "Ube" (or Ubi) and is used as an ingredient in many desserts. In India it is known as "ratalu" or violet yam or the "moraga surprise", while in Hawaii it is known as "Uhi". It was brought to Hawaii by the early Polynesian settlers and became a major crop in the 1800s when the tubers were sold to visiting ships as an easily stored food supply for their voyage (White 2003).

A. T. Ajayi · A. I. Adesoye ) ( University of Ibadan, Department of Botany and Microbiology, Ibadan, Nigeria [email protected] A. T. Ajayi · R. Asiedu · A. Sartie International Institute of Tropical Agriculture (IITA), Yam Breeding Unit, Ibadan, Nigeria

The plant has a long history of asexual propagation. Dioecy, irregular and erratic flowering, asynchrony of sexual seed development and apparent paucity in seed set has led to the species being regarded as sterile (Coursey 1967). However, with the development of artificial pollination under optimized cultural management practices (IITA 1996) and a better understanding of yam flowering biology, large volumes of viable D. alata seeds can now be produced. This has opened up the opportunity for conventional plant breeding in D. alata. The conventional breeding method which has been applied in D. alata is the hybridization method, which may take a very long period for scientists to achieve homozygous genotype. Unfortunately, not much improvement in D. alata has been achieved through conventional breeding methods. They are laborious and time consuming because yam anthers are so small and difficult to pollinate, and also to achieve homozygous genotype will take a very long period of time due to cross pollination. Therefore the use of chemical agents in inducing mutation which can lead to chromosome doubling or polyploidization with possible increase in yield, quality and adaptation (mutation breeding) is desirable (Dewey 1979, Castro et al. 2003, Burun & Emiroglu 2007).

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With the discovery of "colchicine technique" and oryzalin, for inducing polyploidy, breeders have seized upon this unconventional technique as means of plant improvement (Hancock 1997). Colchicine can be used to restore fertility in interspecific hybrids or induce polyploidy as genetic bridge for crossing between species of different ploidy levels (Mehetre et al. 2003). The colchicine technique is attractive for the following reasons: (1) it may enable the isolation of homozygous or near-homozygous genotypes from hybrids more rapidly than the conventional breeding, (2) it may generate new genetic variation by chromosome loss, rearrangement or gene mutation, thereby enlarging the breeder's germplasm base, and (3) it is a simple, inexpensive technique requiring no special equipment or expertise (Luckett 1989). Colchicine mutagenesis at inducing polyploidy has successfully been exploited in various plant species, such as barley (Gilbert & Patterson 1965), azalea (Pryor & Frazier 1968), phaleanopsis (Griesbach 1981), iris (Yabuya 1985), cotton (Luckett 1989), cranberry (Dermen & Henry 1994), cyclamen (Ishizaka & Uematsu 1994, Takamura & Miyajma 1996) and Brachiaria brizantha (Pinheiro et al. 2000), Sorghum bicolor (Ghaffari 2006), Cicer arietinum (Pundir 1983), Acacia species (Blakesley et al. 2002), Colccinia palmate and Lagenaria sphaerica (Nituli & Zobolo 2008), Nicotiana tabacum (Burun et al. 2007), Chamelaucium uncinatum (Yan 2001). It has also been used to overcome interspecific incompatibilities in some Gossipium species (Mehetre et al. 2003, Rauf et al. 2006), to restore fertility in interspecific hybrids of Manihot species (Nassar 2002), Xanthosoma species (Tambong et al. 1997) and Viola x Wittrockiana (Ajalin et al. 2002). Timing and concentration is also important when colchicine is being used to induce polyploidy in plant, since not all plants respond to colchicine in the same way. Colchicine has been used in different concentrations and timing to induce polyploidy. In chickpea, it was most effective at inducing autotetraploidy at 0.25% concentration at 4 h time duration (Pundir et al. 1982). The quantity -1 of 50 mg l of colchicine was effective at inducing polyploidy in Phalaenopsis (Griesbach 1981). Colchicine dropping treatment at a concentration -1 of 2.000 mg l for one day led to the highest rate (14%) of tetraploid formation in the interspecific hybrid between Dianthus caryophllus L. and D. japonicus Thumb. (Nimura et al. 2006). Treating petiole sheaths with 1.25 mM colchicine induces solid polyploids and cytochimeras in cocoyam tissues (Tambong et al. 1997). Tumor-like growths were produced at the nodes of cuttings of Tradescantia poludosa by complete immersion for 48

h in aqueous solution of colchicine (Mc Gowan & Bishop 1953). Fertility of sterile interspecific hybrids between cassava and wild Manihot species was restored by chromosome duplication following application of colchicine at 0.2% aquaeous solution for a period of 24 h and reached 95% of viable pollen in tetraploid types compared to 13% in the diploid forms (Nassar 2002). An enlargement of vegetative organs is a common feature of induced polyploids (Allard 1971). This phenotypic effect has been described in ryegrass (Myers 1939), sorghum (Franzke & Rose 1952), flax (Dirks et al. 1956), cotton (Luckett 1989, Rauf 2006), cassava (Nassar 2002). Studies comparing untreated and colchicine treated ryegrass genotypes, without the occurrence of chromosomal duplication, have shown significant differences in many traits, e.g. leaf area, flowering date and flower number (Hague & Jones 1987), leaf weight (Francis & Jones 1989); mesophyll cell size and chloroplast number (Francis et al. 1990, Hassan et al. 1991). These investigations suggest a mutagenic action independent of changes at ploidy levels. The objectives of this research were to determine the influence of colchicine on: (1) survival of buds of Dioscorea alata genotypes, (2) morphological characters of Dioscorea alata genotypes and (3) survival rate of Dioscorea alata vines exposed to drought. Materials and Methods Tuber collection. Tubers of five accessions of Dioscorea alata, (TDa02/00151, TDa02/00246, TDa98/01176, TDa98/01166 and Tda99/00240) were collected from the yam breeding unit at IITA and were cut into mini sets of about 100 g per set. The mini sets were air dried for 24 hours before planting to prevent decaying from the surface. Planting of sets. The air dried tuber sets were planted in the polyethylene bags of about 2000 cm3, filled with sandy-loam soil in the screen house. The sets were planted according to the arrangement of the polyethylene bags. One set was planted per bag per treatment per accession. Each accession had six treatments, (control, 0.1%, 0.15% 0.2%, 0.25% and 0.3%) of colchicine concentration. Each treatment contained ten plants and was replicated four times. The experimental design was complete randomized design. Colchicine treatments. When the plants were exactly two months old, they were trimmed to two nodes each. Colchicine was dissolved in sterile water in a ventilated weighing station to the

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above mentioned concentrations. Sprouting buds of each plant were plugged with cotton wool. The cotton plugs were held in place with parafilm. Each plugged bud was soaked with appropriate colchicine solution twice a day for 48 hours by the use of a micro-syringe. The control plants were soaked with distilled water. Each sprouting bud was treated four times. Two months after colchicine treatments, data were collected from the vines originated from the treated buds on the survival of buds, number of leaves, leaf length and leaf width.

Vine propagation. In the third month after colchicine treatment, the vines which grew from the treated buds were made into cuttings of two nodes each and transferred to the field for propagation where they were exposed to drought (dry season). Two vines were propagated per plant per colchicine treatment per accession, six colchicine treatments were involved per accession and the total number of accessions was five. The total number of vines transferred to the field was 2400. The experiment was laid out in a complete randomized design with four replications (Fig. 1 and 2). Two

Figure 1. Field plot where the vines exposed to drought were grown Slika 1. Parcela sa gajenim reznicama izlozenim susi

Figure 2. Exposure of vine cuttings derived from colchicine treated sprouted buds to drought Slika 2. Izlaganje reznice dobijene tretiranjem pupoljaka kolhicinom susnim uslovima

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Figure 3. Epidermal cells of control (a and b) and colchicine-treated (c and d) Dioscorea alata obtained from abaxial (a and c) and adaxial (b and d) leaf surfaces Slika 3. elije epidermisa kontrolne (a i b) i tretirane (c i d) varijante Dioscorea alata sa abaksijalne (a i c) i adaksijalne (b i d) povrsine lista

hundred cm3 of water was applied to each plant of both treated and control twice a day. Six months after vine propagation, data were taken on the percentage survival of vines and the stomata number and stomata index were taken from the leaves of the surviving plants. Because of the high death rate of plants on the field, data could only be compared between the control and the 0.2% colchicine treatment for all the accessions, since they were the only remaining survivors on the field. Stomata assessment. To obtain epidermal peels, two matured leaves were obtained from the top of both treated and controls of established plants. Rectangular cuttings were made from the two leaves by the use of scissors. The leaf cuttings were placed in petri-dishes and nitric acid was poured on the leaf cuttings to separate the adaxial part from the abaxial part. The petri-dishes were covered and placed in the sun for about 30 minutes. The transparent epidermal peels were later properly rinsed in water. The strips were stained in safranin, excess stain was rinsed off with water for easy viewing. Painting brush was used to guide the strips onto a clear glass slide in water. The glass slides were gently removed from water and a drop of glycerine was placed on each epidermal strip on each slide. Cover slip was placed over

each slide and the prepared slides were examined under a microscope at 100x magnification. The number of epidermal cells, stomata and stomata indices on two leaves of each treatment and in 40 randomly chosen microscopic fields (area 1 mm2) of each leaf peeling was recorded. Therefore, each figure in the text is a mean based on 80 microscopic fields. Statistical analysis. The results were analyzed using ANOVA and least significant difference (LSD), P 0.05 level of significance was used to compare the differences in means between the treatments. Results and Discussion Results were based on the comparison between only the control and 0.2% colchicine treatment, since they were the only surviving treatments on the field. Colchicine treatment reduced the survival of buds in all the accessions, which resulted in the failure of some plants to induce shoots at the treated nodes. In all five accessions, survival rates of the treated buds were lower in comparison to the control (Tab. 1). Number of leaves. The treated plants of D. alata had more leaves than the control in most of the

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Table 1. Number of survived buds (mean ± standard error and coefficient of variation) of the control and colchicine-treated Dioscorea alata Tabela 1. Broj prezivelih pupoljaka (srednja vrednost ± standardna greska i koeficijent varijacije) kontrole i kolhicinom tretirane Dioscorea alata

Accession Genotip Control Kontrola Treated Tretirane TDa 02/00151 13.25±2.25a 22.20 8.75 ±1.49b 34.37 TDa02/00246 14.75±1.93a 26.20 7.50 ±1.85b 49.00 TDa98/01176 15.00±1.08a 14.00 6.25 ±1.18b 37.00 TDa98/01166 15.25±1.50a 16.00 8.25 ±0.75b 18.00 TDa99/00240 13.75±1.65a 24.00 8.50 ±1.30b 29.00

Figures followed by similar alphabet in a column are not significantly different from each other Brojevi praeni istim slovom u koloni nisu meusobno znacajno razliciti Table 2. Number of leaves (mean ± standard error and coefficient of variation) of the control and colchicine-treated Dioscorea alata Tabela 2. Broj listova (srednja vrednost ± standardna greska i koeficijent varijacije) kontrole i kolhicinom tretirane Dioscorea alata

Accession Genotip Control Kontrola Treated Tretirane TDa 02/00151 31.70 ± 2.74a 17.37 34.42 ± 2.70a 15.69 TDa02/00246 27.25 ±1.14a 8.30 32.85±2.20a 13.36 TDa98/01176 22.90±2.50a 21.83 20.81±1.57a 15.00 TDa98/01166 22.35±1.56a 14.00 25.36±1.90a 13.60 TDa99/00240 14.65±0.46a 6.27 17.43±1.84a 21.20

Figures followed by similar alphabet in a column are not significantly different from each other Brojevi praeni istim slovom u koloni nisu meusobno znacajno razliciti Table 3. Leaf length (mean ± standard error and coefficient of variation) of the control and colchicine-treated Dioscorea alata Tabela 3. Duzina listova (srednja vrednost ± standardna greska i koeficijent varijacije) kontrole i kolhicinom tretirane Dioscorea alata

Accession Genotip Control Kontrola Treated Tretirane Difference Razlika TDa 02/00151 7.13 ± 1.01a 28.20 5.55 ± 1.21b 43.80 -12% TDa02/00246 6.77 ± 0.62b 18.30 8.49 ± 1.10a 26.00 11% TDa98/01176 6.76 ± 0.42a 12.60 6.36 ± 0.16a 5.00 -3% TDa98/01166 7.06 ± 0.68a 17.90 7.35 ± 0.80a 21.80 2% TDa99/00240 6.05 ± 0.73a 24.00 4.95 ± 1.17b 47.00 -10%

Figures followed by similar alphabet in a column are not significantly different from each other Brojevi praeni istim slovom u koloni nisu meusobno znacajno razliciti accessions except for TDa98/01176 where leaf number was fewer in the treated plants. There was a significant (P 0.05) treatment effect for number of leaves but the difference in mean number of leaves between control and the treated plants was not significant for all the accessions (Tab. 2). Leaf length and width. Accessions TDa02/00246 and TDa98/01166 increased in leaf length by 11% and 2% respectively, following colchicine treatment compared to their controls while others had reduced length following colchicine treatment. The difference in means of the treated and the control of TDa02/00246, TDa02/00151 and TDa99/00240 were all significant (P 0.05) while the difference in means between the control and the treated plants of accessions TDa98/01166 and TDa99/01176 were not significant. Moreover, all the treated D. alata had larger leaf width ranging from 0.09% to 13% increase in width. There was significant (P 0.05) colchicine effect on the leaf width for TDa02/00246, TDa98/01166 and TDa99/00240 but not for TDa02/00151 and TDa98/01176 (Tab. 3 and 4). Survival rate of vines on the field. The survival rate of vines of D. alata exposed to the drought condition in the field was low in all the accessions. Within an accession, survival rate was higher in the treated plants compared to the control across all the

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Table 4. Leaf width (mean ± standard error and coefficient of variation) of the control and colchicine-treated Dioscorea alata Tabela 4. Sirina lista (srednja vrednost ± standardna greska i koeficijent varijacije) kontrole i kolhicinom tretirane Dioscorea alata

Accession Genotip Control Kontrola Treated Tretirane Difference Razlika TDa 02/00151 5.30 ±0.55a 20.80 5.46 ±0.69a 25.40 1.5% TDa02/00246 4.91±0.45b 18.00 6.37±0.69a 21.70 13% TDa98/01176 5.34±0.62a 23.60 5.35±0.75a 28.00 0.09% TDa98/01166 5.55±0.46b 16.40 6.37±0.49a 15.50 6.9% TDa99/00240 4.41±0.49b 22.00 5.08±0.39a 15.00 7%

Figures followed by similar alphabet in a column are not significantly different from each other Brojevi praeni istim slovom u koloni nisu meusobno znacajno razliciti Table 5. Percentage of survival of vines of the control and colchicine-treated Dioscorea alata Tabela 5. Postotak prezivelih reznica kontrole i kolhicinom tretirane Dioscorea alata

Accession Genotip Control Kontrola Treated Tretirane TDa 02/00151 7.2 13.8 TDa02/00246 16.69 24 TDa98/01176 14.3 16.7 TDa98/01166 0 0 TDa99/00240 10.3 13.8

Table 6. Number of stomata (mean ± standard error and coefficient of variation) of the control and colchicine-treated Dioscorea alata Tabela 6. Broj stoma (srednja vrednost ± standardna greska i koeficijent varijacije) kontrole i kolhicinom tretirane Dioscorea alata

Accession Genotip Control Kontrola Treated Tretirane Difference Razlika TDa 02/00151 36.25 ±1.19a 19.9 30.27 ±1.34b 10.37 -8.90% TDa02/00246 36.77±0.96a 11.20 22.79±1.17b 8.90 -23.5% TDa98/01176 30.55±0.97a 9.96 29.97±1.11a 13.90 -0.96% TDa98/01166 -- -- -- TDa99/00240 29.71±1.18a 10.8 28.13±0.55a 12.6 -2.7%

Figures followed by similar alphabet in a column are not significantly different from each other Brojevi praeni istim slovom u koloni nisu meusobno znacajno razliciti genotypes. Genotype TDa98/01166 for treated and control did not survive in the field (Tab. 5). Stomata evaluation. Colchicine treatment caused variations in the number of stomata and epidermal cells on the different leaf surfaces. The abaxial surfaces of all the treated plants contain fewer stomata and epidermal cells in comparison to the control (Fig. 3a and 3c). There were no stomata on the adaxial surfaces of all the accessions in both treated and control plants (Fig. 3b and 3d). However, the shapes of the adaxial epidermal cell range from pentagonal to hexagonal. The adaxial epidermal cells of the treated were fewer to the control when compared. The abaxial surfaces of all the accessions both treated and control had irregular shaped cells. The mean number of stomata per 1 mm2 area of leaf and stomata of both treated and control plants of all the accessions are shown in table 6. There were significant (P 0.05) differences in the number of stomata between the treated and the control of TDa02/00151 and Tda02/00246, but stomata number was similar in other accessions. Plant growth inhibition and occasional mortality is a known phenomenon in plants after colchicine treatment (Yan 2001, Rauf et al. 2006). This supports that colchicine can cause death of plants due to its toxic effect (Navarro-Alvarez et al. 1994). The suppression of growth observed among the treated plants at the first month of treatment is common after colchicine treatment

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is caused by the reduced rate of cell division, in plants and it has been reported by many researchers (Blakeslee & Avery 1937, Obute et al. 2007). Successful colchicine treatment results in plants that have gigantic characteristics such as thicker-wider leaves with bigger and fewer stomata number (Uhlik 1981). It was found that the treated plants had wider leaves across the genotypes and also that the stomata number per millimeter leaf area is also related to the leaf width, in that it decreased as the leaf width increased across the genotypes and this is also related to the findings of Yan (2001) who discovered that as the leaf thickness increased in treated wax flower, the stomata density decreased. Fewer stomata number observed in the treated abaxial surfaces of all the treated D. alata were evidence of successful colchicine treatment, and these results are in agreement with the works of SchuizSchaeffer (1985), Allard (1960), Stebbins (1950), Davis & Heywood (1967), Ugborogho & Sodipo (1985), Ugborogho & Obute (1994) and Ndukwu & Obute (2006). Micro-morphological features are rarely affected by environmental factors, thus the changes in these features are due to alteration in chromosome numbers in the treated plants (Ugborogho 1982). Colchicine mutagenesis has been used in horticulture as a breeding tool to enhance ornamental characteristics such as plant size, leaf thickness and increased width to length ratio of leaves (Shao et al. 2003). The increased number of leaves among the treated plants indicates that colchicine can be used to induce bushy habit in plants and this is in support of works by Blakeslee & Avery (1937), Hewawasam et al. (2004) and Obute (2006). The higher survival percentage of vines experienced across the genotypes among the treated plants exposed to dry weather showed that colchicine can induce drought resistance in crops. This is in support with the work of Ntuli & Zobodo (2008) on the effect of water stress on the growth of colchicine induced polyploidy Colccinia palmata and Lagenaria sphaerica and concluded that colchicine technique can be used to make drought resistant plants available to farmers. Conclusions Although there were changes in morphological characters induced by colchicine in all the genoypes of D. alata, nevertheless, further studies must be carried out to elucidate the effects of colchicine on tuber yield, nuclear DNA contents and ploidy level of Dioscorea populations.

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Promene stope prezivljavanja i morfoloskih odlika vodenog jama (Dioscorea alata) izazvane kolhicinom

Abiola T. Adzaji1, 2 · Adenubi I. Adesoje1 · Robert Asijedu2 · Alije Sarti2

1 Univerzitet u Ibadanu, Odeljenje za botaniku i mikrobiologiju, Ibadan, Nigerija Meunarodni institut za tropsku poljoprivredu, Jedinica za oplemenjivanje jama, Ibadan, Nigerija

2

Izvod: Ispitivani su mutageni uticaji 0,2% vodenog rastvora kolhicina na prezivljavanje i morfoloske osobine pet genotipova jama (Dioscorea alata). Pupoljci biljaka starih dva meseca tretirani su sa 0,2% kolhicinom, a promene su praene do zrelosti. Stopa prezivljavanja tretiranih biljaka (6,25%-8,75%) bila je niza u odnosu na kontrolu (13,25-15,25%). Tretirane biljke imale su visu stopu prezivljavanja kada su bile izlozene susi (13,8%-24%) u odnosu na kontrolu (7,2%-16,69%). Vei broj listova, vea sirina lista i manji broj stoma uocen je kod tretiranih biljaka. Vrednosti stope prezivljavanja pupoljaka izmeu tretiranih biljaka i biljaka kontrole znacajno su se razlikovale pri P 0,05. Tretirane i netretirane biljke razlikovale su se pri P 0,05 i za sirinu lista, kod genotipova TDa02/00246, TDa98/00116 i TDa99/00240, i za broj stoma, kod genotipova TDa02/00151 i TDa02/00246. Nasi rezultati pokazuju da kolhicin moze da se koristi za izazivanje mutagenih promena kod jama koje mogu da budu od agronomskog znacaja za ovaj usev i njegovu proizvodnju. Kljucne reci: Dioscorea alata, kolhicin, krtole, morfoloske promene, otpornost na susu, poliploidija

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www.nsseme.com/journal.html

Stare sorte i lokalne populacije paradajza

493 Genetika i oplemenjivanje / Genetics and Breeding Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 493-498 originalni naucni rad / original research article

Korisenje starih sorti i lokalnih populacija paradajza kao izvora geneticke varijabilnosti u oplemenjivanju

Svetlana Glogovac Adam Takac

primljeno / received: 15. 04.2010. prihvaeno / accepted: 12.05.2010. © 2010 IFVC

Izvod: U ovom istrazivanju je analizirano 5 lokalnih populacija i 14 starih sorti paradajza. Ispitivani genotipovi predstavljaju deo kolekcije paradajza Instituta za ratarstvo i povrtarstvo u Novom Sadu. Analizirane su sledee osobine biljke i ploda: tip rasta, boja ploda, indeks oblika ploda, masa ploda, broj komora i sadrzaj suve materije u plodu. Primenjena je analiza grupisanja (klaster analiza) u cilju grupisanja ispitivanih genotipova na osnovu slicnosti i razlika za ispitivana svojstva. Cilj rada je ispitivanje znacaja starih sorti i lokalnih populacija kao izvora geneticke varijabilnosti u oplemenjivanju paradajza. Kljucne reci: klaster analiza, lokalne populacije, paradajz, stare sorte, varijabilnost

Uvod Paradajz (Lycopersicon esculentum Mill.) je nekada smatran otrovnim i korisen je iskljucivo u dekorativne svrhe, a danas s pravom zauzima vodee mesto meu svim povrtarskim vrstama. Savremena proizvodnja paradajza bazira se na upotrebi novoselekcionisanih sorti i hibridnog semena (Takac i sar. 2005). U poreenju sa divljim formama paradajza, kulturne forme se odlikuju uskom genetickom varijabilnosu. Procenjuje se da genom kulturnog paradajza sadrzi < 5% geneticke varijabilnosti svojih divljih predaka (Miller & Tanksley 1990). Pored novoselekcionisanih genotipova, kolekcija paradajza kojom raspolaze Institut za ratarstvo i povrtarstvo u Novom Sadu takoe obuhvata veliki broj starih sorti i lokalnih populacija, koje predstavljaju znacajan izvor pozeljnih gena. S obzirom da su istrazivanja na velikom broju kultura ukazala na postojanje veze izmeu uspeha u oplemenjivanju i genetske divergentnosti pocetnog materijala (Dimova & Krasteva 2007), proucavanje, odrzavanje i prosirivanje kolekcije paradajza je od izuzetnog znacaja.

Materijal i metod Iz kolekcije paradajza Instituta za ratarstvo i povrtarstvo u Novom Sadu odabrano je 19 genotipova za analizu (Tab. 1). Ogled je postavljen po slucajnom blok sistemu u tri ponavljanja na lokalitetu Rimski Sancevi. Setva je obavljena 26.03.2008. u sanducie u staklari. Pikiranje rasada je izvrseno u fazi kotiledonih listia presaivanjem biljaka na razmak 10 cm x 10 cm, u cilju poveanja vegetacionog prostora i dobijanja kvalitetnijeg rasada. Pikiranje u sanducie je izvrseno 09.04. Cupanje rasada (rasad golih zila) prethodilo je rasaivanju, koje obavljeno 27.05. na otvoreno polje. Meuredni razmak je iznosio 1,4 m dok je razmak izmeu biljaka u redu bio 50 cm. Osnovni uzorak se sastojao od 10 biljaka po ponavljanju, na kojima su ispitivane sledee osobine biljke i ploda: 1. tip biljke prema UPOV deskriptorima*: 1-determinantan; 2-indeterminantan 2. boja ploda prema UPOV deskriptorima**: 1-zuta; 2-narandzasta; 4-crvena 3. masa ploda izrazena u g 4. sadrzaj suve materije izrazen u%, odreen refraktometrom 5. broj komora 6. indeks oblika: <1 spljosten do veoma spljosten; 1okrugao; >1 izduzen

Ovo istrazivanje je deo projekta broj 20075: Stvaranje sorti i hibrida povra za proizvodnju u zatvorenom prostoru (2008-2011) Ministarstva za nauku i tehnoloski razvoj Republike Srbije (Tehnoloski razvoj)

S. Glogovac ) ( A. Takac Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

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Glogovac S, Takac A

Tabela 1. Ispitivani genotipovi, osobine biljke i ploda paradajza Table 1. Analyzed genotypes and traits of tomato plant and fruit Masa ploda Fruit weight (g) 98,2 49,3 65,1 95,1 54,0 118,1 91,4 96,5 170,1 149,9 239,9 145,0 149,9 200,0 205,2 172,2 54,9 142,1 125,2 125,2 43,0 Br. komora Locules number 3,9 2,8 2,0 4,5 2,8 10,2 2,2 6,1 4,7 4,4 6,5 4,2 4,8 6,5 4,8 5,8 3,5 4,2 3,9 4,4 40,8 % s.m. Dry matter content 6,2 4,9 6,0 5,9 5,5 5,4 5,4 6,5 6,0 5,9 5,8 6,0 5,9 6,3 6,0 6,3 5,0 5,7 6,0 5,8 7,3 Indeks oblika Fruit shape index 0,8 0,8 1,0 0,7 1,0 0,5 0,9 0,7 0,9 0,9 0,7 0,9 0,9 0,8 0,9 0,9 1,1 0,9 0,9 0,9 15,8 Tip* rasta Growth type 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 2 Boja** ploda Fruit colour 4 4 4 4 4 4 4 4 2 4 4 4 4 4 4 4 4 4 4 -

Genotip Genotype Bitoljski kasni Bitoljski rani evelijski Skopski rani Tetovski Novosadski rani Novosadski export Debrecinski Golden jubilei Gloria di Milano Sunny Brok Valiant Saint Pierre Rutgers Belgijski orijas Moran Keckemeti torpe Hode Novosadski jabucar x ¯ V (%)

U cilju grupisanja ispitivanih genotipova na osnovu slicnosti i razlika za ispitivana svojstva primenjena je analiza grupisanja (klaster analiza). Analiza grupisanja je metod koji se koristi za grupisanje objekata u grupe, tako da su objekti unutar grupe slicniji meu sobom, a izmeu grupa znatno razliciti (Kovaci 1994). Rezultati se izrazavaju dendrogramima, tj. grafickim prikazom grupisanja po slicnosti (Dimitrijevi & Petrovi 2005). Izracunavanje parametara analize grupisanja i konstrukcija dendrograma uraena je pomou CLUSTER programskog modula, statistickog paketa SYSTAT 11 for Windows, Inc. 2004. Rezultati i diskusija Masa ploda ispitivanih genotipova varirala je od 49,3 g do 239,9 g. Prosecna masa ploda iznosila je 125,2 g sa koeficijentom varijacije od 43%. Ovo svojstvo je ispoljilo znacajnu

varijabilnost, kao i prema navodima drugih autora (Sekhar et al. 2008, Singh et al. 2008, Takac i sar. 2005). Dendrogram na slici 1. prikazuje podelu analiziranih genotipova prema masi ploda u tri grupe. Na pocetku dendrograma nalaze se genotipovi prve grupe, tri stare sorte koje se odlikuju najveom masom ploda (200,0 g do 239,9 g). U drugoj grupi se nalazi sest starih sorti mase ploda 142,1 g do 172,2 g. Trea grupa je najbrojnija i deli se na tri manje podgrupe sa masom ploda 49,3 g do 125,2 g. U ovoj grupi se pored starih sorti nalazi i svih pet lokalnih populacija. Prema velicini ploda danas postoje sorte krupnih (120 g-250 g), srednjih (80 g-120 g) i sitnih plodova (60 g-80 g), a u novije vreme sve vise se gaje sorte koktel tipa (30 g-50 g), kao i mini (cherry) paradajz (10 g-30 g) (urovka i sar. 2006).

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Slika 1. Grupisanje genotipova prema masi ploda Figure 1. Grouping of genotypes based on fruit weight

Kvalitet ploda paradajza se odreuje na osnovu kolicine suve materije, seera i organskih kiselina. Sadrzaj organskih kiselina (limunske i jabucne) kao i sadrzaj seera u znacajanoj meri uticu na ukus ploda paradajza (Bletsos & Goulas 2002, Susic et al. 2002). Prosecan sadrzaj suve materije za sve ispitivane genotipove iznosio je 5,8% sa koeficijentom varijacije 7,3%. Variranje ovog svojstva je bilo u granicama 4,9% do 6,5%. Genotip sa najveim sadrzajem suve materije je genotip na samom pocetku dendrograma, stara sorta Debrecinski. Dendrogram na slici 2 prikazuje pet formiranih grupa i pet

genotipova koji im se na odreenim distancama prikljucuju. Kod dve stare sorte prve grupe, Moran i Rutgers, izmereni sadrzaj suve materije iznosio je 6,3%. Od druge ka petoj grupi, sadrzaj suve materije se kretao u intervalu od 6,0% do 4,9%. Visok sadrzaj suve materije, kod veine ispitivanih genotipova, kao jedan od glavnih indikatora kvaliteta ploda, ukazuje na znacaj ispitivanih genotipova. Plod paradajza se sastoji od veeg ili manjeg broja komora, koji odreuju velicinu i oblik ploda, a kod ispitivanih genotipova kretao se od 2,0 do 10,2. Prosecna vrednost za sve is-

Distance

Slika 2. Grupisanje genotipova prema sadrzaju suve materije Figure 2. Grouping of genotypes based on dry matter content

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pitivane genotipove iznosila je 4,4 sa koeficijentom varijacije 40,8%. Prema broju komora genotipovi su svrstani u dve grupe, kao sto je prikazano dendrogramom na slici 3. Stara sorta Novosadski rani je genotip koji se izdvaja sa najveim brojem komora (10,2). Prva grupa se sastoji iz cetiri genotipa sa brojem komora 5,8 do 6,5 dok je broj komora kod 14 genotipova druge grupe, podeljenih u tri podgrupe, iznosio 2,0 do 4,8.

Indeks oblika ploda kod ispitivanih genotipova varirao je od 0,5 do 1,1. Prosecna vrednost je iznosila 0,9 sa koeficijentom varijacije 15,8%. Genotipovi su prema ovom svojstvu grupisani u cetiri grupe (Sl. 4). Dva genotipa se izdvajaju sa najmanjom i najveom vrednosu indeksa oblika. Genotipovi prve, druge i tree grupe odlikuju se spljostenim do blago spljostenim oblikom ploda (indeks oblika 0,7 do 0,9), dok je kod genotipova cetvrte grupe utvren okrugao oblik ploda (indeks

Slika 3. Grupisanje genotipova prema broju komora Figure 3. Grouping of genotypes based on locules number

Slika 4. Grupisanje genotipova prema indeksu oblika ploda Figure 4. Grouping of genotypes based on fruit shape index

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oblika 1,0). Stara sorta Novosadski rani imala je plod veoma spljostenog oblika, dok je stara sorta Keckemeti torpe jedina meu ispitivanim genotipovima imala blago izduzen plod. Tip rasta kod paradajza moze biti indeterminantan, semideterminantan i determinantan. Indeterminantne sorte karakterise neogranicen, visoki rast stabla i bujno grananje (Takac i sar. 2007). U zavisnosti od tipa rasta paradajz se moze proizvoditi na razlicite nacine i koristiti za razlicite namene. Veina ispitivanih genotipova imala je indeterminantan tip rasta, dok je kod dve stare sorte utvren determinantan (ogranicen) tip rasta (Sl. 5). Paradajz i proizvodi od paradajza su glavni izvor likopena (jedinjenja koje ima antioksida-

tivna svojstva) i znacajan izvor ß-karotena u ljudskoj ishrani (Tomlekova i sar. 2007). Crvena boja ploda potice od likopena, dok je kod narandzastih plodova sadrzaj ß-karotena vei. Takoe, plod paradajza moze biti i zute, ljubicaste, pa cak i bele boje. Kod veine ispitivanih genotipova utvrena je crvena boja ploda, osim kod stare sorte, Golden jubilei, koja je imala plod narandzaste boje. Pozeljno je da sorte namenjene industrijskoj preradi imaju sto vei sadrzaj likopena, kako bi preraevine od paradajza imale intenzivniju boju (Schoormans 2006). Za upotrebu u svezem stanju, boja ploda zavisi od zahteva trzista, a nasi potrosaci se u veini slucajeva opredeljuju za paradajz crvene boje.

Slika 5. Grupisanje genotipova prema tipu rasta Figure 5. Grouping of genotypes based on growth type

Slika 6. Grupisanje genotipova prema boji ploda Figure 6. Grouping of genotypes based on fruit color

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Dimova D, Krasteva L (2007): Evaluation of a Large-Fruited Determinate Tomato Collection Using Cluster Analysis and Principal Component Analysis (PCA). Proceedings of the Third Balkan Symposium on Vegetables and Potatoes, 2007. Acta Hortic. 729: 85-88 urovka M, Lazi B, Bajkin A, Potkonjak A, Markovi V, Ilin Z, Todorovi V (2006): Proizvodnja povra i cvea u zastienom prostoru. Univerzitet u Novom Sadu. Poljoprivredni fakultet, Novi Sad Kovaci Z (1994): Multivariaciona analiza. Univerzitet u Beogradu. Ekonomski fakultet, Beograd Miller J C, Tanksley S D (1990): RFLP analysis of phylogenic relationships and genetic variation in the genus Lycopersicon. Theor. Appl. Gen. 80: 437-448 Schoormans M (2007): Tempting consumers with ' lycopene tomatoes'. Fruit Veg. Tech. 6: 18-19 Sekhar L, Prakash B G, Salimath P M, Sridevi O, Patil A A (2008): Genetic Diversity Among Some Productive Hybrids of Tomato (Lycopersicon esculentum Mill). J. Agric. Sci. 21: 264-265 Singh A K, Sharma J P, Kumar S, Chopra S (2008): Genetic divergence in tomato (Lycopersicon esculentum Mill.). J. Res. SKUAST­J 7: 1-8 Susic Z, Pavlovic N, Cvikic D, Sretenovic-Rajicic T (2002): Studies of correlation between yield and fruit characteristics of (Lycopersicon esculentum Mill.) hybrids and their parental genotypes. Acta Hortic. 579: 163-166 Takac A, Gvozdenovi , Bugarski D, Cervenski J (2007): Savremena proizvodnja paradajza. Zbornik radova Instituta za ratarstvo i povrtarstvo 43: 269-281 Takac A, Gvozdenovi , Gvozdanovi-Varga J, Vasi M, Bugarski D (2005): Characteristics of old tomato cultivars. Natura Montenegrina 4: 83-91 Tomlekova N, Atanassova B, Baralieva D, Ribarova F, Marinova D (2007): Study on the Variability of Lycopene and ß-Carotene in Tomato. Proceedings of the Third Balkan Symposium on Vegetables and Potatoes, 2007. Acta Hortic. 729: 101-104

Zakljucak Dobijeni rezultati ukazuju na divergentnost ispitivanog uzorka za sve analizirane osobine. Utvrena geneticka divergentnost olaksae izbor potencijalnih roditeljskih parova u pocetnim fazama selekcionog programa i na taj nacin ubrzati rad na stavaranju novih sorti i hibrida paradajza. Ispitivani genotipovi se mogu koristiti za stvaranje sorti i hibrida za razlicite nacine proizvodnje, kao i za razlicite namene. Genotipovi kod kojih je utvren determinantan tip rasta, visok sadrzaj suve materije i okrugao do blago spljosten i blago izduzen plod crvene boje, pogodni su za stvaranje sorti za industrijsku preradu i mehanizovano ubiranje. Indeterminantni genotipovi, razlicite boje i velicine ploda u zavisnosti od zahteva trzista, sa visokim sadrzajem suve materije, pogodni su za stvaranje sorti i hibrida namenjenih svezoj potrosnji i proizvodnji u zastienom prostoru ili na otvorenom uz oslonac. Literatura

Bletsos F A, Goulas C (2002): Fresh Consumption Tomato Performance of a Local Landrace and Derived Lines. Acta Hortic. 579: 95-100 Dimitrijevi M, Petrovi S (2005): Genetika populacije, adaptabilnost i stabilnost genotipa. Univerzitet u Novom Sadu, Poljoprivredni fakultet, Novi Sad

Heirloom Tomato Cultivars and Local Populations as Sources of Genetic Variability for Breeding

Svetlana Glogovac Adam Takac

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 2100 Novi Sad, Serbia Summary: Five local tomato populations and fourteen heirloom cultivars were analyzed in this study. The analyzed genotypes represent a part of tomato collection of Institute of Field and Vegetable Crops in Novi Sad. The following fruit and plant traits were analyzed: growth type, fruit color, fruit shape index, fruit weight, number of locules and dry matter content. Cluster analysis was performed so as to group the analyzed genotypes by homology and divergence. The aim of this article was to determine the importance of heirloom cultivars and local populations as sources of genetic variability in tomato breeding process. Key words: cluster analysis, heirloom cultivars, local populations, tomato, variability

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www.nsseme.com/journal.html

Vegetable Common Bean Accessions in VIR Collection

499 Genetics and Breeding / Genetika i oplemenjivanje Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 499-504 original research article / originalni naucni rad

Adaptability and Stability of Vegetable Common Bean (Phaseolus vulgaris L.) Accessions from the VIR Collection in Crimea

Margarita A. Vishnyakova Yuliya A.Filimonova

received / primljeno: 22.03.2010. accepted / prihvaeno: 26.04.2010. © 2010 IFVC

Summary: Adaptability and stable pod per plant productivity of 20 varieties of vegetable common bean from the collection of the Vavilov Institute (VIR) were investigated. The accessions were grown on 8 selective backgrounds: four different patterns and density of planting during two years. The most adaptive accessions, i.e. most tolerant to the different density, having the stable productivity, were identified. The level of affection of accessions with bacterial diseases depending on the density of planting was also determined. The optimal pattern and sowing density had been proposed for breeding nurseries as a selective background for breeding of genotypes with stable productivity. Key words: adaptability, selective background, stability, vegetable common bean (Phaseolus vulgaris L.)

Introduction The stability in yield with respect to a range of environments is one of the most desired properties of genotypes to fit the crop under available cropping pattern. It is known that genotypes which can adjust their phenotypic state in response to environmental fluctuations in such a way that it gives maximum economic return, can be termed as stable (Allard & Bardshaw 1964). So, searching for high adaptive and stable genotypes is of prime consideration in formulating efficient breeding programs. Vegetable common bean (Phaseolus vulgaris L.), also known as French beans, is a valuable crop produced mainly in the south of Russia. Most varieties of the crop are intensive, i.e. are quite dependent on the environment. Breeding varieties suitable for semi-intensive cropping and forming stable yield in different environments is very interesting for energy-saving technologies. Plant density ranks are often used as different backgrounds to determine the reaction of genotypes, to reveal the most stable and valuable among them, to select the optimal pattern of cropping and to identify the appropriate selective background for breeding (Kilchevsky & Khotyleva 1989, Zhuchenko 2004).

M. A. Vishnyakova ) ( State Scientific Centre N.I. Vavilov All-Russian Research Institute of Plant Industry of Russian Academy of Agricultural Science (VIR), St. Petersburg, Russia e-mail: [email protected] Y. A. Filimonova Crimean breeding centre "Gavrish" Ltd., Crimsk, Russia

Common beans used to be an object of such kinds of assessment in some regions of Russia and Belarus (Skorina et al. 2004, Mazuka et al. 2005). The aim of our study was to determine the adaptive potential and stability of modern varieties of vegetative common bean at different patterns of sowing and plant density, as well as to assess different options as a selective background for breeding in the Crimea region. Materials and Methods The experiments were carried out in the Krasnodar region, at the Crimean experimental breeding station of the Vavilov Institute in 2002-2004. Weather conditions during the growing seasons were quite different in respect to both air temperature and rainfall, which allowed an insight into rather contrasting growing conditions during the three years of experiment. In 2002 the screening of 100 accessions of vegetable common bean was performed and 20 accessions most adaptive to the local environment were selected for more detailed research. These were modern varieties from the collection of Vavilov Institute. The local variety Dialogue was used as the control (reference variety). The parameters of genotypes` adaptability and the environmental parameters as the background for selection were calculated using the method of Kilchevsky & Khotyleva (1985). There were four different planting pattern variants and plant

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Table 1. Variants of the experiment and selective backgrounds Tabela 1. Varijante ogleda i selekcione osnove

Variants Varijante I (control) kontrola II III IV Sowing pattern* Obrazac setve 42 × 10 40 × 5 20 × 10 20 × 5 Sowing density (plants per ha) Gustina setve (biljaka po ha) 238,000 500,000 500,000 1,000,000 Year Godina 2003 2004 2003 2004 2003 2004 2003 2004 Number of selective background Broj selekcione osnove 1 2 3 4 5 6 7 8

*Row spacing (cm) x spacing between plants in row (cm) *Razmak izmeu redova (cm) x razmak izmeu biljaka u redu (cm) densities within two years (2003-2004), in total corresponding to 8 selective backgrounds. The productivity characteristic, mass of green pods per plant (g) was used as a criterion of the adaptability and the stability of the varieties. Results and Discussion Productivity of Varieties Depending on the Density and Planting Patterns According to the analysis of variance, the highest productivity was revealed in the I variant (pattern 42 cm × 10 cm, control), where the average mass of green pods per plant was 54.25

g and varied among accessions from 24.33 g to 83.13 g (V = 29.7%). The most productive were varieties Vaillant, Empress, Nomad, Slavyanka, Zabava and Sensation with average productivity between 66.2 g and 83.1 g. The rest of the varieties had much poorer productivity than the control variety (Tab. 2). As the density of sowing increased up to 500,000 plants per hectare (II variant of planting 40 cm x 5 cm), the average productivity of the experimental plot decreased by 22% compared with I variant - coming to 41.15 g and varied from 23.72 g to 69.51 g (V = 25.6%). However, the decrease of productivity was largely dependent on the genotypes. Some varieties reduced

Table 2. Yield of green pods of the common bean varieties depending on planting density Tabela 2. Prinos zelenih mahuna sorti boranije u zavisnosti od gustine setve

VIR number VIR broj 14401 15281 15356 15231 15208 14696 15214 14400 15256 13535 15366 13646 15359 15213 15371 13542 15175 15336 15245 14393 Accession varieties Naziv sorte Dialog (reference) standard Nomad Zabava Slavyanka Empress Vaillant Sensation Chaika Zagadka Trelanel Royal Burgundy Purple Pod Oltyn Brilliant Garden Green PI-164093 Doranel Vestochka Tenderwhite Resistant BCMV Oxy Apci Variants of the experiment Varijante ogleda I (control) kontrola g per plant g po biljci 73.7 83.1 80.3 77.9 66.6 66.3 66.2 59.8 51.6 50.8 50.8 50.4 49.7 42.4 39.6 38.9 38.6 37.5 36.7 24.3 54.25 12.3 II g per plant g po biljci 47.6 59.0 62.7 55.5 49.9 34.7 34.4 45.1 69.5 50.0 28.7 46.3 32.7 31.3 24.6 29.5 43.4 26.5 28.0 23.7 41.15 9.7 % 65 71 78 71 75 52 52 75 135 98 56 92 66 74 62 76 112 71 76 98 IV g per plant g po biljci 27.5 28.5 39.0 36.4 27.5 24.0 33.5 22.3 28.0 27.1 23.3 30.4 17.4 22.3 17.0 17.5 24.7 22.2 17.5 17.1 25.14 6.4 % 37 34 49 47 41 36 51 37 54 53 46 60 35 53 43 45 64 59 48 70 -

LSD0.05

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the productivity slightly - 9-25% compared to the reference variety, others by more than 26-35% and the rest showed similar results in both variants, such as Trelanel, Oltyn and Apci (Tab. 2). With increasing the density up to 1,000,000 plants per hectare (IV variant of planting 20 cm x 5 cm), all varieties substantially reduced productivity at an average 52% compared to variant I. The average productivity was 25.14 g and varied from 17.02 g to 38.96 g. Some varieties insignificantly reduced productivity, such as Apci, Tenderwhite Resistant BCMV, Vestochka and Oltyn, while others decreased the yield up to 46% -67%. The most productive were Slavyanka and Zabava exceeding the reference variety up to 36.4 g ­ 39.0 g. Productivity of Varieties Depending on the Planting Patterns Average productivity in the experiment with decreasing spacing between rows from 40 cm to 20 cm decreased by 19% - 33.19 g and varied from 18.70 to 45.05 g (V = 19.8%). Most varieties showed weak reaction on the planting patterns (significant difference at 5% level of significance had not been revealed). 13 varieties slightly re-

duced productivity, the average difference varied from 0.4 to 9.1 g. Nomad, Zabava, Zagadka and Trelanel showed high sensitivity to the compression of the plants in rows (Tab. 3). Thus, different selective backgrounds resulted in large fluctuations in productivity, i.e. different reactions of genotypes on environment. The most productive varieties are of particular interest, reducing productivity insignificantly in all variants of the experiment. Such was variety Oltyn with moderate and stable productivity in all studied environments. Relationship Between Productivity and Stability All studied accessions in all variants of the experiment differed in the properties of adaptability, but all of them decreased productivity at the highest density of the plot (IV variant). The results of variance analysis divided all accessions in two groups in relation to the reference variety: 12 productive varieties and 8 low productive ones. Zabava, Dialog, Nomad, Slavyanka and Zagadka demonstrated the largest effects of general adaptive ability (GAA) among the varieties from the first group. Last three of

Table 3. Yield of green pods of common bean varieties depending on the planting patterns (g per plant) Tabela 3. Prinos zelenih mahuna sorti boranije u zavisnosti od obrasca setve (g po biljci) Variants of experiment Difference between Varijante ogleda the variants VIR number Accession varieties Razlika izmeu VIR broj Naziv sorte varijanti II III 14401 15281 15356 15231 15208 14696 15214 14400 15256 13535 15366 13646 15359 15213 15371 13542 15175 15336 15245 14393 Dialog (reference) standard Nomad Zabava Slavyanka Empress Vaillant Sensation Chaika Zagadka Trelanel Royal Burgundy Purple Pod Oltyn Brilliant Garden Green PI-164093 Doranel Vestochka Tenderwhite Resistant BCMV Oxy Apci LSD0.05 47.6 59.0 62.7 55.5 49.9 34.7 34.4 45.1 69.5 50.0 28.7 46.3 32.7 31.3 24.6 29.5 43.4 26.5 28.0 23.7 41.15 9.7 42.3 40.5 38.3 39.5 38.0 34.7 37.3 44.9 45.1 25.0 29.1 38.0 22.3 27.0 32.2 24.3 35.4 26.3 25.0 18.7 33.19 10.3 5.3 18.5 24.4 16.0 11.9 0 2.9 0.2 24.4 25.0 0.4 8.3 10.4 4.3 7.6 5.2 8.0 0.2 3.0 5.0 9.1 -

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Table 4. The adaptability and stability of common bean varieties Tabela 4. Adaptabilnost i stabilnost sorti boranije VIR Accession varieties number x i GAAi Naziv sorte VIR broj 14401 Dialog (reference) standard 47.7 9.3 15356 Nomad 55.1 16.6 15281 Zabava 52.8 14.4 15231 Slavyanka 52.3 13.9 15208 Empress 45.5 7.0 15256 Vaillant 45.5 10.1 14400 Sensation 43.0 4.6 15214 Chaika 42.9 4.4 13646 Zagadka 41.3 2.8 14696 Trelanel 39.9 1.5 13535 Royal Burgundy Purple Pod 38.2 -0.2 15175 Oltyn 35.5 -2.9 15366 Brilliant 32.9 -5.5 15213 Garden Green 30.8 -7.7 15359 PI-164093 30.5 -7.9 15371 Doranel 28.4 -10.1 15336 Vestochka 28.1 -10.3 13542 Tenderwhite Resistant BCMV 27.6 -10.9 15245 Oxy 26.8 -11.7 14393 Apci 21.0 -17.5 LSD 0.05 12.6 -

Si 17.82 19.6 22.03 17.44 15.61 18.48 14.63 16.09 8.05 20.15 13.32 9.56 10.94 9.57 13.52 9.00 6.33 9.21 10.42 12.40 -

Sgi (%) 37.3 35.6 41.4 33.3 34.3 38.0 33.9 37.5 19.5 50.3 34.8 26.9 33.2 31.1 44.2 31.7 22.4 33.4 38.9 19.0 -

bi 1.24 1.43 1.37 1.36 1.18 1.26 1.12 1.11 1.07 1.04 0.99 0.92 0.86 0.80 0.79 0.73 0.73 0.71 0.70 0.54 -

BVGi i 20.89 25.55 19.93 26.07 21.93 20.70 21.01 18.60 31.14 19.60 18.19 21.16 16.48 16.35 10.18 14.80 18.64 13.69 11.07 19.13 -

* x i ­ average for variety (g plant-1); GAAi ­ general adaptive ability of the variety; SACi - specific

adaptive capacity of the variety; Sgi - relative stability of the variety; bi - plasticity or responsiveness (regression coefficient on the environment); BVGi - breeding value of genotypes (varieties) * x i ­ prosek sorte (g biljka-1); GAAi ­ opsta adaptabilna sposobnost sorte; SACi ­ posebna adaptabilna sposobnost sorte; Sgi ­ relativna stabilnost sorte; bi ­ plasticnost ili odgovor (regresioni koeficijent sredine); BVGi ­ oplemenjivacka vrednost genotipa (sorte) took the third meaning of BVG (breeding value of the genotype) in the sample and revealed the highest productivity - 55.1 g per plant. It also had the highest regression coefficient (bi = 1.43) and more than others responsive on the improvement of cropping conditions. The lowest relative stability (Sg = 33.3%) is typical for Slavyanka, which combines productivity with environmental sustainability and has the second meaning of BVG. Variety Oltyn has high productivity and relative stability and the highest rate BVG. The highest adaptive capacity was identified in variety Apci. Very close to it were varieties Tenderwhite Resistant BCMV, PI-164093 and Oxy, but all of them had low productivity in all variants of the experiment and regression coefficient below 1.0 and were poorly responsive to improvement of cropping conditions. Breeding value of these accessions (BVG) had a low meaning ­ 11.07 ­ 18.64.

them had an average productivity in all variants (Tab. 4). These accessions are the best in the breeding for the general adaptive capacity, but at the same time, they have a high variance specific adaptive capacity (SAC) that determined the lowest stability. These are intensive varieties which provide a guaranteed high yield only in favourable conditions. They are more suitable for semi-intensive technologies with an average level of energy consumption and at the same time are very responsive to the improvement of growth conditions. The best accessions, combining high productivity and its stability, were Oltyn, Slavyanka and Zabava. The last two of them had maximum GAA. Consequently, despite the lack of a close link between productivity and stability, as a result of individual selection from these varieties plants with maximum productivity and high stability can be obtained. Moreover, variety Zabava

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Table 5. Parameters of the environment as a background for the selection Tabela 5. Parametri sredine kao selekcione osnove Selective background u+D* D D Se Ke Selekciona osnova 1 2003-42×10 55.9 17.4 21.09 38.6 9.12 2 2004-42×10 52.6 14.1 15.38 29.5 4.85 Average 54,3 15.8 18.24 34.1 6.99 Prosek 3 2003-40×5 40.3 1.8 17.22 43.7 6.08 4 2004-40×5 42.0 3.5 11.62 27.7 2.77 Average 41,1 2.7 14.42 35.7 4.43 Prosek 5 2003-20×10 32.0 -6.5 9.14 28.6 1.71 6 2004-20×10 34.4 -4.1 6.62 19.2 0.90 Average 33,2 -5.3 7.88 23.9 1.31 Prosek 7 2003-20×5 24.5 -14.0 8.01 32.7 1.31 8 2004-20×5 25.8 -12.7 5.11 19.8 1.32 Average 25,2 -13.5 6.56 26.3 1.32 Prosek

Tk 0.893 0.756 0.825 0.876 0.785 0.831 0.896 0.669 0.783 0.852 0.779 0.816

P 0,345 0,223 0.284 0,374 0,217 0.296 0,256 0,128 0.192 0,279 0,154 0.217

*u+D - the average productivity (g plant-1), D - the productivity of the environment, DCC - variance differentiating ability of the environment, T ­ coefficient of typicality of environment, Se - the relative differentiating ability of the environment, P - coefficient predictable environment, Ke ­ coefficient of compensation environment *u+D ­ prosecna produktivnost (g biljka-1), D ­ produktivnost sredine, DCC ­ varijansa izdvajajua sposobnost sredine, T ­ koeficijent osobenosti sredine, Se ­ relativna izdvajajua sposobnost sredine, P ­ koeficijent predvidivosti sredine, Ke ­ koeficijent kompenzacije sredine Comprehensive Assessment of Planting Density as a Background for the Selection Analyses of the productivity were performed in four environments (pattern and density of planting) during two years, i.e. on 8 selective backgrounds (Tab. 1). The highest productivity (D) was provided on the backgrounds 1, 2, 3, 4, while the lowest was on 7 and 8 (Tab. 5). The highest differentiating ability of the environment and its typicality was observed at backgrounds 1, 2, 3, 4. The rate of their destabilizing effect Ke > 1 corresponded to analyzing background Se > 20%. Backgrounds 5 and 7 are stabilizing, 6 and 8 had a leveling effect (Tab. 6). Typicality of environment (T) in the experiment was quite high, but varied from year to year. The backgrounds 1, 3, 5, 7 had the highest typicality. According to the preliminary analysis, the pattern 40 cm × 5 cm with analyzing ability of the background (Se3;4 = 35.7%) was the most suitable for the selection. It is the best as the combination of typical environment and high productivity of accessions. The pattern 20 cm × 10 cm also suits the requirements for selective backgrounds, but only on the parameters Se, which corresponded to stabilizing background during two years of the experiment. Typicality (T5;6) and productivity (D5;6) of this variant were comparatively low ­ 0.783 and 33.2 respectively. The scheme of 20 cm × 5 cm suited least of all for the selection of genotypes, especially in unfavourable weather conditions. This environment had a weak effect of destabilization (Se7;8 = 26.3%), low typicality (T7;8 = 0,816), the minimum standards of productivity (D7;8 =- 13.5) and weak polymorphism (DCC7,8 = 6, 56). This background may be leveling in unfavourable weather conditions, able to oppress the viability of strains and to smooth the differences between them. Effect of Planting Density on Defeat with Bacterial Diseases The affectation of the accessions with bacteria diseases increases with increasing density of sowing. At the same time, the degree of damage depends on the susceptibility of plant varieties. The varieties slightly affected with bacteriosis (less than 15%), regardless of the pattern and the density of sowing were Zabava, Oltyn, Zagadka, Nomad and Sen-

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Table 6. Characteristics of the selective backgrounds Tabela 6. Karakteristike selekcionih osnova Selective background (Dk) productivity Selekciona osnova Produktivnost high 1 03-42×10 visoka high 2 04-42×10 visoka high 3 03-40×5 visoka high 4 04-40×5 visoka middle 5 03-20×10 srednja high 6 04-20×10 visoka low 7 03-20×5 niska low 8 04-20×5 niska sation. The varieties Dialogue, Trelanel, Doranel, Vestochka, Empress, Garden Green and Slavyanka were ranked as moderately affected (35%). Strong reaction on the density of sowing was characteristic for cultivars Vaillant, Brilliant, Chaika, Oxy, Tenderwhite Resistant BCMV, Royal Burgundy Purple Pod, PI-164093 and Apci, increasing infected plants from 36% to 80% with increased sowing density. Conclusions The significant differences between vegetative common bean varieties in adaptability and stability in different environment were revealed. The varieties that showed the greatest adaptive capacity (tolerance to density of sowing) were identified, as well as the accessions with better ability to combine high productivity and stability. The research on planting patterns as selective background showed that the scheme of 40 cm × 5

(Sek) background Osnova analyzing analizirajua analyzing analizirajua analyzing analizirajua analyzing analizirajua stabilizing stabilizirajua middle srednja stabilizing stabilizirajua middle srednja

(Tk) typicality Osobenost high visoka middle srednja high visoka middle srednja high visoka low niska high visoka middle srednja

cm with a density of 500,000 plants per hectare is the most suitable for breeding and selection of vegetable common bean genotypes: it is the best in providing a more accurate and reliable identification of varieties with high productivity in the Crimea region of Russia. References

Allard R W, Bradshaw A D (1964): Implication of genotype-environmental interaction in applied plant breeding. Crop Sci. 4: 503-508 Kilchevsky A V, Khotyleva L V (1989): Genotype and Environment in Plant Breeding: Science and Technology, Minsk Mazuka E S, Anochina V S, Yankovskaya G P (2005): Phenotypic manifestation of agronomic traits in collection accessions of vegetable common bean. Agrarian Sci. 2: 13-15 Scorina V V, Dobrutskaya E G, Musaev F B (2004): Ecologogeographic evaluation of common bean varieties by productivity and ecological stability. Vestnik Byelorussian State Agrarian Academy 3: 41-46 Zhuchenko A A (1988): Adaptive Potential of Cultivated Plants (Ecological and Genetic Basis). Kishinev

Adaptabilnost i stabilnost populacija boranije (Phaseolus vulgaris L.) iz kolekcije VIR na Krimu

1

Margarita A. Visnjakova1 · Julija A.Filimonova2 Drzavni naucni centar i Sveruski istrazivacki institut biljne proizvodnje N. I. Vavilov Ruske akademije poljoprivrednih nauka, Petrograd, Rusija 2 Krimski oplemenjivacki centar Gavris s.o.o., Krimsk, Rusija

Izvod: Adaptabilnost i stabilnost produktivnosti mahuna po biljci su ispitivane na uzorku od 20 populacija pasulja iz zbirke Instituta Vavilova (VIR). Populacije su gajene u skladu sa 8 razlicitih selekcionih osnova: cetiri razlicita sklopa, odnosno, razmaka izmeu i unutar redova i gustine setve, tokom dve godine. Identifikovane su populacije sa najveom adaptabilnosu, odnosno sa najmanjim uticajem razlicitog sklopa i stabilnom produktivnosu. Takoe je utvren nivo osetljivosti populacija na bakterioze u zavisnosti od sklopa. Predlozeni su optimalni obrazac i gustina setve za oplemenjivacke oglede kao selekciona osnova za oplemenjivanje na stabilnu produktivnost. Kljucne reci: adaptabilnost, boranija, selekciona osnova, stabilnost

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Veza marker-svojstvo kod psenice

505 Molekularna genetika / Molecular Genetics Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 505-510 originalni naucni rad / original research article

Asocijativna analiza izmeu mikrosatelitskih markera i agronomski vaznih svojstava psenice

Ljiljana Brbakli Ankica Kondi-Spika Dragana Trkulja Borislav Kobiljski

primljeno / received: 19.04.2010. prihvaeno / accepted: 05.05.2010. © 2010 IFVC

Izvod: Asocijativna analiza predstavlja najnoviji pristup u genetickim istazivanjima psenice koji omoguava uspesniju implementaciju marker asistirane selekcije (MAS) u oplemenjivacke programe. U cilju utvrivanja veze marker-svojstvo, u radu je izvrsena dvogodisnja fenotipska evaluacija 96 genotipova heksaploidne psenice, kao i molekularna analiza primenom mikrosatelitskih markera (Simple Sequence Repeat - SSR). Na osnovu podataka molekularne analize, u cetiri ispitivana mikrosatelitska lokusa (Xgwm11, Xgwm428, Xpsp3200, Xpsp3071), detektovana je ukupno 31 alelna forma. Asocijativnom analizom su utvrene cetiri statisticki znacajne veze ispitivanjem vaznih agronomskih svojstava i mikrosatelitskih markera. Markeri GWM428 i GWM11 imali su znacajan uticaj na fenotipsku varijabilnost vremena klasanja i vremena cvetanja u toku obe ispitivane godine, sto ih cini pogodnim za prakticnu primenu u MAS. Kljucne reci: agronomska svojstva, asocijativna veza, polimorfnost, psenica, SSR

Uvod Znacajan broj agronomski vaznih svojstava psenice, kao sto su prinos, kvalitet i drugo, determinisan je veim brojem gena, tzv. QTL-a (Quantitative Trait Loci), sto proces oplemenjivanja cini veoma kompleksnim (Sorrells 2008, Neumann et al. 2010). U poslednjih nekoliko decenija, primenom razlicitih molekularnih markera, detektovani su QTL-ovi povezani sa vaznim agronomskim svojstvima, i to najcese u populacijama nastalim ukrstanjem dva roditelja kao sto su rekombinantne inbred linije, dvostruki haploidi, itd. Detektovani QTL-ovi iz ovih biparentalnih populacija validni su samo u odnosu na dve alelne forme koje nose, po jednu od svakog roditelja, sto znacajno ogranicava mogunost za njihovu prakticnu primenu. S druge strane, ispitivanja veze izmeu genetickih i fenotipskih podataka primenom asocijativne analize direktno na oplemenjivackom materijalu (sorte, linije i genotipovi) pruzaju znatno vise informacija o polimorfnosti markera i varijabilnosti fenotipskih svojstava, sto je od velikog znacaja za njihovu prakticnu primenu putem MAS (Kobiljski et al. 2007). U poslednjih nekoliko godina intenzivirana su asocijativna istrazivanja, pre svega asocijativnog mapiranja kompleksnog genoma psenice, cime je poveana dostupnost

Lj. Brbakli ) (· A. Kondi-Spika · D. Trkulja · B. Kobiljski Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

informacija korisnih u procesu oplemenjivanja (Breseghello & Sorrells 2006, Neumann et al. 2010). Cilj rada bila je identifikacija geneticke polimorfnosti mikrosatelitskih lokusa psenice i utvrivanje mogue veze ispitivanih markera i pojedinih vaznih agronomskih svojstava u nasim agroekoloskim uslovima, kao i mogunost prakticne primene utvrenih asocijacija u oplemenjivackim programima psenice. Materijal i metod rada U radu je ispitivano 96 genotipova heksaploidne psenice poreklom iz jedanaest oplemenjivackih centara sveta, koji se nalaze u genetickoj kolekciji Odeljenja za strna zita Instituta za ratarstvo i povrtarstvo u Novom Sadu. Ispitivani oplemenjivacki materijal podeljen je na osnovu dugogodisnjih fenotipskih evaluacija prinosa, na linije/genotipove sa visokim i niskim genetskim potencijalom za prinos. U ovom radu su merena i ocenjena cetiri agronomski vazna svojstva u toku dve vegetacione sezone (2007. i 2008). Ispitivana su sledea svojstva: vreme klasanja i cvetanja, visina stabljike i duzina klasa, koja su prema UPOV deskriptorima za psenicu ocenjena na sledei nacin:

Ovo istrazivanje je deo projekta broj TP -20138: Poveanje genetickih i proizvodnih potencijala strnih zita primenom klasicne i moderne biotehnologije (2008-2010) Ministarstva za nauku i tehnoloski razvoj Republike Srbije / This research results from the project TP-20138 funded by the Ministry of Science and Technological Development of the Republic of Serbia

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· vreme klasanja ­ broj dana od 1. januara do momenta kada je 50% biljaka klasalo · vreme cvetanja ­ broj dana od 1. januara do momenta kada je 50% biljaka cvetalo · visina stabljike (cm) ­ merena od osnove biljke do osnove klasa · duzina klasa (cm) ­ merena od osnove klasa do vrha klasa bez osja Tabela 1. Opis mikrosatelitskih markera Table 1. Description of microsatellite markers

Prajmeri Primers GWM428 GWM11 Sekvenca ponovka Sequence repeat (GA)22 (TA)6CATA(CA)19(TA)6

razdvajani su putem kapilarne elektroforeze na cetvorokapilarnom sekvenceru 3130 Applied Biosystems - ABI Prism 3130. Fragmentna analiza produkata obavljena je uz pomo GeneMapper Software Version 4.0 (Applied Biosystems). Nivo polimorfnosti pojedinacnih lokusa (Polymorfism Index Content - PIC vrednost) utvrena je prema Anderson et al. (1993). Struktura

Sekvence desnog i levog prajmera Sequences of primer pairs 5` TTC TCC ACT AGC CCC GC 3` (NED) 5` CGA GGC AGC GAG GAT TT 3` 5` GTG AAT TGT GTC TTG TAT GCT TCC 3` (6-FAM) 5` GGA TAG TCA GAC AAT TCT TGT G 3` 5` GTT CTG AAG ACA TTA CGG ATG 3` (PET) 5` GAG AAT AGC TGG TTT TGT GG 3` 5` CGT GCC CTA CAC CTC CTT TTC TCT C 3` (VIC) 5` TCC GTA CAT ACT CCG GGA GAC C 3`

Hromozom Chromosome 7D 1B

T (Cº) 62 52

PSP3200 PSP3071

(AAG)16

6D

62

(TC)14

6A

62

Genotipovi su analizirani pomou cetiri mikrosatelitska markera (Tab. 1), za koje je utvrena znacajna povezanost sa agronomski vaznim svojstvima u ranijim istrazivanjima (Quarrie et al. 2003, Kuchel et al. 2007, Li et al. 2007). Izolacija DNK iz klijanaca genotipova izvrsena je prema izmenjenoj metodi po Doyle & Doyle (1990). Lancana reakcija polimeraze vrsena je prema modifikovanom protokolu Röder et al. (1998). Reakciona PCR smesa zapremine 20 l sadrzala je 25 ng genomske DNK, 1 x pufer za Taq polimerazu, 2 mM MgCl2, 0,2 mM svakog dNTP, 2 jedinice Taq polimeraze i po 10 pmol levog i desnog fluorescentnog prajmera. Dobijeni produkti

populacije analizirana je na osnovu molekularnih podataka obraenih u programu Structure v.2.2. (Pritchard et al. 2000). Asocijativna analiza markera i ispitivanih svojstava testirana je prema opstem linearnom modelu (GLM - General Linear Model) u softverskom programu Tassel v. 2.0.1 (Bradbury et al. 2007). Rezultati i diskusija Molekularnom evaluacijom mikrosatelitskih lokusa (Xgwm11, Xgwm428, Xpsp3200, Xpsp3071) kod 96 genotipova psenice detektovana je ukupno 31 alelna forma (Tab. 2.) Prosecan broj alela

Tabela 2. Broj alela po lokusu, velicina alela, PIC vrednost i frekvencija najucestalijeg alela Table 2. Number of alleles per loci, allelic size, PIC value and frequency Broj alela po lokusu Velicina alela PIC vrednost Frekvencija Lokus Number of alleles per Size of allele PIC value Frequency Locus locus Xgwm428 4 121-135 0,377 0,781 Xpsp3200 Xpsp3071 Xgwm11 Ukupan broj Total 6 10 11 31 159-177 148-167 186-212, nulti 0,723 0,831 0,736 0,667 0,417 0,313 0,469 0,495

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po lokusu iznosio je 7,71. Najvei broj alelnih varijanti (11) utvren je u lokusu Xgwm11, dok je u lokusu Xgwm428 broj razlicitih alela bio najmanji (4). Najvea PIC vrednost dobijena je u lokusu Xpsp3071 (0,831), dok je u lokusu Xgwm428 (0,377) bila najmanja. Frekvencije najucestalijih alela bile su u intervalu od 0,313 (Xpsp3071) do 0,781 (Xgwm428). Slican broj alelnih formi i relativno visok nivo polimorfnosti po lokusima detektovani su i u drugim radovima (Kobiljski i sar. 2002, Maccaferri et al. 2007), a predstavljaju pokazatelje znacajne geneticke varijabilnosti ispitivanog materijala. Pored detektovane molekularne divergentnosti, utvrena je i znacajna fenotipska varijabilnost, koja je prikazana u tabeli 3.

