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STUDIES ON LEAF BLIGHT OF CHRYSANTHEMUM CAUSED BY Alternaria alternata (Fr.) Keissler

Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfillment of the requirements for the Degree of

MASTER OF SCIENCE (AGRICULTURE) in PLANT PATHOLOGY

By ARUN KUMAR G. S.

DEPARTMENT OF PLANT PATHOLOGY COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD ­ 580 005 JULY, 2008

ADVISORY COMMITTEE

DHARW AD 5 t h July, 2008 Approved by : Chairman :

(B.C. KAMANNA) CHAIRMAN

___________________________ _ (B.C. KAMANNA)

Mem bers :

1. __________________________ (S. LINGARAJU)

2. __________________________ (V. DEVAPPA) 3. __________________________ (V.S. PATIL)

CONTENTS

Sl. No. CERTIFICATE ACKNOWLEDGEMENT LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF APPENDICES 1 2 INTRODUCTION REVIEW OF LITERATURE 2.1 History 2.2 Survey for leaf blight in chrysanthemum growing areas of northern Karnataka. 2.3 Isolation, identification and proving the pathogenicity of the fungus 2.4 Disease development in relation to environmental factors 2.5 Evaluation of fungicides, botanicals and bioagents 3 MATERIAL AND METHODS 3.1 General procedure 3.2 Survey for disease incidence of leaf blight in chrysanthemum. 3.3 Isolation, identification and proving the pathogenicity of the fungus 3.4 Disease development in relation to environmental factors. 3.5 To evaluate fungicides, botanicals and bioagents against the pathogen under in vitro and in vivo conditions 4 EXPERIMENTAL RESULTS 4.1 Survey for the disease incidence of Alternaria leaf blight of chrysanthemum 4.2 Isolation, identification and proving the pathogenicity of the fungus 4.3 Disease development in relation to weather parameters. 4.4 In vitro and in vivo evaluation of fungicides, botanicals and bioagents 5 DISCUSSION 5.1 Survey for the Alternaria leaf blight of Chrysanthemum in northern parts of Karnataka 5.2 Isolation, identification and proving pathogenicity 5.3 To study disease development in relation to environmental factors 5.4 In vivo and in vitro evaluation of fungicides, botanicals and bioagents 6 SUMMARY AND CONCLUSIONS REFERENCES Chapter Particulars

LIST OF TABLES

Table No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Title Incidence of Alternaria leaf blight of chrysanthemum in different places surveyed during 2007-08 in northern parts of Karnataka Effect of weather parameters on Per cent Disease Index (PDI) of Alternaria leaf blight of chrysanthemum during 2007 Correlation coefficient (r) for leaf blight of chrysanthemum with weather parameters during 2007 Multiple linear regression co-efficient for chrysanthemum leaf blight PDI in relation to weather parameters during 2007 Efficacy of different bio agents in inhibiting mycelial growth of the Alternaria alternata In vitro evaluation of different fungicides in inhibiting the mycelial growth of Alternaria alternata after eight days of inoculation In vitro evaluation of different botanicals in inhibiting mycelial growth of Alternaria alternata after eight days of inoculation Efficacy of different bio agents in inhibiting mycelia growth of the Alternaria alternata Field efficacy of fungicides, botanicals and bioagent inthe management of Alternaria leaf blight of chrysanthemum during 2007 Field efficacy of fungicides, botanicals and bioagent on number of branches of chrysanthemum Field efficacy of fungicides, botanicals and bioagent on the plant height of chrysanthemum Field efficacy of different fungicides, botanicals and bioagent on the number of opened flowers of chrysanthemum Field efficacy of different fungicides, botanicals and bioagent on the number of unopened flowers of chrysanthemum Economic analysis of management of Alternaria blight of chrysanthemum

LIST OF FIGURES

Figure No. 1. Title Development of PDI and meteorological parameters associated during kharif 2007-08 Observed and predicted chrysanthemum PDI of Alternaria leaf blight of

2.

3.

In vitro evaluation of different fungicides against Alternaria alternata Bioefficacy of plant extracts against Alternaria alternata Efficacy of different bio agents against Alternaria alternata Economic analysis of management of Alternaria blight of chrysanthemum

4. 5. 6.

LIST OF PLATES

Plate No. 1. 2.

Title Overall view of the experimental plot Karnataka map showing district-wise average incidence of Alternaria leaf blight of chrysanthemum during kharif 2007 Different fields and symptoms of Alternaria leaf blight observed during survey Proving pathogenicity Photographs showing culture and conidia of A. alternata In vitro evaluation of different fungicides. In vitro evaluation of different botanicals Efficacy of different bioagents against A. alternata. Disease scale for Alternaria leaf bight of Chrysanthemum Treatment proved best with hexaconazole @ 0.1% (T1) Untreated check (T10)

3.

4a. 4b. 5a. 5b. 6a. 6b. 7a. 7b.

LIST OF APPENDICES

Plate No. 1.

Title

Mean monthly meteorological data for the experimental year (2007-08) and the mean of past 57 years (1950 ­ 2006) of Main Agricultural Research Station, University of Agricultural Sciences, Dharwad. List of abbreviations and symbols used Cost of agrochemicals, botanicals, bioagents and labour cost

2. 3.

1. INTRODUCTION

Flowers symbolize purity, beauty, love, peace and passion. They have significant role in beautifying places. Diseased or disturbed flower plants will not serve the purpose. Hence, they have to be protected from diseases or such plants need to be replaced. This involves considerable labour and finance. Chrysanthemum {Dendranthema indicum (= Chrysanthemum indicum L.)}, the golden flower (Chryos=golden; anthus=flower), is one of the most beautiful and perhaps the oldest flowering plants commercially grown in different parts of the world. Chrysanthemum belongs to the family Asteraceae. There are about 30 species of perennial flowering plants in the family Asteraceae. It is commonly known as Queen of East, Autumn Queen and Guldaudi. It is important both as cut flower and as potted plant in the international market. In Dutch, during cut flower action, chrysanthemum ranks second after rose (Sreerama, 1997). Chrysanthemum is preferred practically due to its vast range of shapes and sizes of flowers, brilliance of colour tones, long lasting floret life, diversity of height and growth habit of the plant, exceptionally hardy nature, relative ease to grow all the year round and versatility of use. It is one of the most important traditional flowers of India, mainly used as a potted plant, loose flower, cut flower and as border plant in the garden. Chrysanthemum cinerarifolium inflorescences are of considerable importance in the manufacture of pyrethrin insecticides. The leaves of Chrysanthemum balsamita become leathery on drying and can be used as bookmarks. Fresh young customary leaves can be used in salads and to add as balsam flavor in beer, soups and bread. Chrysanthemum is believed to have originated from China (Carter, 1990). It is an herbaceous perennial and grouped as sub-shrubby plants. Chrysanthemum actually belongs to a group of many small flowers called florets borne on a receptacle called `head' or `capitulum'. The flowers are classified as single and double depending upon the arrangement of florets in the flower head. Chrysanthemum flowers are grown commercially for making garlands and for religious offerings (Kher, 1990). The year 1995-96 was celebrated as "International year of Chrysanthemum." The major chrysanthemum growing states are Karnataka, Tamil Nadu, Maharashtra, Rajasthan, Madhya Pradesh and Bihar. It is also cultivated around big cities like Delhi, Calcutta, Lucknow, Kanpur, Bangalore and Allahabad mainly for the purpose of beautification and exhibition display. In India, Karnataka state ranks first on an area of about 3,729 hectares with a production of 37,290 tons and productivity of 10 tons per hectare (Ramamurthy and Ramakrishnappa, 2000). There has been constant demand for chrysanthemum flowers particularly from European markets during winter months and throughout the year in our country. India is bestowed with varied types of agro climatic conditions besides, the availability of land and cheap skilled labour. Hence, there is a great potential for production of chrysanthemum on a commercial scale in India. However it is difficult to get good quality exportable blooms, higher yields and long lasting post harvest life of the cultivars under open conditions. For these several factors have been identified in India. The most important factors responsible are the diseases like Alternaria leaf blight, Septoria leaf spot, Rust, Wilt, Bacterial blight and non availability of leading varieties which are resistant to biotic and abiotic stresses. Among these diseases Alternaria leaf blight caused by Alternaria alternata (Fr.) Keissler is one of the most destructive diseases, commonly prevailing in almost all chrysanthemum growing pockets of India, which causes heavy losses under field as well as market conditions. During 1987, a severe leaf blight of Chrysanthemum morifolium Ramat. caused by a species of Alternaria was noticed in the fields as well as in home gardens in parts of Dharwad district and spoiled the beauty of the gardens and caused economic losses. The symptoms of the disease were observed on leaves and also on stems. Dark brown spots surrounded by yellow hallow were observed on lower and upper surface of leaves which later lead to blighting of entire leaf and finally defoliation.

A perusal of the literature revealed that three different species of Alternaria cause the leaf blight of chrysanthemum. Several workers reported different species of Alternaria on chrysanthemum. These are all just reports pertaining to symptoms and morphology of the fungus. It is necessary to have information regarding survey of the disease, so that the distribution and extent of spread can be estimated and hot spots may be located which will help in natural screening for host plant resistance. Cochrane (1958) reported that, any consideration of ecology or spread of the economically important fungi must take spore germination into account. To get clear picture of epidemiology of the disease, a clear understanding of different environmental factors like temperature, different humidity levels and rainfall affecting disease development in chrysanthemum are very much essential. Such studies are very much lacking in case of leaf blight of chrysanthemum caused by Alternaria alternata (Fr.) Keissler and also in vitro and in vivo evaluation of fungicides, botanicals and bioagents were lacking but needed urgently (Schmidt, 1958; Rao, 1964, 1965; Srinath and Sarwar, 1965 and Mallikarjunaiah and Rao, 1972). Keeping this in view, the following objectives were choosen for investigation in the present study. 1. To undertake a survey for leaf blight in chrysanthemum growing areas. 2. Isolation, identification and proving the pathogenicity of the fungus. 3. To study the disease development in relation to environmental factors. 4. To evaluate fungicides, botanicals and bioagents against the pathogen.

2. REVIEW OF LITERATURE

Chrysanthemum (Dendranthema indicum L.), is one of the most important traditional flowers of India. Alternaria leaf blight of chrysanthemum caused by Alternaria alternata (Fr.) Keissler is one of the major foliar diseases occurring in all parts of the world, wherever chrysanthemum is grown. The studies with respect to survey, disease development in relation to environmental factors and its management are taken into consideration while reviewing the literature. Accordingly, the literature pertaining to the above aspects is presented here.

2.1 History

Schmidt (1958) for the first time reported a fungal leaf spot of Chrysanthemum maximum L. caused by Alternaria chrysanthemi Simmons and Crosier from Austrian Tyrol. Srinath and Sarwar (1965) for the first time reported Alternaria blight of Chrysanthemum cinerariefolium (Trev.) var. pyrethrum caused by Alternaria tenuissima (Fries) Wiltshire from Bangalore. Rao (1965) reported Alternaria tenuis Auct. (Alternaria alternata (Fr.) Keissler.) causing leaf blight and blossom blight of Chrysanthemum indicum L. from Maharashtra. Hegde (1988) reported the leaf blight of Chrysanthemum morifolium Ramat caused by Alternaria tenuissima from Dharwad in Karnataka. The information on survey for incidence of the disease, development of disease in relation to environmental factors, in vitro and in vivo evaluation of fungicides, botanicals and bioagents against A. alternata is scanty. The research carried out on these aspects has been reviewed and presented. However, to present general a picture on this disease, information which stems mainly from literature on the above species of Alternaria causing leaf blight of chrysanthemum and occurring on different crops are also cited wherever necessary.

2.1.1 Occurrence and symptoms of the disease

Schmidt (1958) for the first time reported a fungal leaf spot of C. maximum L. caused by A. chrysanthemi Simmons and Crosier from Austrian Tyrol. He noticed disease on all green parts of the plants. Initially, round, pale grey spots appeared which enlarged rapidly to their final diameter of one cm when fully developed; they became grey to brownish black, often with a pale fleck at the centre and more or less distinct light and dark zonations. Where the spots were sufficiently numerous entire leaves withered. Sobers (1965) reported A. chrysanthemi from Florida. Srinath and Sarwar (1965) for the first time reported Alternaria blight of C. cinerariefalium (Trev.) var. pyrethrum caused by A. tenuissima from Bangalore. They described the symptoms as brown patches of 2-4 mm diameter near the apical part of the leaves. These patches enlarged irregularly, turning blackish brown in colour. Eventually, the necrosis covered the entire leaf area and proved fatal to the plant. Hegde (1988) reported the leaf blight of C. morifolium Ramat. caused by A. tenuissima (Fries) Wiltshire from Dharwad in Karnataka. He described that initially, yellowish green spots of 3-4 mm diameter appeared on older uninjured leaves, whereas, on injured leaves symptoms appeared after five days. These spots circular in the beginning, enlarged later and became irregular and turned to blackish brown or dark brown color. Eventually such spots covered entire leaf and coalescing of spots caused leaf blight, such blighted leaves were found to defoliate. On upper leaves also similar symptoms appeared flowers remained free from infection.

2.2

Survey for leaf blight in chrysanthemum growing areas of Northern Karnataka

Sudarshan Rao (1975) stated that survey and surveillance form the basis for any successful plant protection strategy. Successful plant protection depends upon early detection of the disease incidence followed by timely adoption and application of preventive measures. Hiremath et al. (1990) reported widespread occurrence of Alternaria leaf blight on sunflower during kharif 1987 from north Karnataka. Regular surveys conducted by AICRP (Sunflower) centers revealed that Alternaria blight was severe during kharif. In Akola the incidence was upto 30 per cent during 1996 to 2000. However, the incidence was less than

10 per cent during 2002-03 and 2004-05. In Coimbatore the disease incidence was moderate (upto 25%) during 1996 to 2000, while, it was severe during 2001 to 2005. Similarly in Bangalore, Latur and Raichur the incidence ranged between 5 to 30 per cent, 5 to 35 per cent and 9 to 60 per cent respectively during 1996 to 2004 (Anon., 1996; Anon., 1997; Anon., 1998; Anon., 1999a; Anon., 2000; Anon., 2001; Anon., 2002a; Anon.,2003; Anon., 2004; Anon.,2005 and Anon.,2006) Mallikarjun (1996) conducted a survey to know the prevalence of Alternaria leaf blight of turmeric in four districts of Karnataka, viz. Belgaum, Bijapur, Dharwad andUttar Kannada. The average incidence of the disease was 27.75 per cent. Patil (1999) carried out intensive survey during kharif 1998 and rabi 1998-99 in northern parts of Karnataka to get precise information on the incidence and intensity of the leaf blight on garlic. It was revealed that incidence of disease was more severe in Dharwad and Gokak taluks. Gorawar (2004) conducted a survey for foliar diseases of turmeric crop in the districts of Belgaum, Bagalkot, Bidar and Gulbarga during November 2003-04. Results indicated that there was higher incidence of Alternaria leaf blight compared to Colletotrichum leaf spot on turmeric crop in all the districts surveyed. The highest incidence of leaf blight caused by A. alternata was observed in Belgaum district (49.39%) wherein Harugeri village recorded the highest incidence (62.50%) followed by ARS, Arabhavi (56.25%). The leaf blight disease recorded in Bagalkot district was 45.81 per cent. In this district highest incidence was seen in Rannabelagoli (66.60%) followed by Hosur (56.25%) and Mudhol (50.00%). Bidar and Gulbarga districts recorded the leaf blight incidence of 28.52 and 26.73 per cent respectively. Mesta (2006) reported that during kharif/rabi 2005-06, the incidence of Alternaria blight in sunflower was more compared to kharif/rabi 2004-05. Highest incidence of Alternaria blight of sunflower was observed in Bagalkot district (50.3%) followed by Raichur (41.0%), Gulburga (39.5%), Bijapur (38.6%) and Koppal (37.8%) districts.

2.2.1 Distribution and economic importance

The Alternaria leaf blight of sunflower has been reported from India and elsewhere (Acimovic, 1969; Shane et al., 1981; Herr and Lipps, 1981; Kolte, 1984; Sackston, 1988). The pathogen is capable of causing the disease over a wide range of atmospheric temperature and thus constitutes a potential threat to sunflower producing regions of the world (Abraham et al., 1976; Islam and Maric, 1978; Sackston, 1988). Reddy and Gupta (1977) reported the loss in sunflower yield ranging from 11.3 to 73.2 per cent. Significant reduction in the yield parameters, viz. height, stem girth, head diameter, number of seeds per head, test weight, yield and oil percentage were observed (Mathur et al., 1978; Balasubramanyam and Kolte, 1980b). The pathogen affected more than 280 ha. of sunflower in June 1980 in Minnesota (Shane et al., 1981). Hiremath et al. (1990) reported 95 to 100 per cent incidence of the disease in north Karnataka. Borkar and Patil (1995) reported severity of this disease to the extent of 52 to 85 per cent in three years of study.