Asocijativnom analizom utvrene su cetiri znacajne veze izmeu ispitivanih fenotipskih svojstva i mikrosatelitskih markera. Mikrosatelitski marker GWM11, koji se nalazi na hromozomu 1B, pokazao je znacajnu vezu sa fenotipskom varijabilnosu QTL-a odgovornog za vreme klasanja u obe ispitivane godine (33,36% i 29,19%) (Tab. 4). Isti marker bio je vezan za fenotipsku varijabilnost perioda cvetanja sa slicnim efektom (oko 32%) u obe godine. Dobijeni rezultati u nasem radu u saglasnosti su sa rezultatima Yang et al. (2002), koji su uocili da je marker GWM11 pokazao znacajnu vezu sa QTL-om za duzinu nalivanja zrna, a ova osobina se nalazi u direktnoj vezi sa vremenom stasavanja.

Tabela 3. Osnovni statisticki parametri ispitivanih agronomskih svojstava tokom dve godine Table 3. Basic statistical parameters of evaluated agronomic traits during two years

Osobine Traits Klasanje Heading Cvetanje Flowering Visina stab. Stem height Duzina kl. Spike lenght Prosek Average 125,7 131 84,4 10,1 Interval poverenja -95% Interval conf. -95% 124,57 129,89 82,21 9,86 Interval poverenja +95% Interval conf. +95% 126,83 132,07 86,71 10,38 Min Min 102 111 19 4,3 Max Max 144 150 132,3 19,7 Std. dev. Std.dev. 7,9 7,5 15,8 1,8 Std. greska Std. err. 0,57 0,54 1,14 0,13

U cilju eliminacije lazno pozitivnih asocijacija izmeu pojedinih kandidat gena i svojstava, neophodno je utvrditi strukturu ispitivane populacije (Pritchard et al. 2000). Na osnovu podataka dobijenih molekularnom evaluacijom, izvrsena je analiza geneticke strukture populacije, a dobijene vrednosti nakon korekcije po Evanno et al. (2005), ukazale su na prisustvo tri subpopulacije (Graf. 1). Ovako divergentan materijal, na osnovu fenotipske i molekularne analize, bio je vazan preduslov asocijativne analize.

U ispitivanom radu, mikrosatelitski marker GWM428 lociran na hromozomu 7D bio je vezan sa varijabilnosu vremena klasanja tokom obe ispitivane godine (14,09% i 10,14%). Slicna veza ovoga markera uocena je i za svojstvo varijabilnost cvetanja (14,3% i 10,79%) tokom obe ispitivane godine (Tab. 5) Groos et al. (2003) su u blizini istog markera identifikovali QTL za masu 1.000 zrna. Ova osobina je u znacajnoj korelaciji sa ranostasnosu, sto implicira da bi primena ovog mikrosa-

Grafikon 1. Graficki prikaz tri subpopulacije procenjene u programu Structure Graph 1. Preview of three subpopulations estimated in programme Structure

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Tabela 4. Asocijativna veza markera GWM11 sa vremenom klasanja i cvetanja Table 4. Associations between GWM11 marker and heading and flowering time Marker Marker Osobina Trait Klasanje Heading GWM11 Cvetanje Flowering Godina Year 2007 2008 2007 2008 P vrednost P value 0,000205** 0,0011** 0,000398** 0,000338** R2 33,36 29,19 32,18 32,09

** nivo znacajnosti / level of significance p<0,01 telitskog markera u MAS za navedena svojstva mogla biti od znacaja u oplemenjivackom radu. U radu Cuthbert et al. (2008), koji su ispitivanja vrsili na mapirajuoj populaciji od 178 linija, utvreni su QTL-ovi za vreme klasanja i sazrevanja, i to u proksimalnim delovima hromozoma 7D i 1B. U odnosu na prethodno definisane linkidz mape, ispitivani mikrosatelitski markeri u nasem radu takoe su smesteni u ovim hromozomskim regionima, te se moze pretpostaviti da se radi o istim QTL-ovima. Takoe, ovo je potvrda da QTL-ovi identifikovani u mapirajuim populacijama, mogu veoma uspesno biti detektovani i asocijativnom analizom oplemenjivackog materijala. Mikrosatelitski markeri PSP3071 i PSP3200 nisu pokazali vezu sa ispitivanim svojstvima, iako su u prethodnim istrazivanjima utvrene znacajne asocijacije (Quarrie et al. 2003), sto moze biti uslovljeno istrazivanjem na malom broju ispitivanih svojstava, potom odabirom oplemenjivackog materijala, kao i mogunosu da odabrani QTLovi nisu ispoljili efekat u datim uslovima spoljasnje sredine.

Tabela 5. Asocijativna veze markera GWM428 sa vremenom klasanja i cvetanja Table 5. Associations between GWM428 marker and heading and flowering time Marker Osobina Godina P vrednost Marker Trait Year P value Klasanje Heading GWM428 Cvetanje Flowering 2007 2008 2007 0,0055** 0,0294* 0,0053** 0,0241*

R2 14,09 10,14 14,3 10,79

2008 * nivo znacajnosti / level of significance p<0,05 **nivo znacajnosti / level of significance p<0,01

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Li S, Jia J, Wei X, Zhang X, Li L, Chen H, Fan Y, Sun H, Zhao X, Lei T, Xu Y, Jiang F, Wang H, Li L (2007): A intervarietal genetic map and QTL analysis for yield traits in wheat. Mol. Breed. 20: 167-178 Maccaferi, M., Stefanelli, S., Rotondo, F., P., Tuberosa, R., Sanguineti, M.C. (2007). Realtionships among durum wheat accessions. I. Comparative analysis of SSR, AFLP, and phenotypic data. Genome 50: 373-384 Neumann K, Kobiljski B, Denci S, Varshney R K, Börner A (2010): Genome-wide association mapping : a case study in bread wheat (Triticum aestivum L.). Mol. Breed. DOI 10.1007/s11032-010-9411-7 Pritchard J K, Stephens M, Donnelly P (2000): Inference of population structure using multilocus genotype data. Genet. 155: 945-959 Quarrie S A, Dodig D, Peki S, Kirby J, Kobiljski B. (2003): Prospects for marker-assisted selection of improved drought responses in wheat. Bulg. J. Plant. Physiol. 28: 83-95 Röder M S, Korzun V, Wendehake K, Plaschke J, Tixier M-H, Leroz P, Ganal M W (1998): A microsatellite map of wheat. Genet. 194: 2007-2023 Sorrells M E (2008): Association breeding strategies for improvement of self-pollinated crops. Conventional and molecular breeding of field and vegetable crops, Novi Sad, 24-27 november, 43-46 Yang J, Sears R G, Gill B S, Paulsen G M (2002): Quantitative and molecular characterization of heat tolerance in hexaploid wheat. Euphytica 126: 275-282

Zakljucak Odabrani mikrosatelitski markeri su pokazali zadovoljavajui nivo polimorfnosti u analiziranom materijalu. Analizom molekularnih podataka dobijeno je grupisanje genotipova u tri subpopulacije. Asocijativnom analizom izmeu mikrosatelitskih lokusa i fenotipskih svojstava, utvrena je znacajna veza izmeu dva ispitivana markera i vremena klasanja i cvetanja. Sa aspekta oplemenjivanja, markeri GWM428 i GWM11 koji su pokazali znacajan uticaj na fenotipsku varijabilnost, mogu se koristiti kao potencijalni kandidat markeri u MAS, dok je za preostala dva markera neophodna dodatna validacija u toku veeg broja godina, na veem broju lokaliteta i na veem broju fenotipski merljivih svojstava. Literatura

Anderson J A, Churchill G A, Autrque J E, Tanksley S D, Sorrells M E (1993): Optimizing parental selection for genetic linkage maps. Genome 36: 181-186 Bradbury P J, Zhang Z, Kroon D E, Casstevens T M, Ramdoss Y, Buckler E S (2007): TASSEL: software for association mapping of complex traits in diverse samples. Bioinforma. 23: 2633-2635 Breseghello F, Sorrells M E (2006): Association analysis as a strategy for improvement of quantitative traits in plants. Crop Sci. 46: 1323-1330 Cuthbert J L, Somers D J, Brule-Babel A L, Broen P D, Crow GH (2008): Molecular mapping of quantitative trait loci for yield and yield components in spring wheat (Triticum aestivum L.). Theor. Appl. Genet. 117: 595-608 Doyle J J, Doyle J L (1990): Isolation of plant DNA from fresh tissue. Focus 12: 13-15 Evanno G, Regnaut S, Goudet J (2005): Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14: 2611-2620 Ganal M W, Röder M S (2007): Microsatellite and SNP markers in wheat breeding. In: Varshney R.K. and Tuberosa R. (eds.), Genomics assisted crop improvement: genomics applications in crops, Springer, The Netherlands, 2:1-24 Groos C, Robert N, Bervas E, Charmet G (2003): Genetic analysis of grain protein-content, grain yield and thousand-kernel weight in bread wheat. Theor. Appl. Genet. 106: 1032-1040 Kobiljski B, Denci S, Hristov N, Mladenov N, Quarrie S, Stephenson P, Kirby J (2007): Potential uses of microsatellites in marker-assested selection for improved grain yield in wheat. In: Buck H T, Nisi J E, Salomón N. (eds.), Developments in Plant Breeding, Wheat Production in Stressed Environments, 729-736 Kobiljski B, Quarrie S, Denci S, Kirby J, Iveges M (2002): Genetic diversity of the Novi Sad wheat core collection revealed by microsatellites. Cell. Mol. Biol. Lett. 7: 685694 Kuchel H, Williams K J, Langridge P, Eageles H A, Jefferies S P (2007): Genetic dissection of grain yield in bread wheat. I. QTL analysis. Theor. Appl. Genet. 115: 10291041

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Association Analysis between Microsatellite Markers and Agronomically Important Traits in Wheat

Ljiljana Brbakli · Ankica Kondi-Spika · Dragana Trkulja · Borislav Kobiljski

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia

Summary: Association analysis is a new approach in wheat genetic studies that enables a more successful implementation of marker assisted selection (MAS) in breeding programs. In this paper, 96 hexaploid wheat genotypes were used for phenotype evaluation during two years, and molecular analysis using four microsatellite markers (Simple Sequence Repeat - SSR), aiming at establishing association between marker and trait. Based on molecular data at four analyzed microsatellite loci (Xgwm11, Xgwm428, Xpsp3200, Xpsp3071), a total of 31 allelic forms were detected. The results of association analysis have indicated four statistically significant associations. Markers GWM428 and GWM11 had significant effects on the phenotypic variability of heading and flowering time during both years, which enables their practical application through MAS. Key words: agronomic traits, association analysis, SSR, polymorphism, wheat

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511 Ispitivanje DNK polimorfizma lucerke primenom RAPD markera Molekularna genetika / Molecular Genetics Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 511-516 originalni naucni rad / original research article

Ispitivanje mogunosti primene RAPD markera u detekciji DNK polimorfizma sorti lucerke

Nevena Nagl Ksenija Taski-Ajdukovi Goran Bara Dragan Mili Slobodan Kati

primljeno / received: 20.04.2010. preraeno / revised: 01.06.2010. prihvaeno / accepted: 03.06.2010. © 2010 IFVC

Izvod: Iako je lucerka najznacajnija gajena biljka za stocnu ishranu u umerenim klimatskim regionima, postizanje znacajnih napredaka u oplemenjivanju je veoma komplikovano zbog njene slozene genetske konstitucije. Usled alogamije i tetraploidije sinteticke sorte lucerke se karakterisu veoma izrazenom varijabilnosu individua unutar same sorte. Poznavanje stepena genetske varijabilnosti sorti i populacija sa pozeljnim agronomskim svojstvima moglo bi znacajno biti unapreeno upotrebom molekularne genetske analize. Stoga je cilj ovog rada bilo ispitivanje mogunosti RAPD markera da detektuju varijacije unutar populacije kod pet sorti lucerke razlicitog porekla. Predstavljeni su rezultati pocetnog ispitivanja 40 slucajnih RAPD prajmera u cilju ispitivanja njihovih mogunosti da sintetisu PCR produkte koji bi omoguili kako detekciju polimorfizma izmeu ispitivanih sorti, tako i unutar njih. Kljucne reci: DNK polimorfizam, Medicago sativa, PCR, RAPD

Uvod Lucerka (Medicago sativa) je najznacajnija gajena biljka za stocnu ishranu u podrucjima sa umerenom klimom. Meutim, uprkos njenom izuzetnom ekonomskom znacaju, napredak u oplemenjivanju kultivisanih formi ove biljne vrste se veoma tesko postize usled njene bioloske i genetske kompleksnosti. Lucerka je autotetraploidna, stranooplodna, entomofilna vrsta sa specificnom graom cveta. Zbog ovih osobina u oplemenjivanju lucerke se koriste relativno jednostavne metode oplemenjivanja (Kati i sar. 2007), sto za rezultat ima veoma visok stepen genetske raznolikosti izmeu pojedinacnih biljaka unutar populacija. Usled toga, genetska struktura sorti lucerke je znatno nepreciznija u odnosu na fiksirane kultivisane forme kao sto su inbred linije ili hibridi, sto znatno otezava njihovu preciznu geneticku karakterizaciju. Iako je takav cilj za sada tesko ostvarljiv, neophodno je preduzeti korake prema njegovom postizanju iz nekoliko razloga: 1) omoguila bi se brza, pouzdana i efikasna diferencijacija sorti, 2) dobila bi se saznanja o genetskoj srodnosti i udaljenosti ekotipova

N. Nagl ) ( · K. Taski-Ajdukovi · D. Mili · S. Kati Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] G. Bara Prirodno-matematicki fakultet Univerziteta u Novom Sadu, Departman za biologiju i ekologiju, Trg Dositeja Obradovia 3, 21000 Novi Sad, Srbija

lucerke sa pozeljnim agronomskim svojstvima, 3) unapredilo bi se znanje o populacionoj genetici vrste a samim tim i njenoj potencijalnoj primeni u oplemenjivanju. Do sada je za odreivanje genetske varijabilnosti i karakterizaciju germplazme gajenog bilja razvijeno nekoliko metoda, od kojih su mnogi primenjeni i u istrazivanjima na lucerki. Kao prvi korisen je metod detekcije polimorfizma izoenzima za utvrivanje komponenti genetske varijabilnosti unutar i izmeu analiziranih populacija (Quiros 1983, Jenczewski et al. 1999). Meutim, stepen polimorfizma koji je detektovan na ovaj nacin nije bio dovoljan, kako za identifikaciju komponenti ispitivane germplazme, tako i za odreivanje stepena njihove srodnosti. Kao prvi metod za detekciju DNK polimorfizma lucerke korisen je polimorfizam duzine restrikcionih fragmenata, odnosno RFLP (Restriction Fragment Length Polymorphism) (Botstein et al. 1980) koji, iako je upotrebljen u odreenom broju istrazivanja (Brummer et al. 1993, Kidwell et al. 1994, Pupilli et al. 1996) nije mogao biti primenjen u ispitivanjima sa velikim brojem uzoraka, usled potrebe za velikom kolicinom DNK, kao i dugotrajnom i komplikovanom procedurom koju je podrazumevao. Sposobnost oligonukleotida duzine 8-10 baznih parova da tokom PCR reakcije omogui sintezu produkata amplifikacije razlicitih duzina,

Ovo istrazivanje je finansirano od strane Ministarstva za nauku i tehnoloski razvoj Republike Srbije

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dovela je do razvitka RAPD (Random Amplifed Polymorphic DNA) markera (Williams et al. 1990, Welsh & McClelland 1990). Ovaj tip dominantnih markera omoguuje veoma laku i jednostavnu detekciju DNK polimorfizma na velikom broju uzoraka, sto je uslovilo njegovu siroku primenu u populacionoj genetici i marker asistiranoj selekciji. U istrazivanjima na lucerki, RAPD markeri su do sada koriseni u ispitivanjima kolekcija razlicite germplazme (Echt et al. 1991, McCoy & Echt 1993), konstrukciji genetskih mapa (Echt et al. 1993, Yu & Pauls 1993), intra- i interspecies varijabilnost jednogodisnjih vrsta roda Medicago (Brummer et al. 1995, Crochemore et al. 1996), kao i ispitivanju polimorfizma tetraploidne lucerke (Mengoni et al. 2000, Gherardi et al. 1998). Iako se u molekularnim istrazivanjima na razlicitim vrstama iz roda Medicago koriste i drugi tipovim markera, kao sto su AFLP (Riday et al. 2003)

i mikrosateliti (Diwan et al. 2000, Sledge et al. 2005), RAPD markeri, zbog jednostavnosti upotrebe i niske cene imaju jos uvek veliki potencijal za primenu u ovoj vrsti istrazivanja. Imajui u vidu krajnji cilj, odreivanje mogunosti primene RAPD markera u selekcionom materijalu oplemenjivackog programa lucerke Instituta za ratarstvo i povrtarstvo u Novom Sadu, cilj ovog rada je bilo ispitivanje mogunosti nasumicno odabranih RAPD prajmera da sintetisu dovoljno polimorfnih PCR produkata koji bi u narednim istrazivanjima omoguili procenu DNK polimorfizma kako izmeu ispitivanih sorti, tako i unutar njih. Materijal i metod rada Kao materijal u istrazivanju odabrano je pet sorti tetraploidne lucerke razlicitog geografskog

Tabela 1. Rezultati ispitivanja prajmera X kita Table 1. Results of X kit primer testing Prajmer Primer X 01 X 02 X 03 X 04 X 05 X 06 X 07 X 08 X 09 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 Sorta Variety 3. Zuzana 0 0 + 0 + 0 + + + +/+ + 0 + + + + 0 0 0

1. Banat 0 0 + 0 + + + + + + + + 0 + + + + 0 0 0

2. Ghareh 0 0 + 0 + 0 + + + + + + 0 + + + + 0 0 0

4. Pecy 0 0 0 0 0 0 + + + +/+/+ 0 + + +/+ 0 0 0

5. RSI 20 0 0 0 0 0 0 + + + +/+ + 0 +/+ + + 0 0 0

(+) polimorfne trake, (+/-) neujednaceni produkti reakcije, (0) nema produkata reakcije

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porekla: 1) NS Banat ZMS II iz Srbije, 2) Ghareh Yon Geh iz Irana, 3) RSI 20 iz Spanije, 4) Pecy iz Francuske i 5) Zuzana iz Ceske. Sve sorte su se znacajno razlikovale u prinosu i komponentama prinosa (Mili 2007). Kod svake sorte su uzorci uzeti sa deset odabranih biljaka iz kojih je kasnije izolovana DNK. Za pocetnu fazu ispitivanja, predstavljenu u ovom radu, ispitivana su po cetiri nasumicno odabrana uzorka od svake sorte. DNK je izolovana iz listova lucerke, prema metodu Somma (2004), dok je za analizu DNK polimorfizma ispitano 40 RAPD prajmera sa 10 nukleotida, iz ROTH®GmbH kitova X i Y. Lancana reakcija polimeraze se odvijala u zapremini od 25 l koja je sadrzavala: 2,5 l pufera (Fermentas), 0,2 mM dNTP, 1,5 mM MgCl2, 0,5 M prajmera, 2 jedinice Taq polimeraze (Fermentas) i 40 ng DNK. Za amplifikaciju produkata reakcije korisen je Biometra Tpersonal PCR aparat, dok

se amplifikacija odvijala po sledeem programu: pocetna denaturacija na 94ºC u trajanju od 4 min, zatim 40 ciklusa sa 94ºC 2 min, 36ºC 1 min i 72ºC 2 min, sa krajnjom elongacijom od 10 min na 72 ºC. Produkti reakcije su analizirani na 1,7% agaroznom gelu sa 0,005% etidijum bromida, pod UV svetlom. Rezultati i diskusija Mogunost detekcije polimorfizma sorti lucerke je ispitivana pomou slucajno odabranih RAPD prajmera. Ispitivanjem prajmera iz kita X (Tab. 1) ustanovljeno je da se u reakcijama sa sedam prajmera (X01, X02, X03, X13, X18, X19 i X20) nisu mogli dobiti produkti reakcije ni kod jedne sorte. U reakcijama sa sest prajmera (X07, X08, X09, X12, X15 i X17) dobijeni su jasno vidljivi produkti amplifikacije koji su bili polimorfni

Tabela 2. Rezultati ispitivanja prajmera Y kita Table 2. Results of Y kit primer testing Prajmer Primer Y01 Y02 Y03 Y04 Y05 Y06 Y07 Y08 Y09 Y10 Y11 Y12 Y13 Y14 Y15 Y16 Y17 Y18 Y19 Y20 Sorta Variety 3. Zuzana +/+ +/+/+ + +/+ + + 0 + 0 + +/+/+/+ 0

1. Banat +/+ +/+/+ + +/+/+ + + 0 + 0 + +/+/+ 0

2. Ghareh +/+ +/+/+/+ +/+/+ + + 0 + 0 + +/+/+/+ 0

4. Pecy +/+ +/+/+ + +/+/+ + + 0 + 0 + +/+/+/+/0

5. RSI 20 +/+ +/+/+ + +/+/+ + 0 + 0 + +/+/+/+ 0

(+) polimorfne trake, (+/-) neujednaceni produkti reakcije, (-) uniformni produkti reakcije, (0) nema produkata reakcije

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na uzorcima svih ispitivanih sorti, dok se pomou preostalih sedam prajmera iz kita (X03, X05, X06, X10, X11, X14 i X16) vidljiv polimorfizam mogao detektovati samo kod pojedinih sorti ili njihovih pojedinacnih uzoraka. Od ispitivanih

sorti, najvei broj polimorfnih prajmera detektovan je kod sorte Banat (13), dok ih je najmanje imala sorta Pecy (7). Ispitivanjem mogunosti detekcije polimorfizma RAPD prajmera iz Y kita (Tab. 2)

Slika 1. Produkti amplifikacije prajmera Y10 kod sorti lucerke: Banat (3,6,8,9), Ghareh (13,16,18,19), Zuzana (23,26,28,29), Pecy (33,36,38,39), RSI 20 (43,46,48,49), GeneRulerTM 100bp DNA Ladder Plus (100 bp) Figure 1. Amplification products of primer Y10 in alfalfa varieties: Banat (3,6,8,9), Ghareh (13,16,18,19), Zuzana (23,26,28,29), Pecy (33,36,38,39), RSI 20 (43,46,48,49), GeneRulerTM 100bp DNA Ladder Plus (100 bp)

Slika 2. Produkti amplifikacije prajmera Y13 kod sorti lucerke: Banat (3,6,8,9), Ghareh (13,16,18,19), Zuzana (23,26,28,29), Pecy (33,36,38,39), RSI 20 (43,46,48,49), GeneRulerTM 100bp DNA Ladder Plus (100 bp) Figure 2. Amplification products of primer Y13 in alfalfa varieties: Banat (3,6,8,9), Ghareh (13,16,18,19), Zuzana (23,26,28,29), Pecy (33,36,38,39), RSI 20 (43,46,48,49), GeneRulerTM 100bp DNA Ladder Plus (100 bp)

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ustanovljeno je da u reakcijama sa tri prajmera (Y12, Y14 i Y20) nije bilo mogue dobiti produkte reakcije, dok je sa jedanaest prajmera (Y01,Y03, Y04, Y05, Y07, Y08, Y09, Y16, Y17, Y18 i Y19) bilo mogue dobiti polimorfne trake samo kod pojedinih sorti ili njihovih pojedinacnih uzoraka. Polimorfni produkti reakcije kod svih ispitivanih sorti su dobijeni u reakcijama sa sest prajmera (Y02, Y06, Y10, Y11, Y13 i Y15) (Sl. 1 i 2). Polimorfizam svih uzoraka sorti Banat i Zuzana je ustanovljen kod reakcija sa devet prajmera, dok se kod uzoraka sorti Ghareh, Pecy i RSI 20 polimorfizam mogao detektovati nakon reakcija sa osam prajmera. U reakciji uzoraka sorte Banat sa prajmerom Y08, kao i uzoraka sorte Zuzana i RSI 20 sa prajmerom Y17, dobijeni su uniformni produkti reakcije. Od 40 ispitivanih prajmera, stabilna i ponovljiva manifestacija DNK polimorfizma kod svih uzoraka ispitivanih sorti se mogla uociti nakon reakcija sa njih trinaest. Reakcije sa 18 prajmera su kao rezultat imale produkte koji su se mogli detektovati samo kod uzoraka pojedinih sorti, pojedinacnih uzoraka ili su bili nezadovoljavajue jasnoe i ponovljivosti. Reakcije sa devet prajmera nisu dale nikakav produkt. Iako e za definitivne zakljucke o stepenu polimorfizma unutar i izmeu sorti, kao i velicini i broju dobijenih fragmenata biti potrebni podaci analize svih uzoraka odabranih sorti, dobijeni preliminarni rezultati amplifikacije pokazuju da velicina i broj produkata reakcije moze znacajno varirati, kao i da postoji verovatnoa da je stepen DNK polimorfizma unutar i izmeu sorti veoma visok. Postojanje polimorfizma unutar sorti ili ekotipova lucerke je do sada u nekoliko navrata navedeno u literaturi (Gherardi et al. 1998, Tucak et al. 2008) sto je posledica kako njenog nacina selekcije tako i njene teraploidne prirode (Mengoni et al. 2000, Noeparvar et al. 2008). Zakljucak Cinjenica da je od nasumicno odabranih RAPD prajmera samo deo njih dao stabilne i polimorfne produkte reakcije, pokazuje da je sprovoenje ovakve vrste preliminarnih istrazivanja veoma korisno, a prethodilo bi bilo kakvom razvoju opseznog programa primene RAPD markera u oplemenjivanju lucerke. Mogunosti izdvajanja polimorfnih prajmera omoguava njihovu kasniju brzu i efikasniju primenu u razlicitim programima marker asistirane selekcije. RAPD markeri mogu biti veoma korisni u ispitivanjima DNK polimorfizma lucerke, sto je

rezultat njihove sposobnosti da detektuju varijabilnost u razlicitim delovima genoma. Iako se, usled slabe ponovljivosti i dominantne prirode, ne mogu koristiti za pouzdanu identifikaciju genotipova, mogu biti veoma korisni u ispitivanju genetske srodnosti i udaljenosti populacija i ekotipova, narocito u kombinaciji sa kodominantnim tipovima markera, kao sto su mikrosateliti. Literatura

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Tucak M, Popovi S, Cupi T, Grljusi S, Bolari S, Kozumplik V (2008): Genetic diversity of alfalfa (Medicago spp.) estimated by molecular markers and morphological characters. Periodicum Biologorum 110: 243-249 Welsh J, McClelland M (1990): Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acids Res. 18: 72137218 Williams J G K, Kubelik A R, Livak K J, Rafalski J A, Tingey S V (1990): DNA polymorphism amplifed by arbitrary primers are useful as genetic markers. Nucl. Acids Res. 18: 6531-6535 Yu P, Pauls K P (1993): Segregation of random amplifed polymorphic DNA markers and strategies for molecular mapping in tetraploid alfalfa. Genome 36: 844-851

Research on the Possibility of RAPD Markers Application in Detecting DNA Polymorphism of Alfalfa Varieties

Nevena Nagl1 Ksenija Taski-Ajdukovi1 Goran Bara2 Dragan Mili1 Slobodan Kati1

1 Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia University of Novi Sad, Faculty of Sciences, Department for Biology and Ecology, 21000 Novi Sad, Serbia

2

Summary: Although alfalfa is the most important forage crop grown in the temperate regions, breeding improvements are not easily achieved due to high genetic complexity of this species. Because of allogamy and tetraploidy, cultivated alfalfa synthetic varieties have a high degree of genetic diversity among individual plants in populations. The knowledge of the extent of genomic variability in cultivars bearing desirable agronomic traits is an area that could benefit from molecular genetic analyses. Therefore, the aim of the present work was to evaluate the ability of RAPD markers to detect intra-population variation in five alfalfa varieties. Presented here are the first results of investigating 40 randomly selected RAPD primers in order to establish whether they were able to synthesize PCR products, which would enable detection of polymorphism between the selected varieties and inside them as well. Key words: DNA polymorphism, Medicago sativa, PCR, RAPD

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www.nsseme.com/journal.html

Uticaj cuvanja i zastite semena na nicanje suncokreta

517 Semenarstvo / Seed Science Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 517-522 originalni naucni rad / original research article

Uticaj hibrida, nacina cuvanja i zastite semena na nicanje suncokreta

Jelena Mra Branislav Ostoji Sinisa Prole Goran Joki Daliborka Butas Karlo ilvesi Vladimir Miklic

primljeno / received: 19.04.2010. preraeno / revised: 20.05.2010. prihvaeno / accepted: 24.05.2010. © 2010 IFVC

Izvod: Ujednaceno nicanje u poljskim uslovima direktno odreuje broj biljaka po hektaru, sto je jedan od najvaznijih uslova za postizanje visokog prinosa suncokreta. Tokom 2007. i 2008. na oglednom polju Instituta za ratarstvo i povrtarstvo iz Novog Sada ispitivan je uticaj hemijskih tretmana na poljsko nicanje tri komercijalna hibrida suncokreta, cuvanih u razlicitim uslovima skladistenja. U proseku, najvea vrednost ispitivanog parametra bila je kod hibrida H1 (88,79%) i hemijskog tretmana fludioksonil + metalaksil + imidakloprid (87,71%). Takoe, najvee poljsko nicanje u proseku pokazalo je seme koje je cuvano u skladistu (87,92%). Kod interakcije uslovi cuvanja x hemijski tretman najbolje rezultate u uslovima nicanja u polju (90,18%) imalo je seme tretirano fludioksonil + metalaksil + imidaklopridom koje je cuvano godinu dana u skladistu, a kod interakcije uslovi cuvanja x genotip seme hibrida H1 i to sveze tretirano seme (91,82%) i seme cuvano godinu dana u skladistu (90,48%). Kod interakcije hemijski tretman x genotip najvee poljsko nicanje je takoe pokazalo seme hibrida H1 tretirano fludioksonil + metalaksil + imidaklopridom (91,84%). Kljucne reci: genotip, hemijski tretman, poljsko nicanje, seme suncokreta, uslovi cuvanja

Uvod Starost do koje seme ne gubi klijavost i moze se koristiti za setvu i proizvodnju zavisi od genetske konstitucije i sorte (Tomi i sar. 1998). Uslovi u kojima se seme cuva, kao i sama duzina cuvanja imaju veliki uticaj na kvalitet semena. Cuvanjem semena pri visim temperaturama i veoj relativnoj vlaznosti vazduha moze doi do opadanja semenskih kvaliteta (Simi i sar. 2006). Pad vigora kod semena suncokreta nakon cetiri godine cuvanja moze iznositi preko 50%, pri cemu je vei gubitak zabelezen na visim temperaturama (Simi i sar. 2005). Da bi u praksi ostvarili visoke prinose pored izbora dobrog hibrida, primene optimalne agrotehnike i navodnjavanja potrebno je za setvu koristititi seme visokih i poznatih (deklarisanih) kvaliteta (Milosevi i sar. 1996). Klijavost semena je osobina koja najcese sluzi kao pokazatelj kvaliteta semena, a predstavlja nicanje i razvoj iz klice semena takve grae, koje ima sposobnost da stvori normalnu biljku u povoljnim uslovima (Milosevi & Zlokolica 1996). Klijavost dobijena

J. Mra ) ( B. Ostoji S. Prole G. Joki D. Butas K. ilvesi V. Miklic Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

u optimalnim laboratorijskim uslovima primenom standardne metode moze odstupati od klijavosti dobijene u polju. Test klijavosti ne odrazava uvek poljsku klijavost ako su uslovi u polju manje povoljni (Vertucci 1991). Pokazatelji vitalnosti semena (energija klijanja, klijavost semena i nicanje u poljskim uslovima) presudno i direktno odreuju broj biljaka po hektaru, koji je jedan od tri osnovne komponente prinosa u biljnoj proizvodnji. Osim toga, kvalitet semena utice i na brzinu i ujednacenost nicanja, kao i na tempo pocetnog rasta biljke (Crnobarac 1992). Partije semena sa visokom klijavosu cesto se razlikuju po poljskom nicanju kada se poseju u isto vreme, na istom polju, a mogu da se razlikuju po istom pokazatelju i posle skladistenja u istim uslovima. Ove razlike izazvane su drugom komponentom kvaliteta semena ­ vigorom semena (Te Crony 1982). Zbog toga se dodatna informacija o kvalitetu semena u tom slucaju dobija primenom nekog od vigor testova (Opoku et al. 1996, Tomer & Maguire 1990). Setvom partija semena sa visokim vigorom dobijaju se zdravije,

Ovo istrazivanje je deo projekta broj 20080: Stvaranje visoko produktivnih genotipova suncokreta (Helianthus annuus L.) (2008-2011) Ministarstva za nauku i tehnoloski razvoj Republike Srbije / This research results from the project 20080: Development of highly productive sunflower (Helianthus annuus L.) genotypes (2008-2011) financed by the Ministry of Science and Technological Development of the Republic of Serbia

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snaznije biljke koje bolje podnose bolesti i druge stresove, a na kraju daju vei prinos (Horlings et al. 1991). Pozitivna veza izmeu vigora i poljske klijavosti, meutim, ne znaci nuzno i visi prinos (Opoku et al. 1996). Ova nedoslednost se uglavnom povezuje sa povoljnim uslovima u kojima su istrazivanja sprovoena (Kulik & Yaklich 1982). Seme je jedan od najvaznijih faktora za zivotnu sposobnost biljke i korisenje kvalitetnog, zdravog, krupnog, zivotno sposobnog semena je izuzetno vazno da se odrzi optimalan sklop biljaka (Ahmad 2001). Cilj ovog istrazivanja je bio da se utvrdi efekat razlicitih hemijskih tretmana, kao i nacina cuvanja semena na nicanje razlicitih genotipova suncokreta u polju. Materijal i metod rada Istrazivanja su vrsena tokom 2007. i 2008. sa semenom tri hibrida suncokreta stvorena u Institutu za ratarstvo i povrtarstvo iz Novog Sada (H1, H2 i H3). Za zastitu semena koriseni su sledei fungicidi: benomil, metalaksil i fludioksonil, kao i insekticidi: tiametoksam i imidakloprid, a za istrazivanje su odabrane sledee kombinacije: kontrola (netretirano seme), benomil + metalaksil (B+M), fludioksonil + metalaksil (F+M), fludioksonil + metalaksil + tiametoksam (F+M+T) i fludioksonil + metalaksil + imidakloprid (F+M+I).

Primenjena je standardna doza preparata koju preporucuju proizvoaci. Za ispitivanje nicanja biljaka ogled je bio postavljen na oglednom polju Instituta za ratarstvo i povrtarstvo na Rimskim Sancevima po split­plot metodu u cetiri ponavljanja na zemljistu tipa karbonatni cernozem u uslovima navodnjavanja. Osnovna parcelica se sastojala od cetiri reda duzine 4,20 m. Setva je obavljena rucno, na rastojanju 70 cm x 30 cm, sa po tri semena u kuicu. Ova vrednost je utvrena prebrojavanjem izniklih biljaka nakon potpunog nicanja suncokreta. Za proveru uticaja uslova cuvanja na nicanje suncokreta deo tretiranog semena je cuvan u skladistu, a drugi deo u hladnoj komori. U hladnoj komori temperatura od 5ºC i relativna vlaznost vazduha od 80% bile su konstantne. U skladistu koje se koristi za cuvanje komercijalnog semena uslovi su zavisili od spoljasnjih uslova, a seme je u oba slucaja cuvano u papirnim vreama. Poljsko nicanje je ispitano neposredno nakon tretiranja i godinu dana nakon tretiranja i cuvanja semena u skladistu, odnosno hladnoj komori. Dobijeni rezultati statisticki su obraeni analizom varijanse trofaktorijalnog split­split-plot ogleda (faktor A ­ genotip, faktor B ­ hemijski tretman, faktor C ­ uslovi cuvanja) pomou statistickog paketa Statistica 8, a rangiranje znacajnosti dobijenih razlika utvreno je testom najmanje znacajne razlike (LSD), na pragovima znacajnosti od 1% i 5% (Mead et al. 1996).

Grafikon 1. Poljsko nicanje suncokreta Graph 1. Sunflower field emergence

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Rezultati i diskusija Analiza varijanse ispitivanih parametara ukazala je na postojanje statisticki visoko znacajnog uticaja genotipa i uslova cuvanja (verovatnoa Ftesta: Fpr=<0,001) na poljsku klijavost i nicanje biljaka. Tretiranje semena fungicidima i insekticidima znacajno je uticalo na poljsku klijavost (Fpr=0,042), dok interakcije genotip x hemijski tretman, genotip x uslovi cuvanja i hemijski tretman x uslovi cuvanja nisu bile znacajne. Rezultati ispitivanja uticaja genotipa, hemijskog tretmana i uslova cuvanja na nicanje suncokreta prikazani su na grafikonu 1. U proseku, najvei procenat izniklih biljaka imao je hibrid H1 (88,79%). Ova vrednost bila je vea u odnosu na hibrid H2 za 4,66%, a u odnosu na hibrid H3 za 6,28%. Ove razlike su statisticki vrlo znacajne, dok izmeu hibrida H2 i H3 nije bilo statisticki znacajnih variranja. Rezultati autora Crnobarac & Marinkovi (1994) potvruju da ukupna klijavost semena zavisi od genotipa, a do slicnih rezultata takoe je dosla ukanovi (1999) koja zakljucuje da je za promene osobina tokom cuvanja samooplodnih linija i hibridnih kombinacija kukuruza najvazniji faktor bio genotip. Kod ispitivanih hemijskih tretmana najvee poljsko nicanje biljaka u proseku bilo je kod tretmana F+M+I (87,71%). Ova vrednost je bila statisticki visoko znacajno vea nego u kontroli (za 4,79%), a znacajno vea nego kod tretmana B+M (za 3,67%). Takoe, vrednost ispitivanog parametra kod tretmana F+M+T (86,29%) bila je znacajno vea nego u

kontroli (za 3,37%). Variranja izmeu nicanja biljaka kod ostalih ispitivanih hemijskih tretmana nisu bila statisticki znacajna. Cilj cuvanja je da se obezbedi sto bolje ocuvanje fizioloskih i fizickih karakteristika semena, jer losi uslovi cuvanja dovode do pada zivotne sposobnosti semena (ukanovi & Sabovljevi 2001). Nicanje biljaka u polju iz semena cuvanog u skladistu (87,92%) i sveze tretiranog semena (86,97%) bilo je statisticki visoko znacajno vee nego semena cuvanog u hladnoj komori (80,54%). Posmatrajui interakciju genotip x hemijski tretman moze se uociti da je procenat izniklih biljaka hibrida H1 bio najvei kod tretmana F+M+I (91,84%) i znacajno vei nego u kontroli (za 6,08%) i kod tretmana F+M+T (za 5,48%). Kod hibrida H3 najvea vrednost ispitivanog parametra zabelezena je kod tretmana F+M+T (85,62%), a bila je znacajno vea nego u kontroli (za 5,61%) i kod tretmana B+M (za 6,40%). Takoe, vrednost zabelezena kod tretmana F+M+I (85,17%) bila je znacajno vea nego u kontroli (za 5,16%) i kod tretmana B+M (za 5,95%). Suprotno rezultatima ovog istrazivanja, Stankovi & Medi (1997) su na osnovu laboratorijskog ispitivanja energije klijanja i klijavosti semena suncokreta i kukuruza tretiranog insekticidima zakljucili da kod svih tretmana dolazi do smanjenja klijavosti koje nije u svim slucajevima bilo znacajno. Isti autori navode da su insekticidi karbosulfan i imidakloprid najmanje negativno uticali na kvalitet semena, pri cemu su svi depresivno delovali nakon cuvanja u trajanju od jedne godine. Kod hibrida H2

Grafikon 2. Poljsko nicanje suncokreta kod interakcije genotip x hemijski tretman Graph 2. Sunflower field emergence at interaction genotype x chemical treatment

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variranja izmeu ispitivanih hemijskih tretmana nisu bila statisticki znacajna (Graf. 2). Mra i sar. (2009) su dosli do istih rezultata ispitujui laboratorijsku klijavost semena hibrida suncokreta Sremac u razlicitim uslovima cuvanja. Nicanje biljaka korisenjem sveze tretiranog semena i semena cuvanog u skladistu hibrida H1 i H2 kod interakcije genotip x uslovi cuvanja bilo je statisticki visoko znacajno vee nego semena cuvanog u hladnoj komori (Graf. 3). Kod hibrida H3 vrednost ispitivanog parametra semena cuvanog u hladnoj komori bilo je visoko znacajno najmanja (77,65%), dok je kod semena cuvanog

u skladistu (87,17%) bila znacajno vea nego kod sveze tretiranog semena (za 4,46%). Slicno ovim rezultatima i seme seerne repe koje je cuvano u nekontrolisanim uslovima bilo je boljeg kvaliteta u odnosu na ono cuvano u laboratoriji na 20°C i vlaznosti vazduha od 65% do 75% (Raji i sar. 2005). Sa druge strane, Simi i sar. (2006) su dosli do zakljucka da su uslovi cuvanja semena kukuruza, soje i suncokreta na temperaturi od 12°C i relativnoj vlaznosti vazduha od 60% imali povoljniji uticaj na kvalitet semena od cuvanja pri temperaturi od 25°C i relativnoj vlaznosti vazduha od 75%. Takoe, Simi i sar. (2005) konstatuju

Grafikon 3. Poljsko nicanje suncokreta kod interakcije genotip x uslovi cuvanja Graph 3. Sunflower field emergence at interaction genotype x storage conditions

Grafikon 4. Poljsko nicanje suncokreta kod interakcije hemijski tretman x uslovi cuvanja Graph 4. Sunflower field emergence at interaction chemical treatment x storage conditions

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da duzim cuvanjem semena na visim temperaturama i veoj relativnoj vlaznosti vazduha dolazi do pada kvaliteta semena. Tati i sar. (2008) su takoe utvrdili razlike u kvalitetu semena u zavisnosti od nacina cuvanja i duzine perioda cuvanja semena soje. Iz interakcije hemijski tretman x uslovi cuvanja moze se uociti da je najvee vrednosti poljskog nicanja pokazalo seme cuvano u skladistu kod svih ispitivanih hemijskih tretmana, osim kod tretmana F+M+T gde je to bilo kod sveze tretiranog semena (89,88%). Najmanje vrednosti ispitivanog parametra imalo je seme koje je cuvano u hladnoj komori (Graf. 4). Ova vrednost je bila visoko znacajno najniza u kontroli (74,90%), dok je kod tretmana F+M (79,32%) bila visoko znacajno niza nego kod semena cuvanog u skladistu (za 9,17%), a kod tretmana F+M+T (82,29%) u odnosu na sveze tretirano seme (za 7,59%). Do slicnog zakljucka dosli su Baca i sar. (2008) koji u cetvorogodisnjem istrazivanju uticaja insekticida imidakloprida i tiametoksama nisu dosli do rezultata o postojanju negativnog uticaja hemijskog tretmana na poljsku klijavost semena kukuruza. Zakljucci Na osnovu rezultata proucavanja uticaja hibrida, tretiranja i nacina cuvanja semena na nicanje biljaka suncokreta mogu se izvesti sledei zakljucci: Hibrid H1 je imao najvei procenat izniklih biljaka. Razlika u odnosu na hibride H2 i H3 bila je statisticki vrlo znacajna, dok izmeu njih nije bilo znacajnih variranja. Seme tretirano preparatima fludioksonil + metalaksil + imidakloprid (F+M+I) bilo je sa najveim procentom izniklih biljaka. Seme cuvano u skladistu i sveze tretirano dalo je statisticki vise biljaka nego seme cuvano u hladnoj komori. Kod hibrida H1 interakcija genotip x hemijski tretman bila je najvea u varijanti fludioksonil + metalaksil + imidakloprid (F+M+I), a kod hibrida H3 u varijanti fludioksonil + metalaksil + tiametoksam (F+M+T). Interakcije genotip x hemijski tretman kod hibrida H2 nisu bile statisticki znacajne. Interakcija genotip x uslovi cuvanja bila je znacajno manja kod semena cuvanog u hladnoj komori u odnosu na druga dva tretmana (cuvanje u magacinu i sveze seme). Seme cuvano u skladistu pokazalo je najbolje rezultate u uslovima nicanja u polju u svim tretmanima, osim kada je zastieno preparatima fludioksonil + metalaksil + tiametoksam (F+M+T).

Sveze seme je najbolje reagovalo na ovu trojnu kombinaciju hemijske zastite. Dezinfekcija i dezinsekcija semena je najnepovoljnije uticala na seme cuvano u hladnoj komori. Na osnovu dobijenih rezultata u ovom istrazivanju moze se zakljuciti da je hladna komora nepovoljno uticala na klijavost semena proucavanih genotipova, sto je posledica poveane vlaznosti vazduha u komori u kojoj je ono cuvano. U nastavku ovih istrazivanja predmet e biti iznalazenje nacina da se ovaj problem resi. Literatura

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Effect of Hybrid, Storage Conditions and Seed Protection on Sunflower Field Emergence

Jelena Mra Branislav Ostoji Sinisa Prole Goran Joki Daliborka Butas Karlo ilvesi Vladimir Miklic

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia Summary: Seed emergence under field conditions decisively and directly determines the number of plants per hectare, which is one of three basic components of yield in the plant world. Influence of chemical treatment on field emergence of three commercial sunflower hybrids stored in different conditions was tested in 2007 and 2008 on experimental field of Institute of Field and Vegetable Crops in Novi Sad. On average, hybrid H1 had the highest value of field emergence (88.79%) and for chemical treatment fludioxonil + metalaxyl + imidacloprid (87.71%). Seed kept in common storage had the highest emergence value in field (87.92%). Seed treated with fludioxonil + metalaxyl + imidacloprid and stored for one year in common storage had the highest field emergence (90.18%). Considering interaction between storage conditions and genotype, hybrid H1 seed sown after chemical treatment had the highest field emergence (91.82%) and seed kept in common storage (90.48%). Hybrid H1 seed compared with other two had the highest field emergence treated with fludioxonil + metalaxyl + imidacloprid (91.84%). Key words: chemical treatment, field emergence, hybrid, storage conditions, sunflower seed

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Klijavost stocnog graska u uslovima zaslanjenosti

523 Semenarstvo / Seed Science Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 523-528 originalni naucni rad / original research article

Uticaj zaslanjenosti na klijanje i parametre porasta ponika stocnog graska (Pisum sativum L.)

Dusica Jovici Milka Vujakovi Mirjana Milosevi ura Karagi Ksenija Taski-Ajdukovi Maja Ignjatov Aleksandar Miki

primljeno / received: 16.04.2010. prihvaeno / accepted: 17.05.2010. © 2010 IFVC

Izvod: Seme stocnog graska (Pisum sativum L.) sadrzi veliku kolicinu proteina, amino kiselina, seera, ugljeno-hidrata, vitamina A i C, kalcijuma i fosfora, te zbog toga nalazi siroku primenu. Iako ima skromne zahteve za uspevanjem, stocni grasak se svrstava meu kulture koje su osetljive na povean sadrzaj soli u zemljistu. Ispitivanje je obuhvatilo osam sorti (Javor, Jantar, Partner, Kristal, Pionir, Junior, Trezor, Dukat) stvorenih u Institutu za ratarstvo i povrtarstvo u Novom Sadu. Simulirani su uslovi zaslanjenosti tako sto su u podloge za naklijavanje dodati rastvori NaCl koncentracija 0, 50, 100 i 150 mM. Odreivani su klijavost semena, duzina ponika, masa svezeg i suvog ponika i masa 1000 semena. Od ispitivanih sorti, sorta Jantar se izdvojila kao najtolerantnija na povean sadrzaj soli u podlogama za naklijavanje u laboratorijskim uslovima. Kljucne reci: duzina ponika, klijavost, masa suvog ponika, masa svezeg ponika, masa 1000 semena, stocni grasak, zaslanjenost

Uvod Seme stocnog graska (Pisum sativum L.) sadrzi veliku kolicinu proteina, amino kiselina, seera, ugljeno-hidrata, vitamina A i C, kalcijuma i fosfora. Pre svega zbog velikog sadrzaja proteina, stocni grasak nalazi siroku primenu, posebno u proizvodnji stocne hrane. Zrno stocnog graska je sastavni deo kvalitetnih krmnih smesa, dok se nadzemni deo biljke moze koristiti kao kabasta stocna hrana (zelena krma, silaza) (Vuckovi 1999). Prednost stocnog graska takoe se ogleda u tome sto se mleveno zrno moze koristiti u ishrani bez prethodne termicke obrade, sto pojednostavljuje i pojeftinjuje proizvodnju hraniva (Karagi i sar. 2007). Takoe, stocni grasak ima veliku ulogu u obezbeivanju zemljista znacajnim kolicinama azota (25kg po jutru) zbog cega se cesto koristi i kao zelenisno ubrivo (Oelke et al. 1991). Zbog svoje siroke primene stocni grasak se gaji na velikim

D. Jovici ) ( M. Vujakovi . Karagi K. Taski-Ajdukovi M. Ignjatov A. Miki Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] M. Milosevi Ministarstvo poljoprivrede, vodoprivrede i sumarstva, Nemanjina 22­26, 11000 Beograd, Srbija

povrsinama u celom svetu. Iako ima skromne zahteve za uspevanjem, stocni grasak se svrstava meu kulture koje su osetljive na povean sadrzaj soli u zemljistu (Maas 1990). Sve cese suse u mnogim regionima sveta i upotreba neadekvatne vode za navodnjavanje dovele su do porasta oranicnih povrsina koje se suocavaju sa problemom zaslanjenosti. Zaslanjenost zemljista je jedan od najznacajnijih faktora koji ogranicava uspesnu proizvodnju gajenih biljaka i samim tim u velikoj meri utice na ekonomske gubitke (Joshi et al. 2009). Salinitet negativno utice na rast i razvoj biljaka stvaranjem osmotskog potencijala u spoljasnjoj sredini koji onemoguava normalno usvajanje vode (Khajeh-Hosseini et al. 2003). Drugi, ali ne manje bitan, negativan efekat zaslanjenog zemljista je toksicnost prevelike kolicine natrijumovih i hloridnih jona, sto dovodi do nedostatka kalcijuma i kalijuma i neravnoteze drugih hranljivih elemenata. Takoe, otezan rast i razvoj biljaka na zaslanjenom zemljistu nastaje usled inhibicije razlicitih enzima koja nastaje usled akumulacije soli u delovima elije. Oertli (1968) smatra da se visak soli nagomilava u apoplastu listova, sto za

Ovaj rad predstavlja rezultat Projekta TR 20090 pod nazivom ,,Multidisciplinarni pristup oplemenjivanja i proizvodnje semena krmnih biljaka za konvencionalne i nove upotrebe" (2008­2010) Ministarstva za nauku i tehnoloski razvoj Republike Srbije.

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posledicu moze imati isusivanje elija i gubitak turgora (Flowers & Yeo 1986, Speer & Kaiser 1991) ili osteenje povrsine elijskih membrana (Cramer et al. 1985, Speer & Kaiser 1991). Ovo su ujedno efekti kojima zaslanjena sredina nepovoljno utice i na klijavost semena (Kaya 2009). Takoe, u zaslanjenoj sredini dolazi do promena u dostupnosti rezervnih materija semena sto otezava klijanje (Machado et al. 2004). Imajui u vidu da je voda presudan faktor za zapocinjanje procesa klijanja semena, moze se rei da je otezano usvajanje vode glavni negativni efekat zaslanjenosti na klijanje. Zbog sve veih potreba u proizvodnji hrane sa jedne strane, te poveanja povrsina zaslanjenog zemljista sa druge strane, ispitivanje tolerantnosti biljaka na povean sadrzaj soli u zemljistu ima sve vei znacaj. Cilj rada je bio da se ispita klijavost semena stocnog graska, kao najvaznije komponente kvaliteta semena, u zaslanjenoj sredini i da se utvrdi koje sorte stocnog graska stvorene u Institutu za ratarstvo i povrtarstvo u Novom Sadu imaju najveu tolerantnost na prisustvo soli u zemljistu.

u laboratorijskim uslovima. Utvrivanje klijavosti semena izvrseno je u cetiri ponavljanja po sto semena. Kao podloga za naklijavanje korisen je pesak, nakvasen rastvorom NaCl koncentracija 0, 50, 100 i 150 mM. Inkubacioni period je trajao osam dana, na temperaturi od 20°C (ISTA 2010), nakon cega je utvrena klijavost semena, duzina i masa svezeg nadzemnog dela i korenovog sistema ponika graska. Masa suvog nadzemnog dela i korenovog sistema izmerena je nakon 24 h susenja biljnog materijala u susnici na temperaturi od 80°C. Rezultati su obraeni u statistickom programu Statistica 7, metodom analize varijanse. Znacajnost razlika utvrena je Dankanovim testom. Rezultati i diskusija Seme sorti Javor i Dukat imalo je istu vrednost klijavosti pri koncentraciji 0 mM NaCl (kontrola) i koncentraciji 50 mM NaCl, dok je klijavost svih ostalih ispitivanih sorti u kontroli bila vea u odnosu na klijavost dobijenu pri ostalim primenjenim koncentracijama NaCl (Graf. 1).