2.3

Isolation, identification and proving the pathogenicity of the fungus

2.3.1 Symptomatology

Rao (1965) reported Alternaria tenuis Auct. (Alternaria alternata (Fr.) Keissler.) causing leaf blight and blossom blight of Chrysanthemum indicum L. from Maharashtra. He described that the spots were oval to irregular or angular, dark brown to black, inciting leaf blight, blossom blight and defoliation. The Alternaria leaf blight of sunflower caused by Alternaria helianthi (Hansf) Tubaki and Nishihara is known to infect all aerial parts of plant viz. leaf, petiole, stem, floral parts and seeds. Initially, the disease appears in the form of small, scattered brown spots on the leaf

lamina. Later, these spots increase in size and coalesce covering larger leaf area (1 to 2.5 cm in diameter), with dark brown margin and yellow halo, linear necrotic lesions also appear on stem, petioles and sepals. In severe cases the head and seeds also get infected (Tubaki and Nishihara, 1969; Narain and Saksena, 1973; Kolte and Mukhopadhyay,1973 and Anilkumar et al., 1974). Severe infection of Alternaria blight on leaf, stem, petiole and inflorescence including petals was described by many workers (Mukewar et al., 1974; Balasubramanyam and Kolte, 1980a and b; Nargund and Nazeer, 1994). Hiremath et al. (1990) observed cracking of stem and petioles along with other symptoms in severely infected plants. Ishiba et al. (1982) reported a new disease of Stevia rebaudiana caused by a hitherto undescribed Alternaria which was found in Kagawa in August 1978. It was characterized by small black spots surrounded by a chlorotic zone, on leaves, stems, petioles and involucral scales. The pathogen was named A. steviae, a new sp., and the disease reproduced experimentally. A. steviae was also pathogenic on wounded leaves of related plants (Eupatorium chinense and E. fortunei) but not on Chrysanthemum spp., Erigeron annuus or Tagetes patula. Culture filtrates of the fungus induced necrotic lesions on leaves of susceptible plants at a dilution of 1:8 and on resistant plants when undiluted. Karlatti and Hiremath (1989) collected marigold flowers heavily infected by Alternaria zinniae from a garden in Dharwad district of Karnataka. Seeds were separated, dried and plated on potato dextrose agar (PDA) medium. Some of the seeds were surface sterilized. Spore suspensions were prepared and inoculated onto seedlings of 10 plants belonging to the Asteraceae. A. zinniae was successfully isolated from apparently healthy and discoloured seeds and from those that had been surface sterilized. The isolated fungus infected Ageratum, Aster, Chrysanthemum, Cosmos and Sunflower seedlings. Safflower, Tridax, Niger and Parthenium were not infected. Mallikarjun (1996) described the symptoms of leaf blight of turmeric caused by Alternaria alternata. The symptoms noticed initially on lower set of leaves later blighting extended to top leaves. The spots were light brown in colour with a clear yellow halo, sub circular to irregular, measuring 0.5 mm to 10 mm wide. Under favourable conditions the plant exhibited a burnt appearance which could be very easily detected from a distance. David (1998) had provided description for leaf spot disease of zinniae caused by Alternaria zinniae. The fungus attacks the leaves, stems of its host and can cause dampingoff of seedlings where both the root and the stems of the plant may be affected. In older plants the fungus attacks the older leaves and then spreads to the younger leaves, and when the attack is severe the spots may become confluent. Ellis (1998) described the leaf blight disease of chrysanthemum caused by Alternaria chrysanthemum .The symptoms on stems, leaves, flowers and seeds of Chrysanthemum maximum; also one record from India on Chrysanthemum indicum. On the leaves, spots were round, at first pale grey, and up to one cm diameter, later grey or brownish black, often with a whitish spot in the centre surrounded by pale and dark concentric rings. Alternaria alternata and Fusarium oxysporum are reported as important diseases of commercial chrysanthemums (C. indicum) in Italy (Cavallini et al., 1992). Sen and Pathania (1997) screened sixty chrysanthemum (Dendranthema grandiflora Tzveler [Chrysanthemum morifolium]) cultivars for resistance to leaf spotting caused by Septoria chrysanthemella and Alternaria sp. in field experiments at the University Research Farm, UHF-Nauni, Himachal Pradesh, India, during 1995 and 1996. Disease severity was calculated on the basis of leaf area covered by the pathogen and per cent disease index was calculated for each variety. None of the 60 cultivars were free from disease. Ten cultivars were grouped as resistant, 13 as moderately resistant and the remaining 37 cultivars were moderate to highly susceptible. Minuto et al. (1997) studied that Dendranthemum frutescens [Chrysanthemum frutescens] is largely produced on the Liguria coast, Italy, as a pot plant and sold in Italy and in north-central Europe. In March 1995 during a cold and wet spell, dark, circular spots with little white areas in the middle were observed on the upper part of leaves of plants with white flowers. Microscopic observations and isolations on PDA carried out at Di.Va.P.R.A.,

University of Turin, showed that the causal agent was an Alternaria sp. Inoculations with the pathogen on some varieties with white, rose and yellow flowers showed that Europa, Carla, Rosa dwarf, Roberta, Yellow australian and a mutant of Camilla were susceptible. Smita et al. (1998) found that ten varieties/lines of sunflower (Helianthus annus) were evaluated against leaf blight caused by Alternaria alternata in pot experiments. Of these, Sungene 90, Peredovik and Sunbred-221 were susceptible to the pathogen while Morden showed a very susceptible reaction against Alternaria leaf blight of sunflower. The fungus was also observed as pathogenic on chrysanthemum and zinnia. Kopacki and Wagner (2003) evaluated ten garden mums (Dendranthema grandiflora [Chrysanthemum morifolium]) cultivars i.e. Barbara, Bravo, Debonair, Frolic, Holly, Linda, Sunny Linda, Nicole, Sarah and Volterra for their winter hardiness and plant pathogens. All cultivars showed good hardiness; however, in the second year of investigations, disease symptoms appeared on the tested plants. Apart from typical disease symptoms, changes in shape and size of plants were observed. There were significant differences in the mean plant diameter and mean plant height between years, while the mean flower diameter did not differ between year 2000 and 2001, but differed significantly between those years and 2002. Flower and plant diameters decreased from year to year. There were no significant differences in mean plant and flower diameters between control plants and plants treated with Topsin. Only mean height of plants differed significantly between combinations. Fusarium spp. predominated among fungi isolated from the infected garden mums. Alternaria alternata seemed to affect plant health status as well. The most susceptible cultivars were Barbara, Bravo, Sarah and Volterra.

2.3.2 Morphology of the pathogen

The genus Alternaria was first described by Nees in 1817 with Alternaria tenuis as the type species. The conidial characteristics of the genus are uniform, attenuated and catenulate. Fries (1832) had proposed the Alternaria alternata as Torula alternata Pers. The reasons why the specific epithet `alternata' should be used instead of the more commonly accepted one `tenuis' was clearly stated by Simmon (1965). Keissler (1912) had given the morphology of Alternaria alternata. According to him the colonies were usually black or olivaceous black and some times grey. Conidiophores produced singly or in small groups, simple or branched, straight or flexuous, sometimes geniculate, pale to mid olivaceous or golden brown, smooth, up to 50 µm long, 3-6 µm thick, with one or several conidial scars. Conidia formed in long often branched chains, obclavate, pyriform, ovoid or ellipsoidal often with short conical or cylindrical beak some times up to but not more than one third the length of the conidium, pale to mid golden brown, smooth or verruculose with upto eight transverse and usually several longitudinal or oblique septa. Over all length 20-63 µm, 9-18 µm thick in the broadest part, beak pale, 2-5 µm thick. The name Alternaria tenuissima was given by Wiltshire (1933) for the fungus earlier identified by Fries (1832) as Macrosporium tenuissimum Fr. Later he also described the morphology of the fungus as, conidiophores solitary or in groups, simple or branched, straight or flexuous, more or less cylindrical, septate, pale or midpale, brown, smooth with one or several conidial scars, measured 115 × 4.6 µm conidia solitary or in short chains, straight or curved, obclavate or with the body of the conidium ellipsoidal, tapering gradually to the beak, swollen at the apex, pale or golden brown, usually smooth, some times minutely verruculose, generally with 4­7 transverse and several longitudinal or oblique septa, slightly constricted at the septa, measured 22-95 × 8­19 µm beak 2-3 µm thick. Srinath and Sarwar (1965) while reporting Alternaria blight of pyrethrum described the morphology of the pathogen (A. tenuissima) as follows: Conidiophores dark brown, septate, septa 8-10 µm apart, sub erect to erect, unbranched, measuring 30 to 110 × 3 to 4 µm. Conidia light brown, produced in short chains of 2 to 4, acrogeneous, smooth, obclavate, 30 to 40 x 11 to 19 µm with 3 to 9 transverse and 2 to 4 longitudinal septa, smooth walled, beak 7 to 14 x 1 to 2 µm with an obtuse end.

Utikar and Padule (1980) reported that conidiophores of A. alternata were light brown, simple, mostly 2-3 septate rarely 4-5 septate and variable in length ranging from 17.10 to 61.56 µm (Average 36.44 µm). Conidia were found to be light to dark brown, uniform with 1-6 transverse septa and 0-2 longitudinal septa, and variable in size and shape, mostly obclavate to oval with rudimentary beak and measured 10.26-77.52 × 4.56-14.82 µm (Average 42.45 x 10.27 µm). Patil and Patil (1992) stated that the mycelium of Alternaria state of Pleospora infectoria was septate, branched, hyaline when young and turning brown or olivaceous brown when old. Conidiophores were found arising singly or in groups, simple or branched, straight or slightly curved, pale to golden brown and 7µm long and 4-6 µm thick having a few or several conidial scars. Conidia were found to form long or branched chains measuring 2476.36 µm in length and 8.30-20.75 µm in breadth. The conidiophores of A. alternata were simple, branched, straight or flexuous, olivaceous or golden brown. Septate conidia were muriform in shape measured 35.21 µm long and 13.47 µm width (Mallikarjun, 1996).

2.4 Disease development in relation to environmental factors

Acimovic (1969) reported that A. helianthi is pathogenic to Leucanthem vulgare Lam. and Ricinus communis L. The pathogen was isolated from infected Xanthium pungens Waller and Xanthium spinosum L. which were the common weeds in sunflower growing in Australia. It is isolated from safflower (Carthamus tinctorius L.) also, (Allen et al., 1983a and Patil et al., 1994). Alternaria helianthi is a serious leaf spot pathogen of sunflower crop especially during kharif season i.e., monsoon grown sunflower crop (Kolte and Mukhopadyay, 1973; Narain and Saksena, 1973; Agrawat et al., 1979; Herr and Lipps, 1981; Allen et al., 1983 a and b). Acimovic (1976) reported that the minimum, optimum and maximum temperatures for infection of sunflower by A. helianthi were about 5oC, 30oC and 35-40oC respectively. The generation time varied from 13 days at 5oC, two days at 20oC, 25oC and 30oC and three days o o o at 35 C.The sporulation occurred between 5 C and 35 C and was most profuse between o o o o 15 C and 20 C. The conidia formed at 5 C, 10 C and 30oC were smaller in size, had fewer o o o septa than those formed at 15 C, 20 C and 25 C. Anilkumar and Urs (1976) noticed that the warm humid conditions were highly congenial for disease build up. Under prolonged warm humid conditions, even the highly resistant Helianthus agrophyllus a wild relative of cultivated sunflower could also get infected by A. helianthi. Islam and Maric (1978) reported that A. helianthi grew at temperature range of 4oC to 32oC, with maximum growth at 20oC to 28oC, and optimum at 26oC. They also found o o o that sporulation was poor below 16 C and abundant between 20 C and 28 C. The minimum dew period required for infection was 12 hrs (Islam et al., 1976). However, Bhaskaran and Kandaswamy (1980) noticed increased incidence of A. helianthi with increase in number of o days of low temperature (< 20 C). Hiremath et al. (1990) reported a positive correlation between disease severity and relative humidity. The high humid conditions prevailed during rainy season caused Alternaria leaf blight of sunflower disease epidemics in many parts of Karnataka. Borkar and Patil (1995) studied the weather in relation to Alternaria leaf blight disease development in Maharashtra. Temperature of 25.9oC to 33.7oC with a relative humidity of 89 to 95 per cent favoured disease development. They further reported that development of the disease was influenced by rainfall. Rajivkumar and Singh (1996) studied the influence of weather factors on development of leaf spot of sunflower caused by A. helianthi under field conditions during kharif 1990 and 1991. The Most important weather factors favoring disease development o o were the temperature and relative humidity ranging from 27 C - 29 C and 78-80 per cent respectively, whereas rainfall did not affect the disease development because it was erratic and abnormal distribution during both the years. The disease intensity was highest in last week of August in both the years. Thereafter there was a gradual decline in disease severity.

Tubaki and Nishihara (1969) noticed the first appearance of disease in the spring and increased rapidly during the rainy season. Allen et al. (1983a) reported that the amount of chlorosis induced by A. helianthi in sunflower was greatest in plants that were inoculated at the vegetative or budding stage. Sunflower plants are more susceptible to infection by A. helianthi during the anthesis and seed filling stages of growth (Anilkumar et al., 1974; Islam et al., 1976; Anilkumar and Urs, 1976; Allen et al., 1983b, Jeffrey et al., 1984). Kong et al. (1995) reported that the susceptibility of sunflower tissue increased with age, so that older leaves were more susceptible than young and expanding leaves. They also reported that planting density in field conditions also influenced the disease development.

2.4.1 Model for prediction of disease

Benagi (1995) developed autoregressive model for late leaf spot of groundnut by incorporating one variable pertaining to environmental factors at a time, viz. RH-I, II, maximum and minimum temperature. It was noticed that RH-I and II of kharif 1993 and RH ­I, maximum temperature of kharif 1994 were found to have negative relationship with the disease severity. The logistic model was also developed for understanding the development of late leaf spot during kharif 1993 and 1994. Amaresh (2000) developed prediction models for Alternaria blight of sunflower using autoregressive and logistic equations. The autoregressive model was given as Yt+1 = 1.233 Y t with autoregressive co-efficient of 0.98, where as logistic model was given as Yt = 100 / 1 + e with R + 0.94.

2.5

Evaluation of fungicides, botanicals and bioagents

2.5.1 In vitro evaluation of fungicides, botanicals and bioagents

2.5.1.1 In vitro evaluation of fungicides Use of fungicides is the best method of controlling the diseases whenever there is out break of disease. Mukewar and Gera (1980) tested nine fungicides in the laboratory for inhibition of the conidial germination of A. helianthi. They reported that Dithane M-45 and Vitavax were more effective followed by Bavistin and Benlate. Singh and Milne (1974) evaluated the efficiency of 15 fungicides against five fungi causing chrysanthemum flower blight, viz. Alternaria alternata, Botrytis cinerea, Itersonilia perplexans, Mycosphaerella ligulicola and Stemphylium vesicarium. No one fungicide was outstanding at low concentrations against all five fungi, but several performed well at higher concentrations. Captafol, Chloroneb, Mancozeb and Thiram appeared the most promising. According to Hegde (1988) Mancozeb @ 2000 ppm was most effective in inhibiting the mycelial growth of A. tenuissima in in vitro studies. Patil (1989) tested eight fungicides by the paper disc method against A. helianthi and found Ziram as the most effective at all the concentrations tested (0.1, 0.2 and 0.3%) followed by Mancozeb and Copper oxychloride. Wadiphasme et al. (1994) tested six non- systemic and three systemic fungicides in vitro against A. helianthi by poison food technique. They found that Dithane M-45 was the most effective followed by Fytalon and Dithane Z-78. Narasimharao and Rajagopalan (1982) evaluated 12 fungicides in vitro against the A. helianthicola causing leaf spot of sunflower. Thiram at 0.15 per cent recorded the highest inhibition of growth and spore germination. Amaresh (2000) reported Propiconazole, while, Rao (2006) reported a combi product Iprodione + Carbendazim as effective fungicide against A. helianthi. Mathur et al. (1971) reported that Dithane M-45 was better than Dithane Z-78 and other fungicides tested against A.solani of potato. Out of the 10 fungicides tested against A. tenuissima, Thiram, Duter, Captan and Dithane M-45 inhibited the growth of the fungus in

vitro studies. At higher concentration of 0.4 per cent, Dithane M-45 was superior to Captan, Thiram and Duter (Hanumanthaiah, 1976). Basavarajaiah et al. (1979) found that the fungicides Thiram, Duter, Brestan, Aureofungin and Captan in decreasing order were effective against Alternaria carthami in vitro, while, Mahabaleshwarappa (1981) reported that Baycor caused the good inhibition of A. carthami under in vitro followed by Deconil, Hexafero, Blitox and Cumin-L. Padmanabhan and Narayanasamy (1976) and Padaganur and Siddaramaiah (1979) reported that, Dithane-Z-78 and Brestan, Duter and Hexafero were more effective against A. macrospora respectively. Natarajan (1980) conducted both in vitro and in vivo fungicidal trials using several fungicides to control Alternaria leaf blight of sesame and obtained maximum control with Dithane M- 45 followed by Dithane Z-78, Duter, Captan and Thiram. Joshi (1981) and Hadagali (1981) reported that Rh-2161 a systemic fungicide gave the best control of Alternaria gompherenae and Alternaria longissima respectively. Karlatti (1983) also observed that RH-2161 was effective at 1000 ppm and above concentration against A. zinniae. Hiremath and Sundaresh (1985) reported that in fungicide tests against A. tenuissima Thiram effectively inhibited the fungal growth in vitro and Mancozeb and Thiram gave excellent control in vivo. With three sprays at 15 day intervals increasing grain yield of soybean. In in vitro evaluation of eight fungicides against A. alternata causing leaf blight of turmeric, Propiconazole (tilt) was found to be superior in inhibiting the growth of the fungus while Ziram a non systemic fungitoxicant found to be the best in inhibiting the growth of fungus (Mallikarjun, 1996). Hwang et al. (1998) reported that among the three fungicides tested (Mancozeb, Iprodine and Pyrifenox), Pyrifenox was the most effective in inhibiting mycelial growth of A. alternata on water agar. Kamble et al. (2000) tested six fungicides against A. alternata under in vitro conditions. They reported that Mancozeb was highly effective in inhibiting the mycelial growth followed by Copper Oxychloride and Iprodine at 1000, 2000 and 3000 ppm. Urbanszki et al. (2003) tested in vitro the efficacy of 16 fungicides against A. alternata. They reported that Tridemorf fungicides proved to be very efficient in controlling the pathogen. 2.5.1.2 In vitro evaluation of plant extracts Amaresh et al. (1998) tested three plant extracts against Alternaria helianthi in vitro condition. Among these neem seed kernel extract (NSKE) was better compared to Ocimum cannum and Tridex procumbense. Ashok Kumar et al. (1998) evaluated 25 botanicals against Alternaria brassicae, among them Solanum xanthocarpum and Datura innoxia at 10 per cent concentration completely inhibited the spore germination. Rao (2006) found neem leaf extract, neem seed kernel extract and Allium sativum bulb extract as effective botanicals against A. helianthi. Shekhawat and Prasad (1971) reported that out of nine plant extracts tested, five viz., Allium cepa L., Allium sativum L., Ocimum sanctum L., Mentha piperita L. and Beta vulgaris L. showed strong inhibitory effect against A. tenuis. Sheik and Agnihotri (1977) studied the antifungal activity of some plant extracts against Alternaria brassicae in vitro. They observed that the extracts prepared from Lawsonia alba, Datura stramonium and Mentha piperita were effective against the test fungi, whereas pieces of the scales of Allium cepa caused maximum inhibition when tested against A. brassicae. Oxygen uptake by the spores of Trichoderma viride was significantly increased with volatile substances from Ocimum basilicum than from Origanum majorana. Volatile substances from O. majorana stimulated the spore germination of T. viride, whereas these spores were not affected by O. basilicum (Afifi and Dowidar, 1978). The toxicity of Allium cepa L., A. sativum L., Ocimum sanctum, Lawsonia inermis, Datura stramonium L. and Thuja orientalis L. has been tested against Alternaria spp. and found to be effective (Tripathi et al., 1978).

Chary et al. (1984) found that phytoextract of Arthobotrytis odoratissimum (leaves and flowers), Delonix regia (flowers, leaves and bark), Euphorbia microphylla (leaves), Oxalis corniculata (petiole and bulb), Physeolus autropurpurens (leaves) were inhibitory to the spore germination of Alternaria alternata (Fr.) Keissler. The leaf extract of Lantana camara L. were effective in inhibiting the spore germination of A. alternata (Saksena and Tripathi, 1985). Garg and Siddiqui (1992) reported the effects of cumaldehyde isolated from seed essential oil of Cuminum cyminum, 1-8-cineole from fruit oil of Luvunga scandens, and caryophyllene and eugenol from leaf oil of Ocimum sanctum on A. alternata and Trichoderma viride. The pure isolate and 1:100 and 1:200 dilutions of cumaldehyde and 1,8-cineole showed very good activity against A. alternata and good to moderate activity against T. viride. Neetha Sharma and Sharma (1992) reported that leaf extract of Clerodendron aculeatum Gaertn. inhibited spore germination of A. alternata. Antifungal activity of the essential oils extracted from the leaves of six plants were tested against A. alternata and T. viride, four oils of which (C. citrinus, E. tereticornis, Ageratum conyzoides and O. kilimandscharicum) inhibited the growth of both the fungi tested (Singh and Gupta, 1992). The toxicity of Allium cepa L., Allium sativum L., Ocimum sanctum, Lawsonia inermis, Datura stramonium L. and Thuja orientalis L. has been tested against Alternaria spp. and found to be effective (Ganapathy and Narayanasamy, 1993). Aqueous extracts from all the plant species tested, significantly reduced conidial germination of Alternaria porri. Maximum reduction was observed with a leaf extract of Polyalthia longifolia, followed by Eucalyptus citriodora, Datura alba, Ocimum sanctum, Punica granatum, Azadirachta indica, Ipomoea carnea, Tridax procumbens and Tabernamontana coronaria (Datar, 1994). Muthulakshmi and Seetharaman (1994) reported that leaf extracts of Aegle marmelos Correca (10%) and Prosopis juliflora (Sw.) DC (10%) were effective in inhibiting the mycelial growth of Alternaria tenuis under in vitro condition. Senthilnathan and Narasimhan (1994) tested extracts from 11 botanical species, among them, the leaf extracts of Aegle marmelos (10%) and Prosophis juliflora L. (10%) were effective in inhibiting the spore germination and mycelial growth of Alternaria tenuissima (Fr.) Wiltshire the causal fungus of leaf blight of onion. Rajamani et al. (1997) studied the in vitro antifungal and antibacterial activities of castor, coconut, gooseberry and groundnut oils against A. tenuis (A. alternata), T. viride, Bacillus subtilis and Pseudomonas sp. The oils inhibited A. tenuis (A. alternata), but T. viride was not sensitive to these oils. Among the bacterial isolates, B. subtilis and Pseudomonas sp. were sensitive to these oils. Karthikeyan and Bhaskaran (1998) reported that neem flower extract is highly inhibitory to Macrophomina phaseolina, but not to Trichoderma spp. Shenoi et al. (1998) evaluated leaf extracts of forty five plants as antifungal agents against A. alternata under in vitro conditions. They obtained best results with Thevetia peruviana K. Schum., Lawsonia inermis L., Leucas aspera Link. and Pongamia glabra Vent. against the pathogen. Turkusay and Onogur (1998) reported that spore germination of A. alternata was inhibited by Hedera helix Lowc. extracts followed by Datura stramonium L. extracts. Ficus carica L. and Avena sativa L. extract also affected colony development. Karade and Sawant (1999) tested different plant extracts on solid and liquid media against A. alternata and reported that Allium sativum L. was found to be more effective. The bishkatali (Polygonum hydropiper L.), garlic (A. sativum L.), ginger (Zingiber officinale Rosc) and neem (Azadirachta indica A. Juss) extracts were effective against A. alternata (Rahman et al., 1999). Ranjan et al. (1999) reported that out of five plant extracts tested against Alternaria alternata in vitro, Aegle marmelos leaf extract (5000 µg/ml) showed minimum colony diameter and maximum growth inhibition of A. alternata in comparison with the control. All the plant extracts were less effective at lower concentrations, there was a positive correlation between concentration and growth inhibition percentage. The bulb extracts of A. sativum and leaf extracts of Ocimum basilicum L. at two per cent were most effective in suppressing radial growth and biomass production of A. alternata (Rashmi and Yadav, 1999). Patni et al. (2005) evaluated methanol extracts of six medicinal plants (Azadirachta indica, Parthenium hysterophorus, Calotropis procera, Datura alba, Eucalyptus globulus and Polyalthia longifolia) against Alternaria blight (Alternaria brassicae) of Indian mustard.

Eucalyptus, ashok and calotropis extracts, in that order, were promising in limiting the growth and sporulation of the pathogen, whereas carrot grass extracts promoted these. Rao (2006) found neem leaf extract, neem kernel extract and A. sativum bulb extract as effective botanicals against A. helianthi. Shekhavat and Prasad (1971) reported that out of nine plant extracts, five, viz. Allium cepa L., Allium sativum L., Ocimum sanctum L. and Mont. Mentha piperata L. and Beta vulgaris L. showed strong inhibitory action against Alternaria tenuis Nees. The leaf extract of Lantana camara L. were effective in inhibiting the spore germination of Alternaria alternata (Fr.) Keissler (Saksena and Tripathi, 1985). Shenoi et al. (1998) evaluated leaf extracts of forty-five plants as antifungal agents against A. alternata under in vitro conditions. They obtained best results with Thevetia peruviana K.Schum., Lawsonia inermis L., Leucas aspera Link. and Pongamia glabra Vent. against the pathogen. Karade and Kalekar (2000) evaluated the effect of solvent extracts (viz., acetic acid, xylene, ethanol, acetone and chloroform) of pudina (Mentha arvensis L.) leaves under in vitro on mycelial growth of A. alternata and inhibition of spore germination on solid and liquid media. Compared with the control treatment, highest percentage inhibition of mycelial growth and spore germination was recorded using acetic acid extract. The next best treatment was xylene extract. Singh and Majumdar (2001) tested water and acetone leaf extracts of neem, datura, tulsi and rhizome or bulb extracts of ginger, turmeric, onion and garlic against A. alternata and found that datura, garlic, ginger, neem and turmeric were effective. The highest inhibition of mycelial growth of A. alternata was observed in ten per cent chromalaena leaf extract, while garlic bulb extract at 10 per cent gave highest inhibition of spore germination of the same fungus (Kota, 2003). 2.5.1.3 In vitro evaluation of bioagents against Alternaria alternata Biological agents provide economical and relatively non polluting delivery systems for protective materials compared to other field application systems. Catska (1987) noted that Psuedomonas putida decreased the number of colony forming unit of A. alternata, in soil rhizosphere of inoculated apple seedlings. It was concluded that the inoculation of some plants with these bacteria could function in the biological control of phytopathogenic, phytotoxic and saprophytic microorganisms. Basim and Katircloglo (1990) studied the antagonistic activity of 12 isolates of Bacillus subtilis Cohn Emend Pras, against A. alternata and Alternaria solani by dual culture technique. Among the isolates tested, B.subtilis AB-2 and AB-27 isolates were the most antagonistic against the pathogen tested. High level of bacterial antagonism by flouroscent Psudedomonas and Bacillus spp. was noticed against Alternaria brassicola (Leifort et al., 1992). Zaspel and Sussir (1992) studied the in vitro inhibition of Pythium ultimum and Alternaria spp. by selected strains of P. putida and Bacillus subtilis. Amaresh (2000) and Rao (2006) found P. fluorescens and T. harzianum as effective bioagents in reducing the mycelial growth of A. helianthi. Culture filtrates of B. subtilis completely arrested the growth ofAlternaria daturae, a serious pathogen causing leaf spot of Datura fastulosa. The bacterial metabolite was thermostable and fungistatic and it caused abnormally abundant septations and formation of chlamydospore like swollen bodies in the hyphae (Rai, 1975). Application of an antifungal complex (Antibiotic F), derived from a local strain of B. subtilis AECL 69, on the cut stem ends (2 cm radius) of citrus before storage gave significant control of black rot caused by Alternaria citri in Valencia oranges and Kinnow mandarins (Farooqi et al., 1982). Dragoescu (1986) found an antagonistic relationship, of moderate value in control of the pathogen, between Alternaria porri f.sp. solani and Trichoderma roseum. Hebbar et al. (1991) reported that bacteria associated with sunflower leaves (actinomycetes and pigmented Gram-positive bacteria) and roots (Pseudomonas fluorescens,

P putida, P. maltophilia, P. cepacia, Flavobacterium sp. and Bacillus sp.) inhibited in vitro growth of A. helianthi. Srivastava et al. (1991) reported that only 5 of 14 fungi screened in vitro for antagonism to onion pathogens showed activity against Alternaria porri. Chawda and Rajasab (1992) observed that undiluted culture filtrate of Myrothecium verrucari inhibited conidial germination in A. porri. The inhibition decreased with increasing dilutions of the culture filtrate. High level of bacterial antagonism by fluorescent Pseudomonas and Bacillus spp. was noticed against Alternaria brassicola (Leifort et al., 1992). Sastrahidayat (1995) found that isolates of Penicillium, Fusarium and Trichoderma were all antagonistic to A. porri in preliminary laboratory tests on PDA. The effect of Pseudomonas spp. on the mycelial growth and sporulation of Alternaria ricini was studied. Inhibition of mycelial growth and sporulation was observed. Antibiosis was shown as a mode of action for Pseudomonas spp. in relation to A. ricini. Ultrastructural studies confirmed the inhibition of spore germination by the bacteria (Silva et al., 1998). Antimicrobial metabolite production was tested by determining the antagonistic activity of B. subtilis isolated from soil samples against A. porri and A. solani. The antagonist was found to be effective in inhibiting both the pathogens under study (Tejeda et al., 1998). Fluorescent and non-fluorescent Pseudomonads and Bacillus spp. were tested in vitro using the dual culture method against the sunflower blight pathogen, A. helianthi. All the fluorescent Pseudomonads (P. fluorescens and P. putida) and P. cepacia showed high antagonistic activity against A. helianthi compared with the Bacillus spp. Reduction in colony diameter of A. helianthi due to Pseudomonas spp. ranged between 50-80 per cent, and due to Bacillus spp. between 37-60 per cent. Among the Pseudomonas spp., P. fluorescens and P. putida were more effective. Among Bacillus spp. B. polymyxa showed maximum inhibition of colony diameter (Prasad and Kulshrestha, 1999). Amaresh (2000) reported that among fungi T. viride and T. harzianum overgrew and inhibited the growth of A. helianthi while the bacterium P. fluorescens produced maximum inhibition zone. The chitinolytic activity of two strains of P. fluorescens and 14 strains of Bacillus sp., antagonists of A. solani, was evaluated in vitro, in which the highest growth of the colonies was observed in nutrient agar + chitin media. There was a significant correlation between colony growth and the transparency of the agar + chitin medium, while no correlation was observed between chitinolytic activity and colony growth in nutrient agar + chitin media. Bacillus sp. strains Q1, Q2, Q3, Q4, Q5, A30 and A31 produced more chitinase than the other strains (Okumoto et al., 2001). Kota (2003) reported that Trichoderma harzianum and Trichoderma virens highly inhibited the growth of A. alternata under in vitro condition. Mandhare and Suryawanshi (2003) conducted laboratory experiments to determine the effect of airborne antagonist B. thermophillus on Alternaria porri (purple blotch of onion) and A. alternata (leaf spot of pomegranate), where conidial germination and mycelial growth of both the fungi were inhibited by Bacillus thermophillus at 2.2x109 and 2.2x108 cfu/ml, respectively. Under ordinary conditions, the bacterium was able to survive for more than 20 months in vitro. However, its population declined after 7 and 34 days of spray on onion and pomegranate leaves. Tejeda et al. (2003) noticed that the bioproducts obtained from B. subtilis in pure and mixed fermentations (BIOBAC and DIMABAC) were antagonistic to A. solani and A. porri. Meena et al. (2004) conducted a study to investigate the efficacy of biological control agent (T. viride isolates H-1, H-2, SI-1, GR, SI-2, B. P and T) in controlling Alternaria brassicae. It was found that, in dual culture, GR isolate of T. viride performed best among the tested isolates of Trichoderma, causing 81 and 82 per cent reduction in mycelial growth of A. brassicae over the control. Performance of isolates SI-2, P and SI-1 of T. viride were at par (P<0.01) with that of GR isolate. Kang-SangJae et al. (2005) isolated nine strains of bacteria for antagonistic activity against plant pathogenic fungi (A. porri) from plant roots and rhizosphere of some weeds. The highest activity of antagonism of the strain was obtained and designated as Bacillus lentimorbus AQ-1. This particular strain exhibited strong antifungal activity against A. porri (11.4 mm).