0 mM NaCl 50 mM NaCl 100 mM NaCl 150 mM NaCl

Grafikon 1. Uticaj zaslanjenosti (NaCl) na klijavost semena Graph 1. Effect of salinity (NaCl) on seed germination

Materijal i metod rada Ispitivano je osam sorti stocnog graska (Javor, Jantar, Partner, Kristal, Pionir, Junior, Trezor, Dukat) koje su stvorene u Odeljenju za krmno bilje Instituta za ratarstvo i povrtarstvo u Novom Sadu. Seme ispitivanih sorti je proizvedeno 2009. Ispitivanje uticaja zaslanjenosti na klijavosti semena i parametara porasta ponika obavljeno je

Klijavost semena u kontroli kretala se od 76% kod sorte Pionir do 95% kod sorte Kristal. Pri koncentraciji 50 mM NaCl klijavost kretala se od 73% kod sorte Pionir do 94% kod sorti Dukat i Kristal. Ispitivani parametar, pri koncentraciji od 100 mM NaCl, kretao se od 71% kod sorte Pionir do 87% kod sorti Junior i Dukat, dok se pri koncentraciji od 150 mM NaCl vrednosti klijavosti znacajno smanjuju i kreu se od 68%

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0 mM NaCl 50 mM NaCl 100 mM NaCl 150 mM NaCl

Grafikon 2. Uticaj zaslanjenosti (NaCl) na duzinu ponika Graph 2. Effect of salinity (NaCl) on lengh of seedlings

kod sorte Pionir do 84% kod sorte Dukat. Ovakvi rezultati su posledica otezanog usvajanja vode zbog prisustva natrijumovih i hloridnih jona i njihovog nakupljanja u eliji. Smanjenje klijavosti sa poveanjem koncentracije NaCl takoe su dobili Al-Thabet et al. (2004) i Radi i sar. (2007). Sorte Pionir (73%) i Jantar (77%) imale su najnizu klijvost pri najmanjoj koncentraciji NaCl, dok su sorte Dukat (89%) i Kristal (79%) imale najvisu klijavost pri najveoj koncentraciji NaCl. Najvisu klijavost pri najnizoj koncentraciji NaCl imale su sorte Kristal (95%) i Dukat (94%), a najnizu pri najveoj koncentraciji imale su Partner (68%), Javor (69%) i Jantar (69%). Kod sorti Dukat, Partner i Kristal izmeu klijavosti na podlozi sa koncentracijom 0 mM NaCl (kontrola) i klijavosti na podlozi sa koncentraciDuzina svezih klijanaca / Fresh seedlings weight (g)

jama 100 i 150 mM NaCl javila se statisticki znacajna razlika. Kod sorti Junior, Pionir, Trezor, Jantar i Javor nije bilo statisticki znacajnih razlika izmeu klijavosti na podlozi sa 0 mM NaCl i 100 mM NaCl, dok se statisticki znacajna razlika javila izmeu koncentracija 0 i 150 mM NaCl. Duzina ponika kod sorte Dukat pri koncentraciji od 0 mM NaCl imala je statisticki znacajnu visu vrednost u odnosu na duzinu ponika pri koncentraciji 100 mM NaCl i 150 mM NaCl. Kod svih drugih ispitivanih sorti statisticki znacajna razlika javila se izmeu koncentracija 0 mM NaCl i svih drugih primenjenih koncentracija (Graf. 2). Masa svezeg ponika kod sorti Junior, Pionir i Javor kod kontrole statisticki se znacajno razlikovala u odnosu na svezu masu ponika kod svih drugih primenjivanih koncentracija. Kod sorti

0 mM NaCl 50 mM NaCl 100 mM NaCl 150 mM NaCl

Grafikon 3. Uticaj zaslanjenosti (NaCl) na masu svezih ponika Graph 3. Effect of salinity (NaCl) on fresh seedlings weight

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0 mM NaCl 50 mM NaCl 100 mM NaCl 150 mM NaCl

Grafikon 4. Uticaj zaslanjenosti (NaCl) na masu suvog ponika Graph 4. Effect of salinity (NaCl) on dry seedlings weight

Partner, Trezor i Kristal javila se statisticki znacajna razliku izmeu kontrole i koncentracija 100 mM NaCl i 150 mM NaCl, dok su sorte Dukat i Jantar pokazivale statisticku znacajnu razliku samo sa najveom koncentracijom NaCl (Graf. 3). Smatra se da je smanjenje mase svezeg ponika sa poveanjem koncentracije NaCl posledica nakupljanja veih kolicina natrijumovih jona. Ovi joni negativno uticu na deobu elija, metabolizam i disbalans drugih hranljivih materija (Mer et al. 2000). Masa suvog ponika kod sorti Partner, Dukat, Pionir, Trezor, Kristal i Javor u konroli se statisticki znacajno razlikovala u odnosu na masu suvog ponika pri koncentracijama 100 mM NaCl i 150 mM NaCl. Kod sorte Junior masa suvog ponika u kontroli statisticki znacajno se razlikovala od mase suvog ponika pri svim drugim primenjivanim koncentracijama, dok se kod sorte Jantar masa suvog ponika u kontroli statisticki znacajno razlikovala

samo od mase suvog ponika pri koncentraciji od 150 mM NaCl (Graf. 4). Masa 1000 semena sorte Jantar (100,60 g) bila je statisticki znacajno niza u odnosu na masu 1000 semena ostalih ispitivanih sorti. Najveu vrednost mase 1000 semena imala je sorta Dukat (254,28 g) (Graf. 5). Sorta Dukat je imala i najveu vrednost za klijavost semena u kontroli. Ova konstatacija moze se objasniti cinjenicom da krupnije seme sadrzi veu kolicinu endosperma i na taj nacin obezbeuje klicu dovoljnom kolicinom hraniva u pocetnim fazama klijanja. Pozitivan uticaj krupnoe semena na klijavost utvrdili su autori Grieve & Francois (1992). S druge strane, sitnije seme, kao sto je kod sorte Jantar, ispoljilo je najveu tolerantnost na poveanje koncentracije NaCl sto se objasnjava cinjenicom da je sitnijem semenu potrebna manja kolicina vode da bi klijalo, pa samim tim usvaja i manju kolicinu natrijumovih i hloridnih jona koji negativno uticu na klijanje.

Grafikon 5. Masa 1000 semena Graph 5. 1000 seeds weight

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Joshi A, Dang H Q, Vaid N, Tuteja N (2009): Isolation of High Salinity Stress Tolerant Genes from Pisum sativum by Random Overexpression in Escherichia coli and Their Functional Validation. Plant Signal Behav. 4: 400-412 Kaya D M (2009): The Role of Hull in Germination and Salinity Tolerance in Some Sunflower (Helianthus annuus L.) Cultivars. Afr. J. Biotechnol. 8: 597-600 Karagi , Mihailovi V, Miki A, Kati S, Malidza G (2007): Tehnologija proizvodnje proteinskog graska, Zbornik radova Instituta za ratarstvo i povrtarstvo 43: 245-254 Khajeh-Hosseini, M, Powell A A, Bingham, I J (2003): The Interaction Between Salinity Stress and Seed Vigor During Germination of Soybean Seeds. Seed Sci. Technol. 31: 715-725 Maas E V (1990): Crop salt tolerance. In: Tanji K.K. (ed) Agricultural Salinity Assessment and Management. ASCE Manuals and Reports on Engineering No. 71, ASCE, NY, 262-304 Machado N N B, Saturnino S M, Bomfim D C and Custodio, C C (2004): Water stress induced by Mannitol and Sodium chloride in Soybean cultivars. Braz. Arch. Biol. Technol. 47: 521-529 Mer R K, Prajith P K, Pandya D H, Pandey A N (2000): Effect of Salts on Germination of Seeds and Growth of Young Plants of Hordeum vulgare, Triticum aestivum, Cicer arietinum and Brassica juncea. J. Agron. Crop. Sci. 185: 209-217 Oelke E A , Oplinger E S ; Hanson C V, Davis D W, Putnam D H, Fuller E I, Rosen C J (1991): Dry Field Pea, Alternative Crops Manual. Oertli J J (1968): Extracellular salt accumulation: A possible mechanism of injury in plants. Agrochimica 12: 461-469 Radi V, Beatovi D, Mra J (2007): Salt Tolerance of Corn Genotypes (Zea mays L.) During Germination and Later Growth. J. Agric. Sci. 52: 115-120 Speer M , and Kaiser W (1991): Ion Relations of Symplastic and Apoplastic Space in Leaves from Spinacia oleracea L. and Pisum sativum L. Under Salinity. Plant Physiol. 97: 990-997 Vuckovi S (1999): Krmno bilje, Institut za istrazivanja u poljoprivredi Srbija, Beograd

Zakljucak Na osnovu dobijenih rezultata mogu se izvesti sledei zakljucci: · Klijavost svih sorti pri najveoj koncentraciji NaCl bila je znacajno manja u odnosu na konrolu. · Duzina ponika, masa svezeg i suvog ponika kod svih ispitivanih sorti se smanjivala sa poveanjem koncentracije NaCl. · Sorta Jantar se izdvojila kao najtolerantnija na povean sadrzaj soli u podlogama za naklijavanje, jer svi ispitivani parametri, osim duzine ponika, u kontroli statisticki su se znacajno razlikovali samo od koncentracije od 150 mM NaCl, te bi se ona mogla preporuciti za setvu na nesto zaslanjenijim zemljistima. Literatura

Al - Thabet, S. S., Leilah A A, Al ­ Hawass I (2004): Effect of NaCl and Incubation Temperature on Seed Germination of Three Canola (Brassica napus L.) Cultivars. Sci. J. King Faisal Univ. 1425: 81-92 Cramer G R, Lauchli A, Polito V S (1985): Displacement of Ca2+ by Na+ from the plasmalemma of root cells. Plant Physiol. 79: 207-211 Grieve C M, Francois L E (1992): The importance of initial seed size in wheat plant response to salinity. Plant Soil 147: 197-205 Flowers T J Yeo A R (1986): Ion relations of plants under drought and salinity. Aust. J. Plant Physiol. 13: 75-91 ISTA Rules (2010): International Rules for Seed Testing. International Seed Testing Association, Switzerland

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The Effect of Salinity on Seed Germination and Growth Parameters of Field Pea (Pisum sativum L.)

Dusica Jovici1· Milka Vujakovi1 · Mirjana Milosevi2 · ura Karagi1 · Ksenija Taski-Ajdulovi1 · Maja Ignjatov1 · Aleksandar Miki1

2

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia Ministry of Agriculture, Forestry and Water Management, Nemanjina 22­26, 11000 Beograd, Serbia

1

Summary: Field pea (Pisum sativum L.) seed contains a large amount of proteins, amino acids, sugars, carbohydrates, vitamins A and C, calcium and phosphorous, and hence it is widely used for many purposes. Although field pea has moderate requirements for its growth, it is sensitive to increased salt content in soil. This research included eight varieties (Javor, Jantar, Partner, Kristal, Pionir, Junior, Trezor, Dukat) developed at Institute of Field and Vegetable Crops in Novi Sad. Sodium chloride solutions of various concentrations (0, 50, 100 and 150 mM) were added in growing media to simulate saline conditions. The following were subsequently determined: seed germination, seedling length, fresh and dry weight of seedlings and 1000-seed weight. Among the analyzed varieties, variety Jantar expressed a high level of tolerance to increased salt content in growing media under laboratory conditions. Key words: field pea, fresh and dry weight of seedlings, germination, salinity, seedling length, 1000-seed

weight

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Kvalitet semena proteinskog graska

529 Semenarstvo / Seed Science Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 529-534 originalni naucni rad / original research article

Kvalitet semena proteinskog graska u zavisnosti od vlaznosti semena u zetvi i sorte

Branko Milosevi ura Karagi Vojislav Mihailovi Aleksandar Miki Sanja Vasiljevi Imre Pataki Milka Vujakovi

primljeno / received: 18.11.2009. preraeno / revised: 27.04.2010. prihvaeno / accepted: 03.05.2010. © 2010 IFVC

Izvod: U rdu je ispitivn uticj vlznosti semen u zetvi n njzncjnije prmetre kvlitet proteinskog grsk. Anlizirn je vlznost semen u momentu zetve, energij klijnj, klijvost, udeo tipicnih ponik i ms 1000 semen kod tri sorte (Jezero, Jvor i NS Junior) pri osm rokov zetve. Prvi rok zetve bio je s sdrzjem vlge od 20,44%, poslednji, osmi, s 11,26%. Njbolji kvlitet semen (energij klijnj 81,78%, klijvost 90,11%) bio je pri sestom roku zetve u kojem je vlg iznosil 12,92%, njlosiji pri prvom roku (energij 68,55%, klijvost 78,56%). Njvisu energiju klijnj (82,00%), klijvost (92,21%) i njmnji udeo tipicnih ponik (1,96%) imla je sorta Jezero koja se odlikuje njkrom vegetcijom, determinntnim porstom stbl i ujedncenim szrevnjem. Njnizu energiju klijnj (70,39%), klijvost (76,54%) i njvei udeo tipicnih ponik (3,58%) imla je sorta NS Junior, s njduzom vegetcijom, nedeterminntnim porstom stbl i neujedncenim szrevnjem. Kljucne reci: klijvost, ms 1000 semen, proteinski grsk, sdrzj vlge

Uvod Stocni grsk (Pisum sativum L.) je biljn vrst koj se odlikuje neujedncenim szrevnjem, posebno sorte indeterminntnog porst stbl. Zbog tog je zetv stocnog grsk pren zncjnim gubicim i osteenjim semen. Gubici i osteenj njmnji su ukoliko se zetv obvi blgovremeno i pri optimlnoj vlznosti semen. Ne postoji jedinstveno misljenje o optimlnoj vlznosti z zetvu, s obzirom n velike rzlike meu sortama proteinskog grsk u morfoloskoj gri biljk, tipu porst stbl, rnostsnosti, uniformnosti szrevnj semen, obliku i krupnoi semen i gri semenjce. Energij klijnj i klijvost su njzncjniji pokztelji kvlitet semen proteinskog grsk. Energij klijnj i klijvost njvei su u fzi fizioloske zrelosti (Copeland & McDonald 1995). U fzi fizioloske zrelosti proteinskog grsk vlg semen vis je od 60% (Ellis et al. 1987), stbljik i mhune su jos zeleni i nemogue je izvrsiti mehnicku zetvu. Energij klijnj semen proteinskog grsk rste posle dostiznj fizioloske zrelosti i nstvlj tj trend krtko vreme posle dostiznj fiziolosB. Milosevi ) ( · . Karagi · V. Mihailovi · A. Miki · S. Vasiljevi · I. Pataki · M. Vujakovi Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

ke zrelosti, ztim opd sglsno odlgnju moment zetve (Siddique & Wright 2003). Njvisi sdrzj vlge u semenu kd je mogu kvlitetn mehnick zetv grsk iznosi oko 18% (Krgi i sr. 2004). Ukoliko se zetv obvi ksnije, pri vrlo niskoj vlznosti semen (nizoj od 13%), kvlitet semen bie losiji usled mehnickih osteenj rotirjuim rdnim orgnim kombjn. Pored tog, odlgnje zetve moze dovesti do smnjenj kvlitet semen i zbog rzlicitih uticj fktor spoljne sredine, ko sto su visok tempertur, visok vlznost vzduh, pdvine neposredno pred zetvu, pojv bolesti i osteenj od insekt (Siddique & Wright 2003). Ako pokisne grsk koji je bio spremn z zetvu, ztim se osusi nekoliko put pre zetve, kvlitet semen e biti umnjen, pre sveg zbog nize klijvosti (Matthews 2008). U ispitivnju sest biljnih vrst iz porodice mhunrki, Ellis et al. (1987) ukzuju d odlgnje moment zetve, posle dostiznj optimlnog sdrzj vlge u semenu, zncjno utice n gubitk klijvosti i veu pojvu tipicnih ponik. Zetv se obicno obvlj pri sdrzju vlge oko 15%. Vlg u semenu moze nglo d opdne

Ovo istrazivanje je deo projekta br. 20090 ,,Multidisciplinarni pristup oplemenjivanju i proizvodnji semena krmnih biljaka za konvencionalne i nove nacine upotrebe" (2008-2010) Ministarstva za nauku i tehnoloski razvoj Republike Srbije.

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u vreme zetve, p stog treb posebnu pznju obrtiti n podesvnj kombjn, pogotovo u uslovim visokih tempertur i smnjenog sdrzj vlge u vzduhu (Shell 1993, Cassells & Armstrong 1998). Izvedeno istrazivanje bilo je usmereno na ispitivnje uticja vlznosti semen u ralicitim rokovima zetve n njzncjnije prmetre kvlitet semena sorti proteinskog grsk. Materijal i metod U cilju resavanja postavljenog zadatka, tokom 2008. i 2009. izvrseno je istrazivanje na oglednom polju i u Lbortoriji z ispitivnje semen Institut z rtrstvo i povrtrstvo u Novom Sadu. Postvljen je dvofktorijlni ogled po modifikovnom blok sistemu, s slucjnim rsporedom prcel u tri ponvljnj. Prvi cinilc je bila sorta i imla je tri tretmn (Jezero, Jvor i NS Junior). Izbrne sorte meu sobom se zncjno rzlikuju po tipu rst, rnostsnosti, tipu list i msi 1000 semen. Determinntn porst imle su sorte Jezero i Jvor koje se koriste iskljucivo za proizvodnju zrna, indeterminntn porast sort NS Junior koj se koristi za kombinovano iskoristavanje (zrn i krma). Sorte Jvor i NS Junior imju obicn tip list, sort Jezero poseduje fil tip list. Duzin vegetcije Jezer iznosi 95 dn, Jvor 105 dn NS Junior 120 dn. Prosecn ms 1000 semen Jezer iznosi 230 g, Jvor 280 g, NS Junior 150 g. Drugi fktor je bio rok zetve. Zetv semen je obvljen u osm rokov. Zetv u prvom roku vrsi se pri vlznosti semen od oko 22%, u poslednjem, osmom roku, pri 8%. Rzlike u sdrzju vlge u semenu izmeu tretmn

zetve iznosile su po 2%. Vremenski period izmeu uzimnj uzork kreto se u intervlu 12-96 sti u zvisnosti od vremenskih uslov. Velicin osnovne prcele iznosil je 10 m2 (5 m x 2 m). Setv je obvljen od pocetk 2008. do polovine mrt 2009. norme setve su bile 150 kg ha-1 z NS Junior i 300 kg ha-1 z Jezero i Jvor. Primenjene su sve grotehnicke mere krkteristicne z svremenu proizvodnju semen proteinskog grsk. Vlg semen je odreena n vlgomeru Mettler neposredno posle zetve. Ispitivnje kvlitet semen vrseno je prem Prvilniku o kvlitetu semen poljoprivrednog bilj (Sluzbeni list SFRJ, br. 47/87) i ISTA prvilim (ISTA 2007). Od prmetr kvlitet semen utvreni su vlg, energij klijnj, klijvost, udeo tipicnih ponik i ms 1000 semen. Kod stndrdne metode nklijvnj proteinskog grsk seme se stvljlo u vlzn sterilisn pesk, ztim u komoru z nklijvnje n temperturu od 20oC. Z svko ponvljnje nlizir se po 100 semen. Nkon pet dn izrcunv se energij klijnj, nkon osm dn klijvost. Rezultti ispitivnj su sttisticki obreni nlizom vrijnse, zncjnost rzlik utvren je testom njmnje zncjne rzlike. Z sttisticku obrdu korisen je progrm Sttistic 8.0. U vreme szrevnj proteinskog grsk smenjivli su se izrzito topli dni s niskom reltivnom vlznosti vzduh u kojim se vlznost semen proteinskog grsk nglo smnjivl i dni s pdvinm i visokom reltivnom vlznosu u kojim je vlznost semen proteinskog grsk bil konstntn.

Tbela 1. Vlznost semen proteinskog grsk u zvisnosti od rok zetve i sorte (%) Table 1. Seed moisture content of dry pea depending on harvest stage and cultivar (%) Sorta Cultivar Jezero Javor NS Junior Prosek Average LSD Rok zetve / Harvest stage 3 4 5 6 18,16 16,20 15,90 13,60 15,57 15,39 13,86 13,17 16,63 16,57 15,33 12,00 16,79 16,05 15,03 12,92 Prosek Average 15,80 15,20 15,23 15,41

1 19,51 21,04 20,77 20,44

2 19,00 17,33 18,80 18,38

7 12,93 12,97 11,30 12,40

8 11,07 12,27 10,43 11,26

Sorta Cultivar 0,05 0,7 0,01 0,9 CV=7,6%

Rok zetve Harvest stage 1,1 1,5

Interakcija sorta x rok zetve Cultivar x harvest stage interaction 1,9 2,6

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Tbela 2. Energij klijnj semen proteinskog grsk u zvisnosti od sorte i rok zetve (%) Table 2. Germination energy of dry pea depending on cultivar and harvest stage (%) Sorta Cultivar Jezero Javor NS Junior Prosek Average LSD Rok zetve / Harvest stage 3 4 5 6 85,33 85,33 86,00 87,33 81,00 83,33 73,00 75,00 67,67 61,67 76,76 83,00 78,00 76,78 78,59 81,78 Prosek Average 82,00 77,38 70,39 76,59

1 76,33 75,33 54,00 68,55

2 84,00 82,67 65,67 77,45

7 78,33 78,00 81,00 79,11

8 73,33 70,67 73,33 72,44

Sorta Cultivar 0,05 3,3 0,01 4,4 CV=7,5% Rezultati i diskusija

Rok zetve Harvest stage 5,4 7,2

Interakcija sorta x rok zetve Cultivar x harvest stage interaction 9,4 12,6

Sdrzj vlge u semenu iznosio je od 20,41% u prvom roku zetve do 11,26% u poslednjem roku. Prosecn vlg z sv tri genotip iznosil je 15,41%. Prosecn sdrzj vlge kod sorte Jezero bio je 15,80%, Jvor 15,20% i NS Junior 15,23% i nije se znacajno rzlikovo. Zncjne rzlike u vlznosti semen utvrene su kod rzlicitih rokov zetve. Njvis vlg bil je kod prvog i drugog rok zetve 20,44%, odnosno 18,38%, visoko znacajno vee u poreenju s svim ostlim rokovim zetve. Njniz vlznost semen utvren je kod poslednjeg rok zetve 11,26% (Tb. 1). Prosecn energij klijnj iznosil je 76,59%. Njvis energij klijnj bil je kod sorte Jezero 82,00%, njniz kod NS Junior 70,39%.

NS Junior odlikuje se njduzim periodom vegetcije, u 2009. neposredno pred zetvu bilo je 10 kisnih dn. Kd pokisne grsk koji je bio spremn z zetvu, ztim se osusi nekoliko put pre zetve, kvlitet semen e biti umnjen, pre sveg zbog nize energije klijnj i klijvosti (Matthews 2008). Rzlike u energiji klijnj izmeu sve tri sorte bile su visoko znacajne. Energij klijnj semen povevl se s smnjenjem vlge od prvog do sestog rok zetve. S dljim smnjenjem vlznosti doslo je do opdnj energije klijnj. Njvis energij klijnj bil je u sestom roku i iznosil je 81,78%, pri sdrzju vlge 12,92%. Visoko znacajno manju energiju klijnj imlo je seme poznjeveno u prvom (68,55%) i poslednjem (72,44%) roku zetve u kojim je vlg iznosil 20,44% odnosno 11,26% (Tb. 2).

Tbela 3. Klijvost semen proteinskog grsk u zvisnosti od sorte i rok zetve (%) Table 3. Germination of field pea depending on cultivar and harvest stage (%)

Sorta Cultivar Jezero Javor NS Junior Prosek Average LSD 1 88,67 89,00 61,00 78,56 2 89,00 87,67 72,00 82,89 Rok zetve / Harvest stage 3 4 5 6 94,00 94,33 96,67 96,00 89,33 91,33 80,67 85,33 73,67 63,33 83,00 89,00 85,67 83,00 86,78 90,11 7 90,67 87,33 89,00 89,00 8 88,33 79,33 81,33 82,99 Prosek Average 92,21 86,25 76,54 85,00

Sorta Cultivar 0,05 2,5 0,01 3,4 CV=5,1%

Rok zetve Harvest stage 4,1 5,5

Interakcija sorta x rok zetve Cultivar x harvest stage interaction 7,1 9,5

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Tbela 4. Atipicni ponici semen proteinskog grsk u zvisnosti od sorte i rok zetve (%) able 4. Abnormal seedling of field pea depending on cultivar and harvest stage (%) Sorta Cultivar Jezero Javor NS Junior Prosek Average LSD Rok zetve / Harvest stage 3 4 5 6 1,33 1,33 1,00 2,00 0,67 0,67 3,33 3,00 3,33 4,33 2,33 2,00 1,78 2,11 2,22 2,33 Prosek Average 1,96 2,29 3,58 2,61

1 2,00 3,00 4,67 3,20

2 1,67 1,67 5,00 2,78

7 2,33 3,67 2,00 2,67

8 4,00 2,33 5,00 3,78

Sorta Cultivar 0,05 0,7 0,01 0,9 CV=43,2%

Rok zetve Harvest stage 1,1 1,4

Interakcija sorta x rok zetve Cultivar x harvest stage interaction 1,9 2,5 Klijvost semen sorte Jezero bil je izmeu 94,00% i 96,67% od treeg do sestog rok zetve pri vlznosti semen od 18,16% do 13,60%. Njvis klijvost semen Jvor iznosil je 89,33% i 91,33%, u treem i cetvrtom roku zetve pri vlznosti semen od 15,57% i 15,39%. Njvis klijvost semen NS Junior (89,00%) ostvren je zetvom u sestom i sedmom roku pri vlzi od 12,00% i 11,30% (Tb. 3). Prosecn broj tipicnih ponik u ogledu iznosio je 2,61%. Njmnji broj tipicnih ponik bio je kod Jezer 1,96%, Jvor je imo 2,29%, NS Junior je imo 3,58% sto je visoko znacajno vee od Jezer i Jvor (Tb. 4). Visoko znacajno vei udeo tipicnih ponik kod sorte NS Junior uslovljen je duzim kisnim periodom u vreme pune fizioloske zrelosti. Pored tog, ov sorta se odlikuje indeterminntnim

Prosecn klijvost n nivou ogled iznosil je 85%. Njvisu klijvost imla je njrnostsnija sorta Jezero (92,21%), visoko znacajno veu od Jvor (86,25%). Njnizu klijvost imla je sorta s njduzim periodom vegetcije, NS Junior (76,54%), visoko znacajno manju od Jvor i Jezer. Ispitivnjem uticj mehnickih osteenj n kvlitet semen proteinskog grsk, Krgi i sr. (2009) utvrdili su prosecnu klijvost semen NS Junior i Jezer od 95,18%, dok je njniz klijvost bil kod Jvor 84,94%. Njvis klijvost (90,11%) bil je u sestom roku zetve. Klijvost semen nije se znacajno rzlikovl u petom i sedmom roku zetve. Vlznost semen u tim rokovim zetve vrirl je od 15,03% do 12,40% iz ceg proizilzi d je to vlznost semen u okviru koje treb urditi zetvu proteinskog grsk.

Tbela 5. Ms 1000 semen proteinskog grsk u zvisnosti od sorte i rok zetve (g)

Table 5. 1000 seed weight of field pea depending on cultivar and harvest stage(g) Sorta Cultivar Jezero Javor NS Junior Prosek Average Rok zetve / Harvest stage 3 4 5 6 292,27 283,23 278,20 286,97 284,93 274,07 266,23 275,73 144,37 149,13 151,13 172,20 240,52 235,48 231,85 244,97 Prosek Average 285,04 273,09 154,39 237,51

1 277,50 262,63 151,77 230,63

2 288,87 284,93 153,53 242,44

7 291,10 280,83 160,9 244,28

8 282,27 255,40 152,13 229,93

Sorta Cultivar 0,05 3,2 0,01 4,2 CV=2,3%

LSD

Rok zetve Harvest stage 5,2 6,9

Interakcija sorta x rok zetve Cultivar x harvest stage interaction 9,0 12,0

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porstom stbl. U uslovim povene vlznosti zemljist, stblo nstvlj rst, pojvljuju se novi cvetovi i formirju se nove mhune. Seme u ovim mhunm ne moze dostii fzu fizioloske zrelosti, sto se odrzv n veem sdrzju tipicnih ponik i nizoj klijvosti semen (Krgi i sr. 2004). Pri prvom i osmom roku zetve jvlj se njvei broj tipicnim ponik sto se vezuje z njvisi (20,44%) i njnizi (11,26%) sdrzj vlge u semenu. Pri visokoj vlznosti semen pri mehnizovnoj zetvi dolzi do gnjecenj semen, dok pri niskoj vlznosti dolzi do pucnj semenjce zbog smnjene elsticnosti (Krgi i sr. 2009). Prosecn ms 1000 semen n nivou ogled iznosi 237,51 g. Njveu msu 1000 semen imla je sorta Jezero 285,04 g, visoko znacajno veu od Jvor 273,09 g i NS Junior 154,39 g. Ms 1000 semen Jvor, u njveem broju godin, zncjno je ve u poreenju s Jezerom (Krgi i sr. 2009). Meutim, u susnim uslovim 2008. posebno 2009. nlivnje semen bilo je bolje kod Jezer, sto se objsnv rnostsnosu ove sorte. Slicne rezultte utvrdili su Mihilovi i sr. (2004) z susnu 2003. kd je ms 1000 semen Jezer iznosil 241,2 g, Jvor 230,9 g. Genotip Jvor imo je visoko znacajno veu msu 1000 semen od NS Junior. Krgi i sr. (2002) utvrdili su msu 1000 semen NS Junior od 165 g (Tb. 5). Zakljucci Njvis prosecn energij klijnj (81,78%) i njvis prosecn klijvost semen (90,11%) ostvrene su zetvom grsk pri sdrzju vlge od 12,92%. Optimln vlznost semen z zetvu proteinskog grsk u ispitivnom periodu nije bil ist z sve sorte. Njve klijvost semen sorte Jezero ostvren je zetvom pri vlznosti od 18,16% do 13,60%, Jvor pri 15,50%, NS Junior od 12,00% do 11,30%. Njvei udeo tipicnih ponik 3,20% i 3,78% utvren je u semenu iz prvog i poslednjeg rok zetve, odnosno pri vlznosti od 20,44% i 11,26%. Udeo tipicnih ponik povevo se s povenjem duzine vegetcije genotip i bio je njvei kod NS Junior 3,58%. Njve prosecn ms 1000 semen 244,97 g i 244,28 g ostvren je zetvom pri vlznosti semen

od 12,00% i 11,30%. Njve ms 1000 semen bil je kod sorte Jezero 285,04 g, kod Jvor 273,09 g. U godinm koje se odlikuju susnim uslovim tokom period nlivnj semen, kod sorti koje u proseku imju slicnu msu 1000 semen, krupnije seme formire rnostsnija sorta. Literatura

Cassells J, Armstrong E (1998): Harvest and Storage, Timing all important in boosting quality, CSIRO, NSW Agriculture, Farming Ahead No. 81 1998 [Elektronski izvor]. dostupno na adresi http://sgrl.csiro.au/storage/commodity/pulses/ Field_peas.pdf Copeland L O, McDonald M B (1995): Seed viability testing. In: Copeland L O et al. (eds), Principles of Seed Science and Technology (3rd ed.), Chapman and Hall, New York, 111-126 Ellis R H, Hong D, Roberts E H (1987): The Development of Dessication- tolerance During Seed maturation in Six Grain Legumes. Ann. Bot. 59: 23-29 ISTA (2007): ISTA Rules, International Seed Testing Association Karagi , Mihailovi V, Miki A (2002): Kvalitet semena domaih sorti jednogodisnjih krmnih leguminoza. Agroznanje 3: 79-90 Karagi , Kati S, Mihailovi V (2004): Proizvodnja i kvalitet NS sorti krmnog bilja. Agroznanje 5: 5-16 Krgi , Vujkovi M, Mihilovi V, Kti S, Miki A, Milosevi B (2009): Kvlitet semen stocnog grsk u zvisnosti od genotip i mehnickih osteenj tokom zetve. Zbornik rdov Instituta z rtrstvo i povrtrstvo 46: 167-173 Matthews S (2008): The effect of harvest on the viability and pre-emergence mortality in soil pea (Pisum sativum L.) seeds. Ann. App. Biol. 73: 211-210 Mihilovi V, Krgi , Kti S, Vsiljevi S, Ptki I, Mili D, Miki A (2004): Prinos i kvlitet semen krmnih biljk u 2003. godini. Zbornik refert XXXVIII Seminr gronom, Nucni institut z rtrstvo i povrtrstvo, Novi Sd, 267-281 NSW (1999): Desication and harvest of field pea, Pulse Point 5, NSW Agriculture [Elektronski izvor], dostupno na adresi http://www.dpi.nsw.gov.au/data/assets/pdf_ file/0004/157099/pulse-point-05.pdf Prvilnik o kvlitetu semen poljoprivrednog bilj. Sluzbeni list SFRJ, 1987, 47: 1156-1167 Santos B M (2007): Seed Quality and Seeding technology. Horticultural Sciences Dept., UF/IFAS, Fla. Coop. Ext. Serv., September, 2007. [Elektronski izvor]. dostupno na adresi http://edis.ifas.ufl.edu/pdffiles/CV/CV10300.pdf Sell R (1993): Field Pea. NDSU Extension Service, Alternative Agriculture Series, Number 16. [Elektronski izvor]. dostupno na adresi http://www.ag.ndsu.edu/pubs/alt-ag/fieldpea.htm Siddique A B, Wright D (2003): Effect time of harvesting Moisture Content on Seed Fresh Weight, Dry Weight, Quality (Viability and Vigour) and Food Reservs of Pea (Pisum sativum L.). Asian J. Plant Sci. 2: 983-992

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Dry Pea Seed Quality Depending on Seed Moisture at Harvest and Cultivar

Branko Milosevi ura Karagi Vojislav Mihailovi Aleksandar Miki Sanja Vasiljevi Imre Pataki Milka Vujakovi Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia Summary: The effect of seed moisture content at harvest on the most important parameters of dry pea was examined. The seed moisture content, germination energy, germination, proportion of abnormal seedlings and 1000-seed weight were analysed in three dry pea cultivars (Jezero, Javor and NS-Junior) and at eight harvest stages. At the first harvest stage, seed moisture content was 20.44%, while at the eighth stage it was 11.26%. The best seed quality, with germination energy of 81.70% and germination of 90.11%, was at the sixth harvest time when seed moisture content was 12.92%. The poorest seed quality one was at the first harvest stage, with germination energy of 68.55% and germination of 78.56%. Cultivar Jezero, with shorter growing season, determinant stem growth and uniform maturity, had the highest germination energy (82.00%), germination (92.21%) and percent of abnormal seedlings (1.96%). Cultivar NS Junior, with the longest growing season, indeterminant stem growth and non-uniform maturity, had the lowest germination energy (70.39%) and germination (76.54%), and the highest percent of abnormal seedlings (3.58%). Key words: dry pea, germination, moisture content, 1000 seed weight

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www.nsseme.com/journal.html

NIR metoda i odreivanje hemijskog sastava semena soje

535 Semenarstvo / Seed Scienc Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 535-538 originalni naucni rad / original research article

Pouzdanost NIR metode u odreivanju sadrzaja ulja i proteina u semenu soje

Svetlana Balesevi-Tubi Vuk orevi Vojin uki Jegor Miladinovi Mladen Tati

primljeno / received: 17.11.2009. prihvaeno / accepted: 25.05.2010. © 2010 IFVC

Izvod: Primarna karakteristika difuzne refleksne spektroskopije je brzo i nedestruktivno odreivanje sadrzaja stvarnih konstituenata u proizvodima. Cilj je da se tacno zabelezi priroda apsorpcije (koja je zavisna od talasne duzine) u odnosu na stabilni standard. NIR spektroskopija je tehnologija koja je uglavnom zasnovana na stvarnom broju molekula svakog pojedinacnog konstituenta i teoretski predstavlja visoko-primenljivu analiticku metodu. Nakon konstrukcije nove kalibracione krive, prilikom transfera kalibracije sa jednog na drugi NIR instrument, potrebno je proveriti nagib i pomeranje kalibracione krive. Dobijeni rezultati visegodisnjih ispitivanja pokazuju pomeranje kalibracione krive, dok je nagib krive ostao nepromenjen, sto znaci da je navedeno pomeranje translatorne prirode. Kljucne reci: kalibracija, NIR spektroskopija, sadrzaj proteina, sadrzaj ulja, soja

Uvod Teorija infracrvene spektroskopije je poznata vise od 160 godina, ali prvi instrument se pojavio u industrijskim laboratorijama 1930. godine. Od 1940. prvi komercijalni infracrveni spektroskop je postao dostupan, a infracrvena spektroskopija je pocela znacajno da napreduje. Danas se NIR (bliska infracrvena) spektroskopija svrstava meu najvaznije analiticke metode. Njene prednosti su joj omoguile da postane jedna od znacajnih alatki za resavanje razlicitih vrsta analitickih problema u razlicitim oblastima, ukljucujui poljoprivredu, prehrambenu, hemijsku, farmaceutsku, tekstilnu, kozmeticku industriju, kao i industriju lekova (Batten 1998, Reeves & McCarty 2001, McClure 2003, Reich 2005). Brojni istrazivaci su koristili ovu metodu za brzo utvrivanje bioloske aktivnosti poljoprivrednog zemljista, kao i za rutinsko ispitivanje teksture i sadrzaja makro i mikro elemenata u zemljistu (Reeves et al. 2000, Confalonieri et al. 2001, Moron & Cozzolino 2003). Proteini, ugljeni hidrati, ulja (ili masti) predstavljaju slozene molekule koji su sastavljeni od prostih hemijskih komponenata, kao sto su amino kiseline, monosaharidi, disaharidi, masne kiseline i glicerol. Molekuli osciliraju na frekvenS. Balesevi-Tubi ) ( V. orevi V. uki J. Miladinovi M. Tati Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

cijama koje odgovaraju talasnim duzinama u infracrvenom regionu elektromagnetnog spektra. NIR spektroskopska analiza je metod za sakupljanje i prevoenje tih oscilacija u uzorke, veoma brzom analitickom procedurom bezbednom za okolinu. Mnogi istrazivaci su zakljucili da je NIR metoda ekonomicna, jednostavna, brza i jedna je od najboljih metoda za analize velikih populacija u ranim generacijama u oplemenjivackim programima (Panford 1987, Robbelen 1990, Tajuddin et al. 2002). Mogunost brzog i nedestruktivnog odreivanja kvaliteta zrna soje predstavlja jedan od glavnih faktora pri uspesnom oplemenjivanju soje na poboljsan hemijski sastav zrna. Prilikom kalibracije i validacije, odreeni broj uzoraka (kalibracioni set) mora biti analiziran korisenjem i referentnog i NIR metoda, sto znaci da se prakticno primenom ove dve procedure analiziraju razliciti poduzorci svakog uzorka (Hruschka 1987). Nakon konstrukcije nove kalibracione krive, potrebno je proveriti nagib i pomeranje kalibracione krive. Uzroci koji dovode do pomeranja kalibracione krive i promene nagiba su razliciti i zavise od same konstrukcije instrumenta, uslova sredine u kojima se instrument nalazi, kvaliteta izvora svetlosti, kao i samih uzoraka koji se ispituju (Osborn & Stivenson 1987, Hong et al. 1994). U poljoprivredi su mogue razlike u nagibu i pomeranju kalibracione krive u zavisnosti od vegetacione sezone (Golic & Walsh 2006). Navedeni faktori, pojedinacno ili zdruzeno, uticu na

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Balesevi-Tubi S i sar.

preciznost merenja, te je neophodno sprovoditi stalnu kontrolu pomeranja i nagiba kalibracione krive kako bi merenja NIR spektroskopijom bila sto pouzdanija. Materijal i metode Kalibraciona kriva za odreivanje sadzaja ulja i proteina u semenu soje konstruisana je po metodologiji Balesevi-Tubi i sar. (2007). Parametri stabilnosti kalibracionih kriva racunati su na osnovu validacionog, nezavisnog seta uzoraka. Raspon vrednosti za sadrzaj proteina u validacinom setu kretao se od 36% do 46%, dok je raspon sadrzaja ulja bio od 15% do 23%. Sadrzaj proteina u uzorcima odreen je standardnom Kjeldahl metodom JUS/ISO 1871 (1992), dok je sadrzaj ulja izrazen preko udela heksanskog ekstrakta metodom JUS/ISO 695 (1991). Standardne hemijske analize sadrzaja ulja i proteina u semenu soje uraene su u duplikatu i validacionom setu prikljuceni su samo oni uzorci cija je standarna laboratorijska greska bila manja od 0,5%. Za sva izracunavanja, laboratorijska vrednost sadrzaja proteina i ulja u semenu soje iskazana je kao prosecna vrednost merenja standardnim tehnikama. Parametri stabilnosti kalibracionih kriva izracunati su na osnovu linearne regresije laboratorijskih i NIR merenja. Pomeraj kalibracione krive izrazen je kao razlika izmeu aritmetickih sredina laboratorijskih i NIR merenja. Nagib linearne regresije i pomeraj kalibracione krive testirani su t-testom. Rezultati i diskusija NIR instrument odreuje sadrzaj proteina i drugih komponenti merenjem logaritamskih vrednosti koje bi morale biti povezane sa kolicinom komponenti ciji je sadrzaj odreen nekim drugim metodom, referentni ili standardni metod. Referentne vrednosti se stavljaju u regresioni odnos sa vrednostima dobijenim NIR metodom da bi se dobile kalibracione konstante. Poveanje tacnosti kalibracionih podataka predstavlja rezultanta granicnih poboljsanja stvarne tacnosti, ukoliko je standardna greska referentnih podataka na pocetku bila manja od dobijene stvarne tacnosti odgovarajue NIR kalibracije. Utvrena je bliska veza izmeu laboratorijskih i NIR vrednosti za ispitivanje sadrzaja proteina i ulja u semenu soje. Opseg kalibracije pokriva sirok dijapazon ocekivanih vrednosti merenja. U svetskoj kolekciji germplazme soje, sadrzaj proNIR

48 46 44 42 40 38 36 34 32 30 30 32 34 36 38 Lab 40 42 44 46 48 y = 0.9676x + 1.2398 R2 = 0.9691

Grafikon 1. Kalibraciona kriva za odreivanje sadrzaja proteina u uzorcima semena soje Graph 1. Calibration curve for protein content determination in soybean seed samples

25 23 21

NIR

y = 0.9324x + 1.267 R2 = 0.9296

19 17 15 13 13 15 17 19 Lab 21 23 25

Grafikon 2. Kalibraciona kriva za odreivanje sadrzaja ulja u uzorcima semena soje Graph 2. Calibration curve for oil content determination in soybean seed samples

teina u zrnu kree se od 30% do 53%, dok je sadrzaj ulja izmeu 10% i 25% (Dong et al. 2004). S obzirom da su uzorci sa ekstremno niskim, odnosno ekstremno visokim sadrzajem proteina i ulja veoma retki, kalibraciona kriva pokriva veliku veinu ocekivanih vrednosti merenja (Graf. 1 i 2). Najpreciznija ocena tacnosti NIR spektroskopije utvrena je kada su standardna greska kalibracije i standardna greska predvianja bile korigovane za nepreciznost referentnih podataka (Sorensen 2002). Visoke vrednosti

3 2 1 0 -1 -2 -3 t-bias korigovan t-bias granicna vrednost p<0.05

Grafikon 3. Promena t-vrednosti za pomeraj krive: oscilacije Graph 3. Changes in bias t-values: oscillations

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 535-538

NIR metoda i odreivanje hemijskog sastava semena soje

10 8 6 4 2 0 -2 -4 -6 t-bias korigovan t-bias granicna vrednost p<0.05

537

Grafikon 4. Promena t-vrednosti za pomeraj krive: skokovite promene Graph 4. Changes in bias t-values: jump changes

koeficijenta determinacije, za ispitivane konstituente u semenu soje, kao i niske vrednosti standardne greske kalibracije, pokazuju da postoji visoka meuzavisnost merenja laboratorijskim tehnikama i NIR tehnikom (Balesevi-Tubi i sar. 2007). Na osnovu visegodisnjeg praenja parametara stabilnosti i preciznosti kalibracione krive za sadrzaj proteina i ulja u zrnu soje, mogue je uociti tri tipa pomeranja kalibracione krive: lagano pomeranje, skokovito i slabe oscilacije oko odreene vrednosti (Graf. 3-5). Skokovito pomeranje kalibracione krive je najlakse uociti i to bez svakodnevnih merenja referentnih uzoraka. Meutim, lagano pomeranje i slabe oscilacije kalibracione krive veoma je tesko detektovati bez svakodnevnih merenja referentnih uzoraka. Pojava da uzorci iz razlicitih godina mogu uticati na pomeranje krive dobro je

3 2 1 0 -1 -2 -3 -4 t-bias korigovan t-bias granicna vrednost

poznata u NIR spektroskopiji (Golic & Walsh 2006, orevi i sar. 2008). Kada kalibraciona kriva osciluje oko granicne vrednosti, neophodno je svakodnevno praenje, kao i pravovremena i precizna korekcija pomeranja krive, cime se eliminise greska pri merenju nepoznatih uzoraka. Za sadrzaj proteina i ulja u semenu soje praen je i testiran nagib kalibracione krive (Graf. 6). Vrednost za nagib kalibracione krive bila je bliska vrednosti jedan i nije zabelezeno statisticki znacajno odstupanje. Ovakav nagib kalibracione krive pokazuje da su navedeni tipovi pomeranja kalibracione krive translatorni. Standardna greska predvianja imala je vrednosti kao i prilikom razvijanja kalibracije, ukrstene provere i eksterne validacije kalibracije (Graf. 7).

0,8 0,7 0,6 0,5 0,4 0,3 0,2 Ulje Proteini

Grafikon 7. Promene standardne greske predvianja kalibracionih kriva za sadrzaj ulja i proteina Graph 7. Calibration standard error of predicting changes of oil and protein content

Zakljucak Promene kalibracione krive za sadrzaj proteina i ulja u semenu soje ukazuju da je jedino svakodnevnim praenjem kalibracione krive mogue pravovremeno uociti translatorno pomeranje krive i obaviti korekciju koja razlike u NIR i standardnim merenjima svodi na slucajno variranje. Svakodnevno merenje referentnih uzoraka i izracunavanje parametara kalibracione krive oduzimaju malo vremena, a veoma efikasno obezbeuju tacnost merenja nepoznatih uzoraka. Drugi nacin koji bi mogao da resi ovaj problem je konstrukcija kalibracione krive na osnovu vise hiljada divergentnih uzoraka i upotreba slozenijih statistickih metoda (Wesley et al. 1999). Ovakvo resavanje problema pomeranja kalibracione krive je svakako trajnije, meutim, daleko je skuplje i prevazilazi potrebe kalibracije jednog NIR instrumenta za analizu hemijskog sastava semena soje.

Grafikon 5. Promena t-vrednosti za pomeraj krive: lagano pomeranje Graph 5. Changes in bias t-values: slow changes

1,2 1,1 1 0,9 0,8 Ulje Proteini

Grafikon 6. Promene nagiba kalibracione krive za sadrzaj ulja i proteina Graph 6. Calibration slope changes of oil and protein content

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Moron A, Cozzolino D (2003): Exploring the use of near infrared reflectance spectroscopy to study physical properties and microelements in soils. J. Near Infrared Spectrosc. 11: 145-154 Osborne B, Stevenson S (1987): Near-infrared resarc in Europe. In: Williams, P and Norris K (Eds), Near-infrared technology in agriculture and food Industries, American association of cereal chemistry, St. Paul, Minesota, USA, 185-200 Panford J (1987): Application of Near-Infrared reflectance spectroscopy in North America. In: Williams, P., Norris, K. (Eds): Near-Infrared Technology in Agriculture and Food Industries. American Association of Cereal Chemistry, St. Paul, Minnesota, USA, 201-212 Reeves J B, McCarty G W, Meisinger J J (2000): Near infrared reflectance spectroscopy for the determination of biological activity in agricultural soils. J. Near Infrared Spectrosc. 8: 161-170 Reeves J B, McCarty G W (2001): Quantitative analysis of agricultural soil using near infrared reflectance spectroscopy and fibre-optic probe. J. Near Infrared Spectrosc. 9: 25-34 Reich G (2005): Near-infrared spectroscopy and imaging: Basic principles and pharmaceutical applications. Adv. Drug Delivery Rev. 57: 1109-1143 Robbelen G (1990): Mutation breeding for quality improvement: A case study for oilseed crops. Mutation Breeding Review, n. 6, FAO/ IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna Sorensen L K (2002): True accuracy of near infrared spectroscopy and its dependence on precision of reference data. J. Near Infrared Spectrosc. 10: 15-25 Tajuddin T, Watanabe S, Masuda R, Harada K, Kawano K (2002): Application of near infrared transmittance spectroscopy to the estimation of protein and lipid contents in single seeds of soybean recombinant inbred lines for quantitative trait loci analysis. J. Near Infrared Spectrosc. 10: 315-325 Wesley I, Uthayakumaran S, Anderssen R, Cornish G, Bekes F, Osborne B, Skerritt J (1999): A curve-fitting approach to the near infrared reflectance measurement of wheat flour proteins which influence dough quality. J. Near Infrared Spectrosc. 17: 229-240

Literatura

Balesevi-Tubi S, orevi V, Tati M, Kosti M, Ili A (2007): Primena bliske infracrvene spektroskopije u odreivanju sadrzaja ulja i proteina u semenu soje. Arhiv za poljoprivredne nauke 246: 5-12 Batten D G (1998) An appreciation of the contribution of NIR to agriculture. J. Near Infrared Spectrosc. 6: 163-172 Confalonieri M, Fornasier F, Ursino A, Boccardi F, Pintus B, Odoardi M (2001): The potential of near infrared reflectance spectroscopy as a tool for the chemical characterisation of agricultural soils. J. Near Infrared Spectrosc. 13: 213-223 Dong Y, Zhao L, Liu B, Wang Z, Jin Z, Sun H (2004): The genetic diversity of cultivated soybean grown in China. Theor. Appl. Genet.108: 931-936 orevi V, Balesevi-Tubi S, uki V, Tati M, Miki A (2008): Parametri stabilnosti NIR merenja hemijskog sastava zrna soje. Multifunkcionalna poljoprivreda i ruralni razvoj, Zemun, Srbija: 4-5 decembar, 2008, 278-284 Golic M, Walsh K (2006): Robustness of calibration models based on near infrared spectroscopy for the in-line grading of stonefruit for total soluble solids content. Analytica Chimica Acta. 555: 286-291 Hong T L, Tsai S J, Tsou S C S (1994): Development of a sample set for soya bean calibration of near infrared reflectance spectroscopy. J. Near Infrared Spectrosc. 2: 223-227 Hruschka W (1987): Data Analysis: Wawelength selection methods. In: Williams, P and Norris K (Eds), Near-infrared technology in agriculture and food Industries, American association of cereal chemistry, St. Paul, Minesota, USA, 185-200 JUS/ISO (1992): Poljoprivredno-prehrambeni proizvodi ­ opsta uputstva za odreivanje azota metodom po Kjeldahlu, 1871:1992 JUS/ISO 695:199 (1991): Odreivanje ulja: Odreivanje heksanskog ekstrakta (ili petroletarskog ekstrakta) nazvanog `'kolicina ulja''. McClure W F (2003): 204 years of near infrared technology: 1800-2003. J. Near Infrared Spectrosc. 11: 487-518

Reliability of NIR Method in Protein and Oil Content Determination in Soybean

Svetlana Balesevi-Tubi Vuk orevi Vojin uki Jegor Miladinovi Mladen Tati

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia Summary: A primary characteristic of diffuse reflectance spectroscopy is fast and non-destructive determination of constituent concentration in products. The main goal is to find the nature of absorption (depending of wave length) and compare it with stable standard. NIR spectroscopy is a technology based on real number of molecules of each constituent and represents highly applicable analytical method. After calibration development or calibration transfer from one to another NIR instrument, it is necessary to verify the slope and bias of calibration. Results from multi year experiments show bias changes, while slope was constant, implicating that calibration changes had translator nature. Key words: calibration, NIR spectroscopy, oil content, protein content, soybean

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www.nsseme.com/journal.html

Prihrana uljane repice

539 Gajenje i agrotehnika / Soil and Crop Management Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 539-544 originalni naucni rad / original research article

Uticaj prihrane na prinos i komponente kvaliteta semena uljane repice

Milka Vujakovi Ana Marjanovi-Jeromela Dusica Jovici Radovan Marinkovi Zorica Nikoli Jovan Crnobarac Ksenija Taski-Ajdukovi

primljeno / received: 16.04.2010. preraeno / revised: 05.05.2010. prihvaeno / accepted: 26.05.2010. © 2010 IFVC

Izvod: Uljana repica je znacajna uljana biljka. Ona se gaji zbog semena koje sadrzi 40­48% ulja i 18­ 25% belancevina. Uljana repica je azotofilna biljka. Ispitivanja su izvrsena na cetiri sorte uljane repice (Banaanka, Valeska, Slavica i Express) proizvedene u vegetacionim sezonama 2007-2008. i 2008-2009. U toku vegetacionog perioda izvrsena je prihrana razlicitim kolicinama KAN-a: kontrola (0 kg N ha-1), 50 kg N ha-1, 100 kg N ha-1, 150 kg N ha-1 a na osnovu balansne metode odreena je kolicina azota (Nmin metoda). Nakon zetve utvreni su prinos semena, masa 1000 semena, klijavost semena, sadrzaj ulja i sadrzaj proteina u semenu. Prinos semena i klijavost semena su zavisili od godine proizvodnje. Masa 1000 semena je zavisila od godine proizvodnje i genotipa. Sadrzaj ulja i proteina u semenu je zavisio od godine proizvodnje, genotipa i primenjenih kolicina azota u prihrani. Kljucne reci: azot, klijavost semena, masa 1000 semena, prinos semena, sadrzaj proteina, sadrzaj ulja, uljana repica

Uvod Uljana repica je znacajna uljana biljka, a po povrsinama koje zauzima u svetu nalazi se na treem mestu, iza soje i pamuka (Marinkovi i sar. 2010). Ona se gaji zbog semena koje sadrzi 40­48% ulja i 18­25% belancevina. Ulje se koristi kao jestivo ili tehnicko, a uljane pogace i nadzemna biomasa u ishrani domaih zivotinja i ljudi. Uspesno gajenje uljane repice zahteva dobro aerirana i duboka zemljista, sa pravilnim rasporedom padavina, jer ima dug vegetacioni period (Marjanovi-Jeromela i sar. 2007). Raznolikost formi (ozime i jare) i sortimenta omoguava joj gajenje u razlicitim agroekoloskim uslovima. Nacin obrade zemljista, vreme setve, zetve i ubrenja treba prilagoditi kvalitetu zemljista i klimatskim uslovima svakog regiona. Za nase proizvodno podrucje, za postizanje prosecnog prinosa semena od 3.000 kg ha-1 potrebno je 210 kg ha-1 azota (N), 75 kg fosfora (P2O5) i 300 kg kalijuma (K2O) (Marinkovi i sar. 2009). Uljana repica je azotofilna biljka.

M. Vujakovi ) ( A. Marjanovi-Jeromela D. Jovici R. Marinkovi Z. Nikoli K. Taski-Ajdukovi Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] J. Crnobarac Poljoprivredni fakultet, Trg Dositeja Obradovia 8, 21000 Novi Sad, Srbija

Azot ulazi u sastav aminokiselina, nukleinskih kiselina, glukozinolata, vitamina, alkaloida itd. Pri nedostatku azota skrauje se sinteza proteina i biljke rano prelaze u period generativnog razvia. U poreenju sa zitima, ozimoj uljanoj repici treba vise NPK hraniva, posebno azota. Ishrana azotom utice na smanjenje sadrzaja ulja, a na poveanje sadrzaja ukupnih proteina (Rathke et al. 2005). Sadrzaj ulja, proteina i glukozinolata u semenu poveava se upotrebom sumpora u ishrani biljaka (Malhi et al. 2007). Pored toga, upotrebljena sumporna hraniva poveavaju efekat unesenog azota (Popovi i sar. 2009). Imajui u vidu znacaj azota u ishrani uljane repice, cilj rada je bio da se utvrdi kako razlicite kolicine azotnih hraniva, upotrebljene u prihrani, uticu na najvaznije proizvodne i semenske kvalitete uljane repice: prinos semena, masu 1000 semena, klijavost semena, sadrzaj ulja i proteina u dve razlicite vegetacione sezone. Materijal i metod Ispitivanje je izvrseno na cetiri komercijalne sorte ozime uljane repice (Banaanka, Valeska, Slavica i Express) proizvedene u vegetacionoj sezoni

Ovo istrazivanje je rezultat projekta TR20081 Ministarstva za nauku i tehnoloski razvoj Republike Srbije / This research results from project TR20081 financed by the Ministry of Science and Technological Development of the Republic of Serbia

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Vujakovi M i sar

2007-2008. i 2008-2009. Ogled je postavljen na oglednom polju Instituta za ratarstvo i povrtarstvo u Novom Sadu u cetiri ponavljanja na zemljistu tipa cernozem. Pred osnovnu obradu (15.8.2007. i 9.7.2008) uneseno je 250 kg/ha NPK ubriva (15:15:15). Krajem avgusta obavljena je rucna setva (29.8.2007. i 26.8.2008). Pocetkom marta (3.3.2008. i 2.3.2009) usevi su prihranjeni razlicitim kolicinama KAN-a: kontrola (0 kg N ha-1), 50 Kg N ha-1, 100 kg N ha-1, 150 kg N ha-1 a na osnovu balansne metode odreena je kolicina azota (N-min metoda, u zavisnosti od prisustva azota u zemljistu u sloju 0­90 cm i razvijenosti biljaka pre prihrane). N-min metodom je odreeno koliko azota u prihrani treba dodati, da bi bilo upotrebljeno 210 kg N ha-1 i kolicina se kretala od 0 do 22 kg N ha-1. Zetva je izvrsena u optimalnom roku (26.6.2008. i 3.7.2009). Nakon zetve utvreni su prinos semena na parcelicama neto povrsine 2,925 m2, masa 1000 semena (ISTA 2008), klijavost semena (ISTA 2008), sadrzaj ulja (NMR metodom) i sadrzaj ukupnih proteina (metodom po Kjeldahl-u). Dobijeni rezultati su statisticki obraeni primenom trofaktorijalne analize varijanse i testa najmanje znacajne razlike za prag znacajnosti 0,05 (Hadzivukovi 1991). Uraen je i koeficijent korelacije za sadrzaj ulja i proteina. Za analizu podataka korisen je statisticki program MSTAT. Rezultati i diskusija Prinos semena u 2007-2008. Kretao se od 0,87 kg po parcelici (Express, N-min metoda) do 1,258 kg po parcelici (Slavica, 100 kg N ha-1) i ova razlika je bila statisticki znacajna (Tab. 1). U 2008-2009. prinos je bio nizi i kretao se od 0,565 kg po parcelici (Slavica, 50 kg N ha-1) do 1,170

temperatura/ temperature (0C)

kg po parcelici (Valeska, 100 Kg N ha-1). Smanjenje prinosa u vegetacionoj sezoni 2008-2009. posledica je nedostatka padavina u toku jeseni, niske temperature i golomrazica tokom januara, susa u vreme intenzivnog porasta biljke i formiranja zrna (april i prva polovina maja meseca) i dugog kisnog perioda u vreme zetve (jun) (Graf. 1). Razlike izmeu sorata i razlicitih varijanti prihranjivanja u istoj godini proizvodnje nisu bile statisticki znacajne, osim izmeu Slavice 100 kg N ha-1 s jedne strane i Banaanke N-min i Valeske 0 kg N ha-1 s druge u 2007-2008. i izmeu Express 50 kg N ha-1 s jedne strane i Slavice 0, 50, 100 kg N ha-1 i Valeske, 0 kg N ha-1 s druge strane u 2008-2009. Za razliku od nasih istrazivanja, autori u publikaciji DowElanco (1991) su utvrdili izrazito poveanje prinosa u uslovima obilne ishrane azotom. Jedan od moguih razloga nepostojanja razlike izmeu razlicitih varijanti prihranjivanja je i u unetoj kolicini ubriva pred osnovnu obradu. Masa 1000 semena je zavisila od godine proizvodnje i ispitivane sorte (Tab. 1). Vise vrednosti ispitivanog parametra dobijene su u 20072008. i one su bile statisticki znacajne u odnosu na 2008-2009. Statisticki znacajno vise vrednosti za masu 1000 semena imala je sorta Valeska u odnosu na druge ispitivane sorte. Primena razlicitih kolicina azota u prihrani nije uticala na ispitivani parametar. Marjanovi-Jeromela i sar. (1999) su utvrdili znacajan uticaj godine na masu 1000 semena, sto je potvreno i u nasim ispitivanjima. Klijavost semena uljane repice u ovim ispitivanjima je zavisila od godine proizvodnje. Statisticki znacajno vise vrednosti kod svih ispitivanih sorata dobijene su u 2007-2008. (94%-98%) u odnosu na 2008-2009. (76%88%) (Tab. 1). Uticaj genotipa i prihrana sa razlicitim kolicinama N ubriva nije uticala na

padavine/rainfall (mm)

25 20 15 10 5 0 -5

8 9 10 11 12 1 2 3 4 5 6 mesec/month

150 100 50 0 8 9 10 11 12 1 2 3 4 5 6

mesec/month 2007/8 2008/9

2007/8

2008/9

Grafikon 1. Srednje mesecne temperature i padavine u vegetacionim sezonama 2007-2008. i 2008-2009. (Rimski Sancevi) Graph 1. Mean monthly temperatures and rainfalls in growing seasons 2007-2008 and 2008-2009 (Rimski Sancevi)

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Tabela 1. Uticaj prihrane na prinos i komponente kvaliteta semena uljane repice Table 1. The influence of top dressing on yield and seed quality components of oilseed rape

Prihrana uljane repice

2007-2008. Masa 1000 semena / 1000 seed weight Klijavost / Germination (%) Sadrzaj ulja/ Oil content (%) Sadrzaj proteina / Protein content (%) Prinos semena/ Seed Yield (kg) Masa 1000 semena / 1000 seed weight Klijavost / Germination (%) Sadrzaj ulja/ Oil content (%) 40,94 40,25 40,14 40,28 40,75 40,21 40,26 39,69 39,11 3,518 3,610 3,565 3,860 3,890 3,765 4,305 4,148 4,265 4,203 37,06 1,395 21,62 0,773 1,060 0,362 4,030 0,362 83 82 82 85 85 86 84 81 83 82 88 5,531 40,17 42,07 41,46 41,46 40,99 41,08 39,21 37,93 38,26 38,23 39,37 1,395 Sadrzaj proteina / Protein content (%) 21,05 22,07 22,67 21,52 21,40 20,01 20,23 19,79 22,34 21,60 20,41 19,75 18,98 20,27 20,18 22,66 23,83 23,12 23,10 22,72 0,773

2008-2009.