Sanjeet kumar et al. (2005) observed that in dual culture, all the three antagonist viz. Trichoderma virens, Trichoderma harzianum and Trichoderma viride overgrew the colony of Alternaria alternata but T. viride parasitized the test fungus earliest. Studies on hyphal interaction between antagonist and test fungus revealed disorganization of protoplasmic content and lysis of host hyphae. Singh et al. (2005) reported the efficacy of Trichoderma viride, T. harzianum, Trichoderma hamatum, Trichoderma koningii and P. fluorescens in controlling Alternaria brassicae under laboratory conditions. All the fungal antagonists inhibited the growth of A. brassicae, with T. viride recording the highest growth inhibition of the pathogen (67.9%). 2.5.1.4 Field evaluation of fungicides, botanicals and bio agents Sobers (1965) reported that the application of Maneb or Zineb to leaves of Chrysanthemum maximum when lesions first appeared at seven to 10 days interval provided adequate control of A. chrysanthemi. Sahn (1989) reported that treatment of chrysanthemum with Thiram, Captalfol or Mancozeb reduced the incidence of A. chrysanthemi. Maximum numbers of flowers were obtained when the plants were treated with Thiram. Abraham et al. (1976) achieved the best control of sesame leaf blight caused by Alternaria sesame by spraying 0.2 per cent Dithane M-45. Singh and Milne (1974) had reported that 17 fungicidal spray treatments were evaluated in the field to determine their efficiency in controlling flower blight of chrysanthemum. The fungi in the disease complex were A. alternata, Botrytis cinerea, Itersonilia perplexans, Mycosphaerella ligulicola and Stemphylium vesicarium. Appreciable disease control was obtained with all the fungicides tested, Chlorothalonil being the most satisfactory individual chemical. Mancozeb and Captafol also gave reasonable control. Combinations of two fungicides did not offer any major advantage. In a field trial conducted by Abraham et al. (1976), the fungicide Miltox was found most effective in controlling Alternaria blight of sunflower followed by Benomyl, Mancozeb and Captan. They have also noticed differences in the reaction of different varieties and fungicides. Kolte et al. (1979) reported that Mancozeb at 0.3 percent, when sprayed four times at an interval of seven to 10 days on sunflower gave a good control of A. helianthi. The fungicide Ziram and Mancozeb were found highly effective in controlling A. helianthi in glass house and field trial (Bhaskaran and Kandaswamy, 1979). Agrawat et al. (1980) found that Carbendazim and Benlate were highly effective against A. helianthi on sunflower. Prathuangwong et al. (1991) observed that, among the seven fungicides used in the control of leaf and stem blight of sunflower caused by Alternaria spp, the greatest reduction in mycelial growth was found when fungicides Iprodione and Imazalil were used, while Benomyl and Copper oxide were least effective. They have also observed the highest 100 seed weight and oil content in the plots treated with Iprodione and Mancozeb. Wadiphasme et al. (1991) conducted field trial for three years and evaluated fungicides against A. helianthi on sunflower. On the basis of pooled data of three years, Mancozeb proved most superior amongst all the fungicides followed by Triadimefon, Chlorothalonil and Ziram, which were on par with each other. For the management of foliar diseases of sunflower, Tilt 25 EC (0.1%) was found best followed by Dithane M-45 (0.2%), herbal antifungal formulation (3%), neem leaf extract (5%) and Captan (0.2%) (Anon.1998). Bavistin was also reported as effective fungicide against Alternaria blight of sunflower (Anon., 1999b). Amaresh (1997) in a field experiment evaluated seven fungicides against Alternaria blight of sunflower. Chlorothalonil (0.2%), Mancozeb (0.2%) and Cyperconzole (0.1%) gave best results among all seven fungicides tested in controlling the diseases under field

conditions. Maximum grain yield was recorded in Chlorothalonil, which was on par with Cyperconazole and Mancozeb. Desai (1998) in field experiment observed that Mancozeb (0.2%) was used to workout optimum protection schedule for rust and blight of rain fed sunflower. Rao (2006) reported Bayleton as effective fungicide against leaf blight of sunflower which increased yield up to 63 per cent. Mesta et al. (2003) reported Propiconazole (0.1%) was effective fungicide against Alternaria blight of sunflower. However, Mancozeb recorded a highest cost benefit ratio. Rao (2006) reported a combi product Iprodione + Carbendazim @ 0.2% as effective fungicide for the management of Alternaria blight of sunflower. Sood and Sharma (2002) evaluated the efficacy of pre-harvest spraying of 500 ppm Carbendazim, Thiabendazole and Thiophenate-methyl and 2000 ppm Mancozeb + Metalaxyl, Copper oxychloride, Mancozeb, Captan and Bordeaux mixture in controlling Alternaria alternata on turmeric. Bordeaux mixture recorded the highest control of the pathogen whereas Carbendazim recorded the lowest. Fokkema and Lorbeer (1974) reported that addition of Aureobasidium pullulans to inoculum of A. porri reduced subsequent infection of onion leaves. The common saprophytes of the onion phyllosphere, Alternaria pullulans, Sporobolomyces roseus, Cryptococcus luteolus and Cladosporium herbarum reduced infection of onion leaves by A. porri by 55, 45, 14 and 18 per cent, respectively. The antagonistic micro-organisms affected superficial mycelial development of the pathogens rather than spore germination. Suppression of infection was neither due to depletion of carbohydrates or amino acids in the phyllosphere nor to pH difference. The applications of Pseudomonas fluorescens strains 679-2 to tomato and alfa-alfa reduced the severity of leaf spot disease caused by A. solani (Casida and Lokizic, 1992). Tyagi et al. (1990) reported that Cladosporium herbarum was the only fungus of five isolated from the onion phylloplane and tested for antagonism against A. porri that could also inhibit the pathogen on contact in vivo. C. herbarum could reduce the infection by 66.6 per cent Penicillium sp. (54 %) and Alternaria pullulans (33.3 %) were much less effective. Sporobolomyces roseus and Cryptococcus luteolus were able to reduce infection by only 20.8 per cent and 12.5 per cent respectively. However, a mixture of all five species caused a reduction of 79.1 per cent in infection by the pathogen. A significant observation was that Penicillium sp., A. pullulans, S. roseus and C. luteolus, which were effective to a greater or lesser degree in reducing the infection by A. porri in vivo, could not inhibit the pathogen in vitro. Casida and Lukezic (1992) reported that P. fluorescens strain 679-2 was able to reduce the severity of the leaf spot disease caused by A. solani. Sastrahidayat (1995) found that in greenhouse trials, two Penicillium isolates and Trichoderma sp. gave some control of the pathogen A. porri on garlic but in the field only the Penicillium isolates were effective, and these for only up to three weeks after inoculation. Martinez and Salano (1995) studied the antagonism of ten Trichoderma strains against A. solani on tomato. Strains L12 and L17 showing three types of antagonism gave 45.7 and 39.77 per cent control of A. solani respectively. Integrated control of purple blotch disease (A. porri) of garlic was tested during January-August 1992 in the laboratory, greenhouse and field. Saprobes isolated from garlic leaves included Fusarium sp., Trichoderma viride, Curvularia sp., Penicillium sp. and Rhizopus sp. T. viride showed high antagonistic ability compared with the other isolates. Treatments including the use of antagonist T. viride (10,000 spores/ml), covering crops with transparent polyethylene sheets and combinations of these treatments inhibited growth of the pathogen in the field (Sastrahidayat, 1995). Liuchienhui et al. (1997) reported that Bacillus megaterium MBS4, Bacillus breveis OBS11 and Bacillus subtilis var. globigii CBS10 were antagonistic to A. solani under field conditions. Silva et al. (1998) studied the potential of fluorescent Pseudomonas spp. to control Alternaria leaf spot on castor bean (Ricinus communis), caused by Alternaria ricini, under greenhouse conditions. Among the antagonists tested, P. fluorescens JA4 and BJ22

were the most effective, showing a reduction in disease severity of 20.9 per cent and 17.8 per cent, respectively, when applied simultaneously with A. ricini. Strains of P. fluorescens were tested in vivo and in vitro for their biological control potential against A. solani in vitro, all six strains of P. fluorescens inhibited A. solani growth by 28-40 per cent compared to the control. Spraying tomato, cv. PKM-1, plants with suspensions of the P. fluorescens strains 48 hours after inoculation with A. solani reduced leaf blight disease by 15-38 per cent compared to the control (Babu et al., 2000). Chethana (2000) indicated that the bioagent P. fluorescens was least effective in controlling the spread of purple blotch disease of onion caused by A. porri. Mathivanan et al. (2000) evaluated two biocontrol agents, viz. T. viride and P. fluorescens were evaluated against foliar disease of sunflower. Their efficacy was compared with that of commonly used fungicides. The results revealed that T. viride was equally effective as that of fungicides in controlling Alternaria blight (A. helianthi) in sunflower. P. fluorescens was ineffective. Seed treatment with T. viride eliminated seed borne infection of pigeon pea by A. alternata (Fr.) Keissler with significant increase in seed germination, vigour index and fresh weight of seedling over untreated control (Pradeep Kumar et al. (2000). Field experiments were conducted in the rabi seasons of 1998 and 1999 to investigate the effects of plant extracts (from Pithecellobium dulce and Prosopis juliflora leaves), plant oils (palmarosa oil at 0.1 and 0.05 % and neem oil at 3 %) and biological control agents (T. viride, P. fluorescens and B. subtilis) in controlling leaf blight of onion cv. CO-4 caused by A. porri. The lowest disease percentage was obtained with palmarosa oil at 0.1 per cent, sprayed at the first appearance of disease symptoms and 15 days after the appearance of the disease. This treatment produced yield at 7650 kg/ha, which was higher than the control. The bioagents used were moderately effective in controlling the disease (Mohan et al., 2001). Experiments were conducted during the 1999-2000, 2000-01 and 2001-02 rabi cropping seasons to determine the effects of soil solarization, biological control agents, organic amendments, fungicides, and cultural methods on fungal pathogens responsible for seedling mortality in onion, brinjal, chilli and cabbage nurseries. All the treatments significantly increased the total plant stand percentage compared to the control in onion, aubergine and cabbage, while it was not significant in chilli (Champawat and Sharma, 2003). Mandhare and Suryawanshi (2003) reported that Bacillus thermophillus produced a thermo-labile metabolite toxic to A. porri (purple blotch of onion) and A. alternata (leaf spot of pomegranate). B. thermophillus restricted the fungal growth and invasion in host plants, and caused morphological changes in the germ tubes or mycelia of these pathogens. Tejeda et al. (2003) reported that the bioproducts obtained from B. subtilis in pure and mixed fermentations (BIOBAC and DIMABAC) were antagonistic to A. solani and A. porri. The pure and mixed bioproducts obtained from Azotobacter chroococcum (DIMARGONA and DIMABAC) stimulated growth in tomato seedlings, increasing root length between 25 and 50 per cent, shoot length by five to 10 per cent, plant height between 13 and 18 per cent and foliar area between 25 and 47 per cent compared to an absolute control. Verzignassi et al. (2003) investigated the effects of effective microorganisms (EM-4, incorporated in the organic matter; and EM-5, applied before planting) on the control of garlic purple blotch (A. porri) for two years. Both EMs were also sprayed on plants. Incubation with EM resulted in reduced disease severity. Chitra (2004) has documented induced systemic resistance in groundnut plants against A. alternata by inoculating groundnut seeds by biocontrol agents. Savitha (2004) observed induced systemic resistance against Alternaria blight in sesame, by seed treatment of salicylic acid and P. fluorescens.

The GR isolate of Trichoderma viride was at par with Mancozeb in checking Alternaria blight (Alternaria brassicae) severity on mustard leaves at Sewar while performance of the bioagent was significantly (P<0.05) inferior to the chemical fungicide at Ludhavai. Performance of the bioagent isolate GR of T. viride in checking the disease severity on pods was at par (P<0.05) with Mancozeb at both Sewar and Ludhavai, the treatment recording the lowest A-value (area under disease progress curve) on pods. While application of bulb extract of A. sativum resulted in highest seed yield at Sewar in 2001-2002, the bioagent isolate GR of T. viride did so at Ludhavai, both the treatments being at par (P<0.05) with Mancozeb and significantly higher than control (Meena et al., 2004).

3. MATERIAL AND METHODS

The present investigations were carried out in kharif/rabi seasons during the year 2007-08 in the farmer's field at Kurubagatti village, since Kurubagatti village is very near to University of Agricultural Sciences, Dharwad. Laboratory experiments were carried out in the Department of Plant Pathology, College of Agriculture, University of Agricultural Sciences, Dharwad, Karnataka. Survey for disease incidence and prevalence of leaf blight disease on chrysanthemum in the parts of northern Karnataka were conducted. Dharwad is situated in northern transitional zone (Zone 8) of Karnataka state at 15º15' N latitude, 75º7' N longitude and at an altitude of 774.0 m above mean sea level. The mean maximum and minimum temperatures, relative humidity of morning and evening along with rain fall data of Main Agricultural Research Station (MARS), University of Agricultural Sciences, Dharwad. For 2007-08 are presented in Appendix I. The details of the materials used and the methodology adopted during the course of investigations are presented in this chapter.

3.1

General procedure

3.1.1 Glassware and Cleaning

For all the laboratory studies Corning and Borosil glassware were used. The glassware were kept submerged overnight in the cleaning solution prepared by dissolving 60 g of potassium dichromate (K2Cr2O7) and 60 ml of concentrated sulphuric acid (H2SO4) in one litre of distilled water. Then, they were washed with vim powder followed by rinsing several times in running tap water and finally when needed for studies, rinsed in distilled water.

3.1.2 Sterilization

All the glassware used in the studies were sterilized in an autoclave at 1.1 kg cm-2 pressure for 20 min. and then dried in a hot air oven at 60°C. Both solid and liquid media were sterilized at 1.1 kg/cm² pressure for 15 min. The soil for pots was sterilized at 20 lb pressure for 30 min.

3.2

Survey for disease incidence of leaf blight in chrysanthemum

A roving survey was conducted to know the per cent disease incidence of leaf blight disease of chrysanthemum in the farmers field of Dharwad, Gadag Haveri and Koppal districts of northern Karnataka during kharif/rabi 2007 when the crop was three to four months old. The leaf blight disease incidence was assessed by recording the number of plants showing disease symptoms and the total number of plants examined. In each village, five fields were selected and in each field ten plants were examined randomly and scored for disease incidence by using following formula. No. of diseased plants Per cent disease incidence = Total no. of plants examined × 100

3.3

Isolation, identification and proving the pathogenicity of the fungus

3.3.1 Collection and isolation of the pathogen

The leaves of chrysanthemum (Dendranthema indicum L.) showing typical symptoms of the disease were collected from a farmer's field of Kurubagatti during the year 2007-08. The standard tissue isolation procedure was followed to isolate the pathogen. The infected leaf bits were surface sterilized with 1:1000 mercuric chloride (HgCl2) solution for 30 seconds and repeatedly washed separately in sterilized distilled water to remove the traces of mercury if any and then transferred to sterilized Petri plates (1-2 leaf bits per Petri dish) containing potato dextrose agar (PDA).

The Petri plates were incubated at room temperature (27±1°C) and observed periodically for the growth of the fungus. Bit of fungal growth developed from the infected tissue was transferred to PDA slants and incubated at 27° ±1°C for 12 days. Then such slants with pure culture were used for further studies.

3.3.2 Single spore isolation

Ten ml of clear, sterilized water agar of two per cent strength was poured into Petri plates and was allowed to solidify. Dilute spore suspension was prepared using sterile distilled water from 12 days old culture. One ml of such suspension was spread uniformly on two per cent water agar plates. The plates were incubated at 27±1°C for eight hours. Then, such plates were examined under microscope to locate germinated conidia. Single isolated and germinated conidia were then marked under the microscopic field with ink on the surface of the plate. These marked agar areas were cut and transferred to PDA slants with the help of cork borer under aseptic conditions and incubated at a temperature of 27±1°C. The growth of fungus in slants was used to study the morphological characters. As single spore isolates were identical and hence they were multiplied further. Pure culture derived from such slants was used for further studies.

3.3.3 Maintenance of the culture

The pathogen were sub cultured on PDA slants and allowed to grow at 27 ± 1°C for ten days and such slants were preserved in a refrigerator at 5°C and renewed once in 30 days.

3.3.4 Identification of the pathogen

The study was undertaken to confirm the identity of the isolated pathogen. Identification of the fungus was made after examining one hundred conidia under microscope (under 10x) from mature pure culture of the fungus obtained from infected leaves of chrysanthemum. Stage and ocular micrometer were used to measure the length, breadth, beak length and number of septa of the fungus. The average length and breadth of the conidial body, beak and septal number were recorded. These observations were compared with those of the standard measurements given by Ellis (1971) to identify the pathogen. Further, the identity of pathogenic isolates of Alternaria was confirmed by the Division of Mycology, Agharkar Research Institute, Pune, Maharashtra. The pathogenic isolate of Alternaria alternata was used for further studies.

3.3.5 Proving the pathogenicity

Chrysanthemum cuttings were raised in earthen pots, size 6" × 5", filled with sterilized soil. Plants were thoroughly cleaned with sterilized distilled water using moist cotton. Later, the plants were sprayed with distilled water. They were covered with polythene bags for 24 hr. The inoculum suspension from ten day old culture was prepared in sterile distilled water and sprayed on to the plants. Similarly control plants were sprayed with sterile distilled water for comparison. The cuttings were covered with polyethylene bags and were incubated for 48 hours to ensure successful penetration of the pathogen into the tissue. The polythene bags were removed after five days and seedlings were kept under greenhouse conditions. Observations were made regularly for the appearance and development of symptoms. After appearance of disease symptoms, re-isolation was made from the diseased tissues of artificially infected plants. The isolate obtained was compared with the original culture for confirmation of fungus under study. Techniques of serial dilution and purification were done as suggested by Sankaram (1961).

3.4

Disease development in relation to environmental factors

The effect of weather factors like temperature (maximum and minimum), relative humidity (morning and evening in per cent) rainfall (mm) and number of rainy days on the

incidence and development of leaf blight were studied in the farmer's field of Kurabagatti village in Dharwad taluk. Since, Kurabagatti village is very near to University of Agricultural Sciences, Dharwad main campus, the meteorological observations at Main Agriculture Research Station, UAS - Dharwad was used for this experiment. This study was undertaken during the year 2007-08 the observations were made on disease incidence and severity starting from first day of its appearance and till the end of the crop. It was correlated with weather parameters by simple correlation. In field 10 plants were examined randomly and scored for disease severity by following 0-5 scale. The details of scales are as shown below. 0 1 2 3 4 5 1969). Sum of numerical ratings PDI = Total number of leaves examined × 100 Maximum grade value No disease symptoms. A few spots towards tip covering 10 per cent leaf area. Several dark brown patchs covering upto 20 per cent leaf area Several patches with paler outer zone covering upto 40 per cent leaf area. Leaf blight covering upto 75 per cent leaf area or breaking of the leaves from center. Complete drying of the leaves or breaking of the leaves from center. Per cent disease index (PDI) was calculated by using the following formula (Wheeler,

3.5

To evaluate fungicides, botanicals and bioagents against the pathogen under in vitro and in vivo conditions

3.5.1 In vitro evaluation of fungicides, botanicals and bioagents against the pathogen

3.5.1.2 In vitro evaluation of fungicides The efficacy of non-systemic fungicides and systemic fungicides against Alternaria alternata were assessed by poisoned food technique. The details of fungicides tested are given below. Required quantities of individual fungicides were added separately into molten and cooled potato dextrose agar so as to get the desired concentration of the fungicides. Later 20 ml of the poisoned medium was poured into sterile Petri plates. Mycelial discs of 5 mm size from actively growing culture of the fungus were cut out by a sterile cork borer and one such disc was placed at the centre of each agar plate. Control was maintained without adding any fungicides to the medium. Each treatment was replicated thrice. Then such plates were incubated at room temperature for eight days and radial colony growth was measured. The efficacy of a fungicide was expressed as per cent inhibition of mycelial growth over control that was calculated by using the formula suggested by Vincent (1947). The per cent values were converted to arc sin transformations, the datd were analysed statistically.

Fungicides Mancozeb Propineb Chlorothalonil Iprodione Carbendazim12%+ Mancozeb 63% WP Carbendazim25%+ Iprodione 25% WP Hexaconazole Propiconazole Difenconazole 3.5.1.3 In vitro evaluation of plant extracts

Trade name Dithane M-45 Antracol 70% WP Kavach 75% WP Rovral 50%WP Saaf 75% WP Quintal 50% WP Contaf 5% EC Tilt 25% EC Score 25% EC

Concentrations (%) 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3 0.1, 0.2 & 0.3

Plant based pesticides which are relatively cheaper, safe and non hazardous can be used successfully against the plant pathogenic fungi. The present investigation was aimed to study the antifungal activity of some plant extracts. The following plant extracts were selected. Botanical name Azadirachta indica A. Juss Ocimum sanctum L. Allium cepa L. Allium sativum L. Tridax procumbens L. Azadirachta indica A. Juss Common name NSKE Holybasil/tulsi Onion Garlic Tridax Neem Family Meliaceae Labiateae Liliaceae Liliaceae Compositae Meliaceae Plant part used Seed Leaf bulb Clove Leaf Leaf Concentrations (%) 2.5, 5 & 10 2.5, 5 & 10 2.5, 5 & 10 2.5, 5 & 10 2.5, 5 & 10 2.5, 5 & 10

3.5.1.3.1 Preparation of cold aqueous extract Fresh plant materials were collected and washed first in tap water and then in distilled water. Hundred grams of fresh sample was chopped and then crushed in a surface sterilized pestle and morter by adding 100 ml sterile distilled water (1:1 w/v). The extract was filtered through two layers of muslin cloth. Finally filtrate thus obtained was used as stock solution. To study the antifungal mechanism of plant extracts the poisoned food technique was used (Nene and Thapliyal, 1982).Ten, five and 2.5 ml of stock solution were mixed with 90, 95 and 97.5 ml of sterilized molten PDA media, respectively so as to get 10, 5 and 2.5 per cent concentration. The medium was thoroughly shaken for uniform mixing of extract. Twenty ml of medium was poured into sterile Petri plates, mycelium of five mm size discs form periphery of actively growing culture were cut out by sterile cork borer and one such disc was placed on the centre of each agar plate. Controls were also maintained by growing the pathogen on PDA plates. Then such plates were incubated at room temperature for eight days and radial growth was taken when maximum growth occurred in the control plates.

The efficacy of plant products or botanicals was expressed as per cent inhibition of radial growth over the control which was calculated by using the formula (Vincent, 1947). (C-T) I= C Where, I C T = Per cent inhibition = Radial growth in control = Radial growth in treatment × 100

3.5.1.4 In vitro evaluation of biocontrol agents Different species of Trichoderma viz. T. harzianum Rifai, T. viride Persex. Fr., T. virens Miller, T. koningii Oudem, Pseudomonas fluorescens Migula and Bacillus subtilis Cohn Emend Pras, procured from Department of Plant Pathology, UAS, Dharwad, were tested in vitro against Alternaria alternata by using dual culture technique (Dennis and Webster, 1971). Twenty ml of sterilized potato dextrose agar medium melted and cooled at 45 C was poured aseptically into sterilized Petri dishes of nine cm diameter. Mycelial discs of five mm diameter cut from the edge of actively growing three days old culture of pathogen and mycelial discs (5 mm) of Trichoderma spp. cut from actively growing colony of the respective fungal species with the help of a sterilized cork borer were placed on the periphery about one cm from the edge of the Petri dish at opposite sides. In case of bacterial antagonist's evaluation, the bacterium was streaked at the centre of the Petri plate and two mycelial discs of the pathogen were placed at opposite ends. The Petri dish containing potato dextrose agar medium inoculated with the pathogen alone served as control. All the treatments were replicated thrice and were incubated at room temperature (27 + 1oC). After incubation when the growth of the pathogen was completed in the control, the colony diameter of Trichoderma spp. and that of the pathogen were measured in each treatment and the per cent inhibition of the pathogen over control was calculated by adopting the formula given by Vincent (1947). The data were analyzed statistically. The following growth media were used in above studies. 1. Potato Dextrose Agar (Hawksworth et al., 1983) Peeled and sliced potatoes Dextrose (C6H12O6) Agar-agar Distilled water Potatoes were boiled in 500 ml of filtering through a muslin cloth. Agar-agar water. The potato extract was mixed in the the mixture. The volume was made upto sterilized at 1.1 kg/cm2 pressure for 15 min. 200.00 g 20.00 g 20.00 g 1000 ml distilled water and the extract was collected by was dissolved separately in 500 ml of distilled molten agar and 20 g of dextrose was added to 1000 ml with distilled water and medium was

o

2. Martin's Rose Bengal Agar (Hawksworth et al., 1983) D-glucose Magnesium sulphate (MgSO4 . 7H2O) Peptone Potassium dihydrogen orthophosphate (KH2PO4) Rose bengal powder Streptomycin Agar-agar Distilled water 10.00 g 0.50 g 5.00 g 1.00 g 0.035 g 0.03 g 18 .00g 1000 ml

Agar-agar was dissolved in 500 ml of distilled water. All the ingredients were added and mixed thoroughly. The volume was made upto 1000 ml and medium was sterilized at 1.1 kg/cm2 pressure for 15 min. 3. Trichoderma Selective Medium (Elad and Chet, 1983) Ammonium nitrate Chloromphenicol Dexon 60 WP Dipotassium ortho-phosphate Glucose Magnesium sulphate Potassium chloride Rose Bengal Agar Agar Distilled water 1.00 g 0.25 g 0.30 g 0.90 g 3.00 g 0.20 g 0.15 g 0.15 g 15.00 g 1000 ml (to make up)

Sodium propionate 500 mg/lit and alkylaryl poly ether alcohol additive gives good growth of Trichoderma spp. in media.

4. Nutrient Agar (Patil and Alagawadi, 1992) Beef extract Peptone Sodium chloride Agar Agar Water pH 3.00 g 5.00 g 1.00 g 18.00 g 1000 ml (to makeup) 7 (to be adjusted)

5. King's `B' medium for isolation of Pseudomonas fluorescens (King et al., 1954) Ampicillin Chloromphenicol Cycloheximide Dipotassium orthophosphate Glycerol Magnesium sulphate Peptone Agar Agar Distilled water 50 ppm 12.50 ppm 100 ppm 1.50 g 10.00 ml 1.50 g 20.00 g 15.00 g 1000 ml (to makeup)

The organisms were cultured on specific media as indicated below.All isolates were purified further by single spore method or hyphal tip method as per procedure given by Rangaswami and Mahadevan (1999). Antagonists Media Reference/Source

Fungal antagonist Trichoderma harzianum Trichoderma specific medium Elad and Chet (1983)

Bacterial antagonists Pseudomonas fluorescens Bacillus subtilis King's B medium Nutrient agar King et al.(1954) Patil and (1992) Alagawadi

3.5.1.5 In vivo evaluations of fungicides, botanicals and bioagents A field experiment was conducted at Kurabagatti village of Dharwad taluk during kharif 2007-08 to know the efficacy of fungicides, botanicals and bio agent against Alternaria blight of chrysanthemum under natural field conditions, the treatment details are given below. Treatment details Hexaconazole Carbendazim 12% + Mancozeb 63% WP Propineb Pseudomonas fluorescens Nimbicidin Garlic clove extract Chlorothalonil Concentrations (%) 0.1 0.2 0.2 5 g/l 0.5 5.0 0.2

Mancozeb Carbendazim 25% + Iprodione 25% Control.

0.2 0.2 -

The experiment was laid out in Randomized Block Design (RBD) with three replications. Five plants in each sub plot were scored for disease and data were converted into Per cent Disease Index (PDI) as explained earlier. Overall view of the experimental plot has been given in Plate 1.

Plate 1. Overall view of the experiment plot.

4. EXPERIMENTAL RESULTS

Among the different foliar diseases of chrysanthemum, Alternaria leaf blight caused by Alternaria alternata (Fr).Keissler. is one of the major disease, causing yield loss up to 80 per cent. Hence, the present investigations on in vitro evaluation of fungicides, botanicals and bioagents against A. alternata were conducted in the laboratory during 2007 in the Department of Plant Pathology, College of Agriculture, UAS, Dharwad. Field evaluations of fungicides, botanicals and bioagents against Alternaria leaf blight of chrysanthemum were conducted in the farmer's field of Kurabagatti village, Dharwad taluk. During the present investigation a field survey was conducted to gather information on the severity of leaf blight of chrysanthemum in selected districts of northern Karnataka. Studied the leaf blight disease development on chrysanthemum in relation to weather parameters. The results thus obtained are presented in different sections under this chapter.

4.1

Survey for the disease incidence of Alternaria leaf blight of chrysanthemum

A roving survey was carried out for recording the incidence of Alternaria leaf blight disease of chrysanthemum during kharif/rabi 2007-08 in four major chrysanthemum growing districts of northern Karnataka viz.6+ Dharwad, Gadag, Haveri and Koppal. The village wise disease incidence has been presented in Table 1 and Plate 2. From the survey it is revealed that the disease was severe in all the districts during kharif /rabi 2007-08 and disease severity ranged from 33.33 to 78.42 per cent in different parts of the districts surveyed. The highest severity (78.42%) of Alternaria leaf blight was noticed in fields of Kurabagatti village in Dharwad district, where as least (33.33%) incidence of the disease was recorded at Lakkundi and Doni villages in Gadag district and Guttala village in Haveri district. The highest district average disease incidence was recorded in Dharwad (62.03%) followed by Haveri (49.70%), Gadag (46.33%) and least incidence was noticed in Koppal (40.93%) district. The highest disease incidence of Alternaria leaf blight in Dharwad district was observed at Kurubagatti (78.42%) of Dharwad taluk, where as least incidence was observed at Serewada (42.85%) in Hubli taluk. In Gadag district, highest disease incidence was recorded at Shiratti (58.38%), whereas least incidence was recorded at Lakkundi (33.33%) in Gadag taluk and Doni (33.33%) in Mundaragi taluk. In Haveri district the highest disease incidence was observed at Byadagi (65.00%), whereas lowest at Guttala (33.3%) of Savanur taluk. In Koppal district the highest disease incidence was recorded at Shivapur (46.66%) in Koppal taluk, whereas, lowest at Hosaslli (35.11%) in Yalaburga taluk. The disease appeared in the form of small, scattered dark brown to black spots, oval to irregular or angular on the leaf. Later these spots increased in size and coalesced covering larger leaf area, with dark brown margin and yellow halo inciting leaf blight and blossom blight and defoliation. The symptoms of the disease in different fields during field survey were photographed and are presented in Plate 3.

4.2

Isolation, identification and proving the pathogenicity of the fungus

4.2.1 Isolation of fungus

Isolation of the pathogen was made from chrysanthemum leaves showing typical symptoms of the disease. Leaves with such symptoms were collected for the isolation purpose. Standard tissue and single spore isolation methods were followed after surface disinfection as described in "Material and Methods". The pure culture of the fungus was obtained after eight days of inoculation which showed whitish growth at initial stage turning later to ash gray color. Such pure culture obtained was again sub cultured in potato dextrose agar slants and kept in the refrigerator at 5°C for further studies.

Table 1: Incidence of Alternaria leaf blight of chrysanthemum in different places surveyed during 2007-08 in northern parts of Karnataka District Taluk Village Kurabagatti Lokur Mangalagatti Narendra Dharwad Hubli Serewada Polykoppa Rayanala Gamanagatti Mavanur District Mean Sambalpur Lakkundi Soratur Adavisomapur Beldadi Athikatti Doni Doni thandya Kadampur Dindoor Shiratti Laxmeshwar District Mean Lingadahalli Nandihalli Halageri Itagi Kakola Byadagi Kadaramundagi Asundi Mannora Savanur Shirabedagi Guttala District Mean Bhosalli Koppal Koppal Yalaburga Kamnoor Hiresindalagi Shivapur Hosalli Bande Bande thande District Mean Variety grown Local variety (Gundi) Local variety (Gundi) Local variety (Gundi) Local variety (Gundi), Karnool Local variety, Karnool Local variety , Raja Local variety, sarwal Local variety, Local variety, Raja Mattur Mattur, Raja Mattur Mattur Mattur, Karnool Mattur, Karnool Mattur Mattur Mattur, Karnool Mattur Mattur Mattur Mattur, Sarwal Mattur(Sevanthige) Mattur, Karnool Mattur Local variety, Mattur Mattur Mattur Mattur Sevanthige (Gundi) Mattur, Raja Mattur, Raja Mattur, Raja Mattur, Raja, Karnool. Mattur, Raja Mattur, Raja, Sarwal. Mattur, Raja Mattur, Raja Mattur, Raja Disease incidence 78.42 68.00 69.90 65.43 42.85 47.05 70.00 60.00 56.66 62.03 46.66 33.30 40.00 46.66 53.33 40.00 33.33 56.66 40.78 50.12 58.38 56.84 46.33 63.33 45.29 61.25 39.41 53.33 65.00 40.12 53.75 53.33 47.50 40.80 33.33 49.70 45.70 39.30 37.50 46.66 35.11 41.12 41.17 40.93

Dharwad

Gadag

Gadag Mundargi

Shiratti

Ranebennur

Haveri

Byadagi

Savanur

Plate 2. Karnataka map showing district-wise average incidence of Alternaria leaf blight of chrysanthemum during Kharif 2007

Plate 3. Different fields and symptoms of Alternaria leaf blight observed during survey

Plate 4a. Proving pathogenicity

Plate 4b. Photographs showing culture and conidia of A. alternata

4.2.2 Pathogenicity test

For proving the pathogenicity on the host plant, the pathogen was artificially inoculated on the leaves of chrysanthemum plants as described in "Material and methods". After ten days of inoculation, the leaves exhibited symptoms of infection. The typical symptoms like small, dark brown to black spots were noticed on leaves of the artificially inoculated plants. The symptoms were photographed and are presented in Plate 4a. The pathogens were re-isolated from such leaves and the morphological character of the re-isolated organism was compared with the original culture of the pathogen which was similar in all respects. Hence, the causal agent of the disease was confirmed as Alternaria alternata.

4.2.3 Identification of the fungus

In culture the fungal colony was initially white, cottony with profuse aerial mycelium which gradually turned greenish grey (Plate 4b). Aged culture appeared completely black with no aerial mycelium. Conidiophores short to long, simple or branched arising singly and measuring 76.2 - 227.6 m long and 9.6 - 22.3 m wide. Conidiophores were hyaline to golden brown coloured with 2-11 septa. Conidia were observed to arise either singly or in chains at the tip of each conidiophore measuring about 63.37 - 197.48 m long and 17.28 48.53 m wide. Conidia are typically muriform, dark brown, thick walled, in long chains (9-15). Majority of conidia are non-beaked few with short rudimentary dark brown beaks, with a range of 11.06 - 55.13 m length and 2.45 - 6.53 m wide, conidia had 6 - 7 transverse septa and 0 - 3 longitudinal septa. Based on the characters of the colony and morphological characters of conidiophores and conidia the fungus was identified as Alternaria alternata.

4.3

Disease development in relation to weather parameters

4.3.1 Disease development (PDI)

In the present investigation, disease development in relation to weather parameters were studied as described in `Materials and Methods'. The planting was taken on the 4th day (July 20th) of 29th standard week during 2007. Observations were taken from 33rd standard week at weekly interval. The data are presented in Table 2 and depicted graphically in Fig. 1. The PDI was lowest during 33rd standard week (5.06) and increased through out the cropping period. It was peak during last stage that is 44th and 45th standard week (83.89 and 84.23) respectively (Table 2). During cropping period maximum temperature ranged from 26.3 C (38 standard week) to 310C (41st standard week), minimum temperature from 180C (45th standard week) to 20.60C (37th standard week), relative humidity (morning) from 66 per cent (42nd standard th week) to 93 per cent (37 standard week) and relative humidity (evening) from 44 per cent th th (45 standard week) to 83 per cent (35 standard week). However, rainfall was very erratic. It st th ranged from 0.8 mm (41 standard week) to 94.8 mm (38 standard week).