Sorta/ Genotype

Prihrana/Top dressing N (kg ha-1)

Prinos semena/ Seed Yield (kg)

4,928 4,823 4,860 5,015 5,053 4,593 4,363 4,343 4,675 4,523 4,430 4,715 4,698 4,723 4,620 5,443 5,538 5,990 5,803 5,673 0,362 97 5,531 97 98 98 98 95 98 97 95 97 94 38,91 41,63 40,28 40,95 40,45 42,75 38,10 36,91 37,22 36,48 18,71 18,83 19,16 19,77 19,46 18,16 21,02 21,29 21,73 23,04 0,865 0,750 0,590 0,970 0,905 0,930 0,705 0,780 1,170 0,860 95 98 96 99 98 97 95 40,89 40,82 39,63 38,86 40,68 40,96 38,53 39,33 37,51 19,24 19,42 18,27 17,88 18,21 19,57 19,99 0,860 1,105 0,850 0,755 0,925 0,630 0,565 0,630 0,925 3,775 3,785 3,923 3,795 3,940 3,578 3,808 3,640 3,610 84 85 83 79 83 76 85 83 82

Banaanka Banaanka Banaanka Banaanka Banaanka Slavica Slavica Slavica Slavica

0 50 100 150 N-min 0 50 100 150

0,920 0,990 1,085 0,930 1,105 1,040 1,020 1,258 1,185

98 97

18,13 19,05

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Slavica Express Express Express Express Express Valeska Valeska Valeska Valeska

N-min 0 50 100 150 N-min 0 50 100 150

1,010 1,075 1,115 1,028 1,190 0,870 0,855 0,950 1,075 0,985

Valeska NZR 0,05 LSD 0,05

N-min

1, 005

541

0,362

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ispitivano svojstvo. Autori Elias & Copeland (2001) su utvrdili da uslovi proizvodnje i godina imaju znacajan uticaj na kvalitet semena, a Sadat-Noori et al. (2007) i Vujakovi i sar. (2008) da klijavost semena zavisi od genotipa, sto nije dobijeno u ovim ispitivanjima. Sadrzaj ulja u semenu u 2007-2008. kretao se od 36,48% (Valeska, 150 kg N ha-1) do 42,74% (Express, N-min metoda), a u 2008-2009. od 37,92% (Valeska, 50 kg N ha-1) do 42,14% (Express, 50 kg N ha-1) (Tab. 1). Izmeu ispitivanih sorti javile su se statisticki znacajne razlike. Statisticki znacajno nize vrednosti za ispitivani parametar dobijene su kod sorte Valeska u obe godine ispitivanja. Primena veih kolicina N ubriva u prihrani dovela je do smanjenja sadrzaja ulja u semenu. Negativan uticaj visokih kolicina azota na sadrzaj ulja takoe su konstatovali Cheema et al. (2001) i Kutcher et al. (2005). Rathke et al. (2005) povezuju ovu cinjenicu sa smanjenom dostupnosu ugljenih hidrata za sintezu ulja usled prisustva visokih kolicina azota. Sadrzaj proteina u 2008-2009. bio je statisticki znacajno visi kod svih ispitivanih sorata u odnosu na 2007-2008. (Tab. 1). Sadrzaj proteina se kretao od 17,88% (Slavica, 0 kg N ha-1) do 23,04% (Valeska, 150 kg N ha-1) u 2007-2008. U vegetacionoj 2008-2009. sadrzaj proteina je bio 18,98% (Express, 100 kg N ha-1) do 23,83% (Valeska, 50 kg N ha-1). U obe godine ispitivanja javile su se statisticki znacajne razlike izmeu ispitivanih sorata. Sorta Valeska je imala statisticki znacajno najvise vrednosti, a sorta Express najnize vrednosti za ispitivani parametar. Uticaj genotipa na sadrzaja proteina u semenu uljane repice utvrdili su Marinkovi i sar. (2010). Pored genotipa na sadrzaj proteina uticu i razlicite doze azotnih ubriva. Prihranom 150 kg N ha-1 kod sorti Slavica, Banaanka i Valeska u 2007-2008. i Express i Slavica u 2008-2009. dobijene su vise vrednosti za ispitivani parametar u odnosu na druge kolicine primenjenog azota. Kod sorti Express 2007-2008. i Banaanka 2008-2009. najvise vrednosti su dobijene pri prihrani od 100 kg N ha-1. Rathke et al. (2005) objasnjavaju da poveano prisustvo azota utice na poveanu sintezu proteina na racun sinteze masnih kiselina, sto utice na smanjenje sadrzaja ulja u semenu. Koeficijent korelacije izmeu sadrzaja ulja i sadrzaja proteina iznosio je -0,50. Negativan koeficijent korelacije izmeu sadrzaja ulja i sadrzaja proteina takoe su utvrdili Hao et al. (2004).

Zakljucci Na osnovu dobijenih rezultata mogu se izvesti sledei zakljucci: · Prinos semena je zavisio od godine proizvodnje, a narocito od rasporeda i kolicine padavina u vreme intenzivnog porasta biljke i formiranja zrna. Razlike izmeu ispitivanih sorata i primena razlicitih kolicina azota u prihrani nisu uticale na ispitivano svojstvo. · Masa 1000 semena nije zavisila od primenjene kolicine azota u prihrani, ali je zavisila od sorte i uslova u vegetacionoj sezoni. · Sadrzaj ulja u semenu se smanjivao sa primenom vee kolicine azota u prihrani. Utvrene su i statisticki znacajne razlike izmeu ispitivanih sorata. · Sadrzaj proteina u semenu se poveavao sa primenom vee kolicine azota. Utvren je negativan koeficijent korelacije izmeu sadrzaja ulja i sadrzaja proteina u semenu. · Klijavost semena je zavisila samo od godine proizvodnje. Razlike izmeu ispitivanih sorti i primenjenih kolicina azota u prihrani nisu konstatovane. · Dobijeni rezultati mogu se primeniti u organizaciji i izvoenju semenske proizvodnje uljane repice.

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Literatura

Cheema M A, Malik M A, Hussain A, Shah S H, Basra S M A (2001): Effects of time and rate of nitrogen and phosphorus application on the growth and the seed and oil yields of canola (Brassica napus L.). J. Agron. Crop Sci. 186: 103-110. Dow Elanco GmbH (1991): Das Rapshandbuch. Selbstverlag, München Elias S G, Copeland L O (2001): Physiological and harvest maturity of canola in relation to seed quality. Agron. J. 93: 1054-1058 Hao X, Chang C, Travis G J (2004): Short communication: effect of long-term cattle manure apllication on ralations between nitrogen and oil content in content in canola seed. J. Plant Nutr. Soil Sci. 167: 214-215 Hadzivukovi S (1991): Statisticki metodi. Drugo prosireno izdanje. Poljoprivredni fakultet, Novi Sad ISTA (2008): International Rules for Seed Testing. International Seed Testing Association, Switzerland Kutcher H R, Malhi S S, Gill K S (2005): Topography and management of nitrogen and fungicide affects diseases and productivity of canola. Agron. J. 97: 533-541 Malhi S, Gan Y, Raney J P (2007): Yield, Seed Quality, and Sulfur Uptake of Brassica Oilseed Crops in Response to Sulfur Fertilization. Agron. J. 99: 570-577 Marinkovi R, Marjanovi-Jeromela A, Mitrovi P (2009): Osobenosti proizvodnje ozime uljane repice (Brassica napus L.) Zbornik radova Instituta za ratarstvo i povrtarstvo 46: 33-43 Marinkovi R, Marjanovi-Jeromela A, Mitrovi P, Milovac Z (2010): Uljana repica (Brassica napus L.) kao proteinska biljna vrsta. Ratar. Povrt. / Field Veg. Crop Res. 47: 157-161 Marjanovi-Jeromela A, Marinkovi R, Crnobarac J (1999): Uticaj rokova setve i ubrenja na komponente prinosa uljane repice (Brassica napus L.). Proizvodnja i prerada uljarica, Savetovanje industrije ulja (zbornik radova) 40: 243-254 Marjanovi-Jeromela A, Marinkovi R, Mitrovi P (2007): Oplemenjivanje uljane repice (Brassica napus L.) pregledni rad. Zbornik radova Instituta za ratarstvo i povrtarstvo 43: 139-148 Marjanovi-Jeromela A, Marinkovi R, Miladinovi D, Miladinovi F, Jestrovi Z, Stojsin V, Miklic V (2010): Uticaj spoljasnje sredine na prinos uljane repice (Brassica napus L.). Ratar. Povrt. / Field Veg. Crop Res. 47: 173-178 Popovi N, Bogdanovi D, Sekuli P (2009): Sumpor ­ dosadasnja saznanja. Zbornik radova Instituta za ratarstvo i povrtarstvo 46: 89-97 Rathke G W, Christen O, Diepenbrock W (2005): Effects of nitrogen source and rate on productivity and quality of winter oilseed rape (Brassica napus L.) grown in different crop rotations. Field Crops Res. 94: 103-113 Sadat-Nori A, Khalaj H, Shirani-Rad H, Alahdadi I, Akbai A, Abadi R L (2007): Investigation of seed vigour and germination of canola cultivars under less irrigation in padding and after it. Pak. J. Biol. Sci. 10: 2880-2884 Vujakovi M, Marjanovi-Jeromela A, Marinkovi R, Nikoli Z, Radi V, Tati M (2008): Genotype specificity of rapeseed (Brassica napus L.) seed quality. Conference Proceedings. International Conference "Conventional and Molecular Breeding of Field and Vegetable Crops". 24-27 November, Novi Sad, 483-486

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Influence of Top Dressing on Yield and Seed Quality Components of Oilseed Rape

Milka Vujakovi1 Ana Marjanovi-Jeromela1 Dusica Jovici1 Radovan Marinkovi1 Zorica Nikoli1 Jovan Crnobarac2 Ksenija Taski-Ajdukovi1

2

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia University of Novi Sad, Faculty of Agriculture, Trg Dositeja Obradovia 8, 21000 Novi Sad, Serbia

1

Summary: Oilseed rape is an agronomically important species grown for seed that contains 40-48% oil and 1825% proteins. Oilseed rape has high demands for nitrogen. The research was performed on four varieties of oilseed rape (Banaanka, Valeska, Slavica and Express) developed at Institute of Field and Vegetable Crops in Novi Sad, during vegetative seasons 2007-2008 and 2008-2009. During vegetation, top dressing was applied with different amounts of KAN: control (0 kg N ha-1), 50 kg N ha-1, 100 kg N ha-1, 150 kg N ha-1 and the N quantity determination were performed applying the balancing method (also known as N-min method). Yield, 1000-seed weight, seed germination, oil and protein content were determined after harvest. Yield and seed germination depended on seed production year. 1000-seed weight depended on seed production year and genotype. Oil and protein content in seed depended on production year, genotype and amount of the N-fertilizer applied by top dressing. Key words: germination, nitrogen, oil content, oilseed rape, protein content, yield, 1000-seed weight

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www.nsseme.com/journal.html

Primena NS-Nitragina i prinos soje

545 Gajenje i agrotehnika / Soil and Crop Management Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 545-548 originalni naucni rad / original research article

Uticaj primene NS-Nitragina na prinos i komponente prinosa kod soje

Jelena Marinkovi Nastasija Mrkovacki Radivoje Aimovi Vuk orevi

primljeno / received: 19.04.2010. prihvaeno / accepted: 24.05.2010. © 2010 IFVC

Izvod: U poljoprivrednim zemljistima Srbije brojnost simbiotskih bakterija soje je mala, a primena bakterioloskih preparata uvedena je kao redovna mera pri gajenju soje. Ogled je postavljen na oglednom polju Instituta za ratarstvo i povrtarstvo u Novom Sadu, na zemljistu tipa cernozem po slucajnom blok sistemu u cetiri ponavljanja. U eksperimentu su primenjena mineralna azotna ubriva u kolicinama 0 kg ha-1, 30 kg ha-1, 60 kg ha-1 i 90 kg ha-1. Ogled je postavljen u dve varijante: sa inokulacijom i bez inokulacije. Uticaj inokulacije i mineralnih azotnih ubriva na prinos i parametre prinosa odreen je preko broja mahuna, broja zrna, mase 1000 zrna i sadrzaja proteina i ulja u zrnu. Statisticki znacajno vei broj mahuna na inokulisanoj varijanti u odnosu na neinokulisanu zabelezen je uz primenu 30 kg N ha-1. Inokulacija NS-Nitraginom pozitivno je uticala na poveanje broja zrna po biljci. Na varijanti bez primene mineralnog azota i uz primenu 30 kg N ha-1 i 60 kg N ha-1 masa 1000 zrna kod inokulisanih biljaka bila je statisticki vea u odnosu na masu zrna kod neinokulisanih biljaka. Inokulacijom je ostvarena statisticki znacajna razlika u prinosu samo na varijanti gde nije primenjen mineralni azot. Inokulacija i primenjene doze mineralnog azota nisu znacajno uticali na sadrzaj proteina u zrnu soje. Kljucne reci: azotofiksacija, mineralni azot, NS-Nitragin, soja

Uvod Azot, kao osnovni biogeni element, gradivna je komponenta mnogih jedinjenja kao sto su strukturni i kataliticki proteini, nukleinske kiseline, jedinjenja koja ucestvuju u prenosu energije u elijama i uticu na procese fotosinteze, disanja (Maksimovi & Petrovi 2008). Nedostatak azota u zemljistu utice na rast i razvoj biljaka, kao i na smanjenje prinosa gajenih biljaka (Zhou et al. 2006). U cilju poveanja prinosa sve vise se primenjuju skupa mineralna azotna ubriva. Meutim, zbog svoje mobilnosti ona se lako ispiraju u dublje slojeve zemljista i podzemne vode zagaujui zivotnu sredinu, dok biljke cesto ne iskoriste vei deo unetog mineralnog azota. U obezbeivanju leguminoznih biljaka azotom znacajnu ulogu ima simbiotska azotofiksacija, koja se ostvaruje u zajednici leguminoza sa zemljisnim bakterijama iz familije Rhizobiaceae (Mrkovacki 2008, Albareda et al. 2009).

J. Marinkovi ) ( N. Mrkovacki V. orevi Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] R. Aimovi Galenika-Fitofarmacija a.d., Batajnicki drum bb, 11080 Beograd -Zemun

Inokulacija semena leguminoznih biljaka je dobro proucena, a eksploatacija ove korisne simbiozne zajednice predstavlja obelezje uspesno primenjene poljoprivredne mikrobiologije (Alves et al. 2003). Zbog izuzetnog hemijskog sastava zrna koje sadrzi oko 40% proteina i oko 20% ulja, soja (Glycine max (L.) Merr.) je jedna od najznacajnijh gajenih leguminoznih biljaka u svetu i kod nas. Sa korenom soje simbiotsku zajednicu mogu da formiraju bakterije iz rodova Bradyrhizobium, Mesorhizobium i Sinorhizobium (van Berkum & Eardly 1998). Veina zemljista u Evropi ne poseduje prirodne populacije simbiotskih bakterija soje, te se one moraju unositi u zemljiste inokulacijom (Catroux et al. 2001, Albareda et al. 2009). Takoe je mala brojnost bakterija iz roda Bradyrhizobium u nasim poljoprivrednim zemljistima, a primena bakterioloskih preparata koji sadrze selekcionisane, visokoefektivne sojeve, uvedena je kao redovna mera pri gajenju soje (Milosevi & Jarak 2005). Cilj rada je bio da se ispita uticaj inokulacije soje NS-Nitraginom na prinos i parametre prinosa. Materijal i metod rada Ogled je postavljen u Backom Petrovcu, na oglednom polju Instituta za ratarstvo i povrtarstvo iz Novog Sada, na zemljistu tipa cernozem po slucajnom blok sistemu u cetiri ponavljanja.

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U eksperimentu je korisena sorta soje Proteinka I grupe zrenja, stvorena u Institutu za ratarstvo i povrtarstvo. Setva je obavljena u optimalnom roku, a na oglednim parcelama primenjena su mineralna azotna ubriva u kolicinama: (1) 0 kg N ha-1, (2) 30 kg N ha-1, (3) 60 kg N ha-1 i (4) 90 kg N ha-1. Ogled je postavljen u dve varijante: (1) varijanta sa inokulacijom i (2) varijanta bez inokulacije. Na varijanti sa inokulacijom, neposredno pre setve, seme soje inokulisano je mikrobioloskim preparatom NS-Nitragin za soju (proizvod Instituta za ratarstvo i povrtarstvo u Novom Sadu). NS-Nitragin sadrzi smesu sojeva simbiotskih bakterija soje Bradyrhizobium japonicum. U fazi tehnoloske zrelosti soje uzeti su uzorci biljaka, po 5 biljaka sa svakog ponavljanja odreenog tretmana. Uticaj inokulacije i primenjenih azotnih ubriva na parametre prinosa odreen je preko broja mahuna po biljci, broja zrna po biljci, mase 1000 zrna. Nakon zetve soje odreen je prosecan prinos zrna po varijantama, kao i sadrzaj proteina i ulja u zrnu. Masa suve materije zrna utvrena je posle susenja do konstantne tezine 24 h na 50°C. Sadrzaj proteina i ulja utvren je spektrofotometrijski na DA-700 FLEXI-MODE NIR/VIS spektrofotometru, na principu NIR (Near Infra Red) tehnike (Balesevi i sar. 2007). Dobijeni rezultati su obraeni statistickom metodom analize varijanse i testirani LSD testom. Rezultati i diskusija Broj mahuna po biljci ukazuje na uspesnost oplodnje jednog genotipa i na taj nacin odreuje visinu prinosa biljke. Najvei broj mahuna po biljci (30,96) zabelezen je na inokulisanoj varijanti sa primenom 30 kg N ha-1, a najmanji (24,24) na inokulisanoj varijanti i primenjenom najvisom dozom mineralnog azota od

90 kg N ha-1. Statisticki znacajno vei broj mahuna na inokulisanoj varijanti u odnosu na neinokulisanu varijantu, zabelezen je sa dodavanjem 30 kg N ha-1. Bez primene mineralnog azota, kao i primenom visih doza (60 kg N ha-1 i 90 kg N ha-1) razlika u broju mahuna po biljci izmeu inokulisane i neinokulisane varijante nije statisticki znacajna (Tab. 1). U istrazivanjima Sable et al. (1998), Hernandez & Cuevas (2003) i Sahid et al. (2009) inokulacija semena soje znacajno je uticala na poveanje broja mahuna po biljci, sto je u saglasnosti sa nasim rezultatima. Broj zrna po biljci visoko je varijabilno svojstvo i zavisi od faktora spoljne sredine koliko i od genotipa. Inokulacija NS-Nitraginom pozitivno je uticala na poveanje broja zrna po biljci, te je najvei broj zrna (118,86) zabelezen na inokulisanoj varijanti sa primenom 60 kg N ha-1. Statisticki znacajna razlika u broju zrna po biljci izmeu inokulisane i neinokulisane varijante ostvarena je bez primene mineralnog azota i sa primenom 90 kg N ha-1 (Tab. 1). Rezultati autora Otieno et al. (2009) pokazali su da razliciti izvori azota (mineralni i bioloski) nisu znacajno uticali na broj mahuna i broj zrna po biljci. Pored toga sto je jedna od osnovnih komponenti prinosa, masa 1000 zrna jedna je od najvaznijih i vrlo stabilnih karakteristika sorte. Rezultati istrazivanja pokazuju da je na inokulisanoj varijanti bez primene mineralnog azota i sa dodavanjem 30 kg N ha-1 i 60 kg N ha-1, masa 1000 zrna bila statisticki vea u odnosu na masu zrna kod neinokulisanih biljaka. Primenom 90 kg N ha-1, inokulisane biljke ostvarile su manju masu 1000 zrna u odnosu na neinokulisane (Tab. 1). U svojim istrazivanjima Agha et al. (2004) takoe su dobili najvei broj mahuna po biljci i najveu masu zrna, primenom inokulacije soje i nizih doza mineralnog azota (50 kg ha-1).

Tabela 1. Uticaj inokulacije i razlicitih nivoa ubrenja azotom na broj mahuna, broj zrna i masu 1000 zrna kod soje Table 1. Effect of inoculation and different nitrogen rates on pod number, nuber of seeds and 1000 sees mass in soybean

Varijanta ubrenja Treatment 0 kg N ha-1 30 kg N ha-1 60 kg N ha-1 90 kg N ha-1 Prosek Average LSD 0,05 LSD 0,01 Broj mahuna po biljci Pod number per plant inokulisano neinokulisano inoculated uninoculated 24,99 24,98 30,96* 24,61 28,58 26,13 24,24 27,25 27,19 6,31 9,57 25,74 Broj zrna po biljci Seed number per plant inokulisano neinokulisano inoculated uninoculated 118,18* 103,90 110,38 112,02 118,86 110,01 111,26* 90,65 114,67 14,27 21,63 104,14 Masa 1000 zrna (g) Mass 1000 seeds (g) inokulisano neinokulisano inoculated uninoculated 183,58* 176,37 188,69** 178,71 188,80* 183,32 187,22 193,09 187,07 5,40 8,28 182,88

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Na osnovu dobijenih rezultata, vidi se da je najvisi prinos zrna soje ostvaren primenom 90 kg N ha-1, na inokulisanoj (4.269,65 kg ha-1) i neinokulisanoj (4.269,28 kg ha-1) varijanti. Bez primene mineralnog azota i bez inokulacije soje ostvaren je najnizi prinos (3.991,60 kg ha-1). Primenom 30 kg N ha-1, 60 kg N ha-1 i 90 kg N ha-1 izmeu inokulisane i neinokulisane varijante nije bilo statisticki znacajnih razlika u prinosu. Inokulacijom semena soje ostvarena je statisticki znacajna razlika u prinosu samo na varijanti gde nije primenjen mineralni azot (Tab. 2). Istrazivanja drugih autora potvrdila su pozitivan uticaj inokulacije na prinos soje (Coutinho et al. 1999, Alves et al. 2003, Kubota et al 2008). Dobijeni rezultati istrazivanja pokazali su da inokulacija i primenjene doze mineralnog azota nisu znacajno uticale na sadrzaj proteina u zrnu soje. Najvisi sadrzaj proteina u zrnu iznosio je 38,88% na neinokulisanoj varijanti sa primenom 30 kg N ha-1. Najvisi procenat ulja u zrnu (21,84%) zabelezen je na varijanti gde je uz inokulaciju primenjeno i 60 kg N ha-1. Statisticki znacajna razlika izmeu inokulisane i neinokulisane varijante utvrena je samo kod sadrzaja ulja sa primenom 90 kg N ha-1 (Tab. 2). Istrazivanja autora Albareda et al. (2009) pokazuju da inokulacija i primena mineralnih azotnih ubriva nije poveala prinos soje u poreenju sa varijantom gde je primenjena samo inokulacija. Takoe, primena visokih doza mineralnog azota (200 kg N ha-1) kod neinokulisanih biljaka, uticala je na smanjenje prinosa u odnosu na inokulisane biljke kod kojih nije primenjen mineralni azot (Albareda et al. 2009a, Hungria et al. 2006). Rezultati Seneviratne et al. (2000) pokazali su da inokulacija soje i primena ubriva poveavaju rast biljaka i prinos zrna. Primena azotnih ubriva u kolicini 23 kg ha-1 pre setve i 23 kg ha-1 na kraju faze cvetanja ne inhibira nodulaciju soje i azotofiksaciju.

Rezultati ogleda Gan et al. (2002) pokazuju da je dodatak visokih doza azota pre setve ili u V4 fazi (vegetativna faza sa cetiri nodije) negativno uticao na prinos zrna soje. Istrazivanja su pokazala da primena manjih kolicina mineralnih azotnih ubriva (u odreenim fazama) pozitivno deluje na rast i razvoj leguminoznih biljaka. U vreme rane faze rasta i formiranja vegetativnih organa, do formiranja kvrzica i uspostavljanja simbiotske zajednice, biljkama je potrebno obezbediti odreene kolicine azota (Wani et al. 1995, Gulden & Vessey 1998, Seneviratne et al. 2000). Takoe, u fazi nalivanja zrna biljka ima vee zahteve za produktima fotosinteze, sto moze izazvati starenje kvrzica (Albareda et al. 2009). U tim fazama, kada fiksirani azot nije dovoljan biljke treba obezbediti mineralnim azotm (Gan et al. 2003, Mahna 2005). Zakljucci Rezultati istrazivanja pokazali su da je inokulacija soje NS-Nitraginom pozitivno uticala na ispitivane parametre prinosa i prinos soje. Najvei broj mahuna zabelezen je na inokulisanoj varijanti sa primenom 30 kg N ha-1, gde je zabelezen i statisticki znacajno vei broj mahuna u odnosu na neinokulisanu varijantu. Inokulacija NS-Nitraginom pozitivno je uticala na poveanje broja zrna po biljci. Na varijanti bez primene mineralnog azota i uz primenu 30 kg N ha-1 i 60 kg N ha-1, masa 1000 zrna kod inokulisanih biljaka bila je statisticki vea u odnosu na masu zrna kod neinokulisanih biljaka. Inokulacijom semena soje ostvarena je statisticki znacajna razlika u prinosu samo na varijanti gde nije primenjen mineralni azot. Inokulacija i primenjene doze mineralnog azota nisu znacajno uticale na sadrzaj proteina u zrnu soje.

Tabela 2. Uticaj inokulacije i razlicitih nivoa ubrenja azotom na prinos, sadrzaj proteina i sadrzaj ulja u zrnu soje Table 2. Effect of inoculation and different nitrogen rates on seed yield, protein content and oil content in soybean seed

Varijanta ubrenja Treatment 0 kg N ha-1 30 kg N ha-1 60 kg N ha-1 90 kg N ha-1 Prosek Average LSD 0,05 LSD 0,01 Prinos (kg ha-1) Yield (kg ha-1) inokulisano neinokulisano inoculated uninoculated 4189,11* 3991,60 4106,25 4183,21 4090,00 4062,14 4269,65 4269,28 4163,75 157,85 239,15 4126,56 Sadrzaj proteina (%) Protein content (%) inokulisano neinokulisano inoculated uninoculated 38,39 38,71 38,52 38,88 38,31 38,67 38,53 38,62 38,44 0,416 0,630 38,72 Sadrzaj ulja (%) Oil content (%) inokulisano neinokulisano inoculated uninoculated 21,68 21,65 21,71 21,52 21,84 21,61 21,73* 21,34 21,74 0,277 0,420 21,53

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Hungria M, Franchini J C, Campo R J, Crispino C C, Moraes J Z, Sibaldelli R N R, Mendes IC, Arihara J (2006): Nitrogen nutrition of soybean in Brazil: contributions of biological N2 fixation and N fertilizer to grain yield. Can. J. Plant Sci. 86: 927-939 Kubota A, Hoshiba K, Bordon J (2008): Effect of fertilizer-N application and seed coating with Rhizobial inoculants on soybean yield in Eastern Paraguay. Rev Bras Cienc Solo 32: 1627-1633 Mahna S K (2005): Soybean cultivation and BNF in China. In: Werner D and Newton W E (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology and the Environment. Springer, Netherlands, 43­66 Maksimovi I, Petrovi N (2008): Mineralna ishrana soje. U: Miladinovi J, Hrusti M, Vidi M (ured.), Soja. Institut za ratarstvo i povrtarstvo, Novi Sad i Sojaprotein, Becej, 243­268 Milosevi N, Jarak M (2005): Znacaj azotofiksacije u snabdevanju biljaka azotom. U: Kastori R (ured.), Azot - agrohemijski, agrotehnicki, fizioloski i ekoloski aspekti. Institut za ratarstvo i povrtarstvo, Novi Sad, 305­352 Mrkovacki N (2008): Azotofiksacija soje. U: Miladinovi J, Hrusti M, Vidi M (ured.), Soja. Institut za ratarstvo i povrtarstvo, Novi Sad i Sojaprotein, Becej, 269­288 Sable S, Sontakey P Y, Tagade R, Deotale R D, Manapure P (1998): Effect of Rhizobium and molybdenum on quality aspects, yield and yield contributing characteristicts of soybean. J. Soils Crops 8: 157-159 Sahid M Q, Saleem M F, Khan H Z, Anjum S A (2009): Performance of soybean (Glicine max L.) under different phosphorus levels and inoculation. Pak. J. Agri. Sci. 46: 237-241 Seneviratne G, van Holm L H J, Ekanayake E M H G S (2000): Agronomic benefits of rhizobial inoculant use over nitrogen fertilizer application in tropical soybean. Field Crop Res. 68: 199-203 Otieno P E, Muthomi J W, Cheminingwa G N, Nderitu J H (2009): Effect of Rhizobia inoculation, farm yard manure and nitrogen fertilizer on nodulation and yield of food grain legumes. J. Biol. Sci. 9: 326-332 Wani S P, Rupela O P, Lee K K (1995): Sustainable agriculture in the semi-arid tropics though biological nitrogen fixation in grain legumes. Plant Soil 174: 29-49 Zhou X-J, Liang Y, Chen H, Shen S-H, Jing Y-X (2006): Effect of rhizobia inoculation and nitrogen fertilization on photosynthetic physiology of soybean. Photosyntetica 44: 530-535

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Agha S K, Oad F C, Buriro U A (2004): Yield and yield components of inoculated and un-inoculated soybean ander varying nitrogen levels. Asian J. Plant Sci. 3: 370-371 Albareda M, Rodriguez-Navarro D N, Temprano F J (2009): Soybean inoculation: Dose, N fertilizer supplementation and rhizobia persistence in soil. Field Crop. Res. 113: 352-356 Albareda M, Rodriguez-Navarro D N, Temprano F J (2009a): Use of Sinorhizobium (Ensifer) fredii for soybean inoculants in South Spain. Eur. J. Agron. 30: 205-211 Alves B J R, Boddey R M, Urquiaga S (2003): The success of BNF in soybean in Brazil. Plant Soil 252: 1-9 Balesevi-Tubi S, orevi V, Tati M, Kosti M, Ili A (2008): Application of NIR in determination of protein and oil content in soybean seed. Arhiv za poljoprivredne nauke 69: 5-14 van Berkum P, Eardly B D (1998): Molecular evolutionary systematic of the Rhizobiaceae. In: Spaink H P, Kondorosi A and Hooykaas P J J (eds.), The Rhizobiaceae, Molecular Biology of Model Plant-Associated Bacteria. Kluwer Academic Publishers, Dordrecht, 1­24 Catroux G, Hartmann A, Revellin C (2001): Trends in rhizobial inoculants production and use. Plant Soil 230: 21-30 Coutinho H L C, Kay H E, Manfio G P, Naves M C P, Ribeiro J R A, Rumjanek N G, Beringer J E (1999): Molecular evidence for shifts in polysaccharide composition associated with adaptation of soybean Bradyrhizobium strains to the Brazilian Cerrado soils. Environ. Microbiol. 1: 401-408 Gan Y, Stulen I, Posthuimus F, Keulen H, Pieter J C (2002): Effects of N management on growth, N2 fixation and yield of soy bean. Nutr. Cycling Agroecosyst. 62: 163-174 Gan Y, Stulen I, van Keulen H, Kuiper P J C (2003): Effect of fertilizer top-dressing at various reproductive stages on growth N2 fixation and yield of three soybean (Glycine max (L.) Merr.) genotypes. Field Crop Res. 80: 147-155 Gulden R H, Vessey J K (1998): Low concentrations of ammonium inhibit specific nodulation (nodule number g-1 root DW) in soybean (Glycine max (L.) Merr). Plant Soil 198: 127-136 Hernandez M, Cuevas F (2003): The effect of inoculating with Arbuscular mycorrhiza and Bradyrhizobium strains on soybean (Glicine max (L.) Merrill) crop development. Cultivos-Tropicales 24: 19-21

Effect of NS-Nitragin Application on Soybean Yield and Yield Components

Jelena Marinkovi1 Nastasija Mrkovacki1 Radivoje Aimovi2 Vuk orevi1

1

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia 2 Galenika-Fitofarmacija, Batajnicki drum bb, 11080 Beograd-Zemun, Serbia

Summary: Agricultural soils of Serbia are low in soybean symbiotic bacteria and application of bacteriological preparations has been introduced as a regular cultivation practice when growing soybean. A trial was set up on experimental field of Institute of Field and Vegetable Crops from Novi Sad on chernozem soil using a randomized block design with four replicates. Mineral nitrogen fertilizers were used in rates of 0, 30, 60, and 90 kg ha-1 in the experiment. Each of the nitrogen treatments had two variations, with and without inoculation. The effects of inoculation and different nitrogen fertilizer rates on yield and yield components were determined based on the pod number, seed number, 1000 seed mass and protein and oil content in seeds. Significantly higher pod number was observed in inoculated plants with the application of 30 kg N ha-1. Inoculation with NS-Nitragin increased seed number per plant. In treatment with no mineral nitrogen applied and with application of 30 kg N ha-1 and 60 kg N ha-1, 1000 seed mass was statistically higher in inoculated plants than in uninoculated ones. Inoculation produced statistically significant difference in soybean yield only in the treatment with no mineral nitrogen applied. Inoculation and applied mineral nitrogen rates had no significant effect on protein content in soybean grain. Key words: mineral nitrogen, nitrogen fixation, NS-Nitragin, soybean

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www.nsseme.com/journal.html

Variranje sadrzaja proteina u zrnu soje

549 Gajenje i agrotehnika / Soil and Crop Management Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 549-554 originalni naucni rad / original research article

Variranje sadrzaja proteina u zrnu soje u zavisnosti od sorte i godine

Vera Popovi · Milos Vidi · Vojin uki · Svetlana Balesevi-Tubi · Miladin Kosti · Aleksandar Ili · Dragana Valan

primljeno / received: 10.02.2010. prihvaeno / accepted: 15.03.2010. © 2010 IFVC

Izvod: U dvogodisnjem poljskom ogledu utvren je sadrzaj proteina u zrnu pet sorti soje razlicite duzine vegetacije (0, I i II grupe zrenja), koje trenutno cine aktuelni sortiment u nasoj zemlji. Utvrene su znatne razlike u sadrzaju proteina u zavisnosti od sorte i godine. Proizilazi da je ovo svojstvo sortna osobina, ali da je takoe pod vrlo jakim uticajem ekoloskih faktora. Rezultati pokazuju da postoje znacajne razlike izmeu genotipova, pri cemu se sadrzaj proteina u proseku kretao od 36,52 % (Venera) do 39,76 % (Proteinka). Za sintezu proteina znatno povoljnija bila je 2008. u odnosu na 2009. Varijabilnost u sadrzaju proteina izmeu genotipova (CV=3,04 %) ukazuje na dalju mogunost poveanja analiziranog svojstva. Kljucne reci: ekoloski faktori, sadrzaj proteina, soja, sorte, varijabilnost

Uvod Soja (Glycine hispida max. (Moench.)) jednogodisnja samooplodna biljka iz porodice Leguminosae. Primarni gen centar porekla soje je severoistocna Kina. Do 15-16. veka preneta je u Indoneziju, Filipine, Vijetnam, Tajland, Burmu, Nepal i severnu Indiju, gde su se razvile lokalne populacije, te se ovi regioni smatraju sekundarnim gen centrom porekla soje (Hymowitz 1988). Soja ima veliki privredni znacaj, prvenstveno zbog siroke upotrebne vrednosti koja proistice iz hemijskog sastava zrna. Celo zrno soje obicno sadrzi oko 40% proteina, 20% ulja, 17% celuloze i hemiceluloze, 7% seera, 5% vlakana i oko 6% pepela na bazi suve mase (Rubel et al. 1972). Soja je apsolutno dominantna proteinska biljka u svetu i kod nas, a kao takva osnovni je izvor biljnih proteina. Soja ima veliki znacaj u proizvodnji ljudske i stocne hrane. Sa poveanjem populacije ljudi u svetu poveava se i potrosnja mesa, cime se pokazala potreba za veim kolicinama kvalitetne i bezbedne hrane za ishranu zivotinja. Sadrzaj proteina je najvazniji pokazatelj kvaliteta. Pored ishrane zivotinja, sojini proteini imaju znacajnu ulogu u ljudskoj ishrani. Konditorski, farmaceutski i mnogi drugi proizvodi sadrze soju ili preraevine

V. Popovi ) ( · M. Vidi · V. uki · S. Balesevi-Tubi · M. Kosti · A. Ili · D. Valan Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected]

od soje, a za dobijanje pojedinih proizvoda neophodna je odgovarajua proteinska struktura. Pored toga, poznat je i pozitivan uticaj ishrane, koja sadrzi soju, na zdravlje ljudi (Pesi 2003). U veini slucajeva, do danas, soja se ne prodaje na osnovu hemijskog sastava, mada se sadrzaj proteina i ulja ve dugo uzima u obzir na trzistu posebnih namena, odnosno hrane. Iako su sadrzaj ulja i proteina kvantitativna svojstva, oba se mogu promeniti oplemenjivanjem (Miladinovi i sar. 2008b). Pored toga, zapadno trziste trazi i dobro izbalansiran odnos sadrzaja proteina i ulja za spravljenje proizvoda za ljudsku ishranu tako da je cilj oplemenjivaca Instituta za ratarstvo i povrtarstvo u Novom Sadu da prilagode sortiment Instituta zahtevima i potrebama trzista (Vidi i sar. 2008). Treba nastojati da se poboljsanjem hemijskog sastava ne umanji prinos zrna, s obzirom da su ova svojstva meusobno u jakoj negativnoj korelaciji, narocito prinos zrna i sadrzaj proteina, kao i sadrzaj proteina i ulja u zrnu soje (Chung et al. 2003). U zavisnosti od sorte i uslova uspevanja, sadrzaj proteina u zrnu varira od 30% do 53%, dok komercijalne sorte najcese sadrze izmeu 38% i 42%. Rezervni proteini semena soje podeljeni su u tri velike grupe na osnovu sedimentacione konstante. Dominantne frakcije cine glicinin (11S frakcija) i beta konglicinin (7S frakcija), dok se u alfa konglicininu (2S frakciji) nalaze inhibitori proteaza. Neki oplemenjivacki programi imaju za cilj smanjenje aktivnosti inhibitora proteaza u zrnu, kako bi se ustedela energija pri preradi soje

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prilikom termicke inaktivacije ovih inhibitora. Inhibitori proteaza imaju povoljan aminokiselinski sastav, pre svega bogati su aminokiselinama koje sadrze sumpor, a kojih inace nema dovoljno u ostalim proteinskim frakcijama (metionin, cistein), te se smanjivanjem ove frakcije u zrnu narusava povoljan aminokiselinski sastav (Pesi 2003). Drugi razlog lezi u cinjenici da sorte sa nizom aktivnosu inhibitora imaju smanjen sadrzaj ukupnih proteina u semenu. Ispitivanja izogenih linija za Kunitz tripsin inhibitor pokazuju znacajno smanjenje sadrzaja ukupnih proteina u zrnu, dok je sadrzaj ulja nepromenjen (Vollmann et al. 2002). Novosaanka i Proteinka su prve sorte sa poveanim sadrzajem proteina uvedene u proizvodnju u nasoj zemlji, a posebno se istice sorta Fortuna koja moze da dostigne 44% proteina u zrnu (Miladinovi i sar. 2008a). Cilj ovog rada je da se utvrdi sadrzaj proteina u zrnu visokoprinosnih genotipova NS soje u zavisnosti od sorte i godine. Materijal i metod rada Dvogodisnji poljski ogled postavljen je po modifikovanom blok sistemu tokom 2008. i 2009. u tri ponavljanja na oglednim poljima Instituta za ratarstvo i povrtarstvo u Novom Sadu uz primenu optimalne agrotehnike. Povrsina osnovne parcelice iznosila je 8 m2. U ogledu je analiziran sadrzaj proteina u pet genotipova soje stvorenih u Institutu za ratarstvo i povrtarstvo u Novom Sadu. Istrazivani genotipovi soje su razlicite duzine vegetacije: Galina, Valjevka i Proteinka (0 grupe zrenja), Balkan (I grupe zrenja) i Venera (II grupe zrenja), a u nasim agroekoloskim uslovima gaje se pri redovnoj setvi. Ogled je izveden u uslovima suvog ratarenja. Sadrzaj ukupnih proteina u semenu odreen je na spektrofotometru, tehnikom infracrvene spektroskopije (Near Infrared Spectroscopy - NIR) na aparatu PERTEN DA 7000, nedestruktivnom metodom, preracunat na apsolutno suvo zrno prema protokolu Balesevi-Tubi i sar. (2007). Osnovni statisticki instrumentarij za utvrivanje varijabilnosti proteina bazirao se na sledeem (Mihailovi 2005): - za izracunavanje stepena variranja odreenih pojava primenjen je koeficijenat varijacije V = 100 / x¯ - kretanje pojava racunato je primenom eksponencijalnog trenda y = Podaci su sistematizovani putem datih statisticko-matematickih metoda. Odreene pojave prikazane su u vidu tabela i grafikona sto omoguava da se uoci kretanje trendova.

Rezultati i diskusija Na sadrzaj proteina u semenu soje uticu nasledne osobine, zemljisno klimatski uslovi i nivo primenjene agrotehnike. Od zemljisno-klimatskih uslova vaznu ulogu imaju temperature vazduha i kolicina raspolozive vlage u zemljistu u fazi nalivanja semena. Sadrzaj proteina kod proucenih genotipova soje obracunat na apsolutno suvo zrno znatno varira i kree se u proseku od 35,83% (Venera) do 39,20% (Proteinka). Razlika u sadrzaju proteina je velika sto navodi na zakljucak da je ovo svojstvo pod uticajem spoljasnjih faktora. Na sadrzaj proteina najvise uticu srednje dnevne temperature i kolicina vlage u zemljistu (Tab. 1). Uticaj faktora spoljne sredine na sintezu proteina uocava se na osnovu minimalnih (35,30%) i maksimalnih vrednosti (41,09%) ovog pokazatelja kvaliteta soje (Graf. 1). Po pravilu gde je nizak sadrzaj proteina, visok je sadrzaj ulja, i obrnuto. Rezultati istrazivanja pokazuju da vremenski uslovi u 2009. nisu favorizovali sintezu proteina u zrnu soje. Za sintezu proteina znatno povoljnija godina bila je 2008. u odnosu na 2009. (Tab. 2). Pocetak vegetacionog perioda soje u 2008. i 2009. praen je povoljnim vremenskim uslovima. Srednje dnevne temperature u aprilu i maju bile su vise u odnosu na visegodisnji prosek (Tab. 1), sto se povoljno odrazilo na ujednaceno i brzo nicanje soje. Godina 2008. je u vegetacionom periodu imala relativno dobre klimatke uslove za rast soje, ukupne padavine (333,2 l m-2) i prosecne temperature (18,8 oC), sto se manifestovalo statisticki znacajno veim sadrzajem proteina svih ispitivanih sorata soje u odnosu na 2009. Od nicanja useva u 2009. do kraja prve dekade jula bilo je dovoljno padavina za rast i razvoj biljaka. U drugoj i treoj dekadi jula i u avgustu, kada su za bujan usev soje bile neophodne vee kolicine vlage radi formiranja i nalivanja zrna, pojavio se deficit vode u zemljistu, sto je rezultiralo statisticki nizim sadrzajem proteina kod ispitivanih sorti. Evidentno je da je prosecan sadrzaj proteina kod svih pet ispitivanih genotipova soje statisticki vei u 2008. (38,78%) u odnosu na 2009. (37,38%) (Tab. 2). Rezultati nasih istrazivanja saglasni su sa rezultatima Vidia i sar. (2010) gde se konstatuje da je sadrzaj proteina u zrnu sortna osobina, ali je takoe pod jakim uticajem ekoloskih faktora sto potvruju visegodisnji rezultati iz mreze makroogleda. Prema Bosnjaku (2008) navodnjavanje u izrazito susnim godinama doprinosi poboljsanju kvaliteta soje. Sadrzaj proteina u uslovima navodnjavanja vei je do 2,5%. Analizirani genotipovi soje pokazali su znacajnu varijabilnost u sadrzaju proteina (Tab. 2).

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Tabela 1. Suma padavina (l m-2) i srednja dnevna temperatura (oC) u 2008. i 2009. Table 1. Precipitation sum (lm-2) and mean daily temperatures (oC) during 2008 and 2009 Padavine Precipitation (l m-2) 2008. april April maj May juni June juli July avgust August septembar September Prosek Average 21,9 46,2 115,9 41,6 14,0 93,6 333,2 2009. 3,6 50,4 127,2 58,1 19,0 13,0 271,3 Temperatura Temperature (oC) 2008. 13,0 18,4 21,8 21,7 22,2 15,7 18,8 2009. 14,6 18,6 19,6 22,8 23,0 20,0 19,7 Padavine Precipitation (l m-2) Temperatura Temperature (oC)

Mesec Month

Visegodisnji prosek (1964-2009) Long-term average 48,8 59,6 85,7 68,2 56,9 45,1 364,3 11,4 16,8 19,9 21,4 21,0 16,8 17,9

Tabela 2. Prosecan sadrzaj proteina u ponavljanjima u NS sortama soje (2008-2009) Table 2. The average protein content in replications in NS soybean cultivars (2008-2009) Prosecna vrednost Average value (%) 38,70 37,28 37,99 38,70 36,50 37,64 40,30 39,20 39,76 38,46 39,01 38,12 38,56 37,20 35,83 36,52 37,85 Stopa promene Rate of change (%) 1,83 8,10 0,74 0,29 1,29 -0,90 2,46 6,34 0,33 2,30 0,65 0,11 0,53 0,58 1,12 0,85 -2,55 CV Coefficient of variation (%) 2,35 0,70 2,58 1,45 1,73 3,40 2,57 1,39 2,42 2,95 0,65 1,16 1,51 0,64 1,29 2,24 3,04

Izvor varijacije Source of variation Galina ­ Replication 2008. Galina ­ Replication 2009. Galina­Replic. Prosek-Average Valjevka ­ Replication, 2008. Valjevka ­ Replication, 2009. Valjevka­Repl.,Prosek-Average Proteinka ­Replication, 2008. Proteinka ­Replication, 2009. Proteinka­Repl.,Prosek-Average Prosek­Average(0 maturity group) Balkan ­ Replication, 2008. Balkan ­ Replication, 2009. Balkan Prosek Venera ­ Replication, 2008. Venera ­ Replication, 2009. Venera, Prosek - Average Prosek - Average

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Prosecna vrednost sadrzaja proteina kod sorte Galina iznosi 37,99%. U istrazivanom periodu doslo je do stagnacije sadrzaja proteina po stopi od 0,74% godisnje sa oscilacijama (CV=2,58%). Znacajno statisticki nizi sadrzaj proteina utvren je kod sorte Galina u odnosu na sorte Proteinku i Balkan. Relativno ujednacen prosecan sadrzaj proteina sorta Galina ostvarila je sa sortom Valjevka (37,64%), dok je u proseku znacajno statisticki vei sadrzaj proteina ostvarila u odnosu na sortu Veneru (36,52%).

Najvei sadrzaj proteina u posmatranom periodu ostvarila je sorta Proteinka u III ponavljanju 2008. (41,09%) dok je najmanji sadrzaj proteina ostvarila sorta Venera, u I ponavljanju 2009. (35,30%). Izmeu maksimalnih i minimalnih vrednosti sadrzaja proteina kod svih sorti ukljucenih u ogled ostvarena je varijabilnost (CV=10,71%) (Graf. 1). Sorta Proteinka pripada kolekciji visoko proteinskih genotipova i ostvarila je statisticki znacajno vei sadrzaj proteina u odnosu na ostale

Grafikon 1. Sadrzaj proteina u NS sortama soje po ponavljanjima (2008-2009) Graph 1. Protein content in NS soybean cultivars by replications (2008-2009)

Prosecna vrednost sadrzaja proteina kod sorte Proteinka iznosi 39,76%. Evidentna je stagnacija sadrzaja proteina po stopi od 0,33% godisnje i varijabilnost (CV=2,42%). Sorta Proteinka ostvarila je znacajno statisticki vei sadrzaj proteina u odnosu na ostale sorte. Analizom svih ispitivanih sorti utvreno je da se prosecna vrednost sadrzaja proteina u zrnu soje kretala u proseku od 36,52% (Venera) do 39,76% (Proteinka). Evidentno je da stabilnost, merena koeficijentom varijacije, ukazuje na variranja u sadrzaju proteina kod ispitivanih sorti (CV=3,04%).

testirane sorte u istrazivanom periodu, dok je statisticki znacajno nizi sadrzaj proteina utvren kod sorte Venera. Sorte Galina, Valjevka i Proteinka pripadaju grupi ranostasnih sorti, 0 grupe zrenja, a zbog svoje dobre adaptabilnosti, izuzetno visokog genetskog potencijala za prinos, iznad 4,5 t ha-1, stabilnosti prinosa u razlicitim uslovima gajenja i visokog stepena otpornosti prema ekonomski znacajnim bolestima, zauzimaju velike povrsine pod sojom u nasoj zemlji. Najvei sadrzaj proteina ostvarila je sorta Proteinka, dok su relativno ujednaceno visok sadrzaj proteina ostvarile sorte

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Galina i Valjevka, koje e se zahvaljujui ovom pozeljnom svojstvu prvenstveno gajiti za specificne namene u industriji za preradu soje. Srednjestasna sorta Balkan, I grupe zrenja, optimalna za nase uslove gajenja, sorta je dobre adaptibilnosti koja u veini regiona gajenja daje izuzetno visoke i stabilne prinose. Pored visokog potencijala rodnosti, Balkan ostvaruje visok stepen otpornosti prema znacajnim bolestima, otporan je na poleganje i odlikuje se visokim sadrzajem proteina u zrnu. Kasnostasna sorta Venera, II grupe zrenja, ima nesto duzi vegetacioni period od trenutno aktuelnih kasnostasnih sorti i ostvaruje visoke i stabilne prinose. Pored visokog potencijala rodnosti i dobre adaptibilnosti, odlikuje se statisticki znacajno nizim sadrzajem proteina i izuzetno visokim sadrzajem ulja u zrnu. Moderno oplemenjivanje podrazumeva i kontinuirano unapreenje tehnoloskog kvaliteta, a narocito povean sadrzaj proteina u sojinom zrnu. Raznovrstan sortiment novosadskih sorti soje omoguuje proizvoacima pravilan izbor sorti razlicitog vremena zrenja i tehnoloskog kvaliteta, sa kompleksom pozitivnih proizvodnih osobina, adaptabilnih razlicitim klimatskozemljisnim uslovima. Posebna paznja, pored prinosa, poklanja se kvalitetu zrna, s obzirom da je preraivackoj industriji potrebna sirovina sa poveanim sadrzajem proteina i ulja u zrnu. Tako je za proizvodnju sojinog mleka upravo ovaj odnos bitan za dobijanje kvalitetnog finalnog proizvoda. Osnovni problem koji se javlja prilikom oplemenjivanja na povisen sadrzaj proteina jeste postojanje negativne korelacije izmeu prinosa i sadrzaja proteina (Miladinovi i sar. 2008b). Ova osobina je od posebnog znacaja za preradu, pa se sorte soje sa visokim sadrzajem proteina mogu koristiti za specificne namene u preraivackoj industriji. Zakljucci Na osnovu rezultata postignutih u ovom ogledu mogu se doneti sledei zakljucci: · Utvreno je da se prosecna vrednost sadrzaja proteina u zrnu soje kod ispitivanih genotipova u istrazivanom periodu kretala u proseku od 36,52% (Venera) do 39,76% (Proteinka). Utvrena je varijabilnost proteinske strukture (CV=3,04%), sto predstavlja vazan preduslov za dalji napredak u oplemenjivanju. · Najvei sadrzaj proteina u posmatranom periodu ostvarila je visokoproteinska sorta Proteinka u III ponavljanju 2008. (41,09%), dok je najmanji sadrzaj proteina ostvarila sorta Venera

u I ponavljanju 2009. (35,30%). Izmeu maksimalnih i minimalnih vrednosti sadrzaja proteina kod ispitivanih sorti ostvarena je varijabilnost (CV=10,71%). · Kao povoljna godina za sintezu proteina pokazala se 2008. u kojoj je zabelezen sadrzaj proteina u proseku od 38,78% za sve ispitivane sorte. Nepovoljnija godina za sintezu proteina bila je 2009. u kojoj je zabelezen sadrzaj proteina u proseku od 37,38% za sve ispitivane sorte. · U narednom periodu trebalo bi poveati broj analiziranih genotipova soje i time prosiriti geneticku osnovu za intenzivniji rad na veem sadrzaju proteina. Literatura

Balesevi-Tubi S, orevi V, Tati M, Kosti M, Ili A (2007): Application of near infrared spectroscopy in determination of protein and oil content in soybean seed. Arhiv za poljoprivredne nauke. (u stampi) Bosnjak (2008): Navodnjavanje soje u redovnoj, drugoj i postrnoj setvi. U: Miladinovi J, Hrusti M, Vidi M (ured.), Soja. Institut za ratarstvo i povrtarstvo, Novi Sad i Sojaprotein, Becej, 339-340 Chung J, Barka H L, Staswick P E, Lee D J, Gregan P B, Shoemaker R C, Specht J E (2003): The seed protein, oil and yield QTL on soybean linkage group I. Crop Sci. 43: 1053-1067 Hymowitz T (1988): Soybeans: The Success Story, Proceedings of the First National Symposium. New Crops: Research, Development, Economics Indianopolis, Indiana, 159-163 Mihailovi B (2005): Marketing u turizmu, Cetinje Miladinovi J, Hrusti M, Vidi M (2008a): Soja. Institut za ratarstvo i povrtarstvo, Novi Sad i Sojaprotein, Becej, 35-36 Miladinovi J, Hrusti M, Vidi M, Balesevi-Tubi Svetlana, orevi V (2008b): Oplemenjivanje soje u Institutu za ratarstvo i povrtarstvo, Zbornik radova Instituta za ratarstvo i povrtarstvo 45: 65-70 Pesi M (2003): Uticaj proteinske molekulske strukture genotipova na tehnoloske funkcionalne osobine soje. Magistarska teza, Univerzitet u Beogradu, Poljoprivredni fakultet Rubel A, Rinne R W, Canvin D T (1972): Protein, oil, and fatty acid in developing soybean seeds. Crop Sci. 12: 739-741 Vidi M, Hrusti M, Miladinovi J, uki V, orevi V (2008): Analiza sortnih ogleda soje u 2007. godini. Zbornik radova Instituta za ratarstvo i povrtarstvo 45: 141-151 Vidi M, Hrusti M, Miladinovi J, uki V, orevi V, Popovi, V (2010): Novine u sortimentu soje. Ratar. Povrt. / Field Veg. Crop Res. 47: 347-355 Vollmann J, Schausberger H, Bistrich H, and Lelley T (2002): The presence or absence of the soybean Kunitz trypsin inhibitor as a quantitative trait locus for seed protein content. Plant Breed. 121: 272-274

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Variation of Protein Content in Soybean Grain Depending on Cultivar and Year

Vera Popovi · Milos Vidi · Vojin uki · Svetlana Balesevi-Tubi · Miladin Kosti · Aleksandar Ili · Dragana Valan

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia Summary: Grain protein content in five soybean cultivars was determined in a two-year field experiment. The soybean cultivars belong to different maturity groups (O, I and II) and they represent the current cultivars in our country. Significant differences were found in protein content depending on cultivar and year. It seems that this is a cultivar property, but is also under a very strong environmental influence. The results show that there are significant differences between cultivars, while the average protein content ranged from 36.52% (Venera) to 39.76% (Proteinka). The year 2008 was significantly more favourable than 2009 for protein synthesis. Considerable variability in protein content among cultivars (CV=3.04%) indicates the possibility of further increase of the analyzed property. Keywords: cultivars, environmental factors, protein content, soybean, variability

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www.nsseme.com/journal.html

Antioksidativna svojstva sociva

555 Kvalitet / Crop Quality Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 555-560 originalni naucni rad / original research article

Antioksidativna svojstva fenolnih komponenata semena sociva (Lens culinaris L.)

Jelena Dragisi Maksimovi Branka Zivanovi Vuk Maksimovi Vesna Hadzi-Taskovi Sukalovi

primljeno / received: 14.04.2010. preraeno / revised: 24.05.2010. prihvaeno / accepted: 26.05.2010. © 2010 IFVC

Izvod: U ovom radu su ispitivana antioksidativna svojstva metanolnih ekstrakata semena sociva (Lens culinaris L.). Skromni literaturni podaci ukazuju na prisustvo razlicitih polifenola u semenu sociva, koji poseduju bioaktivne osobine zahvaljujui sposobnosti da uklanjaju aktivne kiseonicne forme. Kvalitativna i kvantitativna variranja fenola u semenu posledica su razlika izmeu sorti, uslova gajenja/rastenja i primenjenih analitickih metoda. U semenu sociva odreen je sadrzaj i sastav fenola i utvren pojedinacni doprinos identifikovanih komponenata ukupnom antioksidativnom kapacitetu. HPLC analizom je identifikovano sedam fenolnih supstanci. Njihova antioksidativna svojstva, odreena kao sposobnost uklanjanja DPPH radikala imaju sledei redosled opadajue efikasnosti: galna kiselina > epikatehin > katehin > protokatehuinska kiselina > rutin > p-kumarna kiselina > umbeliferon. Veliki antioksidativni kapacitet sociva ukazuje na sposobnost sprecavanja nepozeljnih oksidativnih reakcija izazvanih slobodnim radikalima, pa konzumiranje sociva moze da ima povoljan efekat sa aspekta zdravstvene koristi za ljudski organizam. Kljucne reci: antioksidativni kapacitet, fenolna jedinjenja, socivo, ukupni fenoli

Uvod Fenolna jedinjenja ucestvuju u esencijalnim fizioloskim procesima u biljkama kao sto su npr. rastenje, reprodukcija, odbrana od abiotickih i biotickih stresova, itd. Prisutna su u svim biljnim organima zbog cega su neizostavna u ljudskoj ishrani. Zahvaljujui antioksidativnoj, antimutagenoj i antiradikalskoj aktivnosti, kao i konstataciji da poseduju potencijalni zdravstveno-korisni efekat (Bravo 1998), u skorije vreme se vrse aktivna istrazivanja u smislu identifikacije i kvantifikacije individualnih fenola u namirnicama i utvrivanja njihovog antioksidativnog kapaciteta. Antioksidativna aktivnost se ostvaruje posredstvom nukleofilnih fenolnih grupa za koje se vezuju slobodni radikali, dok pojedina fenolna jedinjenja mogu da imaju ulogu helatora jona metala zadrzavajui antiradikalsku aktivnost i nakon formiranja kompleksa sa metalom (Afanas'ev et al. 1989). Visok sadrzaj polifenolnih jedinjenja kao glavnih antioksidativnih konstituenata smatra se najznacajnijom zdravstveno-zastitnom karakteriJ. Dragisi Maksimovi ) ( B. Zivanovi V. Maksimovi V. HadziTaskovi Sukalovi Institut za multidisciplinarna istrazivanja, Kneza Viseslava 1, 11030 Beograd, Srbija e-mail: [email protected]

stikom leguminoza (zrnastih mahunarki) u koje spada i socivo (Bhatty 1995). Seme sociva sadrzi razlicita fenolna jedinjenja koja predstavljaju vaznu grupu bioaktivnih sastojaka hrane koji mogu da sprece razvoj nekih hronicnih bolesti izazvanih oksidativnim stresom (López-Amorós et al. 2006). Kvalitativna i kvantitativna variranja polifenolnih jedinjenja u semenu razlicitih sorti sociva se objasnjavaju kao posledica genetskih razlika, uslova gajenja/rastenja i cuvanja, kao i primenjenih analitickih metoda. Za potpuno razumevanje znacaja polifenola u ljudskoj ishrani neophodno je detaljno ispitati njihovu (bio)dostupnost, mehanizam delovanja i moguu sinergiju sa drugim sastojcima hrane. Sem polifenola kao bioaktivnih antioksidanasa, socivo je odlican izvor proteina i rastvorljivih i nerastvorljivih vlakana. Takoe sadrzi znacajne kolicine vitamina i minerala. Proizvodnja sociva koje se koristi za ljudsku ishranu najvea je u Aziji i na Bliskom Istoku (60%), u Severnoj Americi (8%), a u Evropi svega 4% od ukupne godisnje svetske proizvodnje (Fernandez-Orozco et al. 2003). Upotreba sociva u ishrani kod nas je zapostavljena usled tradicionalnog pristupa ishrani i neobavestenosti

Ovaj rad je finansiran od strane Ministarstva za nauku i tehnoloski razvoj Republike Srbije (143016B i 143020B).

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potrosaca. Javna afirmacija znacaja fenolnih jedinjenja i njihove zdravstvene korisnosti za ljudski organizam moze doprineti sirenju upotrebe sociva u ishrani. Cilj nasih istrazivanja je bio ispitivanje sadrzaja i sastava fenola, kao i odreivanje antioksidativnog kapaciteta semena sociva. Fenolne komponente su identifikovane i kvantifikovane HPLC metodom, a antioksidativni kapacitet pojedinacnih fenola je odreen merenjem antiradikalske aktivnosti ABTS i DPPH testom i uporeen sa snaznim antioksidansom - askorbinskom kiselinom. U radu je diskutovan doprinos pojedinih fenolnih supstanci ukupnom antioksidativnom kapacitetu sociva. Materijal i metod rada Ekstrakcija fenolnih komponenata iz semena sociva je izvrsena u 80% metanolu u odnosu 1:10 (sveza masa semena sociva:metanol) pomou sistema za pripremu uzoraka Fast Prep-24 (M.P. Biomedicals, Irvine, California, USA). Homogenati su centrifugirani 15 minuta na 10.000 x g, a zatim su supernatanti filtrirani kroz 0,22-mikrometarski filter, nakon cega su koriseni za dalje analize. Antioksidativni kapacitet semena sociva je ispitan ABTS testom po metodi Arnao et al. (1999). Reakciona smesa se sastojala od 2 mM ABTS (2, 2'azino-bis(3-etilbenztiazolin-6-sulfonska kiselina), 15 M H2O2 i 0,25 M HRP (peroksidaza iz rena) u 50 mM fosfatnom puferu pH 7,5. Apsorbancija je merena na 734 nm (UV-vis 2501 PC Shimadzu, Kyoto, Japan) na 25°C. Smanjenje apsorbancije u vremenu, nastalo kao posledica dodavanja ispitivanog uzorka u reakcionu smesu, jeste mera uklanjanja ABTS radikala generisanih peroksidazom. Rezultati su racunati pomou standardne prave konstruisane dodavanjem razlicitih koncentracija L-askorbinske kiseline (0,1-0,8 mM). Ukupna antioksidativna aktivnost ispitivanih uzoraka je izracunata kao kolicina ekvivalenata askorbinske kiseline u miligramima po gramu sveze mase sociva. Antioksidativna aktivnost je merena kao sposobnost ekstrakta da uklanja slobodne radikale 1,1-difenil-2-pikrilhidrazila (DPPH·) (Duan et al. 2006). 1 ml 0,2 mM metanolskog rastvora DPPH je mesan sa istom zapreminom razblazenog metanolskog ekstrakta sociva ili metanolskog rastvora komercijalnih fenolnih supstanci. Posle 30 min inkubacije u mraku na sobnoj temperaturi, citana je apsorbancija rastvora na 517 nm. Optimalna kolicina uzorka koji se analizira treba da smanjuje pocetnu apsorbanciju rastvora za oko 50%. Rezultati su prikazani kao IC50 vrednost koja je definisana kao kolicina uzorka koja

smanjuje apsorbanciju DPPH radikala za 50% u datim uslovima. Manja IC50 vrednost predstavlja veu antiradikalsku aktivnost. L-askorbinska kiselina je sluzila kao pozitivna kontrola, pa su antiradikalske vrednosti prikazane i kao ekvivalent askorbinske kiseline (EAK) i odnose se na kolicinu askorbinske kiseline koja odgovara jedinici mase uzorka. Sadrzaj ukupnih fenola u ekstraktima je odreen spektrofotometrijski (UV-vis 2501 PC Shimadzu, Kyoto, Japan) Folin­Ciocalteu metodom (Singleton & Rossi 1965). U alikvot uzorka je dodato 0,25 ml Folinovog reagensa, a nakon 3 minuta 1,25 ml 20% Na2CO3. Dobijena smesa je inkubirana 60 minuta na sobnoj temperaturi, a zatim je merena apsorbancija na 724 nm. Rezultati su izracunati pomou standardne prave konstruisane sa galnom kiselinom u opsegu 0,1-2 mM i izrazeni u miligramima ekvivalenata galne kiseline (EGK) po gramu sveze mase semena sociva. HPLC analiza fenolnih jedinjenja izvedena je na Waters hromatografskom sistemu (binarna 1525 pumpa, 717+ autosempler, 2996 Diode Array detektor), a razdvajanje je izvrseno na Waters Symmetry RP C-18 hromatografskoj koloni (125 x 4 mm). Korisena je gradijentna elucija (faza A, 0,1% fosforna kiselina i faza B, acetonitril) sa sledeim profilom mobilne faze: u prvih 10 minuta 10-22% faze B, od 10 do 20 minuta 30% faze B i u narednih 5 minuta povratak na 10% faze B uz 5 minuta za ekvilibraciju kolone. Za obradu podataka korisen je racunarski program Waters Empower 2 Software (Waters, Milford, USA). Rezultati su statisticki obraeni koristei program Statistica 6 (StatSoft, Inc. USA) u okviru kojeg su razlike izmeu prosecnih vrednosti rezultata utvrene analizom varijanse (ANOVA), a za procenu statisticke znacajnosti korisen ManVitnijev (Mann-Whitney) neparametarski test. Rezultati dobijeni iz reprezentativnih eksperimenata prikazani su preko srednje vrednosti i standardne greske od tri ponavljanja. Rezultati i diskusija U semenu sociva odreena je kolicina ukupnih fenola od 2,3 mg EGK g-1 sveze mase i antioksidativni kapacitet, meren kao antiradikalska aktivnost od 1,6 mg EAK g-1 sveze mase ABTS testom, tj. 2,2 mg EAK g-1 sveze mase DPPH testom (Tab. 1). Dobijene vrednosti sociva u nasem radu su slicne i u opsegu vrednosti dobijenih iz razlicitih sorti sociva (Fernandez-Orozco et al. 2003,

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Tabela 1. Sadrzaj ukupnih fenola i antioksidativni kapacitet semena sociva (Lens culinaris L.) odreen ABTS (A) i DPPH (B) testom. U tabeli su predstavljene srednje vrednosti i standardna greska od tri ponavljanja. Table 1. Total phenolic content and antioxidative capacity of lentil seed (Lens culinaris L.) determined by ABTS (A) and DPPH (B) tests. Data are means of three replications ± SE. Ukupni fenoli Total phenolic content (mg EGK g-1 sveze mase / fresh weight) 2,302 ± 0,015 (A) (mg EAK g-1 sveze mase / fresh weight) (B) (mg EAK g-1 sveze mase / fresh weight) 2,130 ± 0,170 IC50 (mg) 1,817 ± 0,059

1,589 ± 0,084

Rodríguez et al. 2007). Semena sociva su poznata po velikom sadrzaju antioksidativnih supstanci, a doprinos ukupnih fenola u ukupnoj antioksidativnoj aktivnosti iznosi 92% (Fernandez-Orozco et al. 2003). Veina fenolnih komponenata koje se nalaze u biljnom tkivu potencijalni su antioksidansi jer imaju sposobnost da uklanjaju aktivne kiseonicne i azotne forme. Kako antiradikalska aktivnost zavisi ne samo od kolicine supstance koja uklanja slobodne radikale, ve i od strukture molekula, broja i polozaja OH grupa u molekulu, odredili smo HPLC analizom kvalitativni i kvantitativni profil fenolnih jedi-

njenja (Tab. 2). Od detektovanih jedinjenja, dominantno fenolno jedinjenje je galna kiselina, zatim opadajuim redosledom protokatehuinska kiselina, katehin, epikatehin, p-kumarna kiselina, rutin i umbeliferon. Fenolne komponente zrna uglavnom cine fenolne kiseline i flavonoidi. Od fenolnih kiselina u ispitivanom uzorku sociva detektovane su galna, protokatehuinska i p-kumarna kiselina, a od flavonoida epikatehin, katehin i rutin. Ispitivanja razlicitih sorti sociva (Xu & Chang 2010) pokazala su da postoje znatna variranja u individualnim fenolnim komponentama,

Tabela 2. Koncentracije specificnih fenolnih jedinjenja u metanolskom ekstraktu semena sociva detektovane HPLC tehnikom. U tabeli su predstavljene srednje vrednosti i standardna greska od tri ponavljanja. Razlicitim slovima su obelezene statisticki znacajne razlike (p<0,05). Table 2. Concentrations of specific phenolic compounds in the methanol extract of lentil seeds detected by HPLC. Data are means of three replications ± SE. Different letters within a column denote a significant difference at p < 0.05. Fenolno jedinjenje Phenolic compound: Katehin Catechin Epikatehin Epicatechin Rutin Rutin p-Kumarna kiselina p-Coumaric acid Umbeliferon Umbeliferone Galna kiselina Gallic acid Protokatehuinska kiselina Protocatechuic acid Koncentracija (ng g-1 sveze mase) Concentration (ng g-1 fresh weight) 250,296 ± 24,035b 141,253 ± 2,293a 126,803 ± 6,275a 132,767 ± 7,847a 123,373 ± 9,698a 1241,423 ± 81,436c 288,380 ± 20,469b

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kao i u antioksidativnoj aktivnosti, pri cemu je detektovano devet fenolnih kiselina i njihovih derivata, meu kojima galna, protokatehuinska i p-kumarna kiselina, a (+)-katehin i (-)-epikatehin kao dominantni flavonoidi. Navedene komponente su bile prisutne u svim sortama sociva, sto je u saglasnosti sa nasim rezultatima. Isti autori su ustanovili znacajnu korelaciju izmeu sadrzaja flavonoida (katehina i epikatehina) i sposobnosti uklanjanja peroksidnih radikala. Amarowicz et al. (2009) su u ekstraktu crvenog sociva identifikovali 24 komponente koje pripadaju raznim klasama fenola, meu kojima su dominantne kvercetin diglukozid, katehin, digalat procijanidin i p-hidroksibenzoeva kiselina. Fenolne kiseline u ekstraktu crvenog sociva imaju manji udeo (oko 20%) od ukupnih fenolnih komponenata. Znatne kolicine fenola flavonoidne strukture, monomera i polimera (kondenzovanih tanina) skoncentrisane su u semenjaci (Dueñas et al. 2003) i oni sluze kao odbrambeni mehanizam biljke od parazita i insekata. Seme sociva kao i ostale leguminozne biljke osim fenola male molekulske mase, sadrzi i znatne kolicine polimernih fenolnih komponenata, kondenzovanih tanina. Tanini se tradicionalno smatraju za antinutritivne faktore

usled sposobnosti da se umreze sa proteinima reagujui sa lizinom i metioninom, te uticu na smanjenje svarljivosti proteina. S druge strane, taninski konstituenti imaju visok antioksidativni potencijal (Amarowicz 2007), sto je potvreno i u semenu sociva (Amarowicz et al. 2009). Danas se smatra da efekti pozitivni po zdravlje ili pak antinutritivne osobine zavise od hemijske strukture i kolicine tanina u biljnom tkivu. Da bi se utvrdio doprinos identifikovanih fenolnih komponenata antioksidativnim svojstvima sociva, odreena je antiradikalska aktivnost svake komponente DPPH testom. Rezultati su prikazani u tabeli 3. kao IC50 vrednosti i uporeeni sa askorbinskom kiselinom kao standardom, cija je IC50 vrednost iznosila 3,8 g. Najveu antiradikalsku aktivnost je pokazala galna kiselina, 1 g supstance je ekvivalentan sa 2,3 g askorbinske kiseline koja je poznata kao izuzetno snazan antioksidans. Vee aktivnosti od askorbinske kiseline su dobijene sa epikatehinom, katehinom i protokatehuinskom kiselinom. Visoku antiradikalsku aktivnost epikatehina su odredili Okawa et al. (2001) DPPH testom, cak su potvrdili da postojanje kateholske grupe u strukturi flavonoida u velikoj meri utice na antiradikalsku aktivnost. Rutin, a narocito

Tabela 3. Antioksidativna aktivnost fenolnih komponenata detektovanih u socivu, odreena kao sposobnost uklanjanja DPPH radikala. Aktivnosti su izrazene kao, IC50 i EAK. U tabeli su predstavljene srednje vrednosti i standardna greska od najmanje tri ponavljanja. Table 3. Antioxidative activity of phenolic compounds detected in lentil seeds, determined as DPPH· scavenging activity. Activities are expressed as IC50 and EAK. Data are means of minimum three replications ± SE. Fenolno jedinjenje Phenolic compound: Katehin Catechin Epikatehin Epicatechin Rutin Rutin p-Kumarna kiselina p-Coumaric acid Umbeliferon Umbeliferone Galna kiselina Gallic acid Protokatehuinska kiselina Protocatechuic acid IC50 (g) 2,42 ± 0,13 2,28 ± 0,09 7,32 ± 0,40 267,00 ± 4,50 721,00 ± 1,00 1,69 ± 0,07 2,63 ± 0,38 EAK (g) 1,60 ± 0,08 1,70 ± 0,07 0,53 ± 0,03 0,02 ± 0,00 0,01 ± 0,00 2,30 ± 0,01 1,48 ± 0,21

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p-kumarna kiselina i umbeliferon (7-hidroksikumarin) su slabi antioksidansi. Imajui u vidu odreene kolicine navedenih fenola u semenu, galna kiselina, epikatehin, katehin i protokatehuinska kiselina zajedno cine veliki udeo u kolicini odreenih fenola (oko 83%), mozemo da smatramo da fenolne supstance male molekulske mase znacajno doprinose antioksidativnom kapacitetu semena sociva. Zakljucak Bioaktivne fenolne komponente, detektovane u visokim koncentracijama u semenu sociva, poseduju antioksidativnu aktivnost usled cega mogu biti znacajne kao primarni izvor prirodnih antioksidanasa. Stoga, prikazani rezultati ukazuju na potrebu sire afirmacije korisenja sociva kako za prehrambenu, tako i za farmaceutsku industriju.

Literatura

Afanas'ev I B, Dcrozhko A I, Brodskii A V, Kostyuk V A, Potapovitch A I (1989): Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem. Pharmacol. 38: 1763-1769 Amarowicz R (2007): Tannins: the new natural antioxidants? Eur. J. Lipid Sci. Technol.109: 549-551 Amarowicz R, Estrella I, Hernández T, Dueñas M, Troszyñska A, Kosiñska A, Pegg R (2009): Antioxidant Activity of a Red Lentil Extract and Its Fractions. Int. J. Mol. Sci. 10: 55135527 Arnao M B, Cano A, Acosta M (1999): Methods to measure the antioxidant activity in plant material. A comparative discussion. Free Radic. Res. 31: 89-96 Bhatty R S (1995): Lentils as a dietary cereal complement. Cereal Food World 40: 387-392 Bravo L (1998): Polyphenols: Chemistry, Dietary Sources, Metabolism, and Nutritional Significance. Nutr. Rev. 56: 317333 Duan X J, Zhang W W, Li X M, Wang B G (2006): Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem. 95: 37-43 Dueñas M, Sun B, Hernández T, Estrella I, Sprangeri IM (2003): Proanthocyanidin Composition in the Seed Coat of Lentils (Lens culinaris L.) J. Agric. Food Chem. 51: 7999-8004 Fernandez-Orozco R, Zieliski H, Piskula M K (2003): Contribution of low-molecular-weight antioxidants to the antioxidant capacity of raw and processed lentil seeds. Nahrung/ Food. 47: 291-299 Xu B, Chang S K C (2010): Phenolic Substance Characterization and Chemical and Cell-Based Antioxidant Activities of 11 Lentils Grown in the Northern United States. J. Agric. Food Chem. 58: 1509­1517 López-Amorós M L, Hernández T, Estrella I (2006): Effect of germination on legume phenolic compounds and their antioxidant activity. J. Food Compos. Anal. 19: 277-283 Okawa M, Kinjo J, Nohara T, Ono M (2001): DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of flavonoids obtained from some medicinal plants Biol. Pharm. Bull. 24: 1202-1205 Rodríguez J L, Valdés O, Bilbao T (2007): Antioxidant activity of common legumes. Rev. Cubana Farm. 41: 66 Singleton V L, Rossi J A Jr (1965): Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticult. 16: 144-158

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Antioxidant Activity of Phenolic Compounds in Lentil Seeds (Lens culinaris L.)

Jelena Dragisi Maksimovi Branka Zivanovi Vuk Maksimovi Vesna Hadzi-Taskovi Sukalovi

Institute for Multidisciplinary Research, Kneza Viseslava 1, 11030 Beograd, Serbia Summary: The antioxidant activities of methanol extracts of lentil seeds (Lens culinaris L.) have been investigated in this work. Scarce reference data describe lentil seeds as rich in polyphenols, which are reported to exhibit bioactive properties due to their capability to reduce or quench reactive oxygen species. The content and composition of phenolics is highly dependent of the cultivars, environments/growth conditions and method of analysis. Therefore, this study is an effort in investigation of phenolics content and composition in lentil seeds trying to prove the contribution of identified phenolics to antioxidant capacity. HPLC measurements revealed that lentil seeds contain gallic acid, epicatechin, catechin, protocatechuic acid, rutin, p-coumaric acid and umbeliferone. Their DPPH radical scavenging activity was in descending order from gallic acid to umbeliferone. The presented results contribute to knowledge of the implications in dietary intake of phenolic compounds from lentil seeds. Key words: lentil, phenolic compounds, total antioxidant capacity, total phenolic content

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Mikopopulacija semena kukuruza

561 Zastita useva / Crop Protection Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 561-566 originalni naucni rad / original research article

Mikopopulacija semena kukuruza (2006-2008)

Dragana Petrovi Maja Ignjatov Milka Vujakovi Ksenija Taski-Ajdukovi Zorica Nikoli Mirjana Milosevi Dusica Jovici

primljeno / received: 17.11.2009. prihvaeno / accepted: 24.05.2010. © 2010 IFVC

Izvod: Seme kukuruza parazitira veliki broj mikroorganizama, meu kojima su najznacajnije gljive. Za ispitivanje zdravstvenog stanja 90 uzoraka semena kukuruza u periodu 2006-2008. iz osam doradnih centara, korisen je metod vlaznog filter papira i metoda izolacije na hranljivoj podlozi. Na semenu kukuruza konstatovano je prisustvo gljiva iz rodova Fusarium, Penicillium, Aspergillus i Rhizopus. Stepen zarazenosti semena kukuruza gljivama u sve tri posmatrane godine bio je u dozvoljenim granicama prema vazeim propisima. Procenat zarazenih uzoraka iznosio je 45% u 2006, 40% u 2007. i 32% u 2008. U uzorcima u kojima je konstatovano prisustvo gljiva iz roda Fusarium procenat infekcije je iznosio od 0,25% do 5%, dok se procenat infekcije semena kukuruza gljivama iz grupe saprofita (Penicillium spp., Aspergillus spp. i Rhizopus spp.) kretao u granicama od 0,25% do 15%. Kljucne reci: gljive, kukuruz, mikroorganizmi, seme

Uvod Seme kao zavrsni proizvod zivotnog ciklusa biljke, sluzi za razmnozavanje, prezivljavanje, obnavljanje vrste, kao i za prenosenje zaraze (Milosevi & irovi 1994). Seme gajenih biljaka predstavlja pogodan supstrat za razvoj mikroorganizama, prouzrokovaca oboljenja, meu kojima najznacajnije mesto zauzimaju gljive (Milosevi i sar. 2008). Na semenu kukuruza konstatovano je preko 60 vrsta gljiva, bakterija i virusa (Richardson 1990). Mikroorganizmi (u prvom redu gljive) koji koloniziraju seme kukuruza mogu smanjiti prinos i kvalitet semenskog useva. Njihova stetnost moze se izraziti procentom zarazenog semena, smanjenjem kvaliteta i umanjenjem upotrebne vrednosti (Jovievi & Milosevi 1990). Usled prisustva patogena, na semenu najcese dolazi do smanjenja procenta klijavosti, pa cak i do potpunog propadanja ponika. Pored toga, neke vrste gljiva (Aspergillus spp., Penicillium spp., Fusarium spp.) u zarazenom zrnu stvaraju toksicne metabolite-mikotoksine (Milosevi i sar. 2008).

D. Petrovi ) ( M. Ignjatov M. Vujakovi K. Taski-Ajdukovi Z. Nikoli D. Jovici Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] M. Milosevi Ministarstvo poljoprivrede, sumarstva i vodoprivrede Republike Srbije, Nemanjina 22-26, 11000 Beograd, Srbija

Cilj ovih istrazivanja bio je da se utvrdi prisutnost mikopopulacije semena kukuruza u periodu od tri godine iz osam doradnih centara (Backa Palanka, Kikinda, Sremska Mitrovica, Srbobran, Magli, Vrbas, Zabalj i Backo Gradiste), s obzirom na to da je upotreba zdravog semena osnovni preduslov za dobru vitalnost i produktivnost biljaka. Materijal i metod rada Ispitivanje zdravstvenog stanja semena kukuruza vrseno je u periodu 2006-2008. u Institutu za ratarstvo i povrtarstvo (Laboratorija za ispitivanje semena) u Novom Sadu. Ispitivanje je obuhvatilo odreivanje procenta zarazenosti semena kod 30 uzoraka iz sve tri ispitivane godine (ukupno 90 uzoraka). Uzorkovanje semena izvrseno je u skladu sa ISTA pravilima (ISTA 2006). Zdravstveno stanje semena kukuruza ispitano je metodom filter papira i metodom izolacije na hranljivoj podlozi (PDA i CLA). Za ispitivanje zarazenosti semena kukuruza metodom filter papira koriseno je 400 slucajno odabranih semena od svakog uzorka koja su stavljena u sterilne Petri posude (precnika 9 cm) sa tri filter papira navlazena sterilnom vodom. U svaku Petri posudu postavljeno je po pet zrna. Inkubacija je obavljena na temperaturi od 22±2°C, tokom 7 dana, uz smenu svetla (NUV) i mraka

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svakih 12 sati (Mathur & Kongsdal 2003). Nakon isteka inkubacionog perioda vrsena je determinacija vrsta. Odabrani izolati gljiva iz roda Fusarium zasejavani su na PDA i CLA podlogu i koriseni su za dalja ispitivanja morfoloskih odlika i determinaciju vrste. Determinacija izolata iz roda Fusarium izvrsena je na osnovu karakteristika koje su dali Nelson et al. (1983) i Burgess et al. (1994). Na osnovu utvrenog procenta zaraze dobijen je uvid u zdravstveno stanje ispitivanih uzoraka kukuruza. Rezultati i diskusija Od ukupnog broja ispitanih uzoraka semena kukuruza, procenat zarazenih uzoraka iznosio je 45% u 2006, 40%u 2007. i 32% u 2008. Levi i sar. (2009) navode da su vrste roda Fusarium izolovane sa preko 100 biljnih vrsta. F. graminearum i F. moniliforme u nasoj zemlji predstavljaju najzastupljenije patogene kukuruza koji se prenose semenom. Ove gljive na kukuruzu prouzrokuju trulez stabla i plesnivost klipa odnosno zrna kukuruza. Veoma je bitno istai da ovi patogeni nastavlju sa razvojem i tokom skladistenja kukuruza, posebno u prvim mesecima po berbi (Milosevi & Jovievi 1987). Takoe, trogodisnjim istrazivanjima (2002-2004) ustanovljeno je da Fusarium spp. predstavljaju najzastupljenije patogene semena psenice (Balaz i sar. 2003, Bagi i sar. 2004, Bagi i sar. 2005). Seme zarazeno Fusarium vrstama dovodi do poveanog broja netipicnih ponika i smanjuje klijavost i vigor semena (Richardson 1990). Nasim ispitivanjima zdravstvenog stanja semena kukuruza utvreno je da su od parazitnih gljiva dominantne gljive iz roda Fusarium. Nakon 7 dana razvoja na filter papir podlozi, zarazena

semena bila su prekrivena ruzicastobelom i bledorozom micelijom parazita (Sl. 1a i 1b). Nakon izolacije i determinacije na semenu kukuruza utvreno je prisustvo gljiva Fusarium graminearum i Fusarium moniliforme i nekih saprofita. Gljiva Fusarium graminearum na PDA podlozi obrazovala je bujnu i gustu brzorastuu ruzicastobelu miceliju (Sl. 2a), dok se na CLA podlozi obrazovala ruzicasta vazdusasta micelija. Mikroskopskim pregledom uocene su tanke, vretenaste, srpolike elipticno savijene konidije, na kojima su se uocavale tanke pregrade (septe) ciji se broj kretao od tri do sedam (najcese pet). Apikalna elija makrokonidije je suzena na vrhu, dok je bazalna elija u obliku stopala. Mikrokonidije nisu formirane. Izolacijom gljive F. moniliforme na PDA podlozi doslo je do obrazovanja bledoroze micelije okruglog i ravnog oboda (Sl. 2b). Posmatranjem izolata na CLA podlozi konstatovan je veliki broj mikrokonidija. Makrokonidije su bile veoma retke, izduzene, tanke, skoro prave obicno sa 3-5 septi. Stepen zarazenosti semena kukuruza gljivama iz roda Fusarium u sve tri posmatrane godine bio je u dozvoljenim granicama prema vazeim propisima (Pravilnik o zdravstvenom pregledu semena, rasada i sadnog materijala-"Sluzbeni glasnik RS" 119/07), odnosno zarazenost uzoraka nije prelazila 5%. Procenat prisustva gljiva iz roda Fusarium iznosio je u proseku 3% u 2006, 2,75% u 2007. a 2,5% u 2008. (Graf. 1). Pored gljiva iz roda Fusarium, na semenu kukuruza utvreno je i prisustvo saprofitnih gljiva iz rodova Aspergillus, Penicillium i Rhizopus. Nakon inkubacije semena kukuruza na vlaznom filter papiru, kao i nakon izolacije na PDA podlozi, zapazeno je formiranje zutozelenkaste kolonije koju formira gljiva Aspergillus

Slike 1a i 1b. Seme kukuruza zarazeno gljivom iz roda Fusarium (filter papir metod) Figures 1a and 1b. Maize seeds infected by Fusarium spp. (blotter method)

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Slika 2a. Micelija gljive Fusarium graminearum na PDA podlozi Figure 2a. Mycelium of Fusarium graminearum on PDA medium

Slika 2b. Micelija gljive Fusarium moniliforme na PDA podlozi Figure 2b. Mycelium of Fusarium moniliforme on PDA medium

spp. i crne rastresite micelije karakteristicne za gljivu Aspergillus niger (Sl. 3a i 3b). Mikroskopskim pregledom uocavale su se konidiofore na cijem vrhu su se nalazile okrugle do elipticne konidije. Prosecna zastupljenost gljiva roda Aspergillus iznosila je 1,25% u 2006, 1,75% u 2007, a 1,5% u 2008. (Graf. 1). Pojava kolonija plavicasto zelenkaste boje, kako na semenu kako primenom metode na filter papiru, tako i nakon izolacije na PDA podlozi, ukazivala je na prisustvo gljive iz roda Penicillium (Sl. 4a i 4b). Mikroskopskim pregledom uoceno je prisustvo konidija elipticnog oblika.

Procenat zaraze gljivama iz roda Penicillium iznosio je 2% u 2006, 2,25% u 2007. i 1,75% u 2008. (Graf. 1). Od saprofitnih gljiva u ispitivanim uzorcima, pored gljiva iz roda Aspergillus i Penicillium, najvei broj uzoraka bio je zarazen gljivama iz roda Rhizopus ciji je procenat u uzorcima u kojima je bio zabelezen iznosio 5,25% u 2006, 5,75% u 2007. a 6,75% u 2008. (Graf. 1). Gljive roda Rhizopus najzastupljenije su i na semenu drugih biljnih vrsta, sto potvruju istrazivanja Jevti i sar.. (2008) gde je na semenu psenice ova saprofitna gljiva bila najzastupljenija u ispitivanim uzorcima u tri doradna centra.

Grafikon 1. Procenat zarazenosti semena kukuruza gljivama na filter papiru 2006, 2007. i 2008 Graph 1. Percentage of maize seed infection on blotter in 2006, 2007 and 2008

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Slika 3a. Seme kukuruza zarazeno gljivom Aspergillus niger (filter papir metod) Figure 3a. Seeds of maize infected by Aspergillus niger (blotter method)

Slika 3b. Micelija gljive Aspergillus niger na PDA podlozi Figure 3b. Mycelium of Aspergillus niger on PDA medium

Slika 4a. Seme kukuruza zarazeno gljivom roda Penicillium (filter papir metod) Figure 4a. Seeds of maize infected by Penicillium spp. (blotter method)

Slika 4b. Micelija gljive roda Penicillium na PDA podlozi Figure 4b. Mycelium of Penicillium spp. on PDA medium

Milosevi i sar. (2007a) ukazuju da su od parazitnih gljiva semena kukuruza dominantne gljive iz roda Fusarium, sto odgovara i nasim rezultatima. Pored gljiva iz roda Fusarium, pomenuti autori su uocili i prisustvo gljiva iz roda Helmithosporium, Penicillium i Aspergillus. U cilju prikupljanja i dugorocnog cuvanja cistih kultura gljiva, Milosevi i sar. (2007b, 2008) koristili su metod liofilizacije (freeze drying).

Zakljucci Iz svega navedenog proistice da je potrebno nastaviti kontinuirano praenje nivoa zarazenosti semena kukuruza gljivama koje mogu znacajno varirati iz godine u godinu u zavisnosti od velikog broja cinilaca i time ugroziti kvalitet semena i znacajnije uticati na smanjenje prinosa.

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Literatura

Bagi F, Stojsin V, Balaz F (2004): Mikopopulacija semena strnih zita u 2003. godini. Zito-hleb 31: 75-82 Bagi F, Stojsin V, Balaz F (2005): Mikopopulacija semena sortnih zita u Srbiji. Zbornik Matice srpske za prirodne nauke 108: 189-195 Balaz F, Bagi F, Skrinjar M, Stojsin V (2003): Mikopopulacija semena strnih zita u 2002. godini. Zito-hleb 30: 149-155 Burgess L W, Summerell B A, Bullock S, Gott K P, Backhouse D (1994): Laboratory Manual for Fusarium Research. Fusarium Research Laboratory, Department of Crop Sciences, University of Sydney and Royal Botanic Gardens, Sydney Dragani M, Milosevi M, Matijevi D (1997): Oboljenja kukuruza. Drustvo za zastitu bilja Srbije, Beograd International Rules for Seed Testing (2006): International Seed Testing Association Zürich, Switzerland Levi J T, Stankovi S Z, Krnjaja V S, Bocarov-Stanci A S (2009): Fusarium vrste - pojava i znacaj u Srbiji. Zbornik Matice srpske za prirodne nauke 116: 33-48 Jevti R, Telecki M, Vujakovi M, Ignjatov M, Statki S (2008): Jaca pojava saprofita i crvenila na strni zitima tokom 2007. godine. Biljni lekar 6: 418-425 Jovievi B, Milosevi M (1990): Bolesti semena, Dnevnik, Novi Sad Mathur S B, Kongsdal O (2003): Common Laboratory Seed Health Testing Methods for Detecting Fungi. ISTA, Zürich, Switzerland Milosevi M, Jovievi B (1987): Fitopatolosko ispitivanje zdravstvenog stanja semena u Jugoslaviji. ISTA News Bulletin, 11-12 Milosevi M, irovi M (1994): Seme. Institut za ratarstvo i povrtarstvo, Novi Sad Milosevi M, Ignjatov M, Medi-Pap S (2007a): Maize seedborne diseases. Plant Protect. 18: 22-24 Milosevi M, Medi-Pap S, Ignjatov M, Petrovi D (2007b): Lyophilization as a method for pathogens long term preservation. Zbornik matice srpske za prirodne nauke 113: 203-210 Milosevi M, Ignjatov M, Vujakovi M, Petrovi D, Nikoli Z, Doki V (2008): Svojstva gljiva roda Fusarium sp. i mogunost dugorocnog cuvanja metodom liofilizacije. Arhiv za poljoprivredne nauke 69: 89-95 Nelson P E, Toussoun T A, Marasas W F O (1983): Fusarium Species, an Illustrated Manual for Identification. The Pennsylvania State University Press, University Park and London Pravilnik o zdravstvenom pregledu semena, rasada i sadnog materijala. Sluzbeni glasnik Republike Srbije 119: 2007 Richardson M J (1990): An Annotated List of Seed-Borne Diseases. ISTA, Zürich, Switzerland

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Mycopopulation of Maize Seeds (2006-2008)

Dragana Petrovi1 Maja Ignjatov1 Milka Vujakovi1 Ksenija Taski-Ajdukovi1 Zorica Nikoli1 Mirjana Milosevi2 Dusica Jovici1

1

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia 2 Ministry of Agriculture, Forestry and Water Management, Nemanjina 22-26, 11000 Belgrade, Serbia

Summary: A large number of microorganisms' parasite on maize seed. The most important of them are fungi. Blotter method and method of nutrient medium were used to examine the seed health of 90 maize seed samples from eight seed processing centres in the period 2006-2008. The presence of fungi from the genera Fusarium, Penicillium, Aspergillus and Rhizopus was observed on maize seed. According to current regulations, level of infection caused by the fungi observed in all three years was within the normal limits. The percentage of infected samples was 45% in 2006, 40% in 2007, and 32% in 2008. Percentage of infection ranged from 0.25% to 5% in samples where the presence of fungi of the genus Fusarium was detected, while the percentage of infection on maize seed caused by the fungi from saprophite group (Penicillium spp., Aspergillus spp. and Rhizopus spp.) ranged within the limits of 0.25% to 15%. Key words: fungi, maize, microorganisms, seeds

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Virusi paprike u Srbiji

567 Zastita useva / Crop Protection Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 567-576 originalni naucni rad / original research article

Prisustvo i rasprostranjenost virusa paprike u Srbiji

Dragana Petrovi Aleksandra Bulaji Ivana Stankovi Maja Ignjatov Milka Vujakovi Branka Krsti

primljeno / received: 16.04.2010. prihvaeno / accepted: 24.05.2010. © 2010 IFVC

Izvod: Virusne bolesti paprike mogu ugroziti normalan razvoj biljaka i time znacajno umanjiti prinos i kvalitet plodova. U svetu je opisano vise od 45 virusa infektivnih za papriku, meu kojima su ekonomski najznacajniji virus mozaika duvana (Tobacco mosaic virus, TMV), virus mozaika krastavca (Cucumber mosaic virus, CMV), virus mozaika lucerke (Alfalfa mosaic virus, AMV), virus bronzavosti paradajza (Tomato spotted wilt virus, TSWV) i virus crticastog mozaika krompira (Potato virus Y, PVY). Prisustvo i rasprostranjenost virusnih zaraza paprike u Srbiji ispitivani su tokom 2009. u razlicitim lokalitetima proizvodnje rasada, kao i useva paprike gajene u zastienom prostoru i na otvorenom polju. Sakupljeni uzorci lisa sa simptomima testirani su DAS-ELISA metodom na prisustvo: PVY, AMV, CMV, TSWV, TMV i virusa mozaika krompira (Potato virus X, PVX). Dobijeni rezultati ispitivanja su ukazali na prisustvo cetiri virusa: PVY, CMV, TSWV i AMV, koji su identifikovani u pojedinacnim ili mesanim infekcijama. U testiranim uzorcima paprike iz rasada najzastupljeniji je bio CMV (18,18%), dok je u uzorcima prikupljenim iz plastenika i polja najcesi bio PVY (32,56% i 51,21%). Prisustvo TSWV dokazano je samo na biljkama paprike gajenim u zastienom prostoru. Prisustvo PVX i TMV nije ustanovljeno ni u jednom ispitivanom uzorku paprike. Utvrena znacajna rasprostranjenost i zastupljenost PVY i CMV u proizvodnji paprike u Srbiji ukazuje na potrebu detaljne karakterizacije ova dva virusa na bioloskom i molekularnom nivou, kao i sprovoenje odgovarajuih mera kontrole. Kljucne reci: DAS-ELISA test, paprika, virus bronzavosti paradajza, virus crticastog mozaika krompira, virus mozaika krastavca, virus mozaika lucerke

Uvod Paprika (Capsicum annuum L.) ima veliki privredni znacaj u Srbiji, ubraja se u grupu najznacajnijih povrtarskih useva i gaji se na oko 20.000 ha (Gvozdenovi i sar. 2008), kako u zastienom prostoru tako i na otvorenom polju. Sa ekonomskog stanovista, proizvodnja, prerada i meunarodna razmena, prvenstveno suve i sveze, ali i industrijske zacinske paprike, slatke i ljute, cini ovu kulturu jednim od najznacajnijih useva povra i 2004. ukupno je izvezeno proizvoda u vrednosti preko 1 miliona USD (izvor SIEPA 2005). Brojne biljne bolesti mogu da smanje ili ugroze uspesnu proizvodnju ometajui normalan razvoj biljke i umanjujui prinos i kvalitet plodova. Biljni virusi prouzrokuju znacajne stete na paprici, tako da gubici cesto dostizu 90% (Krsti & Bulaji 2008).

D. Petrovi ) ( M. Ignjatov M. Vujakovi Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] A. Bulaji I. Stankovi B. Krsti Poljoprivredni fakultet Univerziteta u Beogradu, Institut za fitomedicinu, Nemanjina 6, 11080 Beograd - Zemun, Srbija

Zbog znacajnih steta koje cesto prouzrokuju, viroze paprike su predmet proucavanja mnogih autora u svetu (Marchoux et al. 1991, Arli-Sokmen et al. 2005, Choi et al. 2005, Ozaslan et al. 2006). Utvreno je da vise od 45 razlicitih virusa moze da zarazi papriku, ali se smatra da je meu njima svega nekoliko ekonomski znacajno (Green & Kim 1990). Neki virusi su siroko rasprostranjeni, kao sto su virus mozaika krastavca (Cucumber mosaic virus, CMV), virus mozaika duvana (Tobacco mosaic virus, TMV), virus crticastog mozaika krompira (Potato virus Y, PVY) i virus mozaika lucerke (Alfalfa mosaic virus, AMV), dok su drugi jos uvek vezani samo za odreena geografska podrucja (Begomovirusi) (Krsti & Bulaji 2008). Meutim, situacija se vremenom menja, a najbolji primer za to je virus bronzavosti paradajza (Tomato spotted wilt virus, TSWV), koji se ranije cese javljao u suptropskim i tropskim regionima, a danas je opste rasprostranjen

Ovo istrazivanje je deo projekta broj 20077: Stvaranje i korisenje sorata i hibrida povra za otvoreno polje (2008-2011), finansiranog od strane Ministarstva za nauku i tehnoloski razvoj, Republike Srbije / This research results from the project 20077: Development and utilization of vegetable varieties and hybrids for field production, financed by the Ministry of Science and Technological Development of the Republic of Serbia

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Petrovi D i sar.

u svetu, a kod nas je takoe detektovan na brojnim domainima, ukljucujui i papriku (Krsti & Bulaji 2007). Virusi na paprici nisu dovoljno prouceni iako se kod nas javljaju svake godine, a pojedinih godina mogu da izazovu epidemije. U Srbiji su prva proucavanja virusa paprike bila vezana za TMV (Panjan & Prpi 1955), CMV i AMV (Suti 1959, Delevi 1963). Kasnijim ispitivanjima utvreno je i prisustvo sledeih virusa: TSWV, PVY, virusa blagog sarenila paprike (Pepper mild mottle virus, PMMoV), virusa sarenila paprike (Pepper mottle virus, PepMV) i virusa sarenila nerava paprike (Pepper veinal mottle virus, PVMV) (Mijatovi 1986, Mijatovi i sar. 1999, Mijatovi i sar. 2002, Mijatovi i sar. 2007). Poslednja tri virusa se javljaju sporadicno, dok su ostali navedeni virusi ekonomski znacajni (Mijatovi i sar. 2007, Krsti & Bulaji 2008). Istrazivanja sprovedena 2007. na devet lokaliteta gajenja paprike u Srbiji potvrdila su prisustvo virusa iz dve osnovne grupacije: karantinski (TSWV) i ekonomski stetni (CMV, AMV i TMV) (Tomi i sar. 2007). Pojava TMV izaziva smanjenje prinosa paprike i do 80% (Mijatovi i sar. 2007), dok je u slucaju pojave virusnih zaraza prouzrokovanih sa PVY i CMV utvreno smanjenje prinosa do 50% (MnariHattab et al. 1999). S obzirom da virusi svake godine smanjuju prinos i kvalitet paprike u Srbiji, a pojedinih se godina javljaju u epidemijskim razmerama, praenje prisustva, ucestalosti i rasprostranjenosti pojedinih virusa znacajno je za izbor i sprovoenje adekvatnih mera kontrole u proizvodnji paprike. Materijal i metod rada Sakupljanje uzoraka paprike U cilju utvrivanja prisustva, rasprostranjenosti i ucestalosti virusa paprike u Srbiji tokom 2009. pregledano je 15 razlicitih lokaliteta rasadnicke proizvodnje paprike (Tab. 1), 15 plastenicke proizvodnje (Tab. 2) i 16 lokaliteta proizvodnje paprike na otvorenom polju (Tab. 3). Prilikom pregleda sakupljeno je ukupno 457 uzoraka paprike razlicitih sorti, sa simptomima koji su ukazivali na virusnu zarazu. Od ukupnog broja prikuljenih uzoraka, 121 uzorak je poreklom iz rasada paprike, 129 iz plastenicke proizvodnje, a 207 uzoraka je iz proizvodnje paprike u polju. Svaki uzorak se sastojao od vise listova sa simptomima sa jedne biljke. Prilikom pregleda i sakupljanja uzoraka paprike, registrovan je tip i vrsta simptoma za dalju analizu povezanosti ispoljenih simptoma sa vrstom virusa koji je dokazan primenom seroloskih metoda.

Direktna imunoenzimska metoda na ploci (DAS-ELISA) Prikupljeni uzorci paprike sa simptomima koji su ukazivali na virusne zaraze testirani su DASELISA metodom (Clark & Adams 1977) primenom komercijalnih poliklonalnih antiseruma specificnih za detekciju sest najznacajnijih virusa paprike: AMV, PVY, CMV, TSWV, TMV i PVX prema uputstvu proizvoaca (Loewe Biochemica GmbH, Nemacka). Uzorci su pripremani homogenizacijom biljnog materijala u ekstrakcionom puferu u razreenju 1:20. Specificna poliklonalna antitela i poliklonalna antitela konjugovana sa akalnom fosfatazom korisena su u razreenju 1:200 u odgovarajuem puferu. Jedan cas po dodavanju supstrata p-nitrofenil fosfata (1 mg ml-1), reakcija je ocitavana merenjem apsorpcije na 405 nm. Pozitivnom reakcijom ocenjivane su vrednosti ekstinkcije koje su dva i vise puta vee od vrednosti ekstinkcije negativnih kontrola. Rezultati i diskusija Simptomi u polju Pregledom proizvodnje rasada i useva paprike u razlicitim lokalitetima gajenja u zastienom prostoru i u polju, utvrena je pojava niza simptoma koji su upuivali na virusnu zarazu i koji su varirali u zavisnosti od biljke domaina, vremena ostvarene infekcije i vrste virusa. Simptomi na biljkama u rasadu bili su slabo izrazeni, usled cega obolele biljke nisu mogle sa sigurnosu biti razlikovane od zdravih. Ipak, tokom ovih istrazivanja, na odreenom broju biljaka uoceni su simptomi u vidu blagog mozaika i hloroze lisa, nekroticnih lezija kao i razlicite deformacije liske. Pregledom useva paprike i u zastienom prostoru i na otvorenom polju zabelezena je pojava biljaka koje su cesto zaostajale u porastu i usled skraivanja internodija poprimale zbunast izgled. Osim promena opsteg izgleda biljaka simptomi su se manifestovali i u vidu hromatskih i morfoloskih promena na listovima i plodovima. Lise obolelih biljaka bilo je sitnije, slabo naborano, cesto sa razlicitim tipovima mozaika, od blagog do jako izrazenog, sa mozaicnim sarama zukaste boje i hloroticnim povrsinama koje kasnije zahvataju ceo list. Hloroticne promene cesto su bile praene pojavom izumiranja tkiva, uglavnom u vidu nekroze nerava lista i nekroticnih pega na stablu. Na plodovima, koji su bili krzljavi, deformisani i sa nekrozom povrsinskog tkiva, cesto su bile prisutne i linearne belicaste

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ili zukaste pruge i prosaravanja. Simptomi uoceni tokom ovih istrazivanja u saglasnosti su sa ranije opisanim simptomima na paprici u nasoj zemlji (Krsti & Bulaji 2008, Mijatovi i sar. 2007, Tomi i sar. 2007). Ipak, simptomi na inficiranim biljkama cesto mogu liciti na one prouzrokovane gljivama ili bakterijama, osteenjima od herbicida, zagaenja vazduha ili mineralnih nedostataka, tako da dijagnostika viroza u usevu paprike ne moze da se obavlja samo na osnovu simptoma (Ozaslan et al. 2006). Seroloska detekcija virusa Ispitivanjima 121 uzorka sa razlicitih lokaliteta proizvodnje rasada paprike ustanovljeno je prisustvo tri virusa CMV, PVY i AMV, koji se prenose vasima na neperzistentan nacin u 23,97%

uzoraka, dok u 76,03% testiranih uzoraka nije utvreno prisustvo nijednog od ispitivanih virusa. Visok procenat biljaka u kojima nije dokazano prisustvo virusa, uprkos ustanovljenim simptomima, ukazali su na mogunost zaraze rasada virusima koji nisu bili ukljuceni u istrazivanja ili su simptomi posledica nekih drugih faktora. Najzastupljeniji virus CMV bio je detektovan u 18,18% testiranih uzoraka, zatim sledi PVY (3,31%), dok je prisustvo AMV utvreno samo kod izuzetno malog broja uzoraka (2,48%) (Tab. 1). Svi detektovani virusi bili su zastupljeni iskljucivo u pojedinacnoj zarazi. Prisustvo TSWV, PVX i TMV nije ustanovljeno ni u jednom od ispitivanih uzoraka. Rezultati testiranja paprike u rasadu takoe ukazuju na nizak procenat, ili odsustvo, virusa koji se prenose semenom paprike, kakvi su TMV i AMV.

Tabela 1. Prisustvo i procentualna zastupljenost virusa paprike u rasadnickoj proizvodnji Table 1. Presence and percentage of incidence of pepper viruses in transplant production

Lokaliteti Localities Backa Palanka Obrovac Veternik Krusevac I Krusevac II Srbobran Kula Ruma urevo Zabalj Pivnice Gospoinci Rimski Sancevi Aleksinac Sabac UKUPNO Total PVY 0/6* 0% 0/7 0% 2/6 33,33% 0/7 0% 0/7 0% 0/5 0% 1/4 25,00% 0/10 0% 0/10 0% 0/8 0% 0/14 0% 0/8 0% 0/13 0% 0/13 0% 1/3 33,33% 4/121 3,31% CMV 2/6 33,33% 1/7 14,29% 0/6 0% 0/7 0% 0/7 0% 0/5 0% 1/4 25,00% 1/10 10,00% 2/10 20,00% 1/8 12,50% 3/14 21,43% 1/8 12,50 0/13 0% 10/13 76,92% 0/3 0% 22/121 18,18% AMV 0/6 0% 0/7 0% 0/6 0% 0/7 0% 0/7 0% 2/5 40,00% 0/4 0% 1/10 10,00% 0/10 0% 0/8 0% 0/14 0% 0/8 0% 0/13 0% 0/13 0% 0/3 0% 3/121 2,48% Uzorci bez prisustva testiranih virusa The samples without tested viruses 4/6 66,67% 6/7 85,71% 4/6 66,67% 7/7 100% 7/7 100% 3/5 60,00% 2/4 50,00% 8/10 80,00% 8/10 80% 7/8 87,50% 11/14 78,57% 7/8 87,50% 13/13 100% 3/13 23,08% 2/3 66,67% 92/121 76,03%

* broj zarazenih biljaka / broj testiranih biljaka * number of infected plants / number of tested plants

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Petrovi D i sar.

Ispitivanjem 129 uzoraka paprike sakupljenih na 15 razlicitih lokaliteta proizvodnje paprike u zastienom prostoru ustanovljeno je prisustvo virusa u 86,82% uzoraka. Detektovana su cetiri virusa: PVY, CMV, AMV i TSWV, od kojih se prva tri prenose vasima na neperzistentan nacin, a TSWV tripsima. Prisustvo navedenih virusa detektovano je sa relativno ujednacenom ucestalosu. Najzastupljeniji virus, u pojedinacnim ili mesanim zarazama, bio je PVY (32,56%), zatim CMV (31,01%) i TSWV (23,26%), dok je AMV detektovan u nesto manjem broju uzoraka (17,05%) (Tab. 2). Kod najveeg broja uzoraka utvrena je pojedinacna zaraza sa TSWV (23,26%). Kod 17,05% testiranih uzoraka detektovana je pojedinacna zaraza sa PVY ili CMV, a kod 12,40% pojedinacna zaraza sa AMV. Mesana zaraza sa PVY i CMV je detektovana u 12,40% uzoraka, dok je zaraza sa PVY i AMV, kao i CMV i AMV detektovana u malom broju testiranih uzoraka, u 3,10% odnosno 1,55%. Kod 13,18% uzoraka nije utvreno prisustvo nijednog od ispitivanih virusa, mada su simptomi bili tipicni za virusne zaraze, sto moze ukazivati na prisustvo drugih virusa koji nisu ukljuceni u ispitivanja. Prisustvo TMV i PVX nije dokazano ni u jednom testiranom uzorku. U usevima paprike gajenim na otvorenom polju, od ukupno testiranih 207 uzoraka sa simptomima (Tab. 3), ustanovljeno je prisustvo virusa u 83,09% uzoraka, i to dominantno prisustvo PVY (51,21%) u pojedinacnim i mesanim zarazama, za kojim slede CMV (37,20%) i AMV (21,74%). U pogledu pojedinacnih zaraza, najzastupljenije su bile zaraze sa PVY (26,57%), a zatim pojedinacne zaraze sa CMV ili AMV (15,46%). U pogledu mesanih zaraza, detektovane su zaraze sa dva ili tri virusa. Najcese je detektovana mesana zaraza sa dva virusa i to PVY i CMV (19,32%), dok su ostale mesane zaraze dokazane u relativno malom broju uzoraka, PVY i AMV (3,86%), CMV i AMV (0,97%), a samo u tri od 207 uzoraka (1,45%) mesana zaraza sa sva tri detektovana virusa. Kod 16,91% testiranih uzoraka nije utvreno prisustvo ispitivanih virusa, sto je ukazalo na potrebu ispitivanja prisustva sireg kruga virusa infektivnih za papriku u buduim pregledima useva paprike u Srbiji. Prisustvo TMV, TSWV i PVX nije utvreno ni u jednom testiranom uzorku sa otvorenog polja. Ispitivanjem prisustva i rasprostranjenosti virusa u usevu paprike u nasoj zemlji utvreno je prisustvo cetiri virusa: PVY, CMV, AMV i TSWV. Najcese detektovan virus je PVY,

kako u usevu paprike gajene u zastienom prostoru tako i na otvorenom polju, dok je zaraza rasada bila znacajno niza, sa dominantnim prisustvom CMV. Ovako dominantno prisustvo PVY u 2009. znacajno se razlikuje u odnosu na 1999. kada je PVY bio trei po zastupljenosti (Mijatovi i sar. 1999) i 2007. kada njegovo prisustvo nije ustanovljeno (Tomi i sar. 2007). Uporedna analiza zastupljenosti CMV u razlicitim godinama pokazuje njegovu stalnu visoku zastupljenost. U istrazivanjima sprovedenim u ovom radu bio je drugi po zastupljenosti, dok je 1999. (Mijatovi i sar. 1999) i 2007. (Tomi i sar. 2007) bio dominantan u usevu paprike. Ovako znacajna zastupljenost CMV dokazana je i u drugim podrucjima gajenja paprike u svetu (Conti & Masenga 1977, Gaborjanyia et al. 1998, Sepulveda et al. 2005). Iako je AMV stalno prisutan u usevu paprike (Mijatovi i sar. 1999, Tomi i sar. 2007), tokom 2009. dokazan je u manjem broju testiranih uzoraka. Znacajno prisustvo TSWV (23,26%) na paprici gajenoj u zastienom prostoru nije neobicno, imajui u vidu epidemiologiju ovog virusa. TSWV ima sirok krug domaina meu povrem i korovima, a efikasno se siri tripsima (Krsti & Bulaji 2007), za ocekivati je da TSWV bude u daljoj ekspanziji na paprici, narocito u zastienom prostoru. U ispitivanim uzorcima paprike gajene i u zastienom prostoru i na otvorenom polju nije utvreno prisustvo TMV, mada su Mijatovi i sar. (1999) kao i Tomi i sar. (2007) ustanovili da je ovaj virus prisutan i pojedinih godina veoma znacajan na paprici. Analiza dobijenih rezultata ukazuje na tendenciju smanjenja znacaja zaraze paprike sa TMV, verovatno usled dobrog zdravstvenog stanja semena ili dobro sprovedenih higijenskih mera u proizvodnom procesu gajenja paprike. Lista virusa infektivnih za papriku stalno se prosiruje, ne samo zbog pojave novih virusa, ve i zbog nastanka novih patotipova/sojeva u okviru populacije pojedinih virusa. Pojava i rasprostranjenost veine vrsta virusa zavise od brojnih faktora, kao sto su region i lokacija gajenja, vegetaciona sezona, genotip paprike i drugih. Proucavajui prisustvo virusa paprike u Italiji, Conti & Masenga (1977) su utvrdili dominantno prisustvo CMV u 80,2% biljaka paprike gajene na otvorenom prostoru, dok je u zastienom prostoru najzastupljeniji bio TMV sa 84%. Osim ovih virusa utvreno je prisustvo i PVY, AMV, PVX, Broad bean wilt virus, (BBWV) i Tobacco rattle virus, (TRV). Prema istrazivanjima autora Gaborjanyi et al. (1998) najzastupljeniji virusi paprike u Maarskoj su TMV, CMV, PVY, i TSWV, cije

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Tabela 2. Prisustvo i procentualna zastupljenost virusa paprike u pojedinacnim i mesanim infekcijama u plastenickoj proizvodnji Table 2. Presence and percentage of incidence of pepper viruses in single and mixed infections in greenhouse production

Mesana zaraza Mixed infection Ukupna zaraza Total infection

Pojedinacna zaraza Single infection

TSWV

AMV

Lokalitet Locality PVY PVY +AMV PVY+ CMV AMV +CMV TSWV AMV CMV PVY

CMV

Bez testiranih virusa No tested viruses

Virusi paprike u Srbiji

Lukino Selo

Srbobran

Velika Plana

Trstenik

Smederevo

Kikinda

urevo

Selenca

Aleksinac

Sivac

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Sombor

Cacak

Pivnice

Zabalj

Veternik

Ukupno Total

10/10* 100% 0/10 0% 0/10 0% 10/10 100% 0/5 0% 0/10 0% 10/10 100% 0/15 0% 0/10 0% 0/5 0% 0/5 0% 0/6 0% 0/10 0% 0/8 0% 0/5 0% 30/129 23,26%

0/10 0% 0/10 0% 0/10 0% 0/10 0% 0/5 0% 0/10 0% 0/10 0% 4/15 26,67% 0/10 0% 5/5 100% 0/5 0% 0/6 0% 5/10 50,00% 2/8 25,00% 0/5 0% 16/129 12,40%

0/10 0% 0/10 0% 0/10 0% 0/10 0% 5/5 100% 4/10 40,00% 0/10 0% 0/15 0% 7/10 70,00% 0/5 0% 0/5 0% 4/6 66,67% 0/10 0% 2/8 25,00% 0/5 0% 22/129 17,05%

0/10 0% 8/10 80,00% 0/10 0% 0/10 0% 0/5 0% 0/10 0% 0/10 0% 5/15 33,33% 1/10 10,00% 0/5 0% 5/5 100% 1/6 16,67% 0/10 0% 2/8 25,00% 0/5 0% 22/129 17,05%

0/10 0% 0/10 0% 4/10 40,00% 0/10 0% 0/5 0% 0/10 0% 0/10 0% 0/15 0% 0/10 0% 0/5 0% 0/5 0% 0/6 0% 0/10 0% 0/8 0% 0/5 0% 4/129 3,10%

0/10 0% 0/10 0% 4/10 40,00% 0/10 0% 0/5 0% 4/10 40,00% 0/10 0% 0/15 0% 2/10 20,00% 0/5 0% 0/5 0% 1/6 16,67% 0/10 0% 0/8 0% 5/5 100% 16/129 12,40%

0/10 0% 0/10 0% 0/10 0% 0/10 0% 0/5 0% 0/10 0% 0/10 0% 0/15 0% 0/10 0% 0/5 0% 0/5 0% 0/6 0% 0/10 0% 2/8 25,00% 0/5 0% 2/129 1,55%

0/10 0% 2/10 20,00% 2/10 20,00% 0/10 0% 0/5 0% 2/10 20,00% 0/10 0% 6/15 40,00% 0/10 0% 0/5 0% 0/5 0% 0/6 0% 5/10 50,00% 0/8 0% 0/5 0% 17/129 13.18%

10/10 100% 0/10 0% 0/10 0% 10/10 100% 0/5 0% 0/10 0% 10/10 100% 0/15 0% 0/10 0% 0/5 0% 0/5 0% 0/6 0% 0/10 0% 0/8 0% 0/5 0% 30/129 23,26%

0/10 0% 0/10 0% 4/10 40,00% 0/10 0% 0/5 0% 0/10 0% 0/10 0% 4/15 26,67% 0/10 0% 5/5 100% 0/5 0% 0/6 0% 5/10 50,00% 4/8 50,00% 0/5 0% 22/129 17,05%

0/10 0% 0/10 0% 4/10 40,00% 0/10 0% 5/5 100% 8/10 80,00% 0/10 0% 0/15 0% 9/10 90,00% 0/5 0% 0/5 0% 5/6 83,33% 0/10 0% 4/8 50,00% 5/5 100% 40/129 31,01%

0/10 0% 8/10 80,00% 8/10 80,00% 0/10 0% 0/5 0% 4/10 40,00% 0/10 0% 5/15 33,33% 3/10 30,00% 0/5 0% 5/5 100% 2/6 33,33% 0/10 0% 2/8 25,00% 5/5 100% 42/129 32,56%

571

* broj zarazenih biljaka / broj testiranih biljaka * number of infected plants / number of tested plants

Tabela 3. Prisustvo i procentualna zastupljenost virusa paprike u pojedinacnim i mesanim infekcijama u proizvodnji na otvorenom polju Table 3. Presence and percentage of incidence of pepper viruses in single and mixed infections in open field production

572

Mesana zaraza Mixed infection Ukupna zaraza Total infection

Pojedinacna zaraza Single infection

AMV

CMV

PVY

PVY+ AMV

PVY+ CMV

AMV+ CMV

AMV+ PVY+ CMV

Bez testiranih virusa No tested viruses

AMV

CMV

Selenca

Velika Plana

Horgos I

Ravno Selo

Horgos II

Conoplja

Cacak

urevo

Trstenik

Backa Palanka

Senta

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0/14 0% 4/14 28,57% 0/14 0% 0/14 0% 1/14 7,14% 5/14 35,71% 0/9 0% 8/207 3,86% 6/9 6,67% 40/207 19,32% 0/9 0% 2/207 0,97% 1/9 11,11% 3/207 1,45% 0/9 0% 35/207 16,91% 1/9 11,11% 45/207 21,74%

Kraljevo

Smederevo

Inija

5/10* 50,00% 3/15 20,00% 6/10 60,00% 5/15 33,33% 0/5 0% 0/9 0% 0/15 0% 0/15 0% 0/10 0% 3/15 20,00% 5/20 25,00% 0/15 0% 0/15 0% 0/15 0%

0/10 0% 7/15 46,67% 0/10 0% 0/15 0% 1/5 20,00% 0/9 0% 4/15 26,67% 4/15 26,67% 4/10 40,00% 0/15 0% 3/20 15,00% 0/15 0% 7/15 46,67% 0/15 0%

0/10 0% 0/15 0% 2/10 20,00% 3/15 20,00% 0/5 0% 8/9 88,89% 0/15 0% 4/15 26,67% 4/10 40,00% 8/15 53,33% 7/20 35,00% 8/15 53,33% 2/15 13,33% 5/15 33,33%

0/10 0% 0/15 0% 0/10 0% 2/15 13.33% 0/5 0% 0/9 0% 0/15 0% 0/15 0% 0/10 0% 2/15 13,33% 0/20 0% 0/15 0% 0/15 0% 4/15 26,67%

0/10 0% 0/15 0% 0/10 0% 2/15 13,33% 3/5 60,00% 1/9 11,1% 6/15 40,00% 4/15 26,67% 0/10 0% 0/15 0% 0/20 0% 4/15 26,67% 6/15 40,00% 4/15 26,67%

0/10 0% 2/15 13,33% 0/10 0% 0/15 0% 0/5 0% 0/9 0% 0/15 0% 0/15 0% 0/10 0% 0/15 0% 0/20 0% 0/15 0% 0/15 0% 0/15 0%

0/10 0% 0/15 0% 0/10 0% 1/15 6,67% 1/5 20,00% 0/9 0% 0/15 0% 0/15 0% 0/10 0% 0/15 0% 0/20 0% 0/15 0% 0/15 0% 0/15 0%

5/10 50,00% 3/15 20,00% 2/10 20,00% 2/15 0% 0/5 0% 0/9 0% 5/15 33,33% 3/15 20,00% 2/10 20,00% 2/15 13,33% 5/20 25,00% 3/15 20,00% 0/15 0% 2/15 13,33%

5/10 50,00% 5/15 33,33% 6/10 60,00% 8/15 53,33% 1/5 20,00% 0/9 0% 0/15 0% 0/15 0% 0/10 0% 5/15 33,33% 5/20 25,00% 0/15 0% 0/15 0% 4/15 26,67%

0/10% 0% 9/15 60,00% 0/10 0% 3/15 20,00% 5/5 100% 1/9 11,11% 10/15 66,67% 8/15 53,33% 4/10 40,00% 0/15 0% 3/20 15,00% 4/15 26,67% 13/15 86,67% 4/15 26,67% 6/14 42,86%

0/10 0% 0/15 0% 2/10 20,00% 8/15 53,33% 4/5 80,00% 9/9 100% 6/15 40,00% 8/15 53,33% 4/10 40,00% 10/15 66,66% 7/20 35,00% 12/15 80,00% 8/15 53,33% 13/15 86,67% 6/14 42,86% 9/9 100% 106/207 51,21%

Aleksinac

5/14 35,71%

2/14 14,29%

2/14 14,29%

Veternik

Petrovi D i sar.