0 th

4.3.2 Correlation of PDI with weather parameters

The PDI obtained at different stages of crop growth were correlated with weather parameters prevailed during the respective stage. The correlation coefficients are presented in Table 3. The results in Table 3 reveals that during 2007, maximum temperature (r = 0.590) was significantly positively correlated with PDI, minimum temperature (r = -0.824) was significantly negatively correlated with PDI. Morning relative humidity (r = -0.690) and evening relative humidity (r = -0.755) were significantly negatively correlated with PDI. While, rainfall (0.068) was non-significantly positively correlated with PDI. The data are again subjected to multiple linear regression analysis. The regression co efficient for PDI is given in Table 4.

Table 2: Effect of weather parameters on Per cent Disease Index (PDI) of Alternaria leaf blight of chrysanthemum during 2007 Age of Crop (Days) Aug 13-19 Aug 20-26 Aug 27-Sep 2 Sep 3-9 Sep 10-16 Sep 17-23 Sep 24-30 Oct 1-7 Oct 8-14 Oct 15-21 Oct 22-28 Oct 29-Nov 4 Nov 5-11 30 37 44 51 58 65 72 79 86 93 100 107 114 5.06 9.33 12.33 15.00 21.00 40.66 52.66 57.66 68.00 71.00 75.55 83.89 84.23 Temperature ( C) PDI Maximum 27.7 28.8 26.7 26.7 29.3 26.3 27.3 29.6 31.0 30.4 28.1 29.3 30.7 Minimum 20.2 20.4 20.5 20.5 20.6 20.1 20.3 20.2 19.0 19.0 19.3 19.3 18.0 Morning 90 91 92 92 93 92 91 87 70 66 84 84 77 Evening 64 70 83 80 69 76 72 62 56 63 59 55 44

o

Standard week No. 33 34 35 36 37 38 39 40 41 42 43 44 45

Relative humidity (%)

Month and Date

Rainfall (mm) 1.80 30.0 19.2 12.8 32.0 94.8 34.8 3.20 0.80 62.2 8.60 5.80 48.2

PDI

100 90 80 70 60 50 40 30 20 10 0 33 34 35 36 37 38

Max. Temp.

Min. Temp.

RH-I

RH-II

Rainfall (mm)

39

Standard weeks

40

41

42

43

44

45

Fig. 1: Development of PDI and meteorological parameters associated during kharif 2007-08

The regression equation is Y= 594.95 ­ 5.66X1 -9.42 X2 + 7.81X3-1.16 X4-1.58 X5 With R2 =0.70 Where Y= PDI X1 =Maximum temperature (0C) X2 = Minimum temperature ( C) X3 =Rainfall (mm) X4 =Relative humidity (morning) (%) X5= Relative humidity (Evening) (%) The data was again subjected to step down regression by eliminating the nonsignificant factors and including only significant factors. The final equation is Y = 635.14 - 5.13 X1 -1.37 X2 -1.00 X4 -1.29 X5 Including variables maximum and minimum temperature and relative humidity 2 (morning and evening) with R = 0.70. According to these models, the observed and predicted disease severities of Alternaria blight of chrysanthumum during kharif/rabi 2007 are given in Table 5 and depicted in Fig. 2. The results in Table 5 gave actual and predicted PDI values. The mean difference between actual and predicted values is 0.03.

0

4.3.3 Disease prediction models

Weather factors play an important role in disease development when the vulnerable host and virulent pathogen interact. An attempt was made to predict the severity of Alternaria blight of chrysanthemum by using linear model. The disease severity was recorded at weekly interval on susceptible local variety (Gundi) during kharif/rabi 2007. Linear equation of different degree was best fit to predict the disease severity. The best fit linear model was Y = a + b1 t. The calculated value for a = 1.35, b1 = -0.17 with R2 = 0.96. Hence, the model was Yt = 1.35-0.17t.

4.4

In vitro and in vivo evaluation of fungicides, botanicals and bioagents

4.4.1 In vitro evaluation of fungicides

Six non-systemic (including two combi products) and three systemic fungicides were evaluated for their efficacy against A. alternata by poisoned food technique. The data are presented in Table 6. In the present study, it is clear that all the fungicides tested at all concentrations were significantly effective in reducing the mycelial growth of A. alternata (Plate 5a and Fig. 3). The inhibition of the growth over the control of the A. alternata ranged from 61.35 per cent to 100.00 per cent irrespective of the concentrations, Propiconazole and Hexaconazole were proved to be the most effective fungicides and recorded the highest reduction of mycelial growth (100%) which were superior over all other fungicides. The next best treatment was Iprodione (90.00%) which was on par with Difenconazale (88.94%) whereas, Iprodione + Carbendazim was least effective in reducing the fungal growth (61.35%). The effect of concentrations on A. alternata, irrespective of chemicals were found significant and most effective at 0.3% concentration. Maximum reduction of mycelial growth

Table 3: Correlation coefficient (r) for leaf blight of chrysanthemum with weather parameters during 2007

Weather parameters

Correlation coefficient (r)

Maximum temperature ( C)

o

0.590*

Minimum temperature ( C)

o

-0.824**

Relative humidity (morning) (%)

-0.690**

Relative humidity (evening) (%)

-0.755**

Rainfall (mm)

0.068

* = Significant at P=0.05 ** = Significant both at P=0.05 and P=0.01

Table 4: Multiple linear regression co-efficient for chrysanthemum leaf blight PDI in relation to weather parameters during 2007

Constant Parameter (A) X1 X2 X3 X4 X5 R

2

All the weather parameters

594.95

-5.66

-9.42

7.81

-1.16

-1.58

0.7 0

Significant weather parameters

635.14

-5.13

-1.37

-

-1.00

-1.29

0.7 0

X1 =Maximum temperature ( C) X2 = Minimum temperature ( C) X3 =Rainfall (mm) X4 =Relative humidity (morning) (%) X5= Relative humidity (evening) (%)

0

0

Table 5: Observed and predicted PDI of Alternaria blight of chrysanthemum during kharif 2007

Time interval (week) 1 2 3 4 5 6 7 8 9 10 11 12 13

Per cent Disease Index (PDI) Observed 5.06 9.33 12.33 15.00 21.00 40.66 52.66 57.66 68.00 71.00 75.55 83.89 84.23 Predicted 42.39 25.84 14.24 18.48 20.35 37.25 32.49 38.37 70.73 72.50 63.99 63.30 96.42 Deviation -37.33 -16.51 -1.91 -3.48 0.65 3.41 20.17 19.29 -2.73 -1.50 11.56 20.59 -12.19

Observed PDI

120

Predicted PDI

100

80

60

40

20

0 1 2 3 4 5 6 7

Standard weeks

8

9

10

11

12

13

Fig. 2: Observed and predicted PDI of Alternaria leaf blight of chrysanthemum

Table 6: In vitro evaluation of different fungicides in inhibiting the mycelial growth of Alternaria alternata after eight days of inoculation

Fungicides

Per cent inhibition of mycelial growth control Concentrations 0.1 % 0.2 % 76.11 (60.71) 78.36 (62.25) 62.03 (51.94) 89.81 (71.37) 75.36 (60.23) 75.73 (60.81) 100 (89.96) 100 (89.96) 88.69 (70.33) 74.61 (61.76) 0.3 % 77.44 (61.61) 78.36 (62.25) 62.95 (52.49) 90.36 (71.89) 78.77 (62.49) 89.81 (71.37) 100 (89.96) 100 (89.96) 89.99 (71.53) 76.78 (63.38)

over

Mean 75.81 (60.52) 77.24 (61.57) 61.35 (51.54) 90.00 (71.49) 76.94 (61.32) 80.31 (64.13) 100 (89.96) 100 (89.96) 88.94 (70.57) 75.10 (62.11)

Mancozeb Propineb Chiorothalonil Iprodione Mancozeb+carbendazim Iprodione+ carbendazim Hexaconazole Propiconazole Difenconazole Mean

73.88 *(59.24) 74.99 (59.97) 59.07 (50.21) 89.62 (71.2) 74.85 (61.22) 75.36 (60.22) 100 (89.96) 100 (89.96) 88.14 (69.83) 73.79 (61.18)

S. Em + Fungicides (F) Concentrations (C) FxC 0.46 0.25 0.79

CD at 1% 1.73 0.94 2.97

* Figures in parentheses are arcsine transformed values

100 Per cent inhibition of mycelial growth 90 80 70 60 50 40 30 20 10 0

b ze co an

0.10%

0.20%

0.30%

M

b ne pi o Pr

o al th ro lo Ch

l ni

le le le m zim zi zo zo zo da da na na na n o o en ico Ip be ac nc rb ar fe op ex ca c H Pr Di b+ e+ ze on co di an ro Ip M

ne io od r

Fungicides

Fig. 3: In vitro evaluation of different fungicides against Alternaria alternata

Plate 5a. In vitro evaluation of different fungicides

Plate 5b. In vitro evaluation of different botanicals

(76.78%) was observed at 0.3% concentration which was significantly superior over the rest of the concentrations. In case of interaction effect, Propiconazole (100.00%) and Hexaconazole (100.00%) at 0.1, 0.2 and 0.3 per cent were significantly superior to all other treatment combinations.

4.4.2 Effect of plant extracts on the growth of Alternaria alternata under in vitro condition

As plant extracts are cost effective and are available cheaply and in plenty, hold promise for their utilization in increasing crop production and in disease management. Therefore an effort was made to know the efficacy of these botanicals on inhibition of pathogens. The experimental results obtained are presented here under. The plant extracts were screened at three concentrations in the laboratory for their efficacy against the pathogen. Poisoned food technique as detailed in `Material and Methods' was followed. The data are presented in Table 7. Six botanicals were evaluated against A. alternata were found to be significant. NSKE (43.67%) was significantly superior over all other plant extracts evaluated. The next best treatment was neem leaf extract (15.18%). Least inhibition was noticed in case of tulasi (10.09%). Irrespective of the plant species, botanicals were found to be most effective at 10 per cent (18.07%) which was significantly superior over 2.5 per cent (13.59%) and 5 percent (15.66%) concentration. Interactions between botanicals and concentrations were significant. All the plant extracts reduced the mycelial growth with increase in concentrations. Maximum reduction of mycelial growth (50.07%) was noticed in case of NSKE followed by neem leaf extract (18.36%) at 10 per cent and NSKE (43.44%) at 5 per cent concentration. Least reduction of mycelial growth was noticed in case of tulasi (7.93%) at 2.5 per cent concentration (Plate 5b and Fig. 4.)

4.4.3 In vitro evaluation of bioagents through dual culture technique

The antagonistic effects of six antagonists viz. Trichoderma harzianum, T. virens, T.viridae, T. koningii, Bacillus subtilis and Pseudomanas fluorescens on growth of A. alternata was studied in vitro by dual culture method as explained under "Materials and methods". The results are presented in Table 8. The results revealed that the antagonists significantly reduced the growth of A. alternata either by competition (over growing) or by antibiosis (exhibiting inhibition zones). It was noticed that maximum reduction in colony growth of A. alternata was observed in T. harzianum (76.55%) which was significantly superior over all other bio agents tested. Next best was T. koningii (64.35%) and was on par with T. viridae (62.16%) and T. virens (60.49%). Least inhibition was noticed in Pseudomonas fluorescens (36.49%) followed by Bacillus subtilis (36.74%) (Plate 6a and Fig. 5.)

4.4.4 In vivo evaluation of fungicides, botanicals and bioagent

This study was under taken to evaluate the relative efficacy of different fungicides, plant extracts and bioagents as foliar spray against Alternaria leaf blight of chrysanthemum.The experiment was conducted during Kharif /Rabi season of 2007 with seven fungicides, viz. Hexaconazole (Contaf plus), Carbendazim 12% + Mancozeb 63% (Saaf), Propineb (Antracal), Chlorothalonil (Kavach), Mancozeb (Diethane M-45) and Carbendazim 25%+Iprodione 25% (Quintal). One commercial formulation of bioagents Pseudomonas fluorescens (Stanes Biocure- B), two botanicals out of which one commercial formulation of neem i.e., Nimbicidin and one was garlic clove extract were tested as described in "Material and Methods". Totally five sprays were given at 15 days interval starting from the initiation of the disease (30 days after planting). The observations on Alternaria leaf blight were recorded before each spray. Further, these observations were taken by following 0-5 disease scale and converted into per cent disease index (PDI) using the formula given by

Table 7: In vitro evaluation of different botanicals in inhibiting mycelial growth of Alternaria alternata after eight days of inoculation

Botanicals

Botanical name

Plant part used

Per cent inhibition of mycelial growth over control Concentrations Mean 2.5% 5% 10% 37.51 *(37.75) 7.96 (16.38) 12.95 (21.08) 13.08 (21.14) 12.22 (20.45) 11.47 (19.79) 13.59 43.44 (41.21) 9.74 (18.17) 13.37 (21.44) 14.37 (22.26) 13.03 (21.15) 15.70 (23.33) 15.66 50.07 (45.02) 12.59 (20.77) 13.88 (21.87) 15.36 (23.07) 16.25 (23.76) 18.36 (25.36) 18.07 43.67 (41.32) 10.09 (18.44) 13.40 (21.46) 14.25 (22.15) 13.83 (21.79) 15.18 (22.83) 15.77

NSKE

Azadirachta indica A. Juss Ocimum sanctum L.

Seed

Tulasi

Leaf

Onion

Allium cepa L.

Bulb

Garlic

Allium sativum L.

Clove

Tridax Neem leaves

Tridax procumbens L. Leaf Azadirachta indica A. Juss Mean

Leaf

S. Em + Botanicals (B) Concentration (C) B×C * Figures in parentheses are arcsine transformed values 0.27 0.18 0.47

CD at 1% 1.03 0.68 1.79

60

2.50%

5%

10%

Per cent inhibition of mycelial growth

50

40

30

20

10

0

N SK E l Tu as i i On on G li ar c ax id Tr s ve ea l

m ee N

Botanicals

Fig. 4: Bioefficacy of plant extracts against Alternaria alternata

Table 8: Efficacy of different bio agents in inhibiting mycelial growth of the Alternaria alternata under in vitro condition

Bio agents

Per cent inhibition over control 76.55 *(61.01)

Trichoderma harzianum

Trichoderma virens

60.49 (51.03)

Trichoderma viride

62.16 (52.01)

Trichoderma koningii

64.35 (53.32)

Bacillus subtilis

36.74 (37.29)

Pseudomonas fluorescens

36.49 (37.15)

S. Em +

0.80

CD at 1%

3.24

* Figures in parentheses are arc sin transformed values

80 Per cent inhibition of mycelial growth 70 60 50 40 30 20 10 0

um ian z ar ic Tr ire av rm de ho ns e ii rid ng ni vi a ko m a er d rm ho de ric ho T ic Tr b su i til s s en sc e or flu

rm de ho ic Tr

ah

B

i ac

llu

s

as on om ud se P

Bioagents

Fig. 5: Efficacy of different bio agents against Alternaria alternata

Plate 6a. Efficacy of different biogents against A. alternata

Plate 6b. Disease scale for Alternaria leaf bligh of chrysanthemum

Table 9: Field efficacy of fungicides, botanicals and bioagent in the management of Alternaria leaf blight of chrysanthemum during 2007

Per cent Disease Index (PDI) Treatments 30 DAS 45 DAS 60 DAS 75 DAS

Hexaconazole @ 0.1%

2.88 (9.77)* .92 (12.81) 4 4.89 (12.74) 5.27 (13.25) (12.14) 4.49

Carbendazim12%+Mancozeb63@0 .2%

28.47 (32.23) 21.17 (27.36) 17.80 (24.88) 27.27 (31.46) 19.93 (26.49) 17.42 (24.65) 19.66 (26.30) 32.00 (34.43) 46.5 (42.98)

34.73 (36.08) 33.10 (35.10) 36.31 (36.98) 43.17 (41.06) 26.67 (31.06) 21.78 (27.80) 22.57 (28.35) 45.47 (42.38) 56.95 (48.97) 32.57 S.Em +

48.41 (44.09) 51.11 (45.37) 68.88 (56.19) 70.66 (57.19) 46.22 (42.79) 34.66 (35.90) 40.00 (39.14) 68.44 (55.88) 84.55 (66.87) 51.78

58.30 (49.76) 55.51 (48.16) 72.37 (58.39) 74.88 (59.71) 53.73 (47.13) 37.12 (37.49) 44.22 (41.65) 74.72 (59.86) 87.44 (69.22) 56.35

42.48 (40.54) 40.22 (39.00) 48.84 (44.11) 53.99 (47.35) 36.64 (36.87) 27.74 (31.46) 31.61 (33.86) 55.15 (48.14) 68.86 (57.01) 41.00