Ukupno Total

0/9 0% 32/207 15,46%

0/9 0% 32/207 15,46%

2/9 22,22% 55/207 26,57%

7/9 77,78% 77/207 37,20%

* broj zarazenih biljaka / broj testiranih biljaka * number of infected plants / number of tested plants

PVY

Lokalitet Locality

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se infekcije kreu i do 100%. Prema podacima Kovacevskog (1977) (cit. prema Mijatovi i sar. 1999) u Bugarskoj je inficiranost paprike sa CMV iznosila 80% do 90%, a u nesto manjem procentu su identifikovani TMV, TVY i TSWV. Sepulveda et al. (2005) su konstatovali da je oko 30% paprike u severnom Cileu inficirano sa dva ili vise virusa od 7 utvrenih: CMV, TSWV, AMV, PVY, Impatiens Necrotic Spot Virus (INSV), Tomato mosaic virus (ToMV) i TMV. Tokom istrazivanja koja su sprovedena 1998. i 1999. u Turskoj, determinisano je sest virusa: AMV, CMV, PVY, ToMV, TMV i TSWV od kojih su TMV i PVY detektovani u 15,4% uzoraka u najzastupljenijoj mesanoj infekciji (Arli-Sokmen et al. 2005). Simptomi u polju i njihova povezanost sa detektovanim virusima Iako je poznato da virusi paprike mogu da izazivaju veoma razlicite tipove simptoma u zavisnosti od starosti biljke, uslova spoljasnje sredine (narocito temperature), kao i u zavisnosti od soja virusa, ustanovljeno je postojanje izvesne veze izmeu tipa simptoma i detektovanog virusa. Prisustvo PVY utvreno je u uzorcima paprike sa raznolikim simptomima, a najcese u vidu hloroticnog prosvetljavanja nerava, hloroticnog prosaravanja listova sa ili bez deformacije mladih listova (Sl. 1), cesto praeno

nekrozom listova, mozaikom u vidu hloroticnih prstenova i sitnih tamno zelenih povrsina. Veina biljaka u kojima je dokazano prisustvo PVY bile su krzljave i cesto zbunastog izgleda, sto je upuivalo na rane zaraze u toku vegetacije. Simptomi koje izaziva PVY, uoceni u toku ovih istrazivanja, u saglasnosti su sa navodima iz literature (Mijatovi i sar. 2002). Verovatno nijedan drugi biljni patogen ne izaziva takav niz simptoma kao TSWV, a neke biljke ili sorte u usevu mogu biti zarazene i bez ispoljavanja simptoma (Kucharek et al. 2000). U ovim istrazivanjima prisustvo TSWV ustanovljeno je u uzorcima paprike sa simptomima blagog do izrazenog mozaika (Sl. 2). Na nekim biljkama pored mozaika primeena je i hloroza nerava. Krsti i sar. (2008) navode da se infekcija ovim virusom odlikuje simptomima zutila lisa i citavih biljaka, mozaika sa koncentricnim prstenastim pegama ili hloroticnim linijskim mozaikom na starijem lisu, sa cestom pojavom bronzane boje lisa. Slicne simptome tipa mozaicne prstenaste pegavosti i nekroticne prstenaste pegavosti navodi Rusevski (2001). Prisustvo CMV utvreno je u uzorcima paprike sa izrazenim simptomima mozaika i hloroticnog prosaravanja. Karakteristicno za biljke zarazene ovim virusom, uz navedene simptome, je i deformacija ili blaga klobucavost listova (Sl. 3). Krsti & Vico (2004) navode hloroticni mozaik kao pocetni simptom koji ovaj virus izaziva, dok se u kasnijim

Slika 1. PVY: Hloroticno prosaravanje, mozaik i prosvetljavanje nerava Figure 1. PVY: Chlorotic mottling, mosaic and veinbanding

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Slika 2. TSWV: Blagi mozaik na listovima Figure. 2. TSWV: Mild mosaic on the leaves

Slika. 3. CMV: Mozaicno prosaravanje, deformacije i klobucavost listova Figure 3. CMV: Mosaic mottling, malformation leaves that curl downward

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 567-576

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jacu deformaciju, zuto-zeleni mozaik, sarenilo listova, a na nekim biljkama javljale su se i sitne tamno zelene povrsine na liski uz blago kovrdzanje, dok je mesana infekcija AMV i CMV utvena kod biljka sa simptomima svetlo zelenih povrsina i deformacije listova. Mesana zaraza sa PVY, AMV i CMV uglavnom se ispoljavala simptomima hloroticnog prosaravanja, deformacija i zuto-belih povrsina. Kako su na paprici u nasoj zemlji prisutna najmanje cetiri virusa, u pojedinacnim i mesanim zarazama, uoceni simptomi i njihova veza sa virusom prouzrokovacem imaju samo preliminarni dijagnosticki znacaj. Mada je u slucaju pojedinacnih zaraza paprike dijagnosticki znacaj uocenih simptoma vei, ipak postavljanje dijagnoze samo na osnovu simptomatologije nije mogue. Simptomi, koliko god bili izrazeni i karakteristicni, ipak samo ukazuju na postojanje zaraze odreenim virusom, a pouzdana dijagnoza moze se postaviti samo posle odgovarajuih laboratorijskih analiza. Zakljucci

Slika 4. AMV: Hloroticno prosaravanje i belicaste povrsine na listu Figure 4. AMV: Chlorotic mottling and whitish patches on the leaves

fazama javljaju hloroticne pege nepravilog oblika. Cest tip simptoma takoe je nekroza, do koje dolazi u kasnijim fazama razvoja mozaicnih sara i pega. Hloroticne lokalne lezije i sistemicni mozaik takoe su karakteristicni simptomi koje izaziva CMV (Ozaslan et al. 2006). Na paprici zarazenoj virusom AMV najcesi simptomi su hloroticno belicaste pege razlicitog oblika i velicine koje se vremenom spajaju, tako da u nekim slucajevima zahvataju i citave liske (Sl. 4). Nesto drugaciji, ali vrlo slicni, simptomi su rasute bele do zute tackice koje mogu prekriti celu lisku. Simptomi na listovima paprike zarazene sa AMV uoceni tokom ovih istrazivanja bili su slicni simptomima koje navode Mijatovi i sar. (2007). Mesane zaraze sa dva ili vise virusa najcese su menjale tip kao i intezitet ispoljavanja simptoma usled sinergistickog delovanja. Izuzetak su biljke u kojima je konstatovana mesana zaraza sa PVY i AMV, koje su pokazivale simptome tipa hloroticnog prosaravanja i sarenila listova najcese praene prisustvom zutih povrsina koje se cesto javljaju i pri pojedinacnim zarazama sa AMV. Mesana zaraza sa PVY i CMV odlikuje se kompleksnijom slikom simptoma, zbog cega simptomi gube dijagnosticki znacaj. Najcesi simptomi koji su se javljali na biljkama su hloroticno prosaravanje liske uz blagu ili

Sprovedena ispitivanja prisustva virusa u znacajnim proizvodnim podrucjima u Srbiji tokom 2009. pokazala su da od cetiri detektovana virusa: PVY, CMV, TSWV i AMV, najzastupljeniji virusi paprike su PVY i CMV, kako u rasadu tako i u proizvodnji paprike u zastienom prostoru i polju, dok je AMV zastupljen u nesto manjem procentu. Prisustvo TSWV otkriveno je samo na paprici gajenoj u zastienom prostoru. Dobijeni rezultati ukazuju na znacajno prisustvo virusa koji se prenose vasima na neperzistentan nacin u proizvodnji rasada paprike, komercijalnoj proizvodnji paprike u zastienom prostoru i na otvorenom polju, i na prisustvo TSWV u proizvodnji paprike u zastienom prostoru koji se prenosi tripsima. Zbog toga je neophodno ukljuciti odgovarajue integralne mere kontrole virusnih zaraza u proizvodnji paprike, koje obuhvataju i kontrolu vektora detektovanih virusa, biljnih vasi i tripsa, pored primene drugih preventivnih i sanitarnih mera. Literatura

Arli-Sokmen M, Mennan H, Sevik M A, Ecevit O (2005): Occurrence of viruses in field-grown pepper crops and some of their reservoir weed hosts in Samsun, Turkey. Phytopar. 33: 347-358 Clark M F and Adams A N (1977): Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen.Virol. 34: 475-483 Choi S G, Kim H J, Lee H D, Kim S J, Ryu H K (2005): Occurrence and distribution of viruses infecting pepper in Korea. J. Plant Pathol. 21: 258-261 Conti M, Masenga V (1977): Identification and prevalence of pepper viruses in northwest Italy. Phitopath. Z. 90: 212-222

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Delevi B (1963): Viroze paprika u SR Srbiji. Zastita bilja 74: 347-437 Gaborjanyi R, Horvath J, Kovacs J, Kazinczi G (1998): Role of viruses in pepper decline in Hungary. Xth Eucarpia meeting on Capsicum and Eggplant, Avignon, France, 129-132 Green S K, Kim J S (1990): Characteristics and control of viruses infecting peppers. Technical Bulletin No. 18. Asian Vegetable Research and Development Center, 1-60 Gvozdenovi , Bugarski D, Gvozdanovi-Varga J, Vasi M, Cevrenski J, Takac A i Jovievi D (2008): Doprinosi unapreenju povrtarske proizvodnje za 70 godina rada Instituta za ratarstvo i povrtarstvo. Zbornik radova Instituta za ratarstvo i povrtarstvo 45: 113-129 Krsti B, Bulaji A (2007): Karantinski virusi povra i ukrasnih biljaka u zastienom prostoru. Univerzitet u Beogradu - Poljoprivredni fakultet i Ministarstvo poljoprivrede, vodoprivrede i sumarstva, Beograd Krsti B, Bulaji A (2008): Ekonomski znacajni i karantinski virusi paprike u Srbiji. Zbornik radova IX Savetovanja "Savremena proizvodnja povra", Novi Sad, 24-28 Krsti B, Bulaji A, eki I, Berenji J (2008): Virus bronzavosti paradajza ­jedan od najdestruktivnijih biljnih virusa. Pesticidi i fitomedicina 23: 153-166 Krsti B, Vico I (2004): Pregled dosadasnjih saznanja o virusu mozaika krastavca. Biljni lekar 32: 459-467 Kucharek T, Brown L, Johnson F, Funderburk J (2000): Tomato Spotted Wilt Virus of Agronomic, Vegetable, and Ornamental Crops, Plant Pathology Fact Sheet Marchoux G, Gebre-Selassie K, Villevieille M (1991): Detection of tomato spotted wilt virus and transmission by Frankliniella occidentalis in France. Plant Pathol. 40: 347-351 Mijatovi M (1986): Reakcija nekih sorata paprika prema virusu mozaika duvana. Magistarska teza. Poljporivredni fakultet, Beograd

Petrovi D i sar.

Mijatovi M, Obradovi A, Ivanovi M, Stevanovi D (1999): Rasprostranjenost i intenzitet pojave nekih virusa parazita paprika u Srbiji. Zastita bilja 50: 151-159 Mijatovi M, Ivanovi M, Obradovi A, Zecevi B (2002): Potato Virus Y (PVY) on Pepper in Serbia. Proc. 2nd Balkan Symposium on Vegetable and Potatoes. Acta Horti. 579: 545-549 Mijatovi M, Obradovi A, Ivanovi M (2007): Zastita povra. AgroMivas, Smederevska Palanka Mnari-Hattab M, Ezzaier K, Gebre-Selassie K, Marchoux G and Gognalon P (1999): Surveys of viruses affecting pepper (Capsicum annuum L.) in Tunisia. Proceedings of the 7th International plant virus epidemiology symposium on Plant virus epidemiology: Current status and future prospects, Almeria, Spain, 143-144 Ozaslan M, Bas B, Aytekin T, Sigirci Z (2006): Identification of Pepper viruses by Das-Elisa Assays in Gaziantep-Turkey. J. Plant Pathol. 5: 11-14 Panjan M, Prpi Z (1955): O jednoj varijanti mozaika duhana izoliranog iz paprike. Arhiv za poljoprivredne nauke 19: 3-9 Rusevski R (2001): Virus bronzavosti paradajza-patogen paprike u Makedoniji. Doktorska disertacija. Univerzitet u Beogradu, Poljoprivredni fakultet, Beograd-Zemun Sepulveda P, Larrain P, Quiroz C, Rebufel P, Grana F (2005): Identification and incidence of pepper viruses in north central Chile and its association with vectors. Agric. Tecnica 65: 235-245 SIEPA (2005): Vegetable industry in Serbia. Serbia Investment and Export Promotion Agency (SIEPA), Belgrade Suti D (1959): Die Rolle des Paprikasamens bei der Virusubertragung.-Phytopath. Z. 36: 84-93 Tomi , Jeremi S, Simi A, Petkovi N, eki I, Bulaji A, Krsti B (2007): Status viroza paprika u Srbiji. XIII Simpozijum sa savetovanjem o zastiti bilja, Zlatibor, 107-108

Presence and Distribution of Pepper Viruses in Serbia

Dragana Petrovi1 Aleksandra Bulaji2 Ivana Stankovi2 Maja Ignjatov1 Milka Vujakovi1 Branka Krsti2

1

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia 2 Institute of Phytomedicine, Department of Phytopathology, University of Belgrade - Faculty of Agriculture, Nemanjina 6, 11080 Belgrade

Summary: Pepper is a very popular and profitable crop in Serbia and its production is growing rapidly as well as the importance of diseases caused by viruses. Virus infections interfere with development of pepper plants, reducing yield and fruit quality. More than 45 viruses have been isolated from pepper so far, and in our country the following are considered economically very important: Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV) and Potato virus Y (PVY). During 2009, an investigation was conducted regarding the presence and distribution of pepper viruses in Serbia, which included a survey of different localities of transplant production, and both greenhouse and open field pepper crops. Collected samples were tested utilizing a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antisera specific for the detection of economically important pepper viruses: PVY, AMV, CMV, TSWV, TMV and Potato virus X (PVX). In the collected samples, presence of the four viruses (PVY, CMV, TSWV and AMV) was confirmed in single and mixed infections. In tested transplant samples, the most frequently detected virus was CMV (18.18%), while PVY was the most frequent in samples collected in greenhouse and open field pepper crops (32.56% and 51.21% respectively). The presence of TSWV was detected only in greenhouse-grown pepper crops, while the presence of PVX and TMV was not detected during this investigation. The obtained results indicated that PVY and CMV were widely distributed and the most frequent viruses in pepper crops in Serbia. Future investigation should include their detailed biological and molecular characterization, as well as the implementation of appropriate control measures in pepper productions. Key words: Alfalfa mosaic virus, Cucumber mosaic virus, DAS-ELISA test, pepper, Potato virus Y, Tomato spotted wilt virus

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www.nsseme.com/journal.html

Predlog novih diferencijatora populacije Puccinia triticina

577 Zastita useva / Crop Protection Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 577-580 originalni naucni rad / original research article

Efekti primene diferencijatora populacije Puccinia triticina i predlog stvaranja novih

Zoran Jerkovi Marina Putnik-Deli

primljeno / received: 19.04.2010. preraeno / revised: 01.06.2010. prihvaeno / accepted: 01.06.2010. © 2010 IFVC

Izvod: Prikazana su dosadasnja shvatanja interakcija gena Puccinia triticina i psenice. Nova saznanja o stabilnom delovanju gena ispoljivih preko karaktera otpornosti na svojstva rasta omoguuju stvaranje novog seta diferencijatora populacije parazita u Thatcher genetskoj pozadini. Cilj je poreenje podataka u svim uzrastima i uslovima, a samim tim poveanje upotrebljivosti u procesu oplemenjivanja i odreivanju sortimenta. Kljucne reci: diferencijatori populacije, Puccinia triticina, Lr linije

Uvod Agresivnost i virulentnost cine patogenost gljiva (Robinson 1969). Do prvih saznanja o razlikovanju efekta gena domaina prema parazitima doslo se pocetkom dvadesetog veka (Bifin 1905, Stakman & Piemiesel 1917, Chester 1946). Gen za gen model interakcija postavio je Flour (1956), a Read (1970) kao uzrok otpornosti sugerise deljenje fizioloskih mehanizama izmeu razlicitih vrsta. Kiraly et al. (1972) smatraju hipersenzitivnost posledicom, a ne uzrokom otpornosti prema infekciji. Stanoviste je identicno kao kod autora De Wit & Van Kan (1993) koji reakciju povezuju s okidacem parazitnog porekla. Poznati geni za otpornost podrazumevali su 1018 razlicitih kombinacija hemijskih jedinjenja da bi gen za gen model bio adekvatan rezultatima, te je Van der Plank (1982) zakljucio da je osetljivost proizvod specificnog odnosa dva organizma. Gassner & Striab (1932) su primetili da se pojedini geni za otpornost ne ispoljavaju pri razlicitim temperaturama. Produkat simbioze (1ag) po modelu (Loegering 1978, Browder 1985) je bio definitivan (1ap) samo pri odreenim uslovima sredine (Tab. 1). Navedenim modelom preko definitivnih uslova spoljne sredine specificnim su smatrani svi poznati geni otpornosti psenice. Nelson (1978) takoe smatra istim osnove horizontalne i vertikalne otpornosti, a razlicito ispoljivim u zavisnosti

Z. Jerkovi ) ( Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija M. Putnik-Deli Poljoprivredni fakultet Univerziteta u Novom Sadu, Trg Dositeja Obradovia 8, 21000 Novi Sad, Srbija

od genotipa u kojem se nalaze. Browder (1980) je prikazao uticaj spoljne sredine na ekspresiju gena u Lr linijama stvorenim do tad, povratnim ukrstanjem sortom Thatcher i razlicitih izvora otpornosti prema prouzrokovacu lisne re. Specificnost odnosa parazita i domaina nije bila definisana preko jedne od osobina otpornosti (Parlevliet & Kuiper 1977, Ohm & Shaner 1976). Niks et al. (2000) pronalaze specificnost kod kvantitavnih lokusa. Znatno umanjen broj simptoma po lisnoj povrsini pri nekontrolisano velikom broju apliciranih spora (SN = Symptom Number) bio je povezan sa parametrima koji opisuju rast delova genotipova psenice (GR = Growth Ratio) (Jerkovic 2008, Jerkovic & Prijic 2010). Umanjeni SN, te nizi, a najcese mesoviti reakcioni tip (RT = Reaction Type) postignut je i iskljucivim dejstvom gena odgovornih za tolerantnost prema drugim stresovima, pri prosecnim temperaturama vazduha oko 25oC i dnevnoj osvetljenosti preko 12 h (Jerkovi & Priji 2009). Rase Puccinia triticina su odreene preko nizeg RT. Diferencijatori su prvobitno bile sorte, a kasnije izogene Lr linije (Johnston & Browder 1966, Browder et al. 1980). Nakon pojave rase 77 (Negulescu & Ionescu-Cojocaru 1974), setu su bile dodavane pojedine sorte (Bartos 1975). Pronaeno je preko pedeset gena (www.cdl.umn.edu) s efektom na smanjenje razvoja prouzrokovaca lisne re. Broj testiranih gena radi diferencijacije parazitne populacije je bio povean, a interakcije sa genima koji povisavaju SAGR (Adult Steam Growth Ratio = kolicnik maksimalnih duzina izmeu klasa i drugog kolenca od njega i stabla) bile su maksimizirane najuticajnijim Lr 22b. Slicni su bili Lr 3c, Lr 2c, Lr 23, Lr 34. Po novijim saznanjima, bili su indikator uslova spoljne sredine pri testiranju, a ne razlicitosti po genima za virulentnost u

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Tabela 1. Specificnost odnosa faktora parazit : domain : uslovi spoljne sredine Table 1. Specificity of relations parasite : host : environment genotip parazita parasite genotype 1p 1p 1p 1p 0p 0p 0p 0p x x x x x x x x genotip domaina host genotype 1h 1h 0h 0h 1h 1h 0h 0h = = = = = = = = agrikorpus genotip agricorpus genotype 1ag 1ag 0ag 0ag 0ag 0ag 0ag 0ag x x x x x x x x uslovi agrikorpus sredine fenotip environment agricorpus phenotype 1e 0e 1e 0e 1e 0e 1e 0e = = = = = = = = 1ap 0ap 0ap 0ap 0ap 0ap 0ap 0ap

1 = definitivan, odreen (oznaka 1) = definitive 2 = nedefinitivan, neodreen (oznaka 0) = not definitive parazitnoj populaciji. Lr 2c ili Lr Tc efekti mogli su biti proizvod interakcije Lr 22b i gena za otpornost prema niskim temperaturama na 3, 4, 5A ili 4 i 6D hromozomima, sto je demantovano preko Lr 2a linije sa genom lociranim na istom hromozomu kao prethodno pomenuti. Geni koji su se ispoljavajali produzenim latentim periodom (LP) odnosno nizeg RT umanjuju SAGR za oko 0,02, a akumulacije 0,05 (Jerkovi 2010). Navedeno, slican porast korena u odnosu na nadzemni deo te povisen sadrzaj azota iz zrna kod sejanaca gajenih u pesku (Jerkovi & Priji, neobjavljeni podaci), negira ranije navedenu hipotezu o delovanju gena za otpornost samo u prisustvu patogena. Sveobuhvatni model predlozen na osnovu novih saznanja iznet je u tabeli 2. Geni iz y i z ostvaruju nize RT i SN pri visim temperaturama (deset dana od infekcije prosecno oko 25oC). Specificna, odnosno promenljivo ispoljiva u zavisnosti od gena za virulentnost parazita pri svim uslovima sredine je bila kontrolisana x genima psenice. Geni za avirulentnost parazita po modelu iznetom u tabeli 2, koji podrazumeva stalan efekat specificnih Lr gena bez obzira na uslove spoljne sredine, ne postoje (1p) . Obrazlozenje potrebe i nacin stvaranja novog seta diferencijatora Efekti Lr gena u izogenim linijama ne mogu se uporediti preko maksimalnih intenziteta zaraze, a ni RT usled razlicitih uticaja Lr 22b i Lr Tc.

Tabela 2. Rezultati interakcija parazit : domain : uslovi spoljne sredine Table 2. Results of interactions parasite : host : environment genotip parazita parasite genotype 1p x 1p x 1p x xh = yh = zh = genotip domaina host genotype 1ag 0ag 0ag x x x agrikorpus genotip agricorpus genotype 0e = 1e = 1e = uslovi sredine environment phenotype agrikorpus fenotip agricorpus

1ag= RT ili/or LLP 1ag= LRT+LIF + SLP 1ag= LRT+HIF + SLP

x= gen za produzeni latentni period / LP prolongable gene y= gen za snizenje uspesnosti infekcije (gen za tolerantnost prema susi i visokim temperaturama) / SN decreasing gene (gene for drought and heath tolerance) z= gen za otpornost prema niskim temperaturama SLP=kratak latentni period / short LP LLP= produzen latentni period / prolonged LP LSN = niska uspesnost infekcije / low SN HSN= visoka uspesnost infekcije / high SN

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Tabela 3. Razlike izmeu Lr linija Table 3. Differences between Lr lines Genotip Hromozom SAGR (2009) Minimalan RT sejanaca pri 20oC Min. RT of seedlings at 20oC 4 4 4 4 4 0; 0; ; ; ; 1 ; ;N2 Maksimalan intenzitet zaraze P. triticina Max. infection intensity of P. triticina 80 50 80 60-70 60-70 70 60 60 30 50 40 20 60

Genotype

Chromosome

SAGR (2009)

Thatcher Lr Tc ili 2c Thatcher Lr 22b Lr2c Lr 3c Lr 13 Lr1 Lr2a Lr3a Lr9 Lr19 Lr20 Lr24 Lr38

2DS 2DS 2DS 6BL 2BS 5DL 2DS 6BL 6BL 7DL 7AL 3DL 2AL

0,55 0,68 0,55 0,62 0,63 0,53 0,53 0,53 0,66 0,66 0,66 0,66 0,66

Nedostatak prethodnog seta mogue je otkloniti prenosenjem u jednu genetsku pozadinu sa osobinama koje maksimalno odgovaraju prouzrokovacu re. Naime, korisenjem Lr linija kao diferencijatora cesto je izostajao zeljeni efekat oplemenjivanja usled akumulacija gena za otpornost koje su se ispoljavale regionalno nepotrebnim nivoom tolerantnosti prema susi i visokim temperaturama. Potrebni geni za stvaranje novog seta diferencijatora i razlozi odabiranja (2004. godine nije bilo pojave Pyrenophora tritici repentis, koja redukuje razvoj navedenog obligatnog parazi-

ta, te su ostvareni maksimalni intenziteti zaraze) prikazani su u tabeli 3, a plan ukrstanja u tabeli 4. SAGR linija bie 0,57-0,60 i nakon akumulacija dva gena za specificnu otpornost (-0,05). Akumulacija Lr 3C i Lr 13 nije se mogla izbei kod Lr 2a i Lr3a. Novi set diferencijatora omoguuje korisenje gena za otpornost u semiaridnim uslovima bez prethodnih gresaka pri uporeenju efekata (Jerkovi & Priji 2009) te ostvarljivost stabilno maksimalnih prinosa zrna i u budunosti preko povremenog recikliranja.

Tabela 4. Plan ukrstanja radi stvaranja linija bliskih GR vrednosti Table 4. Crosses for achieving Lr lines with near GR values Lr 1, Lr2c Lr 2a, Lr 3a, Lr2c x Lr 9, Lr22b Lr 19, Lr22b Lr 20 Lr22b Lr 24, Lr22b Lr 38 Lr22b

Lr3C Lr13 Lr13

Lr 2C x Lr3C

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Jerkovi Z (2010): Posledice akumulacija gena za otpornost prema Puccinia triticina. Sel. Semen. (u stampi) Jerkovic Z, Prijic Z, Putnik-Delic M (2010): Two new parameters linked with amount of Puccinia triticina. Acta biologica (u stampi) Johnston C O, Browder L E (1966): Seventh revision of physiologic races of Puccinia recondita f. sp. tritici. Plant Dis. Rep. 50: 756 Király Z, Barna B, Érsek T (1972) Hypersensitivity as a consequence, not the cause, of plant resistance to infection. Nature 239: 456-458 Loegering W Q (1978): Current concepts in interorganismal genetics, Ann. Rev. Phytopathol. 16: 309-320 Negulescu F, Ionescu-Cojocaru M (1974): The outbrake of new form of Puccinia recondita tritici occuring on wheat cultivar Aurora in Romania in 1973. Cereal Rusts Bull. 2: 19-22 Nelson R R (1978): Genetics of horizontal resistance to Plant diseases. Ann. Rev. Phytopathol. 16: 369-378 Niks R E, Fernandez B, van Haperen B, Bekele A, Martinez F (2000): Specificity of QTLs for partial and non host resistance of barley to leaf rust fungi. Acta Phytopathol. Entomol. Hungarica 35: 13-22 Ohm H W, Shaner G E (1976): Three components of slow leaf rusting at different growth stages in wheat. Phytopathol. 66: 1356-1360 Parlevliet J E, Kuiper H J (1977): Partial resistance of barley to leaf rust. IV. Effect of cultivar and development stage on infection frequency. Euphytica 26: 249.255 Read C P (1970): Parasitism and Symbiology. Roland, New York Robinson R A (1969): Disease resistance terminology. Rev. Appl. Mycol. 48: 593 Roelphs A P, Singh R P, Saari E E (1992): Rust diseases of wheat: concepts and methods of disease management. CIMMYT, Mexico Stakman E C, Piemiesel (1917): Biologic forms of Puccinia graminis on cereals and grasses. J. Agric. Res. 10: 429-95 Van der Plank J E (1982): Host pathogen interactions in plant disease. Academic Press, New York, London

Literatura

Bartos P (1975): Physiologic specialization of wheat leaf rust (Puccinia recondita) in Czechoslovakia in the years 19701975. Ved. Pr. Vysk. Ustavu Rastl. Viroby, Praha-Ruzyn 20: 7-12 Bifin R H (1905): Mendel's laws of inheritance and wheat breeding. J. Agr. Sci. 1: 4-48 Browder L E (1980): A compedium of information about named genes for low reaction to Puccinia recondita in wheat. Crop Sci. 20: 775-779 Browder L E (1985): Parasite host: environment specificity in the cereal rusts. Ann. Rev. Phytopathol. 23: 201-222 Browder L E, Lyon F L, Eversmeyer M G (1980): Races, pathogenicity phenotypes, and type cultures of plant pathogens. Phytopathol. 70: 581-583 Chester K S (1946): The cereal rusts. Waltham, Mass, USA Flour H H (1956): The complementary genic systems in flax and flax rust. Adv. Genet. 8: 29 Gassner G, Straib W (1932): Zur Frage der Konstanz des Infektionstypus von Puccinia triticina Erikss. Phytopathol. Z. 4: 57-64 De Wit P J G M, Van Kan J A L (1993): Is durable resistance against fungi attainable through biotechnological procedures? In: Jacobs T, Parlevliet J E (Eds), Durability of disease resistance. Kluwer Academic Publishers, 57-70 Priji Z, Jerkovi Z (2009): Common to Puccinia triticina durable resistance genes combinations in Serbia. IV Congress of Serbian Genetic Society, 226 Jerkovi Z, Priji Z (2009): Fitopatoloske posledice gajenja razlicitih tipova sorti psenice u semiaridnom regionu. IV Simpozijum sa meunarodnim ucesema "Inovacije u ratarskoj i povrtarskoj proizvodnji" 23-24. oktobar, Beograd, 34-35 Jerkovi Z, Priji Z (2009): Grow ratio as leaf rust forecasting factor in semiarid region. 12th international cereal rusts and powdery mildews conference. October 13-16. Antalya, Turkey, 96 Jerkovi Z, Priji Z (2010): Osnove trajno umanjenog razvoja Puccinia triticina u semi-aridnom regionu. Ratar. Povrt. / Field Veg. Crop Res. 47: 303-307

Effects of Puccinia triticina Differentials and Suggestion for Creating a New Set

Zoran Jerkovi1 · Marina Putnik-Deli2

2University 1Institute

of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia of Novi Sad, Faculty of Agriculture, Trg Dositeja Obradovia 8, 21000 Novi Sad, Serbia

Summary: Previous explanations of wheat and Puccinia triticina interactions are presented. New ones suggested the stable effects of the resistance genes on growth characters and realized the opportunity for creating the new set of parasite population differentials in Thatcher background comparable in all environmental conditions and growth stages. The main problem was the influence of Lr 22b and Lr Tc in Lr lines. The specific genes Lr1, Lr 2a, Lr 3a, Lr 9, Lr19, Lr 20, Lr 24 and Lr 38 as Lr 2c, Lr 3C and Lr 13 from the opposite growth influential group were chosen. Following growth ratio values could be near the same even after further suggested accumulation of two genes for the specific resistance. New set was directed in regionally higher grain yield achievement as to the stability across temporary recycling. Key words: Lr lines, Puccinia triticina differentials

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Nitrogen and Brassica-Legume Intercrops

581 Intercropping / Zdruzeni usevi Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 581-586 original research article / originalni naucni rad

Niche Separation and Nitrogen Transfer in Brassica-Legume Intercrops

Felipe Alfonso Cortés-Mora · Guillaume Piva · Marie Jamont · Joëlle Fustec

received / primljeno: 13.04.2010. accepted / prihvaeno: 03.05.2010. © 2010 IFVC

Summary: Brassica sp. are interesting late-summer and autumn forages. Since they have high nitrogen requirements, intercropping with legumes may increase yield in low-input systems. However, brassicalegume intercrops still remain poorly documented. Using rhizotrons in a greenhouse, we compared root development of (1) forage rapeseed grown with faba bean to that of forage rapeseed monoculture and (2) fodder cabbage grown with common vetch to that of fodder cabbage monoculture. Legumes were labelled with 15N urea. Seven to eight weeks after sowing, Brassica yield and N content were higher under intercropping than in pure stand. Under intercropping, distribution of root ramifications along the taproot differed from that of monoculture, which reduced the effect of competition. In addition, N transfer from legumes to Brassica was found to be significant. Keywords: facilitation, intercropping, N fixation, niche

Introduction Agriculture is facing new challenges as higher levels of production are expected with less fertiliser. The understanding of the mechanisms involved in agro-ecosystem functioning may help to reach this goal (Altieri 1999). Biological diversity is now recognised as a possible source of productivity. For instance, intercropping has been shown to allow more efficient use of resources than monocultures (Tilman et al. 2002). Interspecific competition may lead to niche separation with more vigorous root systems and more efficient exploration of the soil for nutrients and water, resulting in an increase in productivity (Hauggaard-Nielsen et al. 2001, Hauggaard-Nielsen et al. 2008). Intercropping can improve the use of resources by 10­50% above sole crops grown on the same piece of land, expressed in the Land Equivalent Ratio (LER) (Bulson et al. 1997). LER is the sum of the relative yields of the intercrop components relative to their respective sole crop yield. Most examples of intercrops combine a legume with another crop. In low-input

F. A. Cortés-Mora · G. Piva · J. Fustec ) ( UP-SP LEVA, Ecole Supérieure d'Agriculture - PRES L'UNAM, F-49007 Angers, France Tel +33 2 41 23 55 55, Fax +33 2 41 24 55 65 e-mail: [email protected] M. Jamont UP-SP LEVA, Ecole Supérieure d'Agriculture - PRES L'UNAM, F-49007 Angers, France and Equipe PBI, UMR Bio3P, AgroCampus Ouest-INHP, F-49045 Angers, France

systems, legumes increase the soil N pool throughout their growth, through N symbiotic fixation and rhizodeposition (Jensen 1996a, Fustec et al. 2010). After mineralisation, N can be transferred from the legume to the non-fixing companion crop (Jensen 1996b, Paynel et al. 2008). Most data are based on legume-cereal mixtures. The majority of the time, they reveal a Land Equivalent Ratio higher than 1, indicating that the productivity of legume-cereal intercrops was significantly higher than that of monocultures (de Wit & van den Bergh 1965, Trenbath 1976, Corre-Hellou et al. 2006, Malezieux et al. 2009). Forage Brassica sp. are fast growing, highly productive and digestible crops (Banik et al. 2000). They offer great potential and flexibility for improving stocking rate in late summer and autumn, especially under drought conditions. Brassica sp. have relatively high nitrogen requirements, intercropping with legumes may help to increase yield in low-input systems. However, Brassica-legume intercropping remains poorly documented. The aims of our study were (1) to compare root development, dry matter and N content in fodder rapeseed and fodder cabbage grown either with legumes or in monoculture, and (2) to investigate the N transfer from legumes to the intercropped Brassica sp.

Acknowledgements We thank Marie-Paule Bataillé (University of Caen, France) for performing the mass spectrometer measurements. This study was funded by the Region Pays de la Loire (France).

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Figure 1. Distribution of brassica and legume roots in the upper, middle and lower layers of the rhizotrons: A ­ Rapeseed in monoculture or sown with faba bean, B ­ Cabbage in monoculture or sown with vetch. *, **, *** indicate statistical differences between depth sections at the same date. (a, b) indicate significant differences between brassicas and legumes for a given depth at a given date Slika 1. Raspored korena kupusnjaca i mahunarki u gornjim, srednjim i donjim slojevima rizotrona: A ­ repica u cistom ili zdruzenom usevu sa bobom, B ­ kupus u cistom ili zdruzenom usevu sa grahoricom. *, **, *** ukazuju na statisticke razlike izmeu dubinskih slojeva istog datuma. (a, b) ukazuju na statisticke razlike izmeu kupusnjaca i mahunarki za datu dubinu i dati datum

Materials and Methods Experimental Design In June 2009, seeds were sown in rhizotrons made of Plexiglas® (inner parts: 47 cm length × 19 cm width × 4.6 cm depth) filled with a clay and sandy-rich soil sieved to a 3 mm particle size. We studied four modalities with two plants per rhizotron (N=10): (1) monospecific forage rapeseed (Brassica napus L. cv. `Licapo'), (2) forage rapeseed with faba bean (Vicia faba L. ssp. minor cv. `Gloria'), (3) monospecific fodder cabbage (B. oleracea L. cv. `Proteor'), and (4) fodder cabbage with common vetch (Vicia sativa L. cv. `Pepite'). The rhizotrons were randomly distributed along culture tables in a greenhouse, placed with an inclination of 50 degrees to the horizontal plane, and protected from light with black plastic bags. Soil was kept at field capacity with an automatic watering system.

Root Development Root progression was drawn every two days on the lowest side of the rhizotrons, using different colours at each date. At the end of the experiment, the root length, the number of ramifications and their distribution in the upper, middle and lowest parts of the rhizotrons were recorded. Isotopic Methods In five rhizotrons of each Brassica-legume intercropping, the legume was continuously labelled with 0.2% v/w urea 0.99% atom 15N until harvest (Mahieu et al. 2009). Twenty-five and 28 days after sowing, faba beans and vetches respectively were labelled by cotton wick stem-feeding (Mahieu et al. 2007). Unlabelled rhizotrons were kept as controls (N=5). In all modalities, the aboveground parts were harvested 45 days after sowing, separately

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dried (70°C), weighed and ground before preparation for 15N:14N mass spectrometer measurements. Transfer of N from legume to Brassica (%Ntrans) was estimated based on equation (1) (Høgh-Jensen & Schjoerring 2000). (1)%Ntrans = [N brassica × (B1-B0)] / [N legume (L1-A0) + N brassica × (B1-0.3663)] × 100 where N brassica and N legume denote the aboveground N content of Brassica and legume respectively; B1 is the enrichment (atom%) of labelled Brassica; B0 is the average natural abundance (atom%) of Brassica growing in unlabelled conditions; L1 is the enrichment of labelled Brassica and 0.3663 is the natural abundance of air. Estimation of the legume N derived from atmosphere was calculated as described by the natural abundance method (Hansen & Vinther 2001), and Brassica monocultures were used as

reference. Data were compared with KruskalWallis and Mann-Whitney tests based on median equality (GraphPad Prism 5 Software 2007). Results Niche Separation in the Rhizotrons In the Brassica monocultures, the distribution of root ramifications in the rhizotron parts did not differ between the two sown plants (P > 0.05, data not shown). At 670 degree-days (harvest), rapeseed roots colonised the three compartments similarly (Fig. 1A). In cabbage monocultures, there was no root ramification in the lowest part of the rhizotron (Fig. 1B). When intercropped with faba bean, the proportion of the roots of rapeseed located in the upper part of the rhizotron was significantly lower than in monoculture (P < 0.001, Fig. 1A).

Table 1. Dry matter weight and N content in Brassica grown either with a legume or in a monospecific rhizotron ­ mean (s.e.) Tabela 1. Prosecne vrednosti (i njihove standardne greske) prinosa suve materije i sadrzaja azota kupusnjaca gajenih sa mahunarkama ili u cistom rizotronu Dry matter weight (g plant.1) Prinos suve materije Companion Komponenta Brassica Monoculture Cist usev Common vetch Obicna grahorica Faba bean Bob P Cabbage Kupus 1.77 (0.13) b 2.39 (0.22) a * Rapeseed Repica 4.37 (0.30) b 6.91 (0.80) a ** N content (g plant-1) Sadrzaj azota Cabbage Kupus 2.69 (0.22) b 3.77 (0.33) a * 8.67 (0.94) a ** Rapeseed Repica 4.99 (0.46) b

* P < 0.05 ; ** P < 0.001 ; ­ letters a and b indicate significant differences between lines within a column; N = 10 - Mann-Whitney and Kruskal-Wallis tests * P < 0.05 ; ** P < 0.001 ; ­ slova a i b ukazuju na znacajne razlike izmeu vrednosti unutar kolone; N = 10 ­ Man-Vitnijev i Kraskel-Volisovi testovi

Table 2. Estimation of the part of the legume N derived from fixation (% Ndfa) and of the Brassica N derived from the legume in Brassica-legume intercrops (% Ntrans) ­ mean (s.e.). Tabela 2. Prosecne vrednosti (i njihove standardne greske) procene ucesa azota mahunarki iz fiksacije (% Ndfa) i azota kupusnjaca iz zdruzenog useva kupusnjaca i mahunarki (% Ntrans) % Ndfa Rapeseed - faba bean Repica - bob Cabbage ­ common vetch Kupus ­obicna grahorica 75.0 (5.7) 66.3 (1.9) % Ntrans 12.0 (2.9) 7.8 (0.4)

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The proportion of the roots located in the lowest part of the rhizotron was higher in rapeseed than in faba bean (P < 0.001 at 670 degree-days). Conversely, vetch produced a markedly higher amount of ramifications than cabbage in the upper and the middle part of the rhizotron at 420 and 685 degree-days (P < 0.001, Fig. 1B). At 970 degree-days, most of the root ramifications in the cabbage cultivar were located in the upper section, while there were more vetch roots in the middle section (P < 0.001, Fig. 1B). Dry Matter Weight and Nitrogen Measurements Yield and N content of Brassica cultivars were significantly higher when they were grown with a legume than in monospecific rhizotrons (Tab. 1). In Brassica-legume rhizotrons, the part of the legume N derived from symbiotic fixation was higher than 65% (Tab. 2). About 10% of the Brassica N was derived from the companion legume. Discussion and Conclusions In agreement with previous studies of belowground competition (Gersani et al. 2001), in a monoculture of Brassica, roots competed in all soil layers. However, as demonstrated in pea-barley mixtures by Corre-Hellou et al. (2007), spatial niche separation of roots may be positively involved in yield and N content differences between monoculture and intercropped Brassica sp. From the beginning of biological N fixation, intercro-

pped Brassica and legume cultivars used different N sources, which enhanced the niche separation. Corre-Hellou et al. (2006) showed that because of such complementarity effects, barley meets its N requirement more easily in pea-barley intercrops than in monoculture. In the early stages of root growth, only 20 days of continuous 15N urea labelling were sufficient to reveal substantial N transfer between legume and Brassica. Based on data reported in previous literature, N transfer may represent up to 30-50% of N rhizodeposition of vetch and faba bean (Wichern et al. 2008, Fustec et al. 2010). Jensen (1996b) showed that N transfer between pea and barley may be enhanced by root intermingling. As a consequence, in Brassica-legume intercropping, too different distribution of root ramifications between the plants would not be efficient to combine the effects of niche separation with those of N transfer on the Brassica yield and N content. In conclusion, our results suggest that LER values higher than one can be obtained by cocultivating a legume with a Brassica forage. Further experiments should be undertaken in greenhouse conditions and in field conditions to better characterise the plant-to-plant belowground interactions. A better management of these complex biotic interactions would be useful for increasing yield by optimising the balance between root competition, niche complementarity and N transfer. Root traits of associated genotypes are crucial points for maximising N flux in Brassicalegume mixtures.

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Trenbath B R (1976): Plant interactions in mixed cropping communities. In: R.I. Papendick, A. Sanchez, G.B. Triplett (eds.). Multiple Cropping. ASA Special Publication 27. American Society of Agronomy, Madison, WI, 129-169 Wichern F, Eberhardt E, Mayer J, Joergensen R G, Müller T (2008): Nitrogen rhizodeposition in agricultural crops: methods, estimates and future prospects. Soil Biol. Biochem. 40: 30­48

References

Altieri M A (1999): The ecological role of biodiversity in agroecosystems. Agron. Ecosyst. Environ. 74: 19-31 Banik P, Sasmal T, Ghosal P K, Bagchi D K (2000): Evaluation of Mustard (Brassica compestris var. Toria) and Legume Intercropping under 1 : 1 and 2 : 1 Row-Replacement Series Systems J. Agron. Crop Sci. 185: 9-14 Bulson H A J, Snaydon R W, Stopes C E (1997): Effects of plant density on intercropped wheat and field beans in an organic farming system. J. Agric. Sci. 128: 57-71 Corre-Hellou G, Fustec J., Crozat Y (2006): Interspecific competition for soil N and its interaction with N2 fixation, leaf expansion and crop growth in pea-barley intercrop. Plant Soil 282: 194-208 Corre-Hellou G, Brisson N, Launay M, Fustec J, Crozat Y (2007): Effect of root depth penetration on soil nitrogen competitive interactions and dry matter production in peabarley intercrops given different soil nitrogen supplies. Field Crops Res. 103: 76-85 de Wit CT, van den Bergh JP (1965): Competition between herbage plants. Netherland J. Agric. Sci. 13: 212-221 Fustec J, Lesuffleur F, Mahieu S, Cliquet J B (2010): Nitrogen rhizodeposition of legumes. Agron. Sustain. Dev. 30: 57-66 Gersani M, Brown J S, O'Brien E E, Maina G M, Abramsky Z (2001): Tragedy of the commons as a result of root competition. J. Ecol. 89: 660-669 Hansen J P, Vinther F P (2001): Spatial variability of symbiotic N2 fixation in grass-white clover pastures estimated by the 15 N isotope dilution method and the natural 15N abundance method. Plant Soil 230: 257-266 Hauggaard-Nielsen H, Ambus P, Jensen E S (2001): Temporal and spatial distribution of roots and competition for nitrogen in pea-barley intercrops ­ a field study employing 32P technique. Plant Soil 27: 237-250 Hauggaard-Nielsen H, Jørnsgaard B, Kinane J, Jensen E S (2008): Grain legume­cereal intercropping: the practical application of diversity, competition and facilitation in arable and organic cropping systems. Renew. Agric. Food Syst. 23: 3-12 Jensen E S (1996a): Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea-barley intercrops. Plant Soil 182: 25-38 Jensen E S (1996b): Barley uptake of N deposited in the rhizosphere of associated field pea. Soil Biol. Biochem. 28: 159-168 Høgh-Jensen H, Schjoerring J K (2000): Below-ground nitrogen transfer between different grassland species: direct quantification by 15N leaf feeding compared with indirect dilution of soil 15N. Plant Soil 227: 171-183 Mahieu S, Fustec J, Faure M L, Corre-Hellou G, Crozat Y (2007) :Comparison of two 15N labelling methods for assessing nitrogen rhizodeposition of pea. Plant Soil 295: 193205 Mahieu S, Fustec J, Jensen E S, Crozat Y (2009): Does labelling frequency affect N rhizodeposition assessment using the cotton-wick method? Soil Biol. Biochem. 41: 2236-2243 Malézieux E, Crozat Y, Dupraz C, Laurans M, Makowski D, Ozier Lafontaine H, Rapidel B, De Tourdonnet S, Valantin-Morison M (2009): Mixing plant species in cropping systems: concepts, tools and models. A review. Agron. Sustain. Dev. 29: 43-62 Paynel F, Lesuffleur F, Bigot J, Diquélou S, Cliquet J B (2008): A study of 15N transfer between legumes and grasses. Agron. Sustain. Dev. 28: 281­290 Tilman D, Cassman K G, Matson P A, Naylor R, Polasky S (2002): Agricultural sustainability and intensive production practices. Nature 418: 671-677

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Nisno razdvajanje i prenos azota u zdruzenim usevima kupusnjaca i mahunarki

Felipe Alfonso Kortes-Mora · Gijom Piva · Mari Zamon · Zoel Fistek

Visoka poljoprivredna skola ­ Univerzitet u Nantu, Anzeu i Le Manu, Laboratorija za ekofiziologiju biljaka i agroekologiju, Anze, Francuska

Izvod: Kupusnjace (Brassica sp.) predstavljaju zanimljive kasnoletnje i jesenje krmne biljke. Posto se odlikuju izrazenim zahtevima za azotom, zdruzeni usev sa mahunarkama moze da dovede do poveanja prinosa uz mala ulaganja. Meutim, zdruzeni usevi kupusnjaca i mahunarki i dalje ostaju nedovoljno prouceni. Korisenjem rizotrona u stakleniku, uporedili smo razvie korena (1) krmne repice u zdruzenom usevu sa bobom u odnosu na cist usev krmne repice i (2) krmni kupus u zdruzenoj setvi sa obicnom grahoricom u odnosu na cist usev krmnog kupusa. Mahunarke su bile obelezene 15N ureom. Izmeu sedam i osam nedelja nakon setve, prinos kupusnjaca i sadrzaj azota bili su vei u zdruzenom u odnosu na cist usev. U zdruzenoj setvi, raspored grananja korena duz glavnog korena razlikovao se u odnosu na cist usev, sto je umanjilo uticaj kompeticije. Takoe, prenos azota iz mahunarki u kupusnjace bio je znacajan. Kljucne reci: azotofiksacija, nisno razdvajanje, omoguavanje, prenos azota, prinos, zdruzeni usev

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Sericea Lespedeza Composition

587 Alternative Uses / Alternativna upotreba Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 587-590 original research article / originalni naucni rad

Sericea Lespedeza Biomass Composition for Bioenergy in the Southeastern USA

Jorge A. Mosjidis

received / primljeno: 08.04.2010. accepted / prihvaeno: 04.05.2010. © 2010 IFVC

Summary: Sericea lespedeza (Lespedeza cuneata (Dumont) G. Don) is used for forage or as a soil conservation plant that has shown potential for the production of ligno-cellulosic biomass in the Southeastern USA. Four genotypes of sericea lespedeza were grown at Tallassee, Alabama. Plant canopy of those genotypes was divided into three 10-cm strata. Year of harvest affected NDF, protein and hemicellulose content of leaves and stems. Cut affected NDF, cellulose and hemicellulose content and protein of leaves. No differences were measured among the four genotypes except for protein content in the stems. Leaves had a much higher protein content than stems which makes them undesirable for biofuel use. Large strata effects on stem composition were measured on all traits except lignin which had the same value across the strata. Values of NDF, ADF, cellulose, and hemicellulose increased from the top of the stem to the base whereas protein content was reduced. Key words: biofuel, biomass, Lespedeza cuneata (Dumont) G. Don, sericea lespedeza

Introduction Sericea lespedeza is a forage crop used for grazing, hay, or as a soil conservation plant that has shown potential for the production of ligno-cellulosic biomass in the Southeastern USA (Bransby et al. 1989, Mosjidis 1996). Once established, sericea lespedeza costs are relatively small compared to other plants (Ball & Crew 1995). While most herbaceous plants require nitrogen fertilization, sericea lespedeza fixes its own. Compared to most other crops, relatively few diseases and insect problems are associated with this species (Mosjidis 1997). It is one of the most commonly used species for cultivation on strip mine spoils, road banks, and other disturbed or eroding areas because it can improve important physical characteristics of eroded soils (Campbell et al. 1995). Furthermore, sericea lespedeza is tolerant of aluminum-toxic conditions; therefore, it is especially valuable in soils below pH 5.0 where aluminum toxicity is a problem (Fletcher & Livingston, 1949). This species tolerates drought and low soil fertility (Mosjidis 1997). Agblevor et al. (1994) investigated the conversion of sericea lespedeza biomass into energy

J. A. Mosjidis ) ( Auburn University, Department of Agronomy and Soils & Alabama Agricultural Experimental Station, Auburn, AL 36849-5412, USA e-mail: [email protected]

using pyrolysis. They reported that biomass from plants harvested in December after defoliation produced lower char than switchgrass (Panicum virgatum L.), whereas plants harvested in October had higher char production. Plant biomass is a mixture of structures of differing maturity and composition. Plant cells are composed of two major constituents, cell walls and cell contents. Cell walls make up the ligno-cellulosic material of major importance for biofuel production by pyrolysis whereas cell contents such as protein and carbohydrates contribute to the formation of undesirable residues (Theander & Nelson 1989) A high protein content in the plant tissue increases char formation because of the interaction between amino acids and carbohydrates (Theander & Nelson 1989). The objective of this work was to determine the composition of stems at three canopy levels of four genotypes of sericea lespedeza at different times during the growing season in two years. Materials and Methods Four genotypes of sericea lespedeza (L18, AU Donnelly, 74-24-7, and 79-290-9) were planted in a randomized complete block design with three replications at Tallassee, Alabama, in May 1986. This experiment was located on a Wickham sandy loam (fine-loamy, mixed, semiactive, thermic,

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Typic Hapludults) soil. Plots were single rows 7.6 m long, planted 0.91 m apart. In 1990 and 1991, herbage from each genotype was harvested twice. The canopy of each genotype was divided into three 10-cm strata starting from the tip of the stems. Herbage subsamples were divided into leaves (leaves and petioles) and stems. Herbage samples were taken from each plant structure of each segment, dried at 60 C for 48 h, ground to pass a 1 mm screen, and analyzed for concentration of nitrogen with a LECO combustion analyzer, neutral detergent fiber (NDF), acid detergent fiber (ADF), and permanganate lignin using the methods described by Goering and Van Soest (1970). Hemicellulose content was estimated by the difference between NDF and ADF. Data were analyzed using SAS. Significant differences between treatment were estimated using Waller-Duncan's MSD (minimum significant difference, P<0.05). Results and Discussion Year of harvest affected NDF, protein and hemicellulose content in leaves and stems (Tab. 1). Cut affected NDF, cellulose and hemicellulose content of stems and protein content of leaves.

No differences were measured in stem composition among the four genotypes except for protein content (Tab. 1). Protein content in leaves of the four genotypes ranged from 197 to 231 g kg-1 whereas in stems it ranged from 140 to 156 g kg-1 (Tab. 1). The much higher protein content of leaves makes them undesirable for biomass use because they would increase char production when using pyrolysis. Genotype L18 had lower protein content than the other genotypes which would make it more desirable but it was reported by Mosjidis (1993) to have lower biomass yield than AU Donnelly and 79290-9. Large strata effects on stem composition were measured on all traits except lignin which had the same value across the strata (Tab. 1). Values of NDF, ADF, cellulose, and hemicellulose increased from the top of the stem to the base whereas protein content was reduced. Results indicate that mature stems harvested late in the season would be more advantageous for biofuel use. Therefore, the ideal time to harvest sericea lespedeza plants for biofuel production would be during winter time when the plants are dormant and have lost most leaves.

Table 1. Neutral detergent fiber (NDF), acid detergent fiber (ADF), protein, lignin, cellulose and hemicellulose content (g kg-1) of sericea lespedeza stems as affected by year, cut, genotype, and strata Tabela 1. Sadrzaj (g kg-1) neutralnih (NDF) i kiselih rastvorljivih vlakana (ADF), proteina, lignin, celuloze i hemiceluloze stabla lespedeze u zavisnosti od godine, otkosa, genotipa i sloja NDF Year / Godina 1990 1991 Cut / Otkos 1 2 Genotype / Genotip L18 AU Donnelly 74-24-7 79-290-9 MSD 0.05 Strata / Sloj Top 10 cm / Vrh Central 10 cm /Sred. Lower 10 cm / Dno MSD 0.05 533* 553 532** 554 534 536 559 544 ns 456 550 618 11 ADF 431 418 424 424 406 431 424 437 ns 352 436 483 16 Protein Proteini 163*** 138 153 148 140 152 156 155 5 181 146 125 4 Lignin Lignin 62 82 69 74 65 76 75 72 ns 68 74 74 ns Cellulose Celuloza 324 320 308** 336 306 325 324 334 ns 274 325 366 14 Hemicellulose Hemiceluloza 100* 139 107* 132 126 115 127 109 ns 103 117 138 15

*, **, ***, Significant at probabilities 0.05, 0.01, 0.001, respectively. *, **, ***, Znacajno uz verovatnou od 0,05; 0,01; 0,001; ns ­ nije znacajno.

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Conclusions Sericea lespedeza represents a crop with a relatively non-expensive cultivation for diverse purposes such as forage. Its additional value lays in the possibility of its use as biofuel, as supported by the results of this research, with numerous advantages in comparison to other crops. Composition of sericea lespedeza stems indicated that this plant has the potential to be utilized for biofuel production. Genotypes of sericea lespedeza had the same composition when grown for biofuel use. Thus, genotypes need to be chosen based on biomass yield. Mature stems harvested late in the season would be more advantageous for biofuel use. Harvest of sericea lespedeza plants for biofuel production should be during winter time when the plants are dormant and have lost most leaves.

References

Agblevor F A, Davis M F, Evans R J (1994): Molecular beam mass spectrometric characterization of biomass pyrolysis products for fuels and chemicals [Online] Available at http://rredc.nrel.gov/biomass/ doe/nrel/standards/ (verified 23 September 1999) Biomass Resource Information Clearinghouse, National Renewable Energy Laboratory, Washington, DC, USA Ball D, Crew J R (1995): Comparison of selected Alabama forage crops as pastures for stocker steers. Circular ANR-764, Alabama Cooperative Extension Service, Auburn University, Auburn Bransby D I, Ward C Y, Rose P A, Sladden S E, Kee D D (1989): Biomass production from selected herbaceous species in the southeastern USA. Biomass 20: 187-197 Campbell T A, Nuernberg N J, Foy C D (1991): Differential responses of sericea lespedeza to aluminum stress. J. Plant Nutr. 14: 1057-1066 Fletcher P W, Livingston R B (1949): Structure improvement following legume growth on unfertilized subsoil. Soil Sci. Soc. Am. Proc. 14: 347-350 Goering H K, Van Soest P J (1970): Forage fiber analyses. USDA Agriculture Handbook No. 379. US Gov. Print Off., Washington, DC Mosjidis J A (1996): Variability for biomass production and plant composition in sericea lespedeza. Biomass Bioenergy 11: 63-68 Mosjidis J A (1993): Variability for biomass production and plant composition in sericea lespedeza germplasm. Final Report on a Field and Laboratory Research Program for the Period September 30, 1990 to December 31, 1991, ORNL/90-SG301/1, Oak Ridge National Laboratory, Oak Ridge, Tennessee Mosjidis J A (1997): Lespedeza cuneata. In: Faridah Hanum I, van der Maesen L J G (eds.) Plant Resources of South-East Asia, No. 11, Auxiliary Plants. Backhuys Publishers, Leiden, The Netherlands, 170-173 Theander O, Nelson D A (1989): Aqueous high temperature transformation of carbohydrates relative to utilization of biomass. Adv. Carbohydr. Chem. Biochem. 46: 273-326

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Sastav biomase lespedeze i njeno korisenje za bioenergiju u jugoistocnom delu SAD

Horhe A. Moshidis

Univerzitet u Obernu, Odeljenje za agronomiju i zemljiste i Poljoprivredna ogledna stanica Alabame, Obern, AL 36849-5412, SAD Izvod: Lespedeza (Lespedeza cuneata (Dumont) G. Don) se koristi na vise nacina, prvenstveno kao krmna biljka, ali i za ocuvanje zemljista, uz dokazani potencijal za proizvodnju ligninsko-celulozne biomase u jugoistocnom delu SAD. Cetiri genotipa lespedeza gajena su u Talasiju, Alabama. Usev ovih genotipova bio je podeljen u tri sloja po vertikali od po 10 cm. Godina kosidbe uticala je na sadrzaj kiselih rastvorljivih vlakana (NDF), proteina i hemiceluloze u listu i stablu. Otkos je uticao na sadrzaj NDF, celuloze, hemiceluloze i proteina lista. Nije bilo razlika izmeu genotipova izuzev sadrzaja proteina u stablu. List je imao znacajno vei sadrzaj proteina u odnosu na stablo, sto ga cini nepogodnim za korisenje u vidu biogoriva. Znacajan uticaj sloja utvren je kod svih osobina osim lignina, koji je imao istu vrednost u svim slojevima. Vrednosti NDF, kiselih rastvorljivih vlakana (ADF), celuloze i hemiceluloze rasle su od vrha ka osnovi stabla, dok se sadrzaj proteina smanjivao. Kljucne reci: biogorivo, biomasa, Lespedeza cuneata, lespedeza

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www.nsseme.com/journal.html

Zetveni ostaci kao biomasa i organska materija

591 Kvalitet i plodnost zemljista / Soil Quality and Fertility Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 591-598 originalni naucni rad / original research article

Sadrzaj organske materije u zemljistima AP Vojvodine i mogunost korisenja zetvenih ostataka kao obnovljivog izvora energije

Petar Sekuli Jordana Ninkov Nikola Hristov Jovica Vasin Sran Seremesi Tijana Zeremski-Skori

primljeno / received: 13.04.2010. prihvaeno / accepted: 24.05.2010. © 2010 IFVC

Izvod: Analizom preko 77.000 uzoraka zemljista pod oranicama AP Vojvodine, utvreno je da 39% uzoraka pripada klasi slabo humoznog zemljista sa sadrzajem organske materije od 1% do 3%. U klasi dobre obezbeenosti sa sadrzajem humusa od 3% do 5% nalazi se 59% analiziranih vojvoanskih oranica. Dobijeni rezultati su u suprotnosti sa prirodnim potencijalom visokokvalitetnih vojvoanskih zemljista i posledica su neadekvatne agrotehnike, nedovoljne primene organskih ubriva, neracionalnog odnosenja i spaljivanja zetvenih ostataka. Sadrzaj organske materije u zemljistima Vojvodine pod jakim je antropogenim uticajem, te se bez prethodne analize zemljista ne savetuje iznosenje zetvenih ostataka i njihova upotreba kao biomase. Na osnovu analize sadrzaja organske materije u poljoprivrednom zemljistu u pojedinim opstinama AP Vojvodine, moze se izvrsiti odabir lokacija sa kvalitetnim zemljistem, gde je mogua izgradnja postrojenja za dobijanje energije iz zetvenih ostataka, uz minimalne troskove transporta i ocuvanje zemljista. Kljucne reci: biomasa, obnovljivi izvori energije, organska materija, zemljiste

Uvod Sinteza i transformacija organske materije, odnosno akumulacija humusa u zemljistu, predstavlja opsti pedogenetski proces svakog zemljista. Intenzitet akumulacije humusa zavisi od konstalacije faktora pedogeneze u kojoj je kolicina biljnih ostataka znacajna komponenta. U proseku suva materija biljaka sadrzi oko 45% C, 42% O2, 6,5% H, 1,5% N i 0,5% mineralnih materija (Kastori & Tesi 2006). Prema tome, biljke imaju veoma vaznu ulogu u kruzenju ugljenika jer predstavljaju mesto vezivanja CO2 iz atmosfere, koji se neposredno unosi u zemljiste i cini primarni izvor C u agroekosistemu nakon transformacije u organsku materiju. Les kao maticni supstrat veine zemljista u AP Vojvodini svojim mineraloskim sastavom uslovljava njihovu plodnost u pojedinim elementima, hranljivim za biljke. Plodnost zemljista omoguuje poveanu produkciju prirodne stepske vegetacije, odnosno poveanu sintezu

P. Sekuli ) ( J. Ninkov N. Hristov J. Vasin T. Zeremski-Skori Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] S. Seremesi Poljorivredni fakultet Univerziteta u Novom Sadu, Trg Dositeja Obradovia 8, 21000 Novi Sad, Srbija

humusa u zemljistu u odnosu na neke druge maticne supstrate (prvenstveno recentne aluvijalne nanose). Zivkovi i sar. (1972) navode da se klima Vojvodine moze definisati kao jugozapadni varijetet semiaridne stepske klime. Cernozem Vojvodine, koji je u morfoloskoj i hemijskoj vezi sa cernozemima npr. Pretkavkazja, juzne Ukrajine, Moldavije, za razliku od njih ima nizi sadrzaj humusa. Razlog je vea kolicina padavina i vise prosecne temperature u odnosu na ove regione. Ipak, prirodni sadrzaj humusa u nasim cernozemima je zasigurno visi od granicne vrednosti (3%) koja deli klase zemljista dobro i slabo obezbeenih humusom. AP Vojvodina poseduje visok potencijal za ratarsku poljoprivrednu proizvodnju zahvaljujui kvalitetnom zemljistu, blagoj klimi, obilju vode i dugoj tradiciji. Prema zastupljenosti pojedinih tipova zemljista, cak 60% povrsine Vojvodine cini cernozem koji se po svojim fizicko-hemijskim svojstvima smatra idealnim zemljistem za biljnu proizvodnju. Zatim slede drugi, veoma kvalitetni tipovi zemljista sa znacajnim prostornim ucesem, kao sto su ritska crnica (16%) i aluvijalno zemljiste (9%) (Skori i sar. 1985). Od ukupne povrsine AP Vojvodine (2.150.600 ha) poljoprivredno zemljiste cini cak 1.763.000 ha, odnosno 82%. Prema nacinu korisenja zemljista, Vojvodina predstavlja izrazitu ratarsku oblast budui da oranice i baste

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zauzimaju 90% poljoprivrednog zemljista (Statisticki godisnjak Srbije, 2008). Meutim, korisenju zetvenih ostatka poreklom sa oranica cesto nije pridavan veliki znacaj, a pogotovo ne kao obnovljivim izvorima energije (OIE). U svetskim razmerama, OIE u odnosu na fosilna goriva imaju visestruke prednosti: smanjuje se zavisnost od uvoza, smanjuje se emisija stetnih gasova u atmosferu, obezbeuje se otvaranje novih radnih mesta uz tzv. efekat 3E (energetika, ekologija, ekonomija), a pored korisenja prirodnih resursa (sunce, vetar, itd.) omoguuje se bezbedno i ekoloski prihvatljivo uklanjanje raznih vrsta otpada. Evropska unija obavezala je sve clanice da ucese OIE u ukupnoj proizvodnji energije sa prosecnih 6% u 1997. godini poveaju na 12% u 2010. godini, sto moraju prihvatiti i sve zemlje prilikom pristupanja Uniji. Prema Martinov i sar. (2005) u okviru Nacionalne studije o OIE, izvrseno je ispitivanje kolicine biljnih ostataka s obzirom na prosecan prinos i odnos izmeu osnovnog proizvoda i biljnih ostataka. Rezultati proracuna potencijalnih kolicina biomase pokazali su da je kao energent u Srbiji raspolozivo oko 3.000.000 t biljnih ostataka, od cega je bar polovina u Vojvodini. U ukupnoj potrosnji energije u Vojvodini, OIE ucestvuju sa skromnih 0,3% pri cemu je biomasa - gorivo koje se dobija od biljaka, ili delova biljaka kao sto su slama, stabljike zitarica, drvo i razni ostaci, najvei i najvazniji OIE i cini oko 60% ukupno raspolozivog potencijala. S obzirom na raspolozivu poljoprivrednu povrsinu, dominantnu zastupljenost kukuruza i psenice u setvenoj strukturi i visokoenergetski potencijal sojine slame, uz racionalnu upotrebu zetvenih ostataka kao OIE, na teritotoriji Vojvodine mogue je proizvesti kolicinu energije koja predstavlja ekvivalent 1.000.000 t nafte na godisnjem nivou (http://www.psemr.vojvodina.gov.rs ­ Savet za biomasu). Dugorocno posmatrano, biljna proizvodnja dovodi do smanjenja sadrzaja organske materije u obradivom zemljistu usled spaljivanja zetvenih ostatatka, njihovog odnosenja, neadekvatne obrade zemljista, nedovoljne primene organskih ubriva, erozije i dr. Utvreno je da je zadnjih decenija doslo do smanjenja udela humusa u zemljistima Vojvodine od 0,2% do 0,81% u proseku za 0,38% (Bogdanovi i sar. 1993), pre svega zbog delovanja antropogenog faktora, ali i prirodnih cinilaca. Postavlja se pitanje da li e uz nedomainski odnos prema nuzproizvodima gajenih biljaka, odnosno spaljivanje i svesno odnosenje zetvenih ostataka i njihovo upotreba kao OIE, jos vise osiromasiti zemljiste i samo ubrzati dugorocni degradacioni proces?

Cilj ovog rada je bio da se utvrdi sadrzaj organske materije u zemljistima AP Vojvodine i mogunost korisenja zetvenih ostataka kao OIE, uz konzervacijski uticaj na strukturu i hemijski sastav zemljista. Materijal i metod rada Pocev od 2002. Pokrajinski sekretarijat za poljoprivredu, vodoprivredu i sumarstvo AP Vojvodine pokrenuo je Akciju kontrole plodnosti poljoprivrednog zemljista, a do sada (2002- 2009) analizirano je oko 150.000 uzoraka zemljista u privatnom sektoru od strane Instituta za ratarstvo i povrtarstvo i 13 regionalnih poljoprivrednih sluzbi u Vojvodini. Sistem kontrole plodnosti predstavlja analizu zemljista na parametre plodnosti i davanje adekvatne preporuke za ubrenje za naredne cetiri godine, u zavisnosti od stanja sadrzaja hraniva u zemljistu, potreba za hranivom planiranih biljnih vrsta za gajenje i prognozirane visine prinosa. Kontrola plodnosti je i zakonska obaveza vlasnika, odnosno korisnika poljoprivrednog zemljista prema Zakonu o poljoprivrednom zemljistu (Sluzbeni glasnik Rep.Srbije 62/06, 65/08, 41/09). Poljoprivredni proizvoaci su sami uzorkovali svoje parcele po principima uzorkovanja zemljista za kontrolu plodnosti na osnovu dobijenog uputstva. Pojedinacni uzorak je sacinjen od 20 do 25 pojedinacnih uzoraka sa dubine 0-30 cm i predstavlja povrsinu velicine do 5 ha. Sadrzaj humusa tj. organske materije odreen je modifikovanom metodom ISO 14235:1998 po Tjurin-u, na principu oksidacije organskog ugljenika zemljista sa K2Cr2O7. Rezultati i diskusija U okviru Akcije kontrole plodnosti kao jedan od parametara je analiziran sadrzaj humusa (organske materije). Na osnovu obrade 77.388 podataka za oranice Vojvodine, utvreno je da cak 39% visokokvalitetnog zemljista Vojvodine pripada klasi slabo humoznog zemljista sa sadrzajem organske materije od 1% do 3% (Tab. 1). U klasi dobre obezbeenosti sa sadrzajem humusa od 3% do 5 % nalazi se 59% analiziranih vojvoanskih oranica. Dobijeni rezultati su u suprotnosti sa uvrezenim misljenjem o visokom proizvodnom potencijalu kvalitetnih vojvoanskih zemljista i posledica je neadekvatne agrotehnike, odnosno nedovoljne primene organskih ubriva (nedovoljno razvijeno stocarstvo), kao i spaljivanja i nekontrolisanog odnosenja zetvenih ostataka.

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Tabela 1. Ucese uzoraka u pojedinim klasama obezbeenosti humusa na osnovu analize 77.388 uzoraka oranica Vojvodine u periodu 2002-2009. Table 1: Proportion of samples with different levels of humus as shown by analysis of 77,388 plowland samples taken in the province of Vojvodina in the period 2002-2009 Klasa obezbeenosti zemljista Soil class regarding levels Vrlo slabo humozno Very low in humus Slabo humozno Low in humus Humozno Well provided by humus Jako humozno Very high in humus Postojea klasifikacija sadrzaja humusa je neprecizna u smislu sirokog intervala za slabo i humuzno zemljiste. Radi boljeg razumevanja dinamike sadrzaja organske materije u zemljistu neophodno je redefinisati nivoe obezbeenosti zemljista i uvesti podklase. Na osnovu detaljnije klasifikacije (Tab. 1) u klasi slabo humoznog zemljista dominiraju uzorci sa sadrzajem organske materije od 2% do 3%, dok u klasi humoznog zemljista dominira zemljiste sa sadrzajem organske materije od 3% do 4%. U Vojvodini je dugi niz godina bila praksa spaljivanja zetvenih ostataka (usled poveanih troskova za gorivo i mehanizaciju pri zaoravanju zetvenih ostataka i nedovoljne edukacije individualnih proizvoaca), sto donosi ogromne stete, kako u poljoprivredi, gubitkom organske materije i unistavanjem zemljisnog zivog sveta, tako i znacajno doprinosi zagaenju zivotne sredine emitovanjem ugljen-dioksida i stetnih produkata gorenja u atmosferu. Spaljivanje zetvenih ostataka je zakonski zabranjeno prema Zakonu o poljoprivrednom zemljistu (Sluzbeni glasnik Rep. Srbije 62/06, 65/08, 41/09) i zadnjih godina je pojacana inspekcijska kontrola i sankcionisanje ovakvog nemara. Visokokvalitetno zemljiste je osnova poljoprivredne proizvodnje sto je i najznacajnija delatnost AP Vojvodine, te zastita zemljista ima primaran znacaj za ovu oblast Republike Srbije. Prema globalnoj proceni, zemljiste Vojvodine je pod jakim antropogenim uticajem i odnosenje zetvenih ostataka sa obradivih povrsina nije adekvatno. Meutim, analizom stanja po pojedinim opstinama, moze se izvrsiti odabir lokacija sa Humus % 0-1 1-3 1-3 3-5 3-5 >5 3-4 4-5 1-2 2-3 % ucesa uzoraka zemljista % of soil samples 0,5 39,1 7,1 32,0 59,1 46,5 12,6 1,3

kvalitetnim zemljistem, gde je mogua izgradnja postrojenja za dobijanje energije iz zetvenih ostataka, uz minimalne troskove transporta i ocuvanje zemljisnog kompleksa. Na osnovu posmatranja sadrzaja humusa u zemljistu pojedinih opstina u AP Vojvodini koje se nalaze na razlicitim geomorfoloskim celinama, na najbolji nacin se moze ilustrovati znacaj analize zemljista u odabiru lokacija za izgradnju postrojenja za preradu zetvenih ostataka. Analizom je obuhvaeno sest opstina u Vojvodini: Backa Topola, Vrbas, Bac, Kikinda, Kovin i Irig lociranim na razlicitim geohemijskim podlogama u cilju utvrivanja prirodnog potencijala pojedinih tipova zemljista za sadrzaj humusa (Sl. 1). Prema pedoloskoj karti Vojvodine (Nejgebauer i sar. 1971) prirodno najplodnije zemljiste, pa samim tim i ono sa najvisim sadrzajem humusa, jeste cernozem formiran na geomorfoloskim celinama backa lesna zaravan i backa lesna terasa (Bukurov 1972, Koscal i sar. 2005). Njih predstavljaju opstine Backa Topola i Vrbas. Zemljista sa teritorije ostalih ispitivanih opstina su sa razlicitih geomorfoloskih celina (planina Fruska gora, aluvijalne ravni Dunava, Begeja i Zlatice) i imaju, u manjoj ili veoj meri, manju prirodnu plodnost i nizi sadrzaj humusa (Sl. 1). Na osnovu rezultata ispitivanja zemljista moze se zakljuciti da je u opstinama sa najplodnijim tipom zemljista, cernozemom (Backa Topola i Vrbas), zabelezeno manje ucese zemljista slabo obezbeenih humusom (7%) u poreenju sa opstinama sa zemljistima nize plodnosti (Graf. 3-6) i proseka Vojvodine (Tab. 1). Ovo ukazuje

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Slika 1. Posmatrane katastarske opstine na geoloskoj karti AP Vojvodine po Bukurovu (1972) Figure 1. Location of the studied municipalities on the geological map of the Vojvodina province authored by Bukurov (1972)

%

100 80 60 40 20 0

%

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% humusa

0-1 0

1-3 7

3-5 91

>5 2

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0-1 0

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3-5 92

>5 1

% ucesa uzoraka

% ucesa uzoraka

Grafikon 1. Ucese uzoraka u pojedinim klasama obezbeenosti humusa (%) u opstini Backa Topola (broj uzoraka 2.561) Graph 1. Proportion of samples with different levels of humus (%) in the municipality of Backa Topola (no. of samples: 2,561)

Grafikon 2. Ucese uzoraka u pojedinim klasama obezbeenosti humusa (%) u opstini Vrbas (broj uzoraka 2.241) Graph 2. Proportion of samples with different levels of humus (%) in the municipality of Vrbas (no. of samples: 2,241)

na to da je, pored atropogenog, uticaj maticnog supstrata i tipa zemljista veoma snazan na sadrzaj organske materije u zemljistu. Poveanje ucesa klase zemljista slabo obezbeenih humusom u ostalim opstinama bilo je ocekivano (Graf. 3-6), jer je ova pojava rezultat pedogeneze zastupljenih tipova zemljista.

Na veem delu poljoprivrednih povrsina u opstinama Kikinda i Kovin zastupljena je geomorfoloska celina aluvijalna ravan reka Zlatice i Begeja (opstina Kikinda) i Dunava (opstina Kovin). Na aluvijalnim ravnima su kao maticni supstrati zastupljeni recentni recni nanosi koji su razlicitog sastava i kvaliteta kao podloge za formiranje zemljista. Specifican je sadrzaj organske materi-

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%

100 80 60 40 20 0

%

100 80 60 40 20 0

% humusa

0-1 1

1-3 18

3-5 80

>5 1

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0-1 1

1-3 54

3-5 44

>5 1

% ucesa uzoraka

% ucesa uzoraka

Grafikon 3. Ucese uzoraka u pojedinim klasama obezbeenosti humusa (%) u opstini Kikinda (broj uzoraka 1.877) Graph 3. Proportion of samples with different levels of humus (%) in the municipality of Kikinda (no. of samples: 1,877)

Grafikon 4. Ucese uzoraka u pojedinim klasama obezbeenosti humusa (%) u opstini Kovin (broj uzoraka 2.599) Graph 4. Proportion of samples with different levels of humus (%) in the municipality of Kovin (no. of samples: 2,599)

je u zemljistu na teritoriji opstine Kikinda. Jos uvek dominiraju zemljista sa sadrzajem humusa preko 3% sto je posledica toga sto su obe reke Begej i Zlatica u ovom podrucju na kraju svojih tokova, pa su plavei okolna zemljista nanosile teksturno finije i plodnije cestice u odnosu na Dunav u podrucju Kovina. Na teritoriji opstine Kovin se nalazi i deo geomorfoloske celine Deliblatska pescara, ali ovo nije uticalo na rezultate sadrzaja humusa, jer se pescara u manjoj meri koristi za poljoprivrednu proizvodnju. Poljoprivredna zemljista sa teritorije opstine Bac karakterisu se visokim nivoom podzemne vode prve izdani (podzemne vode akumulirane u prvoj vodonosnoj sredini od povrsine terena) koja je vrlo cesto zaslanjena i/ili alkalizovana. Procesi salinizacije i alkalizacije negativno uticu na produkciju organske materije, kao i na sintezu i akumulaciju humusa u povrsinskim slojevima ovakvih zemljista. Znacajan deo opstine Irig nalazi se na obroncima planine Fruske gore, gde se kao maticni supstrat za formiranje zemljista pojavljuju stene kao sto su serpenti, kristalasti skriljci, glinci i dr. U procesima pedogeneze na njima su formirana zemljista manje plodnosti, usled cega je i formiranje humusa bilo na nizem nivou. Niskom sadrzaju humusa na ovim povrsinama doprinela je takoe aktivnost coveka - voarska i vinogradarska proizvodnja, tj. duboko oranje cime je ubrzan proces mineralizacije, odnosno razgradnje humusa. Pored toga, intenzitet smanjenja sadrzaja organske materije nije isti na svim zemljistima, jer se njen nivo sporije smanjuje na zemljistima koja su slabije obezbeena. Kod zemljista koja su dobro obezbeena organskom materijom (3% do 5%), neadekvatna agrotehnika i nekontrolisana manipulacija zetvenim ostacima moze izazvati brze smanjivanje njenog sadrzaja.

Korisenje zetvenih ostataka kao obnovljivog izvora energije ima niz prednosti, meutim, postoje i suprotna misljenja. Pored nizeg stepena korisnosti sagorevanja biomase, problem uticaja na zivotnu sredinu i visih troskova postrojenja za preradu, postoje i druga ogranicenja pri korisenju biomase kao goriva. Neki agronomi imaju izuzetno negativan stav prema odnosenju biljnih ostataka sa polja, pre svega zbog narusavanja procesa kruzenja materije i nepovoljnog dejstva na plodnost zemljista, odnosno snizavanja prinosa gajenih useva. Malesevi i Jaimovi (2010) navode da se dugogodisnjim zaoravanjem zetvenih ostataka prinos kod psenice poveava za 11,4% a kod kukuruza za 7,2% u odnosu na varijante sa uklanjanjem biljnih ostataka sa parcele. Meutim, isti autori takoe navode da se unosenjem prevelike kolicine biljnih ostatka javlja tzv. azotna depresija, odnosno privremeno blokiranje (vezivanje) azota u telima mikroorganizama, sto se moze nepovoljno odraziti na naredne useve, kao prolazni nedostatak azota. Ako se uzme u obzir da se kod proizvodnje silaznog kukuruza i krmnih biljaka (za seno i silazu), kompletna nadzemna masa svesno gaji za odnosenje sa parcele, namee se zakljucak da se osobine zemljista nee narusiti u duzem vremenskom periodu jedino racionalnim gazdovanjem i odrzivim korisenjem zetvenih ostataka. Tome svakako treba dodati i konzervacijsku obradu, bez prevrtanja zemljista, pri cemu biljni ostaci ostaju na povrsini, a delimicno se u njega unose. Osim agropedoloskih, ovakvom obradom ostvaruju se i druge pogodnosti, poput smanjivanja troskova proizvodnje. Ako se pravilno odredi raspolozivi potencijal biomase i racionalno pristupi odnosenju zetvenih ostataka sa parcele, uz adekvatnu agro-

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%

100 80 60 40 20 0

%

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% humusa

0-1 2

1-3 80

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3-5 8

>5 0

% ucesa uzoraka

% ucesa uzoraka

Grafikon 5. Ucese uzoraka u pojedinim klasama obezbeenosti humusa (%) u opstini Bac (broj uzoraka 2.647) Graph 5. Proportion of samples with different levels of humus (%) in the municipality of Bac (no. of samples: 2,647)

Grafikon 6. Ucese uzoraka u pojedinim klasama obezbeenosti humusa (%) u opstini Irig (broj uzoraka 765) Graph 6. Proportion of samples with different levels of humus (%) in the municipality of Irig (no. of samples: 765)

tehniku (pre svega ubrenje) sadrzaj organske materije u zemljistu moze se odrzavati na zadovoljavajuem nivou. Naime, kod psenice, cija se slama najcese koristi za biomasu, podaci o odnosu zrno-slama racunaju se za nadzemni deo biljke i iznose izmeu 1 i 1,2 u odnosu na zrno. Znacajan je i ostatak slame na strnistu koji e se koristiti za zaoravanje. On pre svega zavisi od visine reza, ali i od klimatskih prilika, odnosno raspolozivih padavina u vegetativnom periodu. Na osnovu izmerenih velicina, ostatak slame na strnistu za visinu reza 15 cm je u ,,vlaznoj" sezoni oko 33%, a u ,,susnoj" oko 39% u odnosu na prinos zrna (Martinov 1984). Tome treba dodati da je i prinos zrna u susnoj sezoni nizi, pa je i ukupno iznosenje hranljivih materija manje. Uzimajui to u obzir, kao i gubitke slame pri presovanju, prosecno raspoloziva kolicina slame je 2,5 t ha-1 do 3 t ha-1, uz prinos zrna od 6 t ha-1. U isto vreme vise od 50% nadzemne biomase mase ostaje u zemlji i na taj nacin poboljsava strukturu zemljista, te u najmanju ruku odrzava sadrzaj organske materije na postojeem nivou (Martinov i sar. 2005). Kukuruzu, kao najzastupljenijem ratarskom usevu na vojvoanskim njivama, mora se posvetiti posebna paznja. Prema izvrsenim merenjima prinosa pojedinih delova biljaka kukuruza u odnosu na zrno, utvreno je da prinos upotrebljive kukuruzovine (lista i stabljike bez najnizih 20 cm) iznosi 60-90% u odnosu na prinos zrna, a prinos oklaska 10-21%. Pri korisenju biljnih ostataka kukuruza javlja se problem vlaznosti kukuruzovine, koja moze da premasi 50%, te tada nije pogodna za sagorevanje i skladistenje. Sa tog polazista sa sigurnosu je mogue koristiti do 20% raspolozive biomase, odnosno ku-

kuruzovinu ranostasnih hibrida FAO 400 grupe. Uzimajui u obzir prosecan prinos kukuruza od 7 t ha-1, cinjenicu da u Vojvodini dominiraju hibridi FAO grupe 600 i 700, kao i efikasnost zetve od 50%, slicno kao i kod psenice i pored svesnog odnosenja zetvenih ostataka kao OIE, za odrzavanje strukure zemljista i sadrzaja organske materije, uz pravilno gazdovanje i dalje ostaje vise od polovine raspolozive biomase (Martinov i sar. 2005). Ekonomska opravdanost korisenja OIE u poljoprivredi jedno je od najcese postavljanih pitanja u cilju definisanja kratkrocne ekonomske koristi i dugorocnog smanjenja produktivnosti zemljista. Procedura za odreivanje kolicine i cene ,,zelene energije", tj. slame kao najcese korisenog OIE u poljoprivredi, relativno je jednostavna i obuhvata (1) odreivanje kolicine zetvenih ostataka na osnovu rezultata proizvedene kolicine zrna, (2) odreivanje kolicine zetvenih ostataka koje bi trebalo ostaviti na polju zbog kontrole erozije i plodnosti zemljista, (3) odreivanje fizicke pristupacnosti zetvenih ostataka, pri cemu je nagnutost terena kriticni element, (4) odreivanje troskova prikupljanja i transporta, (5) odreivanje lokacije i cene skladistenja, (6) odreivanje cene transporta do mesta za konverziju, i (7) odreivanje svih ostalih troskova ukljucujui kupovinu zetvenih ostataka ili hranljivu vrednost slame koja se odnosi sa parcele. Treba uzeti u obzir da ostvareni prinos gajenih kultura, a prema tome i produkcija zetvenih ostataka, izlozena godisnjim varijacijama usled klimatskih promena i zasejane povrsine zavise od uslova trzista i podsticajnih mera drzave.

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Martinov M, Tesi M, Konstantinovi M, Stepanov B (2005): Perspektive u korisenju biomase za grejanje domainstava u seoskim podrucjima. Savremena poljoprivredna tehnika 31: 155-264 Nejgebauer V, Zivkovi B, Tanasijevi , Miljkovi N (1971): Pedoloska karta Vojvodine R 1 : 50.000, Institut za poljoprivredna istrazivanja, Novi Sad Republicki zavod za statistiku (2008): Statisticki godisnjak Srbije, Beograd. Skori A, Filipovski G, iri M (1985): Klasifikacija zemljista Jugoslavije. (knjiga 13) Akademija nauka i umjetnosti Bosne i Hercegovine, Sarajevo Zakon o poljoprivrednom zemljistu. Sluzbeni Glasnik RS broj 62/2006, 65/2008 Zakon o izmenama i dopunama zakona o poljoprivrednom zemljistu. Sluzbeni Glasnik RS broj 41, 2009 Zivkovi B, Nejgebauer V, Tanasijevi , Miljkovi N, Stojkovi L, Drezgi P (1972): Zemljista Vojvodine. Institut za poljoprivredna istrazivanja, Novi Sad

Zakljucak Sve vea potreba za energijom nametnula je potrebu za iznalazenjem alternativnih izvora energije u koje se ubrajaju i zetveni ostaci njivskih biljaka. Korisenje biomase njivskih biljaka u energetske svrhe ima niz prednosti sa ekoloskog stanovista. Sadrzaj organske materije u zemljistima Vojvodine pod jakim je antropogenim uticajem, te se bez prethodne analize zemljista i utvrivanja stanja sadrzaja humusa, ne savetuje iznosenje zetvenih ostataka i njihova upotreba kao biomase. Prema globalnoj proceni, na osnovu analize preko 77.000 uzoraka, u vojvoanskim zemljistima postoji negativan bilans organske materije i moze se ocekivati dalje smanjenje plodnosti i pogorsanje fizicko-hemijskih svojstava zemljista. Istovremeno, na osnovu raspolozivih podataka, mogue je izdvojiti podrucja poljoprivrednih povrsina gde je zemljiste u najveem stepenu dobro obezbeeno organskom materijom (sadrzaj humusa u klasi 3% do 5%). Indentifikacijom ovih podrucja mogue je odabrati najpovoljnije lokacije za izgradnju postrojenja za dobijanje toplotne energije iz zetvenih ostataka uz minimalne troskove prevoza sirovina sa proizvodnih parcela. Korisenje zetvenih ostataka kao obnovljivog izvora energije mora biti u skladu sa savremenim naucnim principima zastite zemljista. Visokokvalitetno zemljiste je osnova poljoprivredne proizvodnje sto je i najznacajnije delatnost AP Vojvodine, te zastita zemljista ima primaran znacaj za ovu oblast Republike Srbije. Literatura

Bogdanovi D, Ubavi M, Dozet D (1993): Hemijski sastav i obezbeenost zemljista Vojvodine neophodnim elementima. U: Kastori R (ured.), Teski metali i pesticidi u zemljistu - Teski metali i pesticidi u zemljistima Vojvodine, Poljoprivredni fakultet, Institut za ratarstvo i povrtarstvo, Novi Sad, 197-215 Bukurov B (1972): Geomorfoloske karakteristike podrucja SAP Vojvodine. regionalni prostorni plan SAP Vojvodine, Novi Sad Kastori R, Tesi M (2006): Ekoloski aspekti primene zetvenih ostataka njivskih biljaka kao alternativna goriva. Zbornik radova Instituta za ratarstvo i povrtartvo 42: 3-13 Koscal M, Menkovi Lj, Knezevi M, Mijatovi M (2005): Geomorfoloska karta Vojvodine sa tumacem. Geozavod - Gemini Beograd, Republika Srbija - AP Vojvodina - Izvrsno vee AP Vojvodine Pokrajinski sekreterijat za energetiku i mineralne sirovine, Novi Sad Malesevi M, Jaimovi G (2010): Agronomski i ekoloski aspekti razlicitih postupaka sa zetvenim ostacima u poljoprivredi. U: Poljoprivrednikov poljoprivredni kalendar 2010, Dnevnik-Poljoprivrednik, 243-245 Martinov M (1984): Mogunosti korisenja slame kao izvora toplotne energije. Magistarska teza, Poljoprivredni fakultet, Univerzitet u Zagrebu

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Organic Matter Content in Vojvodina Soils and the Possibility of Using Harvest Residues as Renewable Source of Energy

Petar Sekuli1 Jordana Ninkov1 Nikola Hristov1 Jovica Vasin1 Sran Seremesi2 Tijana Zeremski-Skori1

1

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia 2 Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovia 8, 21000 Novi Sad, Serbia

Summary: An analysis of over 77,000 samples of plowland in AP Vojvodina indicated that 39% of the samples belong to the category of low humus soils with the organic matter content ranging from 1% to 3%. The category of soils well provided with humus (3% to 5%) included 60% of the analyzed plowland of Vojvodina. The obtained results are in contradiction with the natural potential of high quality land of Vojvodina and they are consequence of inadequate agricultural practices, insufficient application of organic fertilizers, excessive removal and burning of harvest residues. Organic matter content in Vojvodina soils is under strong anthropogenic influence, and it is not advisable to remove harvest residues and use them as biomass without prior soil analysis. Based on the analysis of organic matter content in agricultural soils in individual municipalities of AP Vojvodina, it is feasible to select locations with quality soils, where it would be possible to construct plants for energy production from harvest residues, while securing minimum transportation costs and preserving the soil quality. Key words: biomass, organic matter, renewable sources of energy, soil

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 591-598

www.nsseme.com/journal.html

Uticaj nacina primene Azotobacter chroococcum

599 Mikrobiologija zemljista / Soil Microbiology Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 599-606 originalni naucni rad / original research article

Uticaj nacina primene Azotobacter chroococcum na mikroorganizme u rizosferi i prinos seerne repe

Nastasija Mrkovacki Nikola Caci Janja Kuzevski Lazar Kovacev Snezana Mezei Nevena Nagl Dragana Bjeli

primljeno / received: 18.11.2009. preraeno / revised: 03.03.2010. prihvaeno / accepted: 07.05.2010. © 2010 IFVC

Izvod: Ispitan je efekat tri razlicita nacina inokulacije seerne repe sa sojevima Azotobacter chroococcum na parametre prinosa seerne repe i mikrobioloski status zemljista. U ispitivanjima je korisena sorta Drena, a eksperiment je bio lociran na Rimskim Sancevima u toku 2008. i 2009. Kao mikrobiolosko ubrivo koriseno je pet sojeva Azotobacter chroococcum. Inokulacija je izvrsena na tri nacina: (A) inkorporacijom sojeva u zemljiste pre setve, (B) inkorporacija sojeva u zemljiste pred prvu meurednu kultivaciju i (C) nanosenjem inokuluma na seme pre setve. Najvee poveanje u prinosu korena od 6,25 t ha-1 i u prinosu kristalnog seera od 0,91 t ha-1 dobijeno je sa sojem 10 na varijanti inokulacije zemljista pre prve meuredne kultivacije. U obe godine dobijen je pozitivan efekat na ukupan broj mikroorganizama i broj azotobaktera u rizosferi. Kljucne reci: Azotobacter chroococcum, prinos korena, prinos seera, sadrzaj seera, seerna repa, ukupan broj mikroorganizama

Uvod Rizobakterije koje pospesuju biljni rast (PGPR - Plant Growth Promoting Rhizobacteria) predstavljaju vrlo raznovrsne zemljisne bakterije (Pseudomonas, Klebsiella, Citrobacter, Azospirillum, Azotobacter, Bacillus, Azorhizobium) koje u asocijaciji sa biljkom domainom rezultiraju u stimulaciji rasta domaina. Takve asocijacije sa PGPR su veoma vazne za odrzivu poljoprivredu (Mrkovacki & Mili 2001, Mrkovacki & Mezei 2003). Teznja odrzive poljoprivrede je da se smanji upotreba azotnih ubriva na racun bioubriva. Termin ,,bioubriva" je skorasnji i najcese se odnosi na korisenje zemljisnih mikroorganizama u poveanju dostupnosti i usvajanja mineralnih hraniva za biljke. Komercijalna bioubriva slobodnih PGPR obezbeuju moguu alternativu korisenja ubriva i pesticida za razlicite biljne vrste, mada je njihova primena za sada oskudna (Glick et al. 1999). Veina ovih proizvoda su biokontrolne materije koje indirektno doprinose poboljsanju rasta biljaka (Chet & Chernin 2002).

N. Mrkovacki ) ( · N. Caci · L. Kovacev · S. Mezei · N. Nagl · D. Bjeli Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija e-mail: [email protected] J. Kuzevski Institut za primenu nauke u poljoprivredi, Bulevar despota Stefana 68b, 11000 Beograd, Srbija

Kratkorocni pozitivan efekat PGPR rezultira u poboljsanom razviu biljaka. Ovaj efekat obuhvata ubrzano razvie korena, bolji pristup hranljivim materijama i vodi, a time brzi inicijalni rast, sto omoguuje biljkama iskljucivanje korova (kao konkurenata) iz dostupnih izvora hraniva, tj. smanjuje se potreba za herbicidima. Dugotrajan pozitivan efekat PGPR moze rezultirati u poboljsanju rasta biljaka, zdravlju i opstanku, vodei do odrzive proizvodnje hrane sa pozitivnim odnosom prema zivotnoj okolini i ekonomicnosti proizvodnje. Vazno istrazivacko pitanje je nacin inokulisanja biljaka u polju. Nekoliko opcija je na raspolaganju: inokulacija semena ili sadnica i inokulacija tretiranjem zemljista inokulumom ili direktno na biljke. Drugi izazov je formulacija inokuluma da bi se obezbedilo odgovarajue mikrostaniste koje bi sprecilo brz pad u broju bakterija unesenih u zemljiste. Stoga je cilj nasega rada bio da se ispitaju tri nacina aplikacije Azotobacter chroococcum inokuluma kod seerne repe, u dve godine ispitivanja, putem odreivanja brojnosti azotobaktera i ukupnog

Ovo istrazivanje je deo projekta broj 20020: Poboljsanje germplazme seerne repe u cilju poveanja prinosa i smanjenja gubitaka nastalih uticajem biotskih i abiotskih faktora (2008-2011) Ministarstva za nauku i tehnoloski razvoj Republike Srbije/ This research results from the project no. 20020: Improvement of sugar beet germplasm with aim to increase yield and reduce losses caused by biotic and abiotic factors (2008-2011) financed by the Ministry of Science and Technological Development of the Republic of Serbia

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broja mikroorganizama u rizosferi, kao i utvrivanjem prinosa korena i kvaliteta seerne repe na kraju vegetacije. Materijal i metod Istrazivanja su izvedena u toku 2008 i 2009. na lokalitetu Rimski Sancevi, Novi Sad sa pet sojeva Azotobacter chroococcum (1, 5, 8,10 i 14) sa sortom Drena stvorenoj u Institutu za ratarstvo i povrtarstvo u Novom Sadu. Sojevi su gajeni na podlozi Fjodora, u tecnoj kulturi. Gustina tecne kulture bila je 109 elija po cm3. Ispitana su tri nacina inokulacije: (A) tretiranje zemljista neposredno pred setvu, (B) tretiranje zemljista pred prvu meurednu kultivaciju i (C) inokulacija semena. Zemljiste je tretirano sa 2 l ha-1 inokuluma korisenjem 300 l ha-1 vode. Inokulacija semena je obavljena sa 300 ml sj-1 (sj=100.000 semenki). Ogledi su postavljani u cetiri ponavljanja. Duzina osnovne parcelice bila je 10 m a sirina 2 m. Povrsina obracunske parcelice je 9,6 m2 (dva srednja reda bez rubnih repa). Setva je obavljena masinski na razmak 50 cm x 10 cm, a po nicanju je izvrsena korekcija gustine na razmak u redu od 20 cm.

Uzorci za mikrobioloske analize uzeti su u tri roka (kraj maja, kraj jula i kraj septembra) u obe godine i iz sva cetiri ponavljanja. Ukupan broj mikroorganizama odreivan je na zemljisnom agaru (razreenja 106), a broj azotobaktera na Fjodorovoj podlozi (razreenja 102) (Jarak & uri 2004). Vaenja ogleda obavljena su u prvoj polovini oktobra, pri cemu je utvrena masa korena i broj biljaka. Na uzorku od dvadeset repa iz svakog ponavljanja u laboratoriji Instituta za ratarstvo i povrtarstvo u Novom Sadu za analizu korena seerne repe ,,Venema" utvren je sadrzaj seera i sadrzaj neseera (K, Na i amino N). Iz dobijenih podataka izracunat je prinos korena po jedinici povrsine, prinos polarizacionog seera, iskorisenje seera i prinos kristalnog seera. Dobijeni podaci su obraeni analizom varijanse trofaktorijalnog ogleda. Rezultati i diskusija Prinos korena Primenom azotobaktera u ovim ispitivanjima ostvaren je prosecni prinos korena od 70,26 t ha-1 sto je za 1,71 t ha-1 vise od kontrole. Pozitivan

Tabela 1. Efekat inokulacije sa Azotobacter chroococcum na prinos korena seerne repe (t ha-1) Table 1. Effect of inoculation of Azotobacter chroococcum on the yield of sugar beet roots Nacin primene Type of Application (NA) A Godina Year (G) 2008 2009 Prosek Average 2008 2009 Prosek Average 2008 2009 Prosek Average Kontrola Control 64,17 70,84 67,51 67,49 73,67 70,58 63,97 71,18 67,58 68.55 G LSD 0,05 0,01 CV (%) 1,10 1,40 4,75 A 1,90 2,52 Azotobacter Soj Strain 1 64,93 68,61 66,77 67,70 79,03 73,37 64,99 70,66 67,83 69,32 NA 1,35 1,78 Soj Strain 5 64,84 70,63 67,74 66,63 78,79 72,71 66,16 68,64 67,40 69,28 G/A 2,69 3,56 Soj Strain 8 66,54 67,33 66,94 67,91 77,56 72,75 69,43 76,00 72,72 70,80 G/NA 1,90 2,52 c h r o o c o c c u m (A) Soj Strain 10 66,49 70,61 68,55 67,65 74,75 71,20 67,72 79,94 73,83 71,19 Soj Strain 14 64,45 70,25 67,35 67,71 80,08 73,90 65,45 76,34 70,89 70,71 A/NA 3,29 4,36 Prosek Average 65,45 69,49 67,48 67,52 78,04 72,79 66,75 74,32 70,54 70,26 G/A/NA 4,66 6,16

B

C Prosek A, B, C Average A, B, C

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uticaj na prinos korena ispoljili su svi primenjeni sojevi azotobaktera, a poveanje u odnosu na kontrolu kretalo se od 0,73 t ha-1 kod soja 5 do 2,64 t ha-1 kod soja 10. Maksimalno poveanje prinosa korena od 6,25 t ha-1 ostvareno je inokulacijom semena sa sojem 10. Primenom azotobaktera preko zemljista neposredno pred setvu nije bilo poveanja prinosa korena. Inokulacijom semena i primenom azotobaktera preko zemljista pred prvu meurednu kultivaciju prinos korena povean je za 2,96 t ha-1, odnosno za 2,21 t ha-1 (Tab. 1). U svojim istrazivanjima, Mrkovacki i sar. (2007) su dobili poveanje prinosa korena seerne repe kod tri ispitivane sorte na tri lokaliteta od 7% tj. 5 t ha-1. Na proizvodnim parcelama na podrucju Vojvodine, prinos korena korisenjem smese sojeva NS-Betafixin povean je za 3,08 t ha -1 u 2007. a za 6,18 t ha-1 u 2008. Raniji rezultati istrazivanja (Caci i sar. 2003) pokazali su poveanje prinosa korena kod tri hibridne sorte seerne repe primenom azotobaktera od 0,65 t ha-1 do 3,7 t ha-1. Fikretin et al. (2004) navode da je poveanje prinosa seerne repe inokulacijom bilo od 6% do 11% u odnosu na kontrolu.

Sadrzaj seera Primenom azotobaktera u ovim ispitivanjima ostvareno je poveanje sadrzaja seera od 0,14%. Svih pet primenjenih sojeva azotobaktera ispoljilo je pozitivan uticaj na sadrzaj seera, a poveanje u odnosu na kontrolu bilo je od 0,04% kod soja 14 do 0,24% kod soja 5. Najizrazeniji pozitivan uticaj na sadrzaj seera, od 0,37% bio je ostvaren tretiranjem zemljista pred prvu meurednu kultivaciju sa sojem 10. Tretiranjem zemljista azotobakterom pred prvu meurednu kultivaciju ostvaren je najizrazeniji pozitivni efekat na sadrzaj seera. Poveanje u odnosu na kontrolu iznosilo je 0,18% (Tab. 2). Rezultati autora Geci i sar. (2007) pokazuju poveanje sadrzaja seera za sve tri ispitivane sorte, kako tretiranjem semena tako i tretiranjem zemljista smesom sojeva azotobaktera. Prinos polarizacionog seera Primenom azotobaktera u ovim ispitivanjima ostvaren je prosecni prinos polarizacionog seera od 11,65 t ha-1, sto je poveanje u odno-

Tabela 2. Efekat inokulacije sa Azotobacter chroococcum na sadrzaj seera (%) Table 2. Effect of inoculation of Azotobacter chroococcum on sugar content (%) Nacin primene Type of Application (NA) A Godina Year (G) 2008 2009 Prosek Average 2008 2009 Prosek Average 2008 2009 Prosek Average Kontrola Control 16,85 16,38 16,62 16,50 16,43 16,47 16,84 16,07 16,46 16,45 G LSD 0,05 0,01 CV (%) 0,13 0,17 2,31 A 0,22 0,29 Azotobacter Soj Strain 1 16,85 16,29 16,57 16,92 16,18 16,55 16,80 16,30 16,55 16,56 NA 0,15 0,20 Soj Strain 5 17,07 16,36 16,72 16,82 16,80 16,81 16,85 16,23 16,54 16,69 G/A 0,31 0,41 c h r o o c o c c u m (A) Soj Strain 10 16,84 16,65 16,75 17,30 16,38 16,84 16,66 15,97 16,32 16,63 Soj Strain 14 16,85 16,17 16,51 16,90 16,27 16,59 16,89 15,85 16,37 16,49 Prosek Average 16,95 16,35 16,65 16,94 16,35 16,65 16,80 16,15 16,48 16,59 G/A/NA 0,54 0,71

Soj Strain 8 17,12 16,28 16,70 16,78 16,12 16,45 16,80 16,41 16,61 16,58 G/NA 0,22 0,29

B

C

Prosek A, B, C Average A, B, C

A/NA 0,38 0,51

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Tabela 3. Efekat inokulacije sa Azotobacter chroococcum na prinos polarizacionog seera (t ha-1) Table 3. Effect of inoculation of Azotobacter chroococcum on polarized sugar yield (t ha-1) Nacin primene Type of Application (NA) A Godina Year (G) 2008 2009 Prosek Average 2008 2009 Prosek Average 2008 2009 Prosek Average Kontrola Control 10,82 11,36 11,09 11,16 12,10 11,63 10,77 11,44 11,11 11,27 G LSD 0,05 0,01 CV (%) 0,21 0,28 5,54 A 0,37 0,49 C 0,26 0,34 Azotobacter Soj Strain 1 10,95 11,18 11,07 11,47 12,79 12,13 10,92 11,52 11,22 11,47 Soj Strain 5 11,07 11,56 11,32 11,20 13,23 12,22 11,14 11,13 11,14 11,56 G/A 0,52 0,69 Soj Strain 8 11,41 10,96 11,19 11,40 12,50 11,95 11,69 12,47 12,08 11,74 G/NA 0,37 0,49 c h r o o c o c c u m (A) Soj Strain 10 11,20 11,76 11,48 11,71 12,23 11,97 11,26 12,77 12,02 11,82 A/NA 0,64 0,84 Soj Strain 14 10,86 11,36 11,11 11,43 13,03 12,23 11,06 12,10 11,58 11,64 Prosek Average 11,10 11,36 11,23 11,44 12,76 12,10 11,21 12,00 11,61 11,65

B

C

Prosek A, B, C Average A, B, C

G/A/NA 0,90 1,19

su na kontrolu od 0,38 t ha-1. Pozitivan uticaj na prinos polarizacionog seera ispoljili su svi primenjeni sojevi, a poveanje je bilo od 0,20 t ha-1 kod soja 1 do 0,55 t ha-1 kod soja 10. Znacajno vei prinos polarizacionog seera u odnosu na kontrolu ostvaren je primenom sojeva 8, 10 i 14. Poveanje prinosa polarizacionog seera iznad 0,90 t ha-1 ostvareno je inokulacijom semena sojevima 8 i 10. Primenom azotobaktera preko zemljista neposredno pre setve, ostvaren je njegov najslabiji uticaj na prinos polarizacionog seera. Poveanje u odnosu na kontrolu iznosilo je svega 0,14 t ha-1. Znatno izrazeniji pozitivan uticaj na prinos polarizacionog seera ispoljen je inokulacijom semena azotobakterom i njegovom primenom preko zemljista pre prve meuredne kultivacije, u datim slucajevima prinos polarizacionog seera povean je za 0,50 t ha-1, odnosno za 0,47 t ha-1 (Tab. 3). Navedene razlike statisticki su znacajne. Primenom azotobaktera povean je prinos polarizacionog seera kod sve tri sorte u obe godine ispitivanja. Najvei procenat poveanja bio je kod sorte Sara 6,57% ili 0,66 t ha-1 (Geci i sar. 2007).

Prinos kristalnog seera Primenom azotobaktera ostvaren je prosecni prinos kristalnog seera od 10,01t ha-1. Poveanje u odnosu na kontrolu bilo je 0,30 t ha-1. Pozitivan uticaj na prinos kristalnog seera ispoljen je kod svih pet sojeva azotobaktera, a poveanje u odnosu na kontrolu bilo je 0,07 t ha-1 kod soja 5 do 0,50 t ha-1 kod soja 10. Znacajno vei prinos kristalnog seera od kontrole ostvaren je primenom sojeva 8, 10 i 14. Maksimalno poveanje prinosa kristalnog seera od 0,86 t ha-1 ostvareno je inokulacijom semena sa sojem 8. Tretiranjem zemljista azotobakterom neposredno pre setve osvaren je prosecni prinos kristalnog seera od 9,70 t ha-1, sto je poveanje u odnosu na kontrolu od 0,19 t ha-1. Mnogo izrazeniji pozitivni uticaj azotobaktera na prinos kristalnog seera ispoljen je inokulacijom semena i tretiranjem zemljista pred prvu meurednu kultivaciju. Poveanje je iznosilo 0,46 t ha-1 odnosno 0,35 t ha-1 (Tab. 4). Mrkovacki i sar. (2007) dobili su poveanje prinosa kristalnog seera od 6% tj. 600 kg ha-1 inkorporacijom azotobaktera u zemljiste. Na proizvodnim parcelama na podrucju Vojvodine, korisenjem NS-Betafixina, prinos kristalnog seera

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Tabela 4. Efekat inokulacije sa Azotobacter chroococcum na prinos kristalnog seera (t ha-1) Table 4. Effect of inoculation of Azotobacter chroococcum on crystal sugar yield (t ha-1) Nacin primene Type of Application (NA) A Godina Year (G) 2008 2009 Prosek Average 2008 2009 Prosek Average 2008 2009 Prosek Average Kontrola Control 9,53 9,49 9,51 9,86 10,33 10,10 9,54 9,53 9,54 9,71 G LSD 0,05 0,01 CV (%) 0,19 0,26 5,85 A 0,33 0,44 C 0,24 0,31 Azotobacter Soj Strain 1 9,62 9,40 9,51 10,14 10,78 10,46 9,64 9,77 9,71 9,89 G/A 0,47 0,63 Soj Strain 5 9,75 9,74 9,75 9,86 11,28 10,57 9,83 9,41 9,62 9,78 c h r o o c o c c u m (A) Soj Strain 10 9,87 10,03 9,95 10,37 10,44 10,41 9,89 10,64 10,27 10,21 A/NA 0,58 0,76 Soj Strain 14 9,55 9,63 9,59 10,05 11,07 10,56 9,75 10,22 9,99 10,04 Prosek Average 9,77 9,62 9,70 10,09 10,82 10,45 9,88 10,11 10,00 10,01

Soj Strain 8 10,07 9,28 9,68 10,02 10,51 10,27 10,30 10,49 10,40 10,11 G/NA

B

C

Prosek A, B, C Average A, B, C

G/A/NA 0,82 1,09

0,33 0,44

Tabela 5. Efekat inokulacije sa Azotobacter chroococcum na ukupan broj mikroorganizama u rizosferi seerne repe Table 5. Effect of inoculation of Azotobacter chroococcum on total number of microorganisms in sugar beet rhizosphere Nacin primene Type of Application (NA) A Godina Year (G) 2008 2009 Prosek Average 2008 2009 Prosek Average 2008 2009 Prosek Average Kontrola Control 214,17 198,62 206,40 179,04 133,58 156,31 205,49 182,53 194,01 185,57 Azotobacter Soj Strain 1 226,88 205,12 216,00 258,94 297,63 278,29 191,42 244,59 218,01 237,43 Soj Strain 5 249,25 230,37 239,81 168,70 214,07 191,39 246,58 205,44 226,01 219,07 c h r o o c o c c u m (A) Soj Strain 10 212,27 248,82 230,55 239,83 180,05 209,94 224,87 249,95 237,41 225,97 Soj Strain 14 233,68 234,00 233,84 226,47 208,28 217,38 235,05 268,60 251,83 234,35 Prosek Average 228,05 221,51 224,78 224,21 211,06 217,64 227,34 231,17 229,26 223,89

Soj Strain 8 218,16 189,22 203,69 227,13 155,25 191,19 238,79 187,25 213,02 202,63

B

C

Prosek A, B, C Average A, B, C

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Tabela 6. Efekat inokulacije sa Azotobacter chroococcum na broj azotobaktera u rizosferi seerne repe Table 6. Effect of inoculation of Azotobacter chroococcum on number of azotobacter in sugar beet rhizosphere Nacin A z o t o b a c t e r c h r o o c o c c u m (A) primene Godina Kontrola Soj Soj Soj Soj Soj Type of Year Control Strain Strain Strain Strain Strain Application (G) 1 5 8 10 14 (NA) 2008 22,03 33,75 31,32 40,70 41,12 28,97 2009 19,52 38,33 31,72 30,68 26,37 29,84 A Prosek 20,78 36,04 31,52 35,69 33,75 29,41 Average 2008 27,96 40,30 45,24 40,06 40,97 46,65 2009 25,78 34,80 30,41 39,33 26,47 40,05 B Prosek 26,87 37,55 37,83 39,70 33,72 43,35 Average 2008 22,29 34,24 47,16 37,55 28,52 55,17 2009 20,60 36,98 49,77 47,24 33,65 43,69 C Prosek 21,45 35,61 48,47 42,40 31,09 49,43 Average Prosek A, B, C 23,03 36,40 39,27 39,26 32,85 40,73 Average A, B, C povean je u 2007. za 680 kg ha-1, a u 2008. za 1.050 kg ha-1 (Mrkovacki i sar. 2009). Rodelas et al. (1999) su pokazali da azotobakter poveava prinos kod seerne repe, sargarepe i kupusa oko 10%. Ukupan broj mikroorganizama Ukupan broj mikroorganizama inokulacijom je bio vei od kontrole. Prosecan ukupan broj mikroorganizama bio je 223,89 sto je za 20,6% vei od kontrole. Pozitivan uticaj na ovo svojstvo pokazali su svi ispitivani sojevi, a poveanje u odnosu na kontrolu kretalo se od 9,1% kod soja 8 do 27,9% sa sojem 1. Aplikacija sojeva pred prvu meurednu kultivaciju (B) imala je najvei uticaj na poveanje ukupnog broja mikroorganizama, sa poveanjem u odnosu na kontrolu od 39,2%. Manji pozitivan efekat pokazalo je dodavanje inokuluma na seme (C) (18,16%) i inkorporacija (A) (8,9%) (Tab. 5). Nasa ranija istrazivanja (Mrkovacki i sar. 2006, Mrkovacki i sar. 2008) pokazala su poveanje ukupnog broja mikroorganizama u rizosferi seerne repe inokulisanih varijanti sa sojevima azotobaktera za 45,7% i od 35­118% u odnosu na kontrolu. Takoe, rezultati Mrkovacki i sar. (2009) sa proizvodnih parcela na podrucju Vojvodine, inokulisanih sa smesom sojeva NS Betafixin, pokazuju povean ukupni broj mikroorganizama za 57% u 2007. a za 63% u 2008. Broj azotobaktera

Prosek Average 35,16 31,39 33,28 42,64 34,21 38,43 40,52 42,27 41,40 37,70

Broj azotobaktera povean je inokulacijom sa svim ispitivanim sojevima. U proseku, nezavisno od nacina inokulacije, bio je 37,7 sto je za 63,7% vei od kontrole. Poveanje se kretalo od 76,8% sa sojem 14 do 42,6% sa sojem 10. Najvei pozitivan uticaj na broj azotobaktera ostvaren je na varijanti (C), tj. nanosenjem na seme od 93%, a zatim inkorporacijom (A) od 60,15% i sa varijantom (B) (Tab. 6). Slicne rezultate, tj. poveanje broja azotobaktera u rizosferi seerne repe inokulacijom sa sojevima Azotobacter chroococcum od 17% (Mrkovacki i sar. 2006) u odnosu na kontrolu, a od 34% u 2007. i 47% u 2008. dobijeno je na proizvodnim parcelama na podrucju Vojvodine (Mrkovacki i sar. 2009). Ebrahimi et al. (2007) zakljucuju da PGPR ukljucujui i azotobakter imaju prednost u poreenju sa mineralnim ubrivima cime opravdavaju svoju primenu. Zakljucci Na osnovu dvogodisnjih ispitivanja uticaja pet sojeva Azotobacter chroococcum na prinos i kvalitet seerne repe, kao i na ukupan broj mikroorganizama i azotobaktera u rizosferi, pri razlicitim nacinima aplikacije, mogu se izvesti sledei zakljucci: ·Azotobacter chroococcum je ispoljio veoma izrazen pozitivan uticaj na prinos korena seerne

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chroococcum. Zbornik radova Instituta za ratarstvo i povrtarstvo 45: 241-245 Mrkovacki N, Mezei S, Caci N, Kovacev L, Nagl N (2009): Efekat primene mikrobioloskog ubriva za seernu repu. Zbornik radova Instituta za ratarstvo i povrtarstvo 46: 175180 Rodales B, Gonzales-Lopez J, Pozo C, Salmeron V, MartinezToledo M V (1999): Response of faba been (Vicia faba L.) to combined inoculation with azotobacter and Rhizobium leguminosarum bv.Viciae.Al. Soil Ecol. 12: 51-59

repe, pozitivan uticaj na sadrzaj seera i veoma izrazen pozitivan uticaj na prinos seera. · Inokulacijom je povean ukupan broj mikroorganizama i broj azotobaktera u rizosferi seerne repe. Najvei efekat pokazali su sojevi 1 i 14 u poveanju ukupnog broja mikroorganizama, a sojevi 10 i 14 u broju azotobaktera. · Izmeu ispitivanih sojeva su utvrene razlike u delovanju na prinos i kvalitet seerne repe, a izrazenijim pozitivnim uticajem na ova svojstva izdvajaju se sojevi 8 i 10. · Bolji efekat na prinos i kvalitet seerne repe ispoljen je pri inokulaciji semena i pri tretiranju zemljista pred prvu meurednu kultivaciju. · Najbolji efekat u poveanju brojnosti mikroorganizama dobijen je na varijanti tretiranja zemljista pred prvu meurednu kultivaciju. · Najbolja preporuka za primenu Azotobacter chroococcum kod seerne repe je tretiranje zemljista pred prvu meurednu kultivaciju. Literatura

Chet I, Chernin L (2002): Biocontrol, microbial agents in soil. In: Bitton G (ed), Encyclopedia of Environmental Microbiology. John Willey and Sons Inc., New York, USA, 45-465 Ebrahimi S, H Iran Nejad, A H Shirani Rad, G Abbas Akbari, R Amiry, Modarres Sanavy S A M (2007): Effect of Azotobacter chroococcum Aplication on Quantity and Quality Forage of Rapeseed Cultivars. Pak. J. Biol. Sci. 10: 3126-3130 Caci N, Mrkovacki Nastasija, Mezei Snezana, Kovacev L (2003): Efekat primene Azotobacter chroococcum u seernoj repi. Zbornik radova Instituta za ratarstvo i povrtarstvo 38: 271-280 Sahin F, Cakmakci R, Kantar F (2004): Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria. Plant Soil 265: 123-129 Geci J, Mrkovacki N, Caci N (2007): Primena razlicitih tipova inokulacije seerne repe sa Azotobacter chroococcum. Letopis naucnih radova Poljoprivrednog fakulteta u Novom Sadu 1: 47-54 Glick B R, Patten C L, Holguin G, Penrose D M (1999): Biochemical and genetic mechanisms used by plant growthpromoting bacteria. Imperial College Press, London, UK Jarak M, uri S (2004): Praktikum iz mikrobiologije, Poljoprivredni fakultet, Novi Sad Mrkovacki N, Mili V (2001): Use of Azotobacter chroococcum as potentially usefull in agricultural application (Review). Ann. Microbiol. 51: 145-159 Mrkovacki N, Mezei S (2003): Primena sojeva Azotobacter chroococcum NS-Betafixina u gajenju seerne repe. Zbornik radova Instituta za ratarstvo i povrtarstvo 39: 49-58 Mrkovacki N, Mezei S, Caci N, Kovacev L (2006): Efekat inokulacije na biogenost rizosfere seerne repe. Letopis naucnih radova Poljoprivrednog fakulteta u Novom Sadu 1: 48-52 Mrkovacki N, Mezei S, Caci N, Kovacev L (2007): Efekat primene razlicitih tipova inokulacije seerne repe. Zbornik radova Instituta za ratarstvo i povrtarstvo 43: 201-207 Mrkovacki N, Mezei S, Caci N, Kovacev L, Nagl N (2008): Biogenost rizosfere seerne repe inokulisane sa Azotobacter

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Effect of Azotobacter Chroococcum Application Type on Microorganisms in the Rhizosphere and Sugar Beet Yield

Nastasija Mrkovacki1 · Nikola Caci1 · Janja Kuzevski2 · Lazar Kovacev1 · Snezana Mezei1 · Nevena Nagl1 · Dragana Bjeli1

1

Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia 2 Institute for Application of Science in Agriculture, Bulevar despota Stefana 68 b, 11000 Beograd, Srbija

Summary: Plant growth promoting rhizobacteria (PGPR) are a group of diverse soil bacteria which stimulate the growth of the host plant. It has been shown that Azotobacter chroococcum may be used as biofertilizer for increasing the yield and improving technological characteristics of sugar beet. The effect of three different ways of inoculation of sugarbeet with Azotobacter chroococcum on the production features of sugar beet and microbiological status of rhizosphere soil has been tested. One variety of sugar beet, Drena, was included in the tests in the region Rimski Sancevi, Novi Sad, during 2008 and 2009. Five strains of Azotobacter chroococcum (1, 5, 8, 10 and 14) were used as microbiological fertilizers. There were three variations of inoculation: (A) incorporation in the soil, (B) before the first cultivation, and (C) applying the liquid culture of strain on the seed before sowing. The highest increase in yield of sugar beet roots 6.25 t ha-1 and yield of white sugar 0.91 t ha-1 was achieved with strain 10 with variant of inoculation of soil before the first cultivation. In both years, a positive effect of Azotobacter chroococcum was observed on the total number of microorganisms in the rhizosphere. Key words: Azotobacter chroococcum, root yield, sugar beet, sugar content, total microbial number, yield

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www.nsseme.com/journal.html

Kokurentnost osnovnih ratarskih useva u Vojvodini

607 Ekonomska analiza / Economic Analysis Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 607-612 originalni naucni rad / original research article

Kokurentnost osnovnih ratarskih useva u Vojvodini

Danica Bosnjak · Vesna Rodi

primljeno / received: 16.03.2010. prihvaeno / accepted: 28.04.2010. © 2010 IFVC

Izvod: Ekonomski polozaj i unapreenje svake proizvodnje u najveoj meri su definisani ekonomskim efektom koji se u njoj postize. U 2009. visok nivo ulaganja s jedne i niska prodajna cena s druge strane uslovili su pozitivan finansijski rezultat (dobit) u poljoprivrednim preduzeima samo u proizvodnji seerne repe (922 ha-1) i soje (167 ha-1), dok je gubitak evidentiran u proizvodnji kukuruza (206 ha-1), psenice (121 ha-1) i suncokreta (162 ha-1). Na porodicnim gazdinstvima, proizvoacima sredstva za dalju reprodukciju obezbeuje nivo dohotka ostvaren u proizvodnji seerne repe (924 ha-1) i soje (594 ha-1), ali ne i kukuruza (105 ha-1), suncokreta (60 ha-1) i psenice (33 ha-1). Pored visine prinosa, presudni uticaj na ostvareni rezultat imaju uslovi realizacije. U tom pogledu skladistenje i kasnija realizacija, kao i ubiranje useva sa manjim sadrzajem vlage, mogu znacajno da poveaju vrednost proizvodnje, a samim tim i zaradu proizvoacu. Kljucne reci: dobit, dohodak, gubitak, osnovni ratarski usevi, troskovi, vrednost proizvodnje

Uvod Poljoprivredna proizvodnja u Vojvodini odvija se na preko 1,7 miliona hektara poljoprivrednog zemljista. Blizu 90% poljoprivrednih povrsina (1.557.000 ha) koristi se u vidu oranica, koje predstavljaju osnovni kapacitet i uslov za organizovanje ratarske proizvodnje. Ova oranicna povrsina se koristi za setvu useva razlicite namene. U periodu od 1999. do 2008. pet ratarskih useva (kukuruz, psenica, seerna repa, suncokret i soja) angazuju prosecno godisnje oko 82% oranica u Vojvodini, sto im daje epitet osnovnih ratarskih useva. Evidentno je da u pojedinim godinama dolazi do promena u pozetim povrsinama. Te promene su posebno izrazene kod soje, suncokreta i psenice, dok su kod kukuruza i seerne repe te oscilacije manje izrazene. Za razliku od soje i suncokreta, cije povrsine se u Vojvodini poveavaju, psenica ustupa mesto drugim usevima. Ranija ispitivanja (Bosnjak 1997, Bosnjak & Rodi 1999, Bosnjak i sar. 2003, Bosnjak & Rodi 2006) pokazala su da mesto pojedinih useva u strukturi setve odreuju pre svega ekonomski faktori. Mnogi autori, meu kojima Jovanovi i sar. (1997), Brodt et al. (2006) isticu da je za donosenje upravljackih odluka vazno poznavati ne

D. Bosnjak ) (· V. Rodi 1 Univerzitet u Novom Sadu, Poljoprivredni fakultet, Trg Dositeja Obradovia 8, 21000 Novi Sad, Srbija e-mail: [email protected]

samo proizvodna, ve i ekonomska obelezja proizvodnje, jer upravo od ekonomskog efekta koji se u nekoj proizvodnji postize u najveoj meri zavise polozaj i unapreenje proizvodnje. Uvazavajui navedene cinjenice, u radu je na bazi izvrsene ekonomske analize uslova i rezultata u proizvodnji utvrena meusobna konkurentnost osnovnih ratarskih useva u Vojvodini u 2009. godini. Materijal i metod rada Ekonomska analiza proizvodnje osnovnih ratarskih useva izvrsena je na osnovu obracunskih kalkulacija poljoprivrednih preduzea i evidencije o troskovima i vrednosti proizvodnje posmatranih porodicnih poljoprivrednih gazdinstava na podrucju Vojvodine u 2009. Znacajan obim informacija dobijen je metodom intervjua kao oblika usmenog prikupljanja podataka. Predmet analize su osnovni ratarski usevi u Vojvodini: kukuruz, psenica, seerna repa, suncokret i soja. Primenjena je prostorna analiza, koja se odnosi kako na pomenute useve, tako i na subjekte u organizovanju proizvodnje (poljoprivredna preduzea i porodicna gazdinstva).