Propineb @0.2%

Pseudomonas fluorescens @ 5 g/l

Nimbicidin @0.5%

Garlic clove extract @5%

Chlorothalonil @0.2%

Mancozeb @0.2% Carbendazim 25%+Iprodione 25% @0.2% Control. Mean

23.31

CD at 5% 3.26 2.06 6.52

Treatments (T) DAS (D) T×D

1.16 0.73 2.32

* Figures in parentheses are arc sin transformed values

wheeler (1969). The data on PDI of Alternaria leaf blight is presented in Table 9 and disease scale is photographed in Plate 6b. From the data it is observed that at 30, 45, 60 and 75 DAS all the treatments differ significantly over unprotected check. Maximum per cent disease index of Alternaria leaf blight was noticed in untreated check (68.86%). It was followed by Carbendazim 25% + Iprodione25% (55.15%) and least PDI was observed in Hexaconzaole (4.49%) which is followed by Chlorothalonil (27.74%). The days after spraying also differ significantly. Maximum PDI was noticed in 75 DAS (56.35%) and minimum was noticed in 30 DAS (23.31%). Interaction between the treatments and number of days after spraying were significant. Maximum PDI was observed in unprotected check (87.44%) at 75 DAS which was on par with unprotected check (84.55%) at 60 DAS minimum PDI was observed in Hexaconazole (2.28%) of 30 DAS which was on par with 45, 60 and 75 DAS. 4.4.4.1 Effect of fungicides, botanicals and bio agent spray on number of branches per plant Number of branches per plant were observed and presented in Table 10. As it can be seen from the Table 10, the highest number of branches per plant was observed from Hexaconazole (5.29) which is on par with Carbendazim 12% + Mancozeb 63% (5.13) and Propineb (4.12) treatments. The no. of branches per plant was least in untreated check (3.07) which was on per with Carbendazim 25% + Iprodione 25% (3.42). In case of number of days after spraying, the maximum number of branches per plant at 75 DAS (4.70) which was on per with 60 DAS (4.51) and least number of branches per plant at 30 DAS (3.66). In case of interaction effect in all the treatments. It was increase in the number of branches per plant with increase in the number of days after spraying. The highest branches per plant was observed in Hexaconazole (5.73) at 75 DAS which was on par with Hexaconazole (5.67) at 60 DAS, 45 DAS (5.13), Carbendazim 12% + Macozeb 63% (5.00, 5.40 and 5.57) at 45, 60 and 75 DAS, Propineb (5.00, 5.33 and 5.47) at 45, 60 and 75 DAS, Mancozeb (4.80 and 5.07) at 60 and 75 DAS. 4.4.4.2 Effect of fungicides, botanicals and bio agent spray on plant height Plant height was observed and presented in Table 11. Plant height was significantly superior in Carbendazim 12% +Mancozeb 63% (28.86 cm) which was on par with Propineb (28.72 cm). Untreated control (23.66cm) recorded least plant height which was on par with Chlorothalonil (24.43 cm). The Plant height was more at 75 DAS (30.41 cm) and least at 30 DAS (21.14cm). Interaction between treatments and number of days after spraying differ significantly. The highest height was observed in Carbendazin 12% + Mancozeb 63% (32.93 cm) at 75 DAS which was on par with Carbendazim 12% + Mancozeb 63% (32.27 cm) at 60 DAS, P. fluorescenes (30.67 cm) at 75 DAS, nimbicidin (31.04 cm) at 75 DAS, Garlic clove extract (30.67 cm) at 75 DAS, Carbendazim 25% + Iprodione 25% (30.33cm) at 75 DAS. The lowest was in untreated check (18.57cm) at 30 DAS which was on par with untreated check (21.71 cm) at 45 DAS, Chlorothalonil (18.63 cm) at 30 DAS, Nimbicidin (20.17 cm) at 30 DAS, Mancozeb (20.50 cm) at 30 DAS. 4.4.4.3 Effect of fungicides, botanicals and bio agent spray on number of opened flowers per plant The yield parameter like opened flowers at different spray intervals were recorded and presented in the Table12. The treatments were differ significantly over un protected check. Maximum opened flowers were from Propineb (23.87) which was on par with Hexaconazole (23.87), Mancozeb (23.34) and Garlic clove extract (21.82). Minimum numbers of opened flowers from untreated check (9.39). Number of flowers increased significantly with increase in the number of days after spraying. The highest number of opened flowers at 75 DAS (59.76) and lowest number of opened flowers at 30 DAS. Interaction Effect also differs significantly. Maximum number of opened flowers from Propineb (82.53) at 75 DAS which was on par with Garlic clove extract (76.67) at 75 DAS and Mancozeb (79.93) at 75 DAS.

Table 10: Field efficacy of fungicides, botanicals and bioagent on number of branches of chrysanthemum

No. of branches per plant Treatments 30 DAS 4.63 4.57 4.67 3.07 3.00 3.40 3.67 3.93 3.10 2.53 Mean 3.66 45 DAS 5.13 5.00 5.00 3.34 3.87 3.77 4.00 4.37 3.40 3.00 4.10 S.Em + Treatments (T) DAS (D) T×D 0.17 0.10 0.33 60 DAS 5.67 5.40 5.33 4.13 4.40 4.13 4.33 4.80 3.53 3.33 4.51 75 DAS 5.73 5.57 5.47 4.33 4.67 4.47 4.63 5.07 3.67 3.43 4.70 CD at 5% 0.46 0.29 0.93 Mean 5.29 5.13 5.12 3.74 3.98 3.94 4.16 4.54 3.42 3.07 4.24

Hexaconazole @ 0.1% Carbendazim12%+Mancozeb 63%@ 0.2% Propineb @0.2% Pseudomonas fluorescens @5 g/l Nimbicidin @ 0.5% Garlic clove extract @5% Chlorothalonil @0.2% Mancozeb @ 0.2% Carbendazim 25%+Iprodione 25%@ 0.2% Control.

Table 11: Field efficacy of fungicides, botanicals and bioagent on the plant height of chrysanthemum

Plant height (in cm) Treatments 30 DAS 20.43 23.57 24.30 21.04 20.17 22.60 19.57 20.50 21.57 18.57 Mean 21.14 45 DAS 26.03 26.67 28.07 25.17 25.03 25.77 22.44 24.73 26.03 21.71 25.16 S.Em + Treatments (T) DAS (D) T×D 0.40 0.26 0.81 60 DAS 30.20 32.27 30.90 29.37 29.33 29.93 27.87 29.33 29.87 26.73 29.58 75 DAS 30.73 32.93 31.63 30.67 31.04 30.67 27.77 29.71 30.33 27.63 30.41 Mean 26.85 28.86 28.72 26.56 26.39 27.24 24.43 26.07 26.95 23.66 26.57

Hexaconazole @ 0.1% Carbendazim 12% + Mancozeb 63%@0.2% Propineb @ 0.2% Pseudomonas fluorescens @ 5 g/l Nimbicidin @ 0.5% Garlic clove extract @ 0.5% Chlorothalonil @ 0.2% Mancozeb @ 0.2% Carbendazim 25% + Iprodione 25% @0.2% Control.

CD at 5% 1.14 0.72 2.28

Table 12. Field efficacy of different fungicides, botanicals and bioagent on the number of opened flowers of chrysanthemum

No. of opened flowers Treatments 30 DAS Hexaconazole @ 0.1% Carbendazim 12% + Mancozeb 63% @ 0.2% Propineb @ 0.2% Pseudomonas fluorescens @ 5 g/l Nimbicidin @ 0.5% Garlic clove extracts @ 5% Chlorothalonil @ 0.2% Mancozeb @ 0.2% Carbendazim 25% + Iprodione 25% @ 0.2% Control. Mean 3.53 3.00 2.00 0.67 1.33 1.20 1.97 1.83 2.50 1.67 1.98 45 DAS 6.27 6.87 4.33 4.00 4.00 2.87 4.03 4.00 4.07 4.00 4.44 S.Em + Treatments (T) DAS (D) T×D 1.12 0.71 2.24 60 DAS 12.20 14.33 7.73 10.13 6.20 6.53 6.60 7.60 7.20 7.98 8.64 75 DAS 73.50 32.10 82.53 66.87 46.07 76.67 68.33 79.93 47.67 23.97 59.76 Mean 23.87 14.07 24.15 20.42 14.40 21.82 20.23 23.34 15.36 9.39 18.70 CD at 5% 3.15 1.99 6.30 Total 95.5 56.3 96.59 81.67 82.33 87.27 80.93 93.36 61.44 37.62 -

Table 13: Field efficacy of different fungicides, botanicals and bioagent on the number of unopened flowers of chrysanthemum

No. of unopened flowers Treatments 30 DAS 14.47 17.53 10.00 10.00 11.33 10.67 19.33 18.27 9.17 5.33 Mean 12.61 45 DAS 62.73 15.90 38.67 38.67 22.33 42.67 41.67 36.47 24.53 10.00 33.36 S.Em + Treatments (T) DAS (D) T×D 1.66 1.05 3.31 60 DAS 67 32.20 81.27 71.13 65.06 88.83 74.53 89.73 54.47 24.75 64.90 75 DAS 8.4 17.73 8.00 5.00 3.00 8.33 6.33 7.47 4.53 0.33 6.91 Mean 38.15 20.84 34.48 31.20 25.43 37.62 35.47 37.98 23.17 10.10 29.45 CD at 5% 4.66 2.95 9.33 Total 152.6 83.36 137.94 124.8 101.72 150.5 141.86 151.94 92.7 40.41 -

Hexaconazole @ 0.1% Carbendazim 12% + Mancozeb 63% @ 0.2% Propineb @ 0.2% Pseudomonas fluorescens @ 5 g/l Nimbicidin @ 0.5% Garlic clove extract @ 5% Chlorothalonil @ 0.2% Mancozeb @ 0.2% Carbendazim 25% + Iprodione 25% @ 0.2% Control

4.4.4.4 Effect of fungicides, botanicals and bio agent spray on number of unopened flowers or number of flower buds per plant Number of unopened flowers per plant were recorded and presented in Table 13. Number of flower buds differs significantly in all the treatments. The highest number of flower buds were observed in Hexaconazole (38.15) treatment which was on par with Mancozeb (37.98), Garlic clove extract (37.62), Chlorothalonil (35.47) and Propineb (34.48). Untreated control has obtained (10.10) least. Days after spraying differ significantly. Maximum number of flower buds obtained at 60 DAS (64.90) and least was at 70 DAS (6.91). Interaction between the treatments and days after spraying also differ significantly. The highest number of flower buds in Mancozeb (89.73) at 60 DAS which was on par with Garlic clove extract (88.83) at 60 DAS and Propineb (81.27) at 60 DAS. 4.4.4.5 Economics of fungicides, botanicals and bio agent evaluation The economics of cost benefit ratio has been worked out for different fungicides, botanicals and bioagent are presented in Table 14 and depicted in the Fig. 6. The highest yield (76.25 Q/ha) and incremental cost benefit ratio was obtained by Hexaconazole (7.16) followed by Chlorothalonil over control.

Table 14: Economic analysis of management of Alternaria leaf blight of chrysanthemum

Treatments

PDI 4.49 *(12.14) 42.48 (40.54) 40.22 (39.00) 48.84 (44.11) 53.99 (47.35) 36.64 (36.87) 27.74 (31.46) 31.61 (33.86) 55.15 (48.14) 68.86 (57.01) 1.16 3.26

Yield (Q / ha.) 76.25

C:B ratio

Hexaconazole @ 0.1% Carbendazim 12% + Mancozeb 63% @ 0.2% Propineb @ 0.2%

7.16

63.5

4.25

63

4.09

Pseudomonas fluorescens @ 5 g/l

60.5

3.09

Nimbicidin @ 0.5 %

60

3.02

Garlic clove extract @ 5%

63

4.82

Chlorothalonil @ 0.2%

72

5.55

Mancozeb @ 0.2% Carbendazim 25% + Iprodione 25% @ 0.2% Control. S. Em+ C D at 5%

66

5.46

58

2.01

55 3.17 9.43

2.02 -

* Figures in parentheses are arcsine transformed values

80

70

60

50

40

1. Hexaconazole @ 0.1% 2. Carbendazim 12%+Mancozeb 63% @ 0.2% 3. Propineb @ 0.2% 4. Pseudomonas fluorescens @ 5 g/l 5. Nimbicidin @ 0.5 % 6. Garlic clove extract @ 5% 7. Chlorothalonil @ 0.2% 8. Mancozeb @ 0.2% 9. Carbendazim 25 +Iprodione 25% @ 0.2% 10. Control.

30

20

10

0 1 2 3 4 5 6 7 8 9 10 Bioagents

Fig. 6: Economic analysis of management of Alternaria blight of chrysanthemum

Plate7a. Treatment proved best with Hexaconazole (T1).

Plate 7b. Untreated check (T10)

5. DISCUSSION

Chrysanthemum is one of the most important commercial floriculture crops of India. The crop is subjected to attack by a number of diseases, of which Alternaria leaf blight caused by Alternaria alternata is serious and also a major limiting factor in cultivation of chrysanthemum (Cavallini et al., 1992). It is most conspicuous by its severe blighting on leaves and thus destroying most of the photosynthetic area of the plant. Disease was reported for the first time by Rao during 1965 on chrysanthemum plant, as Alternaria tenuis Auct (Alternaria alternate (Fr.) Keissler) from Maharastra. The disease appeared in an epiphytotic form during kharif 2007, damaging the major photosynthetic area of leaves on chrysanthemum at Dharwad and surrounding area. It caused a heavy destruction in the yield of chrysanthemum. It is possible that due to continuous growing of chrysanthemum throughout the year, which is the main host of the pathogen, the disease might have appeared in epiphytotic form because of inoculum buildup. In recent years, this disease has become a menace to chrysanthemum growers in Karnataka. Alternaria leaf blight of chrysanthemum is one of the most serious diseases in India. Three species of Alternaria have been reported on chrysanthemum in India. These are Alternaria chrysanthemi on Chrysanthemum indicum (Rao, 1964). Alternaria tenuis Auct (Alternaria alternata (Fr.) Keissler) on Chrysanthemum indicum (Rao, 1965), A. tenuissima on Chrysanthemum cinerariefolium (Trev.) var. pyrethrum (Srinath and Sarwar, 1965) and on Chrysanthemum indicum (Mallikarjunaiah and Rao, 1972). In the present study, leaf blight of Dendrathema indicum L. (Chrysanthemum indicum) caused by Alternaria alternata was observed in the farmer's field of Kuraubagatti village in Dharwad district. No detailed investigations of this serious disease has been made in Karnataka, though, for the past few years the disease is spreading in epiphytotic proportions in areas wherever chrysanthemum is being grown. Though few studies have been carried out on the life cycle of the pathogen and management of the disease, information regarding the intensity of disease, weather relation with disease development and effective biological and chemical management studies are scanty. Hence there is a need to study all these aspects in detail to suggest an effective management strategy to the farmers. Therefore to throw a light on the present pathogen in question on chrysanthemum, keeping the above objectives in view, in the present study an endeavor was made to gather more information on leaf blight of chrysanthemum. Several aspects considered and studied include identification and proving its pathogenicity on crop, survey for disease incidence, disease development in relation to weather parameters, in vitro and in vivo evaluations of fungicides, botanicals and biocontrol agents. The results obtained on the above aspects have been discussed hereunder.

5.1

Survey for the Alternaria leaf blight of chrysanthemum in northern parts of Karnataka

A detailed roving survey was undertaken during kharif/rabi 2007 in parts of northern Karnataka to gather information on the incidence, distribution and spread of Alternaria leaf blight on chrysanthemum from different localities. This information is highly useful to identify the hot spots of this disease in Dharwad, Gadag, Haveri and Koppal distrcts where chrysanthemum is extensively grown as commercial floriculture crop. From the survey it was revealed that the incidence of this disease varied from locality to locality depending on the type of chrysanthemum variety cultivated. The severity of disease was also dependent on inoculum load, agro-climatological situations prevailing in different localities. This is in agreement with Anonymous (2003 and 2006) wherein large variations in severity of Alternaria blight of sunflower was observed over a wide geographical tract. Among the districts surveyed, the incidence of disease was more in Dharwad district (62.03%) and less in Koppal district (40.93%) indicating that this disease was not consistent in all localities. This is in agreement with Patil (1999), who carried out intensive survey during Kharif 1998 and Rabi 1998-99 in northern parts of Karnataka to get precise information on the

incidence and intensity of leaf blight disease on garlic caused by Alternaria spp. It was revealed that incidence of disease is more severe in Dharwad and Gokak taluks. Hiremath et al. (1990) reported 95 to 100 per cent incidence of the Alternaria blight of sunflower disease in north Karnataka. The disease incidence was observed more in Dharwad district compared to all other districts surveyed. This may be due to farmers normally do not practice effective fungicidal spray against Alternaria leaf blight. Also the rainfall was continuous throughout the season that it did not allow for the fungicidal spray at least to those farmers who normally practiced it. Higher rainfall and relative humidity was reported to cause severe epidemics of Alternaria blight of sunflower (Kolte, 1984). With respect to individual talukas, Kurabagatti village (78.42%) of Dharwad taluk recorded highest incidence. This may be due to susceptibility of the cultivars and / or favorable environmental conditions like temperature and continuous rainfall experienced during the period under study. Another reason for the outbreak of high disease in Dharwad taluk may be continuous cropping of chrysanthemum in farmers field year after year without following crop rotation system. So that the infected debris left in the field serves as major source of infection, thus causing epidemic throughout the season. Continuous cultivation of any crop over the season and years will build up inoculum level to such an extent that the epidemic will become a common phenomenon (Chaube and Singh, 2001). This is clearly evidenced by looking at the disease incidence of Lakkundi and Doni villages of Gadag district and Guttala village of Haveri district which are lesser (33.30%) than the other parts of northern Karnataka. In these places majority of farmers are following their own cropping pattern and taking suitable management practices at suitable interval by foliar spraying of new molecules like triazoles.