Rad je deo istrazivanja na projektu: ,,Unapreenje i razvoj oplemenjivanja i tehnologije proizvodnje soje", broj TR20094, koji je finansiran od strane Ministarstva za nauku i tehnoloski razvoj Republike Srbije. Deo rezultata prikazanih u radu saopsten je na 44. Savetovanju agronoma Srbije, Zlatibor 2010, u okviru predavanja po pozivu pod naslovom ,,Ekonomska analiza proizvodnje osnovnih ratarskih useva"

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Primenom kalkulativnog metoda izvrsena je ekonomska analiza u cilju izracunavanja osnovnih pokazatelja ekonomske efektivnosti i efikasnosti proizvodnje. Primenjen je i komparativnoanaliticki metod, a rezultati istrazivanja prikazani su tabelarno. Rezultati i diskusija Prinosi osnovnih ratarskih useva Ostvareni prinosi svih osnovnih ratarskih useva u periodu od 1999. do 2008. imaju tendenciju porasta. Najintenzivniji porast imaju prinosi seerne repe (stopa 3,71%) i suncokreta (stopa 3,18%). Priblizno istim tempom se poveavaju prinosi kukuruza (stopa 2,55%) i psenice (stopa 2,75%) dok prinosi soje u posmatranom periodu beleze rast po nesto nizoj prosecnoj godisnjoj stopi od 2,08%. Prosecni nivo prinosa osnovnih ratarskih useva u Vojvodini moze se oceniti relativno zadovoljavajuim ako se uporedi sa prosecnim nivoom prinosa u svetu ili u Evropi (Tab. 1). Ova konstatacija proizilazi iz cinjenice da je prosecni nivo ostvarenih prinosa osnovnih ratarskih useva u Vojvodini, izuzev seerne repe, vei u odnosu na prosecno ostvarene u svetu.

sa prosecno ostvarenim na nivou EU ili pak sa prinosima pojedinih zemalja clanica EU, onda se mogu konstatovati znatne razlike pre svega u prinosima kukuruza, seerne repe i psenice. Naime, proizvoaci u Vojvodini prosecno proizvedu 47% manje kukuruza, 21% manje psenice i 22% manje seerne repe po jedinici povrsine u odnosu na prosek EU. Efektivnost i efikasnost proizvodnje osnovnih ratarskih useva Za potrebe upravljanja, kontrole i unapreenja proizvodnje potrebna je ekonomska analiza troskova i ostvarenih rezultata, nastalih kao rezultat ucinjenih ulaganja. Ekonomskom analizom se sagledava doprinos obima proizvodnje sa jedne strane i prodajnih cena sa druge strane, ostvarenoj vrednosti proizvodnje (efektivnosti) i utvruju razni merni odnosi, kako bi se ocenio kvalitet ucinjenih ulaganja (efikasnost). Rezultati proizvodnje osnovnih ratarskih useva u poljoprivrednim preduzeima U posmatranim poljoprivrednim preduzeima, kao rezultanta ostvarenih prosecnih prinosa

Tabela 1. Razlike u prinosima osnovnih ratarskih useva u Vojvodini i nekim zemljama (prosek 19982007) Table 1. Differences in basic field crops yields in Vojvodina and some other countries (1998-2007 average)

Drzava/Country Kukuruz Maize t ha-1 Razlika Differencee Psenica Wheat t ha-1 Razlika Difference Se. repa Sugar beet t ha-1 Razlika Difference Suncokret Sunflower seed t ha-1 Razlika Difference Soja Soybean t ha-1 Razlika Difference 2,29 1,79 2,07 2,32 2,40 2,41 +0,11 +0,61 +0,33 +0,18 -

Svet / World Evropa/Europe EU - 27 Srbija*/Serbia Vojvodina* Vojvodina**

4,67 5,49 7,37 4,45 5,02 5,95

+0,35 +0,47 -2,35 +0,57 -

2,82 3,44 4,62 3,44 3,82 3,88

+1,0 +0,38 -0,8 -0,38 -

43,87 42,38 49,53 40,23 40,75 45,62

-3,12 -1,63 -8,78 -0,52 -

1,26 1,22 1,71 1,90 1,89 2,43

+0.63 +0,67 +0,18 -0,01 -

* (1999-2008) Izvor: www.fao.org; http://webrzs.stat.gov.rs i obracun autora ** ( 2009) Izvor: Informacija Pokrajinskog statistickog zavoda od 30.12. 2009. U odnosu na evropski prosek, proizvoaci u Vojvodini ostvaruju nesto nize prinose kukuruza i seerne repe, ali se ta razlika ne moze oceniti znacajnom. Meutim, ako se uporede prosecno ostvareni prinosi ratarskih useva u Vojvodini

i aktuelnih prodajnih cena u momentu realizacije daleko najveu vrednost proizvodnje po jedinici povrsine obezbeuje seerna repa (194.616 RSD ha-1 ­ 2.070 ha-1), a najmanju psenica (45.885 RSD ha-1 ­ 488 ha-1) (Tab. 2).

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Tabela 2. Rezultati proizvodnje osnovnih ratarskih useva u poljoprivrednim preduzeima u 2009. Table 2. Results of basic filed crops production in agricultural enterprises in 2009 Elementi troskova Costs elements Kukuruz Maize Psenica Wheat ha-1 Suncokret Sunflower seed Seer.repa Sugar beet Soja Soybean 677 20,9 26 844 167 3,05

Ukupni troskovi Total costs Cena kostanja Costs of produc. (RSD kg-1) Prodajna cena Selling price (RSD kg-1) Vrednost proizvodnje Value of production Finansijski rezultat Finacial results Prinos Yield (t ha-1)

609 11,9 9,5 488 -121 4,83

843 9,2 7,0 637 -206 8,56

1.148 1,5 2,7 2.070 922 72,08

717 20,7 16,0 555 -162 3,26

* 1 = 94 RSD Uz nivo troskova dat u tabeli 2 (Bosnjak & Rodi 2010b) moze se konstatovati da je pozitivan finansijski rezultat (dobit) ostvaren u proizvodnji seerne repe (922 ha-1) i soje (167 ha-1), dok je u proizvodnji kukuruza (206 ha-1), psenice (121 ha-1) i suncokreta (162 ha-1) evidentiran gubitak Ako se ima u vidu da je finansijski rezultat utvren kao razlika vrednosti proizvodnje i ukupnih troskova, kao i da su ostvareni prinosi posmatranih gazdinstava iznad prosecno ostvarenih u Vojvodini, jasno je da je gubitak u proizvodnji posledica sa jedne strane niskih otkupnih cena, a sa druge visokih troskova proizvodnje (Tab. 2). Moze se rei da presudan uticaj ipak ima niska otkupna cena jer je i proizvodnja seerne repe i soje organizovana uz visoke troskove, ali je otkupna cena za ove useve formirana na nivou koji proizvoacima obezbeuje odreenu dobit, neophodnu za dalju reprodukciju. Pri navedenim prodajnim cenama i ucinjenim troskovima, granica rentabilnosti u proizvodnji psenice je na nivou od 6.026 kg ha-1, kukuruza 11.320 kg ha-1, suncokreta 4.212 kg ha-1, sto je vise od prosecno ostvarenih prinosa kako posmatranih proizvodnih jedinica, tako i prosecno ostvarenih prinosa ovog sektora u Vojvodini. Posledica je nerentabilnost proizvodnje kukuruza, psenice i suncokreta u 2009. Granica rentabilnosti u proizvodnji seerne repe na nivou od 39.967 kg ha-1 i soje od 2.448 kg ha-1 je za 32.113 kg ha-1, odnosno 602 kg ha-1

manja od prosecno ostvarenih prinosa, tako da su ove proizvodnje u posmatranim preduzeima bile rentabilne. Mora se konstatovati da je granica rentabilnosti i kod ovih proizvodnji dosta visoka i uslovljena pre svega visokim cenama inputa, meutim povoljniji odnos cena realizacije i cena inputa omoguio je proizvoacima povoljan finansijski rezultat i ekonomicnu proizvodnju ovih useva. Kolicina proizvoda koja ostaje nakon pokria troskova proizvodnje, u ovom slucaju u proizvodnji seerne repe oko 32.000 kg ha-1 i soje oko 600 kg ha-1 predstavlja za proizvodne jedinice takozvanu granicu sigurnosti koja ukazuje na ekonomski polozaj useva; to je ona vrednost koja ostaje za dalju reprodukciju. Sa poveanjem granice sigurnosti poboljsava se ekonomski polozaj useva i njegova konkurentnost. Rezultati proizvodnje osnovnih ratarskih useva na porodicnim gazdinstvima Analiza ostvarenih rezultata na porodicnim gazdinstvima u 2009. potvruje ranije konstatacije (Bosnjak & Rodi 2006) da je proizvodnja ratarskih useva kod ovih subjekata efektivnija i efikasnija, sto je posledica pre svega cinjenice da ova gazdinstva proizvodnju organizuju uz nize troskove, a u pogledu prosecno ostvarenih prinosa sve manje zaostaju za poljoprivrednim preduzeima (Jovanovi i sar. 2007).

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Ispitivana gazdinstva su u 2009. ostvarila najveu vrednost proizvodnje u proizvodnji seerne repe (162.996 RSD ha-1 ­ 1.734 ha-1). Slede soja (86.762 RSD ha-1 ­ 923 ha-1), kukuruz (57.810 RSD ha-1 ­ 615 ha-1), suncokret (47.564 RSD ha-1 ­ 506 ha-1) i psenica (34.552 RSD ha-1 ­ 368 ha-1) (Tab. 3). Razlike u ostvarenoj vrednosti proizvodnje, ali i razlike u troskovima, obezbeuju ovim proizvoacima razlicit dohodak po jedinici povrsine pojedinih useva. Ako se analiza ogranici na ona porodicna gazdinstva koja delimicno plaaju usluge (samo kombajniranje), a takvih je najvise u Vojvodini, ostvaren je dohodak od 924 ha-1 u proizvodnji seerne repe do svega 33 ha-1 u proizvodnji psenice (Tab. 3).

Otvarena vrednost dohotka ne moze se takva u celosti upotrebiti za dalju reprodukciju. Ovu vrednost treba umanjiti za iznos amortizacije, troskove sopstvenog rada, kamate i fiksne troskove. Ako se sve ovo ima u vidu, moze se rei da ostvareni nivo dohotka u 2009. obezbeuje proizvoacima zaradu samo u proizvodnji seerne repe i soje, dok je veina proizvoaca u proizvodnji psenice, kukuruza i suncokreta, zahvaljujui pre svega niskim otkupnim cenama, ostala bez zarade, pa samim tim i sredstava za dalja ulaganja. Ranija istrazivanja (Bosnjak i sar. 2003, Bosnjak & Rodi 2006, Pejanovi i sar. 2003) pokazuju da ekonomski rezultati proizvodnje na porodicnim gazdinstvima zavise od opremljenosti gazdinstva mehanizacijom (sa po-

Suncokret Sunflower seed

Seer.repa Sugar beet

Tabela 3. Rezultati proizvodnje osnovnih ratarskih useva na seljackim gazdinstvima u 2009. Table 3. Results of basic field crops production on family farms in 2009 Elementi troskova ha-1 Costs elements

Kukuruz Maize Psenica Wheat

Ukupni troskovi 335 510 810 446 329 Total costs Prinos 3.637 8.260 60.375 2.973 3.337 Yeald (kg ha-1) Prodajna cena bez premije (RSD kg-1) 9,5 7,0 2,7 16,0 26 Selling price without subsidies Vrednost proizvodnje 368 615 1 734 506 923 Value of production Finansijski rezultat 33 105 924 60 594 Financial result * 1 = 94 RSD ** U obracun troskova nije ukljuceno osiguranje jer mali broj proizvoaca osigurava ratarske useve zbog niske profitabilnosti. Zarada iz koje proizvoac treba da pokrije socijalne i penzijske doprinose je mala, tako da mnogi proizvoaci ne plaaju doprinose fondu a samim tim, prema vazeim propisima ostaju bez subvencija. / The calculation does not include costs of insurance since a small number of producers ensures agricultural crop, due to low profitability. Earnings from which the manufacturer should cover the social and pension contributions are small; consequently many manufacturers do not pay contributions to the fund and therefore, according to the current regulations, remain without subsidies. Ostvarivanje subvenicija u iznosu od 12.000 d ha-1 (128 ha-1) doprinelo je poveanju ukupnog prihoda i dohotka kod onih gazdinstava koja su imala pravo na ovu vrstu drzavne pomoi, pri cemu treba znati da je uslov za dobijanje subvencija bilo izmirenje obaveza prema Fondu za penzijsko i invalidsko osiguranje za prethodnu godinu. rastom nivoa mehanizovanosti raste ostvarni dohodak). Kod istog nivoa opremljenosti mehanizacijom dohodak proizvoaca zavisi od nivoa intenzivnosti, merenog u prvom redu postignutim nivoom prinosa. Krajnji rezultat, meutim, uvek je u velikoj meri determinisan uslovima pod kojima je izvrsena realizacija gotovog proizvoda. Misli se pre svega na vreme

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Soja Soybean

Kokurentnost osnovnih ratarskih useva u Vojvodini

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realizacije i ostvarene prodajne cene. Takva kretanja su uslovila da se u 2009. skladistenje i cekanje povoljnije cene u proizvodnji soje (Bosnjak & Rodi 2010a), a i drugih useva pokazalo izuzetno opravdanim.

Odluka o vremenu zetve posebno je znacajna za useve koji se ubiru kasno u jesen. U tom pogledu istice se kukuruz, kod koga niza vlaznost zrna u berbi obezbeuje znatno vee ekonomske efekte pri realizaciji, pri istoj prodajnoj ceni (Tab. 4).

Tabela 4. Uticaj vlaznosti zrna u berbi na ekonomske efekte u proizvodnji kukuruza (po 1 ha) Table 4. The impact of the grain humidity at harvest on economic effects in maize production (per hectare) Vrednost Ulazna vlaga * Predata kolicina zrna (kg) Neto prinos Prodajna cena realizacije Humidity level Delivered grain quantity Neto yield Selling price Realization (RSD kg-1) value (RSD) < 14,5 14,6 - 15,0 15,1 ­ 15,5 : 18,1 ­ 18,5 : 8.260 8.260 8.260 : 8.260 : 8.260 7.513 7.394 : 6.722 : 7 7 7 : 7 : 57.820 52.591 51.758 : 47.054 :

* Obracun izvrsen prema podacima preuzetim od jedne zemljoradnicke zadruge, iz tablica o uslovima prijema, susenja i razmene merkantilnog kukuruza roda 2009/ The calculation has been done considering data taken from one cooperative about the condition of maize storage and drying in 2009 Meutim, nisu svi proizvoaci podjednako spremni i opremljeni da skladiste gotove proizvode. Samo oni proizvoaci koji imaju sredstva za dalju reprodukciju i/ili izgraene skladisne kapacitete, a takvih je kod nas, na zalost malo, mogu da odlazu realizaciju i tako poveaju dohodak. Svi ostali su prisiljeni da proizvode realizuju neposredno nakon zetve kada su cene po pravilu najnize. S obzirom na cinjenicu da je nizi nivo intenzivnosti najcese posledica upravo nedostatka sredstava, desava se da pojedini proizvoaci u istoj godini ostvare i nekoliko puta vei dohodak zbog intenzivnije proizvodnje i mogunosti skladistenja, odnosno kasnije realizacije. Pored cene, koja je najznacajniji uslov realizacije, proizvoaci pri planiranju proizvodnje, razmatrajui ekonomsku isplativost useva, dosta paznje posveuju izboru sorte i hibrida. Pored visoke rodnosti, kao tradicionalnog faktora od uticaja na odluke, intenzitet otpustanja vlage u zrenju i vlaznost zrna pri zetvi ili berbi osobine su kojima se poslednjih godina pridaje sve vea paznja. Razlozi za to su pre svega ekonomske prirode jer ako se pri ubiranju useva ulazi sa manjom vlagom, zrno je lakse i jeftinije dosusiti. Stoga se odluka proizvoaca o izboru sorte ili hibrida i momentu kombajniranja useva (tj. pri kojoj vlaznost e se ubirati usev) direktno odrazava na ekonomske efekte. Ako se pretpostavi da proizvoaci predaju istu kolicinu zrna kukuruza sa razlicitim sadrzajem vlage onda npr. vlaznost zrna vea za 1% u odnosu na propisani uzans-kvalitet, pri istoj prodajnoj ceni, smanjuje vrednost realizacije za nesto vise od 10%. Vlaznost vea za 4% smanjuje vrednost realizacije za cak 19%, sto je svakako dobar razlog da se pri izboru hibrida vodi racuna o sadrzaju i intenzitetu otpustanja vlage u periodu berbe. Ovo je ujedno i dobar signal za selekcionere, koji menjaju pravce u selekciji, imajui u vidu da se sve vei broj proizvoaca opredeljuje za berbu u zrnu, a ne u klipu. Stoga, prethodno konstatovane granice rentabilnosti treba prihvatiti sa odreenom rezervom, jer u uslovima znacajnog variranja cena, sto je ispoljeno u 2009. treba biti veoma oprezan u njenom definisanju. Analiza troskova i efekata u proizvodnji osnovnih ratarskih useva pokazuje da iako postoje relativno povoljni uslovi za poljoprivrednu proizvodnju (kvalitetno zemljiste, povoljni klimatski uslovi, hibridi i sorte znacajnih genetskih mogunosti), potrebno je stvoriti uslove u kojima e poljoprivreda biti i ekonomski isplativa. U prilog tome govore i iskustva drugih razvijenih zemalja (Diamond 2007, Landes 2003, Tracy 1996) cit. po Bogunoviu (2009), koje nisu ekonomski napredovale dok nisu resile pitanja svoje poljoprivrede i snabdevanje hranom.

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Efikasna agrarna politika treba da ponudi proizvoacima pre svega takvu bazu podataka koja e smanjiti neizvesnost u pogledu osnovnih ekonomskih uslova jer je poljoprivreda, a posebno biljna proizvodnja, sama po sebi u velikoj meri neizvesna i rizicna. Na samim je proizvoacima da sagledaju drustveno-ekonomske uslove u kojima posluju, iznau mogunosti za njihovo korisenje i izvrse maksimalnu racionalizaciju troskova proizvodnje. Zakljucak Proizvodnju osnovnih ratarskih useva u 2009. karakterise visok nivo ulaganja po jedinici povrsine, koja su uslovljena visokim cenama inputa, a ne poveanjem upotrebljenih kolicina faktora. U proizvodnji seerne repe i soje u 2009. ostvaren je pozitivan finansijski rezultat kod svih proizvoaca. U proizvodnji kukuruza, suncokreta i psenice u poljoprivrednim preduzeima je ostvaren gubitak, a dohodak ostvaren na porodicnim gazdinstvima ne obezbeuje proizvoacima sredstva za dalju reprodukciju. Razloge za ovakvo stanje treba traziti pre svega u visokim cenama inputa sa jedne strane i niskoj prodajnoj ceni u momentu zetve s druge strane. Nestabilan drustveno-ekonomski ambijent nepovoljno se odrazava na primarnu poljoprivrednu proizvodnju. U rukama je samih proizvoaca da maksimalno iskoriste sve mogunosti, kako bi proizvodnju ucinili efikasnijom. U tom pogledu skladistenje i kasnija realizacija, kao i ubiranje useva sa manjim sadrzajem vlage mogu znacajno da poveaju vrednost proizvodnje, pa samim tim i zaradu proizvoacu.

Literatura

Bogunovi M (2009): Profitni model hrvatske poljoprivrede: Kako i gde ga primeniti? Ekonomija/Economics 16: 75-110 (www.rifin.com) Bosnjak D (1997): Organizaciono-ekonomska obelezja ratarske proizvodnje u Srbiji, Agroekonomika 26: 315-326 Bosnjak D, Rodi V (1999): Obelezja ratarske proizvodnje u Vojvodini i mogunosti njenog prestruktuiranja. Agroekonomika 28: 93-105 Bosnjak D, Tica N, Rodi V (2003): Ekonomska obelezja proizvodnje kukuruza, Zbornik radova Naucnog instituta za ratarstvo i povrtarstvo Novi Sad, 38: 121-130 Bosnjak D, Rodi V (2006): Ekonomska obelezja proizvodnje soje, Zbornik radova Naucnog instituta za ratarstvo i povrtarstvo Novi Sad, 42: 117-127 Bosnjak D, Rodi V (2010a): Ekonomska analiza proizvodnje soje, Zbornik radova Naucnog instituta za ratarstvo i povrtarstvo Novi Sad, 47: 193-202 Bosnjak D, Rodi V (2010b): Komparativna analiza troskova proizvodnje osnovnih ratarskih useva u Vojvodini, Ekonomika poljoprivrede (u stampi) Brodt S, Klonsky K, Tourte L (2006): Farmer goals and management styles: Implications for advancing biologically based agriculture. Agric. Syst. 89: 90-105 Jovanovi M, Bosnjak D (2001): Organizaciono-ekonomska obelezja osnovnih ratarskih useva. Agroekonomika 30:120131 Jovanovi M, Vukoje V, Maleti D (2007): Uporedna analiza proizvodnih rezultata ratarske proizvodnje u Vojvodini. Agroekonomika 36: 40-50 Pejanovi R, Bosnjak D, Rodi V, Tica N (2003): Ekonomska analiza proizvodnje psenice, Zito-hleb 6: 225-233 Food and Agricultural Organisation of the United Nations: The FAO on-line Statistical Database [Elektronski izvor]. dostupno na adresi http://faostat.fao.org/site/339/default. aspx (citirano 12.11.2009, verifikovano 22.01.2010). FAO, Rome Republika Srbija, Republicki zavod za statistiku, Baza podataka statistike poljoprivrede [Elektronski izvor]. dostupno http://webrzs.stat.gov.rs/axd/poljoprivreda/index. na php?ind=1 (citirano 15.11.2009, verifikovano 22.01.2010). Republicki zavod za statistiku, Beograd

Competitiveness of Basic Field Crops in Vojvodina

Danica Bosnjak · Vesna Rodi

University of Novi Sad, Faculty of Agriculture, Trg Dositeja Obradovia 8, 21000 Novi Sad, Serbia Summary: The economic position and improvement of any kind of production is largely defined by the economic effect that it achieves. In agricultural enterprises in 2009 the high costs of production on the one hand and low commodity prices on the other have assured profit only in sugar beet ( 922 per hectare) and soybean production ( 167 per hectare), while loss has been recorded in the production of maize ( 206 per hectare), wheat ( 121 per hectare) and sunflower ( 162 per hectare). On family farms, the income achieved in the production of sugar beet ( 924 per hectare) and soybean ( 594 per hectare) provides the funds for further reproduction, but in maize ( 105 per hectare), sunflower ( 60 per hectare) and wheat production ( 33 per hectare) it does not. In addition to the yield level, the conditions under which products have been sold have the decisive influence on the achieved results. In this regard, storage and deferred sale, as well as the collection of crops with lower moisture content, can significantly increase the value of production, and consequently the producer's revenue. Key words: basic field crops, costs, income, loss, profit, total revenue

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Big Bang in the Early Iron Age

613 Archaeobotany / Arheobotanika Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 613-616 original research article / originalni naucni rad

Charred Crop Storages and Agriculture of the Iron Age Settlement Gradina upon Bosut in Syrmia

Aleksandar Medovi Aleksandar Miki

received / primljeno: 08.04.2010. preraeno / revised: 26.04.2010. accepted / prihvaeno: 03.05.2010. © 2010 IFVC

The "Big Bang" in the Early Iron Age

Summary: The Early Iron Age granaries of Tell Gradina upon Bosut exploded in a fire inferno in the 8th century B.C. The result of this catastrophe is 2-5 cm thick layer with mixed carbonized seeds and fruits. Recently, eight samples were taken from Gradina's profile for archaeobotanical analysis. The goal was to obtain basic information on land use and on major crops and weeds of that period. The most abundant were cereals, followed by millets, pulses and oil/fibre plants. The dominant cereals were einkorn (Triticum monococcum) and hulled barley (Hordeum vulgare vulgare). Broomcorn millet (Panicum miliaceum) was also very important. Pulses were represented with six and oil/fibre plants with three species. Among weeds and ruderals, most common are rye brome (Bromus secalinus), fat hen (Chenopodium album), darnel ryegrass (Lolium temulentum), hairy crabgrass (Digitaria sanguinalis) and corncockle (Agrostemma githago). Key words: archaeobotany, burnt layer, Early Iron Age, sampling, Syrmia, Tell Gradina upon Bosut

Introduction Catastrophes in which whole settlements were burnt down and entire crop reserves exploded were quite common in the past. Archaeobotanical sampling of carbonized plant remains through the entire burnt layer of an archaeological site differ from usual archaeobotanical work. It requires less work onsite and disproportionally more work offsite. Therefore, the answer to "when is enough?" can be very helpful. If our goal is to reveal major cultivated plants and weeds, then we need to analyze 10-20 samples per burnt layer. Tell Gradina upon Bosut with its up to 4 m thick Early Iron Age layers in Syrmia (Serbia) is from an archaeobotanical point of view one big lost chance. Twenty years after excavations had ended, we were able to collect only eight samples from the eldest building horizon at site's profile. Could we "correct the past"? Is there enough material to say which crops were cultivated and which were the major weeds? Plant seeds and fruits have different chances of getting carbonized in standard archaeological contexts. The term "carbonization" is widely used in archaeology and archaeobotany to

A. Medovi ) ( Museum of Vojvodina, Dunavska 35, 21000 Novi Sad, Serbia e-mail: [email protected] A. Miki Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia

describe the most common mode of survival of archaeological plant remains. Carbonization occurs upon exposure to high temperatures due to fire. Such heating, under a limited supply of oxygen, converts the plant's organic compounds into charcoal. Since charcoal is not affected by bacteria, fungi, or other decomposing organisms, the carbonized plant remains survive in most environments for centuries. Because of everyday activities and practices in archaeological settlements, some plants are more likely to get in contact with fire than others, e.g. some of them need to be roasted or cooked before eating. Consequently, they have more chances of accidental carbonizing. Differences in this matter exist even within one plant-group. Glume cereals need to be dehusked before consumed. Dehusking is aided if grains are dry, and this is sometimes affected by exposure to fire (parching). Free-threshing cereals are mostly underrepresented in the usual archaeological record because parching is not required for separating grains from their husks. But, if whole settlements catch fire, all seeds and fruits obtain equal opportunity to get in contact with it. The "only" things that could affect their carbonization are differences in their chemical composition, consistency and texture (thickness of the seed coat, moisture content), their amount and size, temperature height, etc. It is needless to mention that after successful carbonization, seeds and fruits should be recovered during archaeological excavations

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in the way which can provide realistic picture of either human diet in ancient times, or reveal past subsistence strategies, or make contribution to the history of plant domestication, or just to give an overview of main crops and their weeds in a specific moment of time in one settlement. Materials and Methods Is it possible for a settlement to "explode"? Amount of cereal-grains (barley, wheat, rye, triticale) that can produce heat energy equivalent to 1 litre of fuel oil is 2.1-1.8 kg. But, one needs only 1.4-1.3 kg of oil plant seeds to achieve the same effect. Therefore, archaeological settlements could be seen as time bombs. A retired archaeology professor from Berlin is an eyewitness of Dresden bombardment from 1945. He still remembers the explosions and "lightshow" coming from the faraway grain silos hours after air raid. Similar examples happen today as well. For instance, a silo in Secanj, near Zrenjanin, full of sunflower seed that was inadequately dried and managed, exploded in December 2003, with devastating effects since

the heat potential of sunflower oil is equal to that of petrol (Radmilo 2005). Gradina upon Bosut is a multi-period Tell in Syrmia, Serbia. Its Early Iron Age layers are up to 4 m thick. The eldest building horizon, Bosut IV a (Kalakaca Culture, ca. 850-750 B.C.) ended in a big fire inferno with exploding grain storages. Witness of this catastrophe is a 2-5 cm thick layer of charred seeds and fruits, which is still visible in Gradina's profile. Twenty years after archaeological research, only eight samples could be recovered from the layer for archaeobotanical analysis. The samples (350 ml soil each) cover 24 m range in more or less equal intervals. Is there enough material to say which crops were cultivated and which were the major weeds? After Medovi (2004) one needs ten to twenty samples for this to find out. If we also want to record rare weeds, we need up to 50 samples. Results and Discussion A total of 63 plant taxa could be identified from 24.260 recovered charred plant items (Medovi, in press). On average, one sample contained 7.88

Graph 1. Horizontal mapping of the weight of cereals, millets and weeds in the burnt Early Iron Age layer of Gradina upon Bosut Grafikon 1. Horizontalna distribucija koncentracija zitarica, prosa i korovsko-ruderalnih biljaka u ugljenisanom sloju starijeg gvozdenog doba sa Gradine na Bosutu

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different plant taxa. Among seed remains, the most abundant were crops. Eight different cereals, two millets, three oil / (fibre) plants and even six different pulses were identified. Only three cereals (einkorn, barley and broomcorn millet) exceeded the number of one thousand records. Among pulses, the most dominant was lentil (Lens culinaris Medik.), followed by cultivated grass pea (Lathyrus sativus L.) and bitter vetch (Vicia ervilia (L.) Willd.). The weeds and synanthropic vegetation, including ruderals, were represented by 35 taxa. Most common were rye brome, fat hen, darnel ryegrass, hairy crabgrass and corncockle. The amount of weeds was rather small and varied from 0.42% to 4.21% (average 2.16%). Although the percentages by weight of oil/fibre plant remains was 0,01%, their frequencies (found in 7 of 8 samples) indicate much higher significance of gold-ofpleasure, opium poppy (Papaver somniferum L.) and flax (Linum usitatissimum L.) in diet/"manufacture" of Iron Age inhabitants of Gradina. Lately, much effort has been made in understanding processes of carbonization (Braadbaart et al. 2004, Braadbaart 2008, Guarino & Sciarrillo 2004, Märkle & Rösch 2008. Some results of those experiments were

striking: cereal grains show a lower resistance to high temperatures, whereas pulses become completely carbonized at higher temperatures. While reducing conditions usually enlarge the temperature range at which seeds carbonize without getting destroyed, broomcorn millet (Panicum miliaceum) behaves exactly the opposite way. The chances of carbonization for Linum usitatissimum are quite good. And some results were expected: Papaver somniferum has only very little chance of becoming carbonized at all, because the temperature range at which this happens is very small. Could this information be somehow applied in the case of Gradina upon Bosut? There are too many unknown variables and too much chaos caused by explosion for this information to be used. Nevertheless, a horizontal mapping of the weight of crops reveal that pulses concentrate to the right side and that the cereals have their peak in the middle of the investigated layer Weight proportions of crop assemblages found in the material are 85.41% (cereals) : 13.94% (millets) : 0.65% (pulses) : 0% (oil/fibre plants). The ranking of crops changes only for millets and cereals if we compare the number of

Graph 2. Horizontal mapping of the weight of pulses in the burnt Early Iron Age layer of Gradina upon Bosut Grafikon 2. Horizontalna distribucija koncentracija variva u ugljenisanom sloju starijeg gvozdenog doba sa Gradine na Bosutu

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recorded items 46.55% (cereals) : 52.75 (millets) : 0.61 (pulses) : 0.09 (oil/fibre plants). Are these proportions in accordance with real conditions before the moment the catastrophe occurred? We cannot know. But, according to results of archaeobotanical analysis of remarkably preserved human faeces from the Iron Age salt mine on the Dürrnberg Mountain near Hallein/Austria (Boenke 2005), our proportions fit almost perfectly. The miners' diet was dominated by cultivated plants and based mainly on cereals. Pulses and oil plants were far less important. These results may change our idea that pulses and oil plants were underrepresented in archaeobotanical record. Conclusions With only eight archaeobotanical samples from one burnt layer of Tell Gradina upon Bosut, valuable information was obtained on land cultivation in Early Iron Age in Syrmia: Inhabitants cultivated several cereals, millets and even six pulse crops. They also grew two oil plants and one oil/fibre plant. Major weeds in the assemblages were determined, and concentration of crops in the layer was also tracked. These results confirm the statement that one needs only ten samples from burnt layer of a settlement to gain basic information on its land use.

References

Boenke N (2005): Ernährung in der Eisenzeit ­ Ein Blick über den Tellerrand. In: Karl R, Leskovar J (eds.) Interpretierte Eisenzeiten. Fallstudien, Methoden, Theorie Tagungsbeiträge der 1. Linzer Gespräche zur interpretativen Eisenzeitarchäologie. Studien zur Kulturgeschichte von Oberösterreich 18. Oberösterreichisches Landesmuseum, Linz, 241-256 Braadbaart F (2008): Carbonisation and morphological changes in modern dehusked and husked Triticum dicoccum and Triticum aestivum grains. Veg. Hist. Archaeobot. 17: 155166 Braadbaart F, Boon J J, Veld H, David P, van Bergen P F (2004): Laboratory simulations of the transformation of peas as a result of heat treatment: changes of the physical and chemical properties. J. Archaeol. Sci. 31: 821-833 Guarino C, Sciarrillo R (2004): Carbonized seeds in a protohistoric house: results of hearth and house experiments. Veg. Hist. Archaeobot. 13: 65-70 Märkle T, Rösch M (2008): Experiments on the effects of carbonization on some cultivated plant seeds. Veg. Hist. Archaeobot. 17: 257-263 Medovi A (2004): Zum Ackerbau in der Lausitz vor 1000 Jahren. Der Massenfund verkohlten Getreides aus dem slawischen Burgwall unter dem Hof des Barockschlosses von Groß Lübbenau, Kreis Oberspreewald-Lausitz. In: Haffner A, Müller-Wille M (eds.), Starigard/Oldenburg. Hauptburg der Slawen in Wagrien 5. Naturwissenschaftliche Beiträge. Offa-Bücher NF 82. Wachholtz Verlag, Neumünster, 185­ 236 Medovi A (in press): Biljna privreda Gradine na Bosutu (ili Savi?) u starijem gvozdenom dobu. In: Medovi P, Medovi I (eds) Gradina na Bosutu. Naselje starijeg gvozdenog doba Radmilo M (2005): Fire extinction experience with grain dryer. J. Process. Energy Agric. 9: 65-66

Eksplodirana skladista ratarskih useva i poljoprivreda starijeg gvozdenog doba u naselju Gradina na Bosutu u Sremu Aleksandar Medovi1 Aleksandar Miki2

Muzej Vojvodine, Dunavska 35, 21000 Novi Sad, Srbija 2 Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, 21000 Novi Sad, Srbija

1

,,Veliki prasak" u starijem gvozdenom dobu

Izvod: Na utvrenom preistorijskom naselju Gradina na Bosutu iz sloja sa ugljenisanim ,,zitaricama" starijeg gvozdenog doba uzeto je osam uzoraka za arheobotanicku analizu. Sloj debljine 2­5 cm nastao je kao posledica velikog pozara i eksplozije skladista ratarskih useva. Analizom je ustanovljeno da se u momentu eksplozije u naselju cuvalo nekoliko vrsta zitarica, mahunarki i uljarica. Od zitarica najzastupljenije su bile jednozrna psenica (Triticum monococcum), jecam (Hordeum vulgare vulgare) i proso (Panicum miliaceum). Od sest identifikovanih vrsta variva najzastupljenjie je socivo (Lens culinaris), dok inventar uljarica cine obicni lanik (Camelina sativa), mak (Papaver somniferum) i lan (Linum usitatissimum). Sem kulturnih biljaka izdvojeno je 35 korovsko-ruderalnih vrsta od kojih su najzastupljenije Bromus secalinus, Chenopodium album, Lolium temulentum, Digitaria sanguinalis i Agrostemma githago. Kljucne reci: arheobotanika, Gradina na Bosutu, sloj sa ugljenisanim ,,zitaricama", starije gvozdeno doba, Srem

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Genetic Resources of Grain Legumes in Albania

617 Genetic Resources / Geneticki resursi Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 617-622 Research Note / Prethodno saopstenje

Review of Genetic Resources of Grain Legumes in Albania

Sokrat Jani Agim Canko

primljeno / received: 22.03.2010. prihvaeno / accepted: 03.05.2010. © 2010 IFVC

Summary: Activities regarding grain legumes genetic resources in Albania started in 1963, at first collecting, conservation, evaluation and utilization of landraces and old cultivars, later importing cultivars from European countries. Initially, all the work was done by the Agricultural Research Institute (ARI) of Lushnja, funded by the Ministry of Agriculture, with a clear plan to select and produce the commercial seeds for daily needs of agricultural cooperatives. About 180 accessions of common beans (Phaseolus vulgaris L.) were collected until the end of the 1980s, 53 of which were landraces and old cultivars. During 1990-2000 another project was accomplished, where several collecting missions took place, especially in the central and south-eastern regions of Albania. In 1998 the National Gene Bank (NGB) of Tirana was established to conserve the Albanian plant genetic resources. Key words: accession, Cicer arietinum, grain legumes, landrace, Lens esculenta, old cultivar, Phaseolus vulgaris, Phaseolus coccineus

Introduction Pulses have been cultivated in Albania for a long time, mainly for human consumption. Among the major food legumes, common bean (Phaseolus vulgaris L.) is the most important in Albania. Landraces are very diverse populations. They have particular properties or characteristics such as good adaptation to local climatic conditions, resistance or tolerance to diseases and pests which allow them to have high yield stability under a low input farming. For this reason, the landraces must be conserved for future generations. Genetic erosion is widely present in Albania. A number of landraces have been lost due to former almost semi-centennial system of the concentrated economy and use of modern cultivars developed by our institution or introduced from foreign countries. Another factor of erosion in the previous years is emigration of the population from rural zones to large cities. These factors of the purest genetic erosion have affected all the crops. Farmers are the main contributors and curators of agro-biodiversity, with landraces

S. Jani ) ( Agricultural Technologies Transfer Center (ATTC) of Lushnja, Albania e-mail: [email protected] A. Canko Agricultural Technologies Transfer Center (ATTC) of Fushe-Kruje, Albania

in most countries being one of the most threatened components of biodiversity. Unless an appropriate action is taken, the trend is likely to continue or even worsen, in spite the fact that landraces constitute an insurance guarantee for food security and our future ability to deal with climate change, users' needs and meeting local consumers demands. Genetic erosion factors in Albania are already very active. Nevertheless, a wide variation of environmental factors makes possible safeguard of genetic variation of species in levels, by interfering with particular programs, with objective not only for collection of some species' germplasm, whatever they may be, but also for becoming aware of the real situation, identifying the problems and putting forth proposals and solutions in order to reduce the danger of genetic erosion and protecting important species from their irrevocable extinction. This report analyzes the recent status of the main Albanian collection of the following grain legumes: common beans, runner bean, big white bean or Spain bean (Phaseolus coccineus L.), lentil (Lens esculenta Gross el Germ.), and chickpea (Cicer arietinum L.). Economic Importance of Grain Legumes Grain legumes are important staple in many areas of the world, especially common bean (Phaseolus vulgaris L.) used in human diet, which has

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Table 1. Cultivated area of grain legumes in Albania (ha) Table 1. Povrsina pod zrnenim mahunarkama u Albaniji Years Species Godina Vrsta 2000 Phaseolus vulgaris 22,500 Phaseolus coccineus 300 Cicer arietinum 70 Lens esculenta 25 Total 22,900 Ukupno

2004 17,200 400 80 20 17,700

2006 15,100 200 85 15 15,400

2008 14,300 200 85 15 14,600

Table 2. Cultivated area (ha) and production (t) of common beans in Albania Tabela 2. Povrsina (ha) i proizvodnja (t) obicnog pasulja u Albaniji Years Godina 2000 2004 2006 Area 22,500 17,200 15,100 Povrsina Production 25,200 22,400 24,300 Proizvodnja high protein content and is rich in phosphorus, iron, vitamin B1 and fiber, while containing no cholesterol. In Albania 4.71% of area is planted with grain legumes, 97.12% of which belong to four species given in this report (Tab. 1 and 2). Common bean is the prime most important crop in terms of planted area and production. In Albania, legumes produce is used for the home market, but the possibilities are to produce much more and for exporting. Most cultivated varieties are advanced cultivars, due to their higher yields and resistance to more races of Colletotrichum lindemuthianum (Sacc. et Magn) Br. Et Cav., such varieties are cv. Shijaku, Kallmeti, etc. Major producers are located in the lowland of west and southwest of Albania. The purpose of the work at this time is to maintain and make available to the public high quality seeds of grain legumes varieties that are produced, conditioned, and distributed as to insure proper identity and genetic purity. This process of maintaining genetic purity is performed through a nationally recognized seed certification system. Some seed quality factors, like seed-born diseases, are as important as genetic purity in the case of common beans. Common bean producers that have planted small areas have used local varieties and populations, mostly for self-consumption in old people's households. Most of these native landraces and old local cultivars are distinguished

2008 14,300 21,800

for some special characteristics, such as protein content, good taste, color, high tolerance or resistance to diseases and pests, etc. Lentil, chickpea, and runner bean, among grain legumes species, are less interesting for production, but are always present at market. The major pests of grain legumes in Albania are aphids (Aphis spp.), bruchid (Bruchus pisorum), and less spider mite (Tetranychus spp). The widespread fungal and bacterial diseases are respectively root rot (Fusarium ssp (Burch) Sn. et Hn), anthracnose (Colletotrichum lindemuthianum(Sacc. et Magn) Br. Et Cav), and halo blight (Pseudomonas phaseolicola (Burk.) Young et al). Status of the National Collections The active preservation of genetic variability of local varieties (old varieties and landraces) plays an important role in crop improvement and food production. A clear understanding of genetic resources is an important key for their practical conservation and utilization. The conservation work concerning legume crops from the grain legumes group was conducted in the ex-Agricultural Research Institute (ARI) in Lushnja, now called Agriculture Technologies Transfer Center (ATTC) of Lushnja. Albania has a small collection of legumes, 133 of which are landraces grain legumes accessions stored and conserved in NGB-Tirana and ATTCLushnja.

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Table 3. Number of accessions per grain legumes species stored in the ATTC-Lushnja Tabela 3. Broj uzoraka zrnenih mahunarki po vrstama u ATTC u Lusnji No. of accessions Broj uzoraka Species Vrsta Phaseolus vulgaris Lens esculenta Cicer arietinum Phaseolus coccineus Total Ukupno 430 Total Ukupno A B C 95 88 220 5 11 4 6 1 105 105 220 Albanian Albanski B 15 15 Foreign Inostrani A B C 73 207 11 6 90 207

A 95 5 4 1 105

C 13 13

A - landraces and old cultivars; B - advanced cultivars; C - breeder's lines A ­ lokalne i stare sorte; B ­ savremene sorte; C ­ oplemenjivacke linije Table 4. Foreign common bean accessions countries of origin Tabela 4. Zemlje porekla inostranih uzoraka obicnog pasulja No. of accessions Broj uzoraka Total Ukupno 108 38 10 5 5 3 1 67 43 280 Cultivars Sorte 22 27 10 5 5 3 1 73 Lines Linije 86 11 67 43 207

Country of origin Zemlja porekla USA SAD Italy Italija Greece Grcka Bulgaria Bugarska China Kina Romania Rumunija Denmark Danska FAO Unknown Nepoznato Total Ukupno

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Jani S, Canko A

These will soon be available for distribution or exchange, since during the period 2009-2010 all Albanian accessions are being regenerated. The Seeds Department of ATTC holds a working and active collection, which has widely been used in the last thirty years, especially in basic research to develop varieties with higher yield potential and with greater yield stability. This grain legumes working collection includes 430 accessions. The largest group is common bean with 403 accessions (95 landraces, 88 cultivars and 220 breeder's lines) as the most important (mainly they are white bean accessions), following by lentil with 16 accessions (5 landraces and 11 cultivars), chickpea with 10 accessions (4 landraces and 6 cultivars), and runner bean with 1 accession (1 landrace) (Tab. 3). The accessions collected originate from 7 countries and FAO, plus anonymous (Tab. 4). After 2005, agricultural research funding was severely reduced, leading to a decrease in some activities related to collection, evaluation and regeneration of grain legumes genetic resources. Even as a result of merging of the agriculture research institutes, the breeding activities for developing new cultivars will not be carried out for some time. Storage and Regeneration The seed samples are deposited and conserved in short-term storage at +4 to +6°C and 30% RH in a cooling room (ATTC-Lushnja), whereas the seed samples of accessions originated from Albania (landraces, old varieties and breeder's lines) are stored and conserved at -18ºC as duplicates in NGB-Tirana. Once they are harvested, seed samples are thoroughly dried before they are packed in paper bags. Last year, seed samples were packed in plastic box according to the international standards (Painting et al. 1993). Regeneration is the main goal for NGB-Tirana and ATTC at this time, since several accessions need regeneration and characterization. Every year about 1/4 to 1/5 of grain legumes collection is regenerated. During the regeneration process, international standards are adhered to and biological requirements of individual species are taken into account. All grain legumes accessions are regenerated in the open field. Each accession is represented by about 100 plants. Open-pollination method is commonly applied, by isolating each accession with a corridor 1.2 m apart. Rows were placed 0. 7 m apart and in-row spacing was 0.15-0.2 m.

It is planted with two rows wide by 10 m long per each accession. Documentation and Characterization The data on common bean and other grain legumes collections were gathered from breeders. Passport/collecting and storage data have already been documented and computerized. The evaluation and characterization work on grain legumes species within the framework of the National Program for Plant Genetic Resources is mainly conducted at the exAgricultural Research Institute (Hyso & Canko 2001, Canko et al. 2003, Canko et al. 2006). Characterization has been done for all accessions of grain legumes, according to IPGRI descriptor list, slightly modified (IBPGR 1983, Salillari et al. 2007), including morphological and agronomic traits, disease resistance and reaction to stress conditions. A computerized grain legumes database is available and operational. Collecting Missions and Research Activities The last uttermost collecting mission was organized during 1999-2003 in some areas of eastern and southeastern regions of Albania. A total of 63 common bean accessions were collected, including 39 seed colored accessions and 24 white beans, according to international standards (Hawkes 1980, Guarino 1995, Adham 1999). From the collected landrace germplasm, the common beans are mainly used in the genetic breeding activities. Among them only white beans can be mentioned. During the last 10 years 12-13& of white bean accessions have been used. The main objectives of genetic breeding activities were focused in the developing new varieties with specific characteristics as the disease resistance, tolerance to stress conditions, etc. In ex-Agricultural Research Institute of Lushnja four new varieties have been developed and registered (Canko et al. 2004). The relations between genetic conservation, plant breeding, seed production and production need to be improved. In this context, the institutional reform has to be completed (including qualified staff of ex-Agricultural Research Institutes in this process), and the way of the barrier financial and technical overcoming which actually restrain the activities in those fields.

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 617-622

Genetic Resources of Grain Legumes in Albania

621

Figure 1. Examples of the common bean landraces in the districts of Shkodër and Skapar Slike 1. Neke od sakupljenih lokalnih sorti pasulja u okruzima Skadar i Skapar

Owing to its conditions, Albania is a rich country with a wide diversity of plant genetic resources. There are some micro-zones with a lot of grain legume landraces. During 2009, in a short time collecting mission funded by SeedNet project, in the district of Skrapar (situated in the middle part of Albania) 28 samples of landraces were collected (Fig. 1). These samples will be characterized and evaluated to avoid synonymous accessions. Some of local grain legumes varieties are not yet included in the collection and are under pressure of genetic erosion. The activities of the NGB of Albania and ATTC-Lushnja are being started, but organizational and financial problems make it impossible to launch an expedition in order to collect new accessions, especially in the northern and north-eastern regions. Therefore we invite friends and colleagues to send seeds of old varieties and local populations.

Conclusions Grain legumes for food (especially common bean) are crops with a great potential for high, quality and stable yields and the results of its evaluation show that there is a considerable genetic variation. It is important to know that there are a lot of districts that have a rich diversity of grain legume crops which have not yet been explored. In some of those districts, common bean is the most important food resources for their population, e.g. Dibra, Puka, etc. There is a need to collect the diversity of dry bean in those districts, because a lot of people are leaving their old settlements for the urban zones, and these landraces will be lost forever. Therefore, we need to have a chance to undertake some collecting missions on those districts to get as much as possible of the common bean diversity before it is lost. On the other hand, the facts express the importance of these materials not only for conservation, but also for their use in breeding programs and need for their improvement.

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Jani S, Canko A

Canko A, Celami A, Kashta F, Gjondeda F (2006): Koleksionimi dhe studimi i germoplazmes autoktone të fasules [Collecting, characterization and evaluation of autochthon accessions on common bean]. Buletini i Shkencave Bujqësore 1: 17-22 Guarino L, Ramanatha V, Reid R (1995): Collecting Plant Genetic Resources. Chapman and Hall, London Hawkes J G (1980): Crop Genetic Resources Field Collection Manual. IBPGR ­ EUCARPIA, Wageningen Hyso M., Canko A (2001): Koleksionimi dhe studimi i popullatave të fasules. Studime Biologjike 5-6: 370-373 Painting K A, Perry M C, Dennige R A, Ayad W G (1993): Guidebook for Genetic Resources Documentation. FAO, Rome Salillari A, Hyso M, Faslia N, Rusinovci I (2007): Resurset gjenetike të bimëve. Tirana

References

Adham J J (1999): Sampling Strategies and Collection Method. Saragossa, Spain Canko A, Kashta F, Celami A, Caullari F, Kycyku K (2003): Pasurimi, studimi, rigjenerimi dhe ruajtja ex-situ e germoplasmës së bimëve të arave [Collecting, characterization, evaluation, regeneration and ex-situ conservation of field crops germoplasm]. Workshop i organizuar nga Banka Gjenetike Shqiptare në bashkëpunim me Projektin e Shërbimeve Bujqësore [Edition of Workshop organized by NBG-Tirana and Agricultural Services Project], Tirana, Albania. Canko A, Shehu R, Hyso M (2004): Fasulja. Tirana

Pregled genetickih resursa zrnenih mahunarki u Albaniji

Sokrat Jani1 · Agim Canko2

Centar za transfer poljoprivrednih tehnologija (ATTC) u Lusnji, Albanija Centar za transfer poljoprivrednih tehnologija (ATTC) u Fuse-Kruji, Albanija

1

2

Izvod: Aktivnosti na genetickim resursima zrnenih mahunarki u Albaniji pocele su 1963. godine, najpre sakupljanjem, cuvanjem, evaluacijom i korisenjem lokalnih i starih sorti, a potom i uvozom sorti iz evropskih zemalja. U pocetku je ovo sprovodio Poljoprivredni istrazivacki institut (ARI) u Lusnji, osnovan od strane Ministarstva poljoprivrede, sa jasnim planom da odabere i proizvede komercijalno seme za svakodnevne potrebe poljoprovrednih kooperanata. Do kraja osamdesetih godina proslog veka, sakupljeno je oko 180 uzoraka obicnog pasulja (Phaseolus vulgaris L.), od kojih su 53 bili lokalne i stare sorte. U razdoblju 1990-2000, izveden je drugi projekat, u okviru kojeg je obavljeno nekoliko sakupljackih ekspedicija, posebno u sredisnjim i jugoistocnim oblastima zemlje. Godine 1998. u Tirani je osnovana Nacionalna banka gena, kako bi organizovala rad na ocuvanju biljnih genetickih resursa u Albaniji. Kljucne reci: Cicer arietinum, genotip, Lens esculenta, lokalna sorta, Phaseolus vulgaris, Phaseolus coccineus, stara sorta, zrnene mahunarke

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 617-622

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Kokurentnost osnovnih ratarskih useva u Vojvodini

623 Prikaz knjige / Book Review Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 623

Janko Cervenski: Gajenje kupusa Institut za ratarstvo i povrtarstvo, Novi Sad, 2010, 199 str.

Monografija ,,Gajenje kupusa'' namenjena je pre svega proizvoacima kupusa, ali i strucnjacima, studentima, naucnim radnicima i svima onima koji zele da prosire znanje o kupusu ili zapocnu jedan od ponuenih nacina gajenja kupusa. Pisui ovu knjigu, zelja autora je bila da svoja licna saznanja, ali i iskustva savremene nauke i proizvodne prakse objedini na jednom mestu, kako bi sto korisnije posluzila pre svega novim proizvoacima, ali i iskusnim proizvoacima kupusa, da otkriju nesto sto nisu znali. Ova knjiga, prva do sada stampana monografija o gajenju kupusa u nasem okruzenju, nesumnjivo e predstavljati pouzdanog saveznika svakom proizvoacu kupusa, a moze biti interesantno stivo i za neke budue proizvoace kupusa. Autor je tokom pisanja koristio svoje bogato iskustvo, rezultate vlastitih istrazivanja, ali i rezultate ogromnog broja istrazivaca, kako iz nasih zemalja tako i iz inostranstva, harmonizirajui ih u jednu homogenu celinu, uveavajui vrednost pojedinacnog rezultata i svih rezultata zajedno. Autor je odbranio magistarsku tezu iz oblasti selekcije kupusa, doktorsku disertaciju iz oblasti oplemenjivanja kupusa, napisao je strucno popularnu knjigu pod nazivom ,,Kupus", ucestvovao je u izradi elaborata o brendiranju Futoskog kupusa, bio je rukovodilac nekoliko projekata iz oblasti oplemenjivanja kupusa i povra, koautor je dve sorte kupusa, te je autor ili koautor preko 150 radova iz oblasti povrtarstva. U knjizi su obraena sledea poglavlja: Poreklo kupusa, Znacaj kupusa, Proizvodnja kupusa u republici Srbiji, Botanicke karakteristike, Uslovi uspevanja, Agrotehnika kupusa, Najcese gajene sorte i hibridi, Rana proizvodnja kupusa, Letnja proizvodnja kupusa, Proizvodnja kasnog kupusa, Gajenje kupusa u zastienom prostoru, Proizvodnja kupusa direktnom setvom, Suzbijanje korova u proizvodnji kupusa, Stetocine kupusa i njihovo suzbijanje, Bolesti kupusa i njihovo suzbijanje, Berba kupusa, Cuvanje kupusa, Proizvodnja semena kupusa, Prerada i kiseljenje kupusa. Autor je takoe uneo i recepte za jela od kupusa cime je gajenje kupusa priblizio i potrosacima, a ne samo proizvoacima. Sva navedena poglavlja su strucno opisana i ilustrovana korisnim grafickim prilozima. Knjiga je napisana na 199 strana B5 formata, sadrzi 119 slika koje u velikoj meri dopunjavaju tekst, 15 preglednih tabela, 2 grafikona i 1 histogram. Na osnovu podataka i informacija koje nam autor daje u ovoj knjizi, mozemo zakljuciti da je koristio znacajne izvore literature. Literatura citirana u ovoj monografiji sastoji se od 72 izvora tekstova, knjiga, clanaka. Od pomenutog broja sam autor je prvi autor jedne treine, tj. 24 rada, a koautor 19 radova, iz cega zakljucujemo da je autorovo iskustvo iz ove oblasti potkrepljeno sa dosta prakse. prof. dr uro Gvozdenovi

Janko Cervenski: Cabbage Growing (in Serbian) Institute of Field and Vegetable Crops, Novi Sad, 2010, 199 pages

First of its kind in the region, this monograph is intended for cabbage producers, agronomy experts and students. It comprises 199 pages (B5 format), 119 photographs, 15 tables, two graphs and one histogram. Various sources are cited in the text with 72 references, more than half of which are author's own research references. The monograph offers chapters on these topics: cabbage origin and importance, botanical characteristics, growing conditions and cultivation practices, most often grown domestic varieties and hybrids, cabbage production (early, summer and late), indoor cabbage growing, direct drilling production, weed, pest and disease management, cabbage harvesting and storing, seed production, processing and souring, as well as some cabbage dish recipes.

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624

Bosnjak D, Rodi V Prikaz knjige / Book Review Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 624

uro Gvozdenovi i Sandra Cveji: Oplemenjivanje paprike Institut za ratarstvo i povrtarstvo, Novi Sad, 2009, 162 str.

Monografija «Oplemenjivanje paprike» u izdanju Instituta za ratarstvo i povrtarstvo iz Novog Sada prvi je do sada napisan rukopis ovog tipa o paprici kod nas. Sadrzi 162 stranice teksta, 15 tabela, 20 grafikona, oko 55 ilustracija i slika veinom od samih autora. Od oko 100 navoda literature 20 navoda su samocitati. Uvodni deo sadrzi obraene teme o istorijatu i poreklu paprike, kada ju je Kolumbo 1493. na svom drugom putu preneo iz Amerike u Evropu. Date su povrsine i prinosi na kojima se paprika gaji u svetu i kod nas, kao i najvei svetski proizvoaci. Privredni znacaj paprike lezi u ishrani stanovnistva i meunarodnoj trgovini. Naravno, obraen je hemijski sastav sa datim vrednostima za pojedina jedinjenja, a poznato je da paprika sadrzi najvise C vitamina od svi gajenih biljaka. Zatim je detaljno obraena botanicka klasifikacija, kao i biologija paprike, gde je posebno istaknuta biologija cvetanja i oplodnja, s obzirom da od toga u mnogome zavisi i proces oplemenjivanja. Autori su posebno obradili poglavlje Istrazivanja na paprici, od imena i porekla paprike, egzoticne germplazme do oplemenjivanja na pojedina svojstva i korisenja molekularnih markera u stvaranju sorti i proizvodnji semena. Takoe su ukljucena ispitivanja ljutine paprike i korisenje kapsavicina u lecenju odreenih oboljenja. U poglavlju Oplemenjivanje paprike, autori su na osnovu konvencionalnih metoda hibridizacije i selekcije i koncepta izbora roditeljskih parova postavili jasan cilj oplemenjivanja odgovarajueg modela sorte, kod nas i na osnovu bogatog iskustva i rezultata vlastitih istrazivanja dosli do odgovarajuih rezultata u proucavanju nasleivanja prinosa i komponenti prinosa, kao i morfoloskih osobina ploda i pojave heterozisa, a kao krajnji rezultat jesu stvorene i priznate sorte paprike koje se danas gaje u sirokoj povrtarskoj proizvodnji. Na kraju monografije slikovito je prikazano tridesetak sorti paprike koje se danas najvise gaje u Srbiji, sa datim morfoloskim karakteristikama ploda i cele biljke, kao i nacinom gajenja i upotrebe. Napisana je lakim stilom, za sve citaoce razumljivim tecnim jezikom, tako da je pristupacna sirem krugu citalaca, kako strucnjacima iz ove oblasti tako i drugim korisnicima u produbljivanju njihovih znanja iz ove tematike, a pre svega studentima master i doktorskih studija poljoprivredne i bioloske struke. prof. dr Jan Boanski

uro Gvozdenovi and Sandra Cveji: Pepper Breeding (in Serbian) Institute of Field and Vegetable Crops, Novi Sad, 2009, 162 pages

This monograph is the first on breeding peppers published in Serbian language. Intended for breeding experts and graduate students, it contains 162 pages, 15 tables, 20 graphs, around 55 illustrations and photographs, mainly taken by the authors, and it cites about 100 references, 20 of which are authors' own. History and origins of peppers, current acreage and yields with economic importance are given in the text. Further chapters supply chemical importance and nutritive value of peppers, as well as botanical classification and morphology of the plant. Last, but certainly not the least, readers are informed on pepper research, exotic germplasm and various breeding methods and goals. Finally, thirty commercial varieties grown in Serbia are described in detail.

Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 624

www.nsseme.com/journal.html

Kokurentnost osnovnih ratarskih useva u Vojvodini

625 Prikaz knjige / Book Review Ratar. Povrt. / Field Veg. Crop Res. 47 (2010) 625

Nada Hladni: Geni i prinos suncokreta / Genes and Sunflower Yield Zaduzbina Andrejevi, Beograd i Institut za ratarstvo i povrtarstvo, Novi Sad, 2010, 114 str.

Monografija ,,Geni i prinos suncokreta" ima naucni i prakticni znacaj, a predstavlja vazan doprinos genetskim istrazivanjima suncokreta u oblasti populacione genetike. Objavljena je dvojezicno, na srpskom i engleskom jeziku. U monografiji nas autor upoznaje sa suncokretom kao ratarskom kulturom koja ima velik ekonomski znacaj za proizvodnju jestivog ulja u Srbiji i svetu. Saznajemo njegovo poreklo, rasprostranjenost, morfoloske karakteristike, nacine korisenja kao i specificnosti ove znacajne uljane kulture. Posebna paznja u monografiji posveena je osnovama kvantitativne genetike, metodama oplemenjivanja suncokreta, efektu gena i nacinu nasleivanja agronomski vaznih svojstava suncokreta. Monografija sadrzi sledea poglavlja: Poreklo i privredni znacaj suncokreta; Rod Helianthus; Morfoloske osobine cvetanje, oprasivanje i oplodnja suncokreta; Oplemenjivanje suncokreta; Oplemenjivanje na morfofizioloske komponente prinosa suncokreta; Prosecan doprinos inbred linija i njihove interakcije u ekspresiji kvantitativnih svojstava; Meuzavisnost morfofizioloskih svojstava sa prinosom semena i ulja suncokreta. Autor je uspeo da poveze svoje visegodisnje naucnoistrazivacke rezultate dobijene primenom metoda populacione genetike sa najnovijim radovima iz oblasti genetike i oplemenjivanja suncokreta domaih i stranih istrazivaca, sto se vidi u pregledu korisene literature od 195 citata, od cega su 52 autocitati autora. Ova monografija je namenjena naucnim i strucnim radnicima koji se bave naucnoistrazivackim radom kako u Srbiji tako i u drugim zemljama, a moze da posluzi i studentima i svima onima koji zele da vise saznaju o oplemenjivanju suncokreta. prof. dr Mile Ivanovi

Nada Hladni: Geni i prinos suncokreta / Genes and Sunflower Yield Zaduzbina Andrejevi, Belgrade and Institute of Field and Vegetable Crops, Novi Sad, 2010, 114 pages

Published in Serbian and English, this monograph is both scientifically and practically important, since it represents a significant contribution to sunflower genetic research in the area of population genetics. The author introduces sunflower as a field crop of economic significance for the production of edible oil both in Serbia and in the world. We learn about its origin, distribution, morphological characteristics, different ways of usage as well as specific traits of this important oil crop. Special attention was paid to basics of quantitative genetics, sunflower breeding methods, gene effects and manner of inheritance of the agronomically important sunflower traits. The book contains following chapters: Origin and Agricultural Importance of Sunflower Species Helianthus; Morphological Traits, Flowering, Pollination, Fertilization; Sunflower Breeding; Breeding for Morphophysiological Traits; Average Contribution Inbred Lines and Their Interaction in the Expression of the Quantitative Traits; Interdependence of Morphophysiological Traits with Seed and Oil Yield. The author has brought together years of her scientific and research results gained by the application of the population genetics methods with the most recent papers on genetics and sunflower breeding written both by domestic and foreign researchers, citing over 195 references, 52 of which are author's own papers. This monograph is of great value not only for scientists and experts both from Serbia and other countries, but also for students and others interested in finding out more on sunflower breeding.

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Bosnjak D, Rodi V

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