5.2

Isolation, identification and proving pathogenicity

The symptoms of Alternaria leaf blight on chrysanthemum at Kurabagatti village of Dharwad taluk showed oval to irregular or angular, dark brown to black, blightened appearance on leaves and infected leaves dried leading to defoliation. In the present study, A. alternata was isolated from the leaves of chrysanthemum showing typical symptoms of leaf blight using tissue isolation and purified using single spore isolation method. The pure culture of the fungus obtained from these two methods was used in various laboratory studies. Working on artificial creation of Alternaria leaf blight epiphytotics in chrysanthemum for identifying sources of resistance. Dhiman and Chadha (1986) obtained pure culture of the fungus using tissue isolation method and described it as a new technique for inoculum preparation. Morphological characters are important tool in identification and classification of the fungus. In the present study, identification of fungus was made on the basis of important morphological characters such as shape, size, septation of conidiophores and conidia in the culture medium. The characteristics of the fungus in context were compared with all Alternaria alternata reported on the host. The conidiophores were short to long, simple or branched, arising singly and measuring 76.2 - 227.6 µm long and 9.6 - 22.3 µm wide. Conidiophores were hyaline to golden brown coloured with 2-11 septa. Conidia were observed to arise either singly or in chains at the tip of each conidiophore measuring about 63.37 - 197.48 µm long and17.28 48.53 µm wide. Conidia are typically muriform, dark brown, thick walled, in long chains (9-15). Majority of conidia are non-beaked few with short rudimentary dark brown beaks, with a range of 11.06 -55.13 µm and 2.45 - 6.53 µm wide, conidia had 6 - 7 transverse septa and 0- 3 longitudinal septa. In the present study identity of the causal organism was further confirmed as A. alternata. Schmidt (1958) for the first time reported a fungal leaf spot of Chrysanthemum maximum L. caused by Alternaria chrysanthemi Simmons and Crosier from Austrian Tyrol. Srinath and Sarwar (1965) for the first time reported Alternaria blight of Chrysanthemum

cinerariefolium (Trev.) var. pyrethrum caused by Alternaria tenuissima (Fries) Wiltshire from Bangalore. Rao (1965) reported Alternaria tenuis Auct. (Alternaria alternata (Fr.) Keissler.) causing leaf blight and blossom blight of Chrysanthemum indicum L. from Maharashtra. Hegde (1988) reported the leaf blight of Chrysanthemum morifolium Ramat caused by Alternaria tenuissima from Dharwad in Karnataka. In the present study also, A. alternata was consistently isolated from the diseased leaves of chrysanthemum confirms the above report. However, the identity of A. alternata was further confirmed by the Division of Mycology, Agharkar Research Institute, Pune, Maharashtra. The pathogenicity of the fungus was established by artificial inoculation of chrysanthemum plantings with the conidia of the pathogen. For proving the pathogenicity of a given organism, different methods are in practice. Comparing the efficiency of different forms of inoculum, Dhiman and Chadha (1986) concluded that spore/conidial suspension is the most effective inoculum. In the present study spray inoculation with conidial suspension method was tried which resulted in symptoms development. Typical symptom of the disease was observed on the leaves after ten days of inoculation. Initially, yellowish green spots of 3-4 mm were appeared on older uninjured leaves where as on injured leaves symptoms appeared after five days. These spots enlarged oval or irregularly turned to blackish or brown color. Eventually such spots covered entire leaf and leaf blightened. David (1998) had provided description for leaf spot disease of zinniae caused by Alternaria zinniae, in older plants the fungus attacks the older leaves and then spreads to the younger leaves, and when the attack is severe the spots may become confluent. In the present study, symptoms of the disease mentioned above and inoculation technique were found to be in agreement with the typical symptoms of the disease described earlier by many workers, (Schmidt,1958; Sobers, 1965; Srinath and Sarwar, 1965; Roa, 1965; Hegde, 1988; Karlatti and Hiremath, 1989; Cavallini et al., 1992; Ellis, 1998).

5.3

To study disease development in relation to environmental factors

The present investigation of Alternaria leaf blight on chrysanthemum was undertaken at Kurbagatti village of Dharwad taluk. The mean rainfall at Kurbagatti was 139.36 mm during the experimental period. The mean minimum and maximum temperature during this period ranged from 19.8oC to 28.1oC. The mean relative humidity ranging from 53 to 85 per cent was favourable for the disease development. The rainfall during experimental period might have favoured conidial germination, multiplication and disease development. In general the environmental conditions were favourable for the outbreak of disease in kharif 2007. Hence, Alternaria leaf blight was severe. The result obtained is in confirmation with the results obtained by the following workers, (Kolte and Mukhopadyay, 1973; Narain and Saksena, 1973; Agrawat et al., 1979; Herr and Lipps, 1981; Allen et al., 1983 a and b), on Alternaria blight of sunflower. In the weather studies the per cent disease index at weekly interval were also calculated. This was progressing at linear rate as the age of the plant was increasing. Kong et al. (1995) observed that susceptibility of sunflower tissues increased with age so that older leaves were more susceptible than young and expanding leaves. The minimum temperature had significant negative effect on the PDI. The Alternaria blight of sunflower was mainly depending on the weather, excessive sporulation of the conidia and given good incubation temperature, which has resulted in progressive increase of the disease during the study period. Bhaskaran and Kandaswamy (1980) have reported that prolonged minimum temperature of 20oC is very much congenial for the development of the disease. The relative humidity (morning) had negative correlation with PDI. The result obtained is against to the previous studies conducted by the workers like Kolte (1984), Hiremath et al. (1990), Rajiv kumar and Singh (1996) and Das et al (1998) in case of Alternaria blight of Sunflower. The reason for this may be due to variations in relative humidity that prevailed during morning hours in the cropping period.

In the present investigations rainfall had non significant positive effect on PDI. This may be due to highly erratic nature of the rainfall during the cropping year. Several workers (Hiremath et al., 1990 and Anilkumar et al., 1974) reported influence of rainfall on diseases development. But during the period of present investigation, the rainfall was too erratic. During kharif 2007 in 41st standard week rainfall was 0.8 mm, while in 38th standard week it was 94.8 mm. Such an erratic nature of rainfall does not give clear picture in correlation studies. The present situation was very much similar to the findings of Rajivkumar and Singh (1996) who reported that rainfall was not correlated with disease development of Alternaria blight of sunflower because of its erratic nature. The multiple linear regression equation 2 developed for kharif 2007 was 594.95 ­ 5.66 X1 ­ 9.42 X2 +7.81 X3 ­ 1.16 X4 -1.58 X5 with R value of 0.70 i.e., the weather factors put together influence PDI to the extent of 70 per cent. Step down regression method was followed to find out a meaningful result. The significant weather factors included were maximum and minimum temperatures and relative humidity (morning and evening) which gave regression equation of 635.14 ­ 5.13 X1- 1.37 X2 ­ 1.00 X4 2 ­ 1.29 X5 with R of 0.70. In the present investigation, to establish the relation of PDI with that of weather 2 parameters, only multiple regression method is employed. The R values are bit low because of erratic weather parameters. However, models other than the multiple regressions may give higher R2 values. Benagi (1995) noticed that RH-I and RH-II of kharif 1993, RH-1 and maximum temperature of kharif 1994 were found to have negative relationship with severity of late leaf spot of groundnut. In the present investigation, the weather studies lead to development of predication model for the estimation of per cent disease index (PDI) at any given time. The model 2 developed was linear i.e., Y =a +b t, where a = 1.35 and b = -0.17 with high R value of 0.96. This confirms the validity of the model in estimating the per cent disease index when the observed and predicted values were tabulated (Table 5), there was not much difference observed. However, Amaresh (2000) gave auto regression and logistic model for estimation of PDI of Alternaria blight of sunflower. In the present investigation, the logistic model was tried but did not fit well.

5.4

In vivo and in vitro evaluation of fungicides, botanicals and bio-agents

5.4.1 In vitro evaluation of fungicides

The use of fungicides has became an inevitable method in the management of plant diseases particularly in chrysanthemum in the absence of resistant cultivars to Alternaria blight. Foliar sprays give maximum protection against foliar diseases. Out of nine fungicides used, six were non-systemic (including two combi-products) and three were systemic. These were evaluated by means of poisoned food technique. The results revealed that Propiconazole and Hexaconazole at all the concentrations used (0.1%, 0.2%, and 0.3 %) were highly effective resulting in 100% inhibition of mycelial growth. The results obtained are in confirmation with the works of Mallikarjun (1996), Amaresh (2000), Anonymous (2000 and 2001), Mesta et al. (2003) and Gorawar (2004). Iprodione (0.3%) was also found effective to some extent. The efficacy of Iprodione alone was previously reported by several workers (Amaresh, 1997 and Anon., 2002b).

5.4.2 In vitro evaluation of botanicals

Contrary to the problems associated with the use of synthetic chemicals, botanicals are environmentally non pollutive, indigenously available, easily accessible, non phytotoxic, systemic ephemeral, readily biodegradable, relatively cost effective and hence constitute a suitable plant protection in the strategy of biological management of diseases. Hence, screening of plant products for its effective antifungal activity against the pathogen is essentially required to minimize the use of fungicides and to consider as one of the components in the integrated disease management (Khadar, 1999 and Nagesh, 2000).

In the present investigation all the six plant extracts tested at 2.5%, 5% and 10% concentrations were significantly effective in reducing the growth of A. alternata NSKE (50.07%) at 10 per cent proved to be the most effective botanical. This was followed by the NSKE (43.44%) at 5 per cent and neem leaves extract (18.37%) at 10 per cent concentrations. Extracts from neem contains a number of chemical compounds viz., nimbin (0.04%), nimbicidin (0.4%), nimibicidin (0.001%), nimbosterol (0.03%), essential oil (0.02%) tannin (6.0%) and margosine. Neem oil yields various acids, sulphur, etc. Meliantiol and azadiractin are obtained from seeds and decatylimbin also contains quecetin and sitosterol. The fungicidal spectrum of Azadirachta indica has been attributed to azadiractin which belongs to C25 terpenoides (Subramaniam and Shrinivas, 1993). Inhibition of A. alternata in the present investigation by neem products may also be because of the same reasons. The present investigation of various botanicals inhibiting the growth of A. alternata is in line with the earlier findings (Shekhawat and Prasad, 1971; Sheik and Agnihotri, 1977; Chary et al., 1984; Saksena and Tripathi, 1985; Garg and Siddiqui, 1992; Neetha and Sharma, 1992; Ganapathy and Narayanasamy, 1993; Datar, 1994; Amaresh et al., 1998; Rahman et al., 1999; Rashmi and Yadav, 1999; Amaresh, 2000; Singh and Majumdar, 2001; Rao, 2006; Pramod Kumar 2007). In the present investigation, though complete inhibition of the pathogen was not observed in any of the plant extract tested but considerable amount of inhibition was noticed in some of them and holds promise with their use in management of Alternaria leaf blight of chrysanthemum.

5.4.3 In vitro evaluation of biocontrol agents against Alternaria alternata

Biological control through the use of antagonistic microorganisms is a potential, non chemical means of controlling plant diseases by reducing inoculum levels of pathogens. Such a management would help in preventing the pollution and also health hazards. In the present investigation, the antagonistic effect of different bio-agents was assessed against A. alternata by dual culture technique. Maximum reduction in colony growth of A. alternata was observed in T. harzianum which was significantly superior to all the other bioagents tested. Next best was T. koningii and T. viride. In general, species of Trichoderma, viz. T. harzianum, T. koningii, T. viride and T. virens showed more mycelial inhibition of organism compared to bacterial antagonists. This could be obviously attributed to several possibilities of existence of microbial interactions such as higher competitive ability, stimulation, antibiosis by these Trichoderma isolate over test pathogen. This has been enumerated by many workers (Porter, 1924; Ghaffar, 1969 and Naik and Sen, 1995). The antagonism of Trichoderma spp. against many fungi is mainly due to production of acetaldehyde, a carbonyl compound (Robinson and Park, 1966; Dennies and Webster, 1971). This may also be the reason for its antagonistic effect on A. alternata. Similar results wherein efficacy of Trichoderma spp. against Alternaria species was previously reported by Deshmukh and Raut (1992), Leifort et al, (1992), Rukmani and Mariappan (1994), Amaresh (2000), Kota (2003), Savitha (2004), Mesta (2006) Rao (2006) and Pramod Kumar (2007).

5.4.4 In vivo evaluation of fungicides, botanicals and bio-agents

The fungicides which were found effective in the laboratory conditions, along with Nimbicidin (azadirachtin 1500 ppm @ 0.5%), garlic clove extract (5%) and Bio-cure (Pseudomonas fluorescence) were evaluated under field conditions. Five sprays were given at 15 days interval starting from disease development. Experimental results revealed that Hexaconazole (0.1%) was effective in minimizing the per cent disease index and getting higher yields. This chemical not only increased yield by giving protection against the disease, but also increased number of branches per plant, plant height, number of opened flowers as well the flower buds than other fungicides. Mesta et al. (2003), Mesta (2006) and Anonymous (2002b) has reported triazoles as effective fungicides against Alternaria blight of sunflower. Chlorothalonil (0.2%) and Mancozeb (0.2%) were next to Hexaconazole in terms of efficacy. However, Mancozeb has been reported to be effective fungicide against A. alternata

(Abraham et al., 1976; Singh and Milne, 1974; Anon, 1998; Amaresh, 1997; Desai, 1998 and Sood and Sharma, 2002). But from the farmer's point of view, the economics of disease management is important. In the present investigation Hexaconazole has given highest total returns, net returns and additional returns over control than any other fungicides. Hexaconazole has also recorded highest incremental benefit: cost of 7.16. Since present day economists are advising for net return concept, Hexaconazole can be recommended to the farmers for the efficient management of Alternaria blight of chrysanthemum.

Future line of work

1. The survey data can well be interpreted by using softwares of Geographical Information System (GIS), which can reveal the facts about direction and intensity of movement of disease over time and space, which help in adopting management measures in a large scale. Use of remote sensing and satellite technology for possible crop loss due to diseases can also be done. 2. The weather studies can be continued for at least 7-8 years, so that a software package can be developed for prediction of disease at any given time using input variables. The different prediction models can be tested over a period of time for validity. 3. Field screening of more number of fungicides, botanicals and bio-agents and possibility of their integration need to be studied. 4. Screening of chrysanthemum genotypes / varieties for resistance to Alternaria leaf blight. 5. Developing integrated management practices. 6. Studies on persistence of fungicides.

6. SUMMARY AND CONCLUSIONS

Chrysanthemum is the important flower crop. It is one of the most important commercial floricultural crops of India, cultivated both for domestic market and export purposes. Chrysanthemum is top second cut flower in the world floricultural trade. The maintenance of quality and quantity of the crop is deteriorated by certain diseases. One of the most important diseases is the Alternaria leaf blight caused by Alternaria alternata (Fr) Keissler; the disease causes extensive damage to flower yield and quality. In view of the destructive nature of Alternaria leaf blight of chrysanthemum the present investigations were carried out planned with the following objectives. Survey for disease incidence of leaf blight of chrysanthemum. Isolation, identification and proving pathogenicity. To study the disease development in relation to weather parameters. In vitro and in vivo evaluations of fungicides, botanicals and bio-agents. Survey carried out during kharif /rabi 2007 revealed that incidence of Alternaria leaf blight of chrysanthemum was found in 4 districts of northern Karnataka. The highest per cent disease incidence was noticed in Kurabagatti village in Dharwad district. While the lowest was recorded in Doni and Lakkundi villages in Gadag district and Guttala village in Haveri district. On the basis of isolation and morphological studies, the pathogen was identified as Alternaria alternata (Fr) Keissler. Identity of the fungus was further confirmed by the Division of Mycology, Agharkar Research Institute, Pune, Maharashtra. The weather studies revealed that per cent disease index (PDI) was progressing at linear rate throughout the plant growth and it was negatively correlated with minimum temperature, relative humidity (morning and evening). While, positively correlated with maximum temperature and rainfall. The multiple regression model developed for PDI is Y = 594.95 ­ 5.66 X1 - 9.42 X2+7.18 X3 -1.16 X4 -1.58 X5 with R2 value of 0.70. Step down regression has modified the equation was Y = 635.14 -5.13X1 -1.37 X2 1.00 X4- -1.29 X5 with R2 value of 0.70 including the independent variables of maximum and minimum temperatures and relative humidity (morning and evening). The prediction model developed for PDI was linear i.e., Y =a +b, t, where a = 1.35 and b = -0.17; with high R2 value of 0.96. Out of nine different fungicides tested in vitro, Propiconazole and Hexaconazole at all the concentrations completely inhibited the mycelial growth of A. alternata. Among the six bio-control agents tested against A. alternata under laboratory condition in dual culture, Trichoderma harzianum recorded highest inhibition of radial growth. T. koningii, T. viridae, and T. virens were next in order. Bacterial antagonists proved to be least effective. Out of six plant extracts tested against A. alternata, NSKE followed by neem leaf extract and garlic clove extracts were highly inhibitory to A. alternata, while tulasi leaf extract proved to be least inhibitor. In case of field evaluation of fungicides, botanicals and bio-agent Hexaconazole (0.1%) effectively controlled the disease incidence which recorded very less per cent disease index. followed by Chlorothalonil (0.2%) and Mancozeb (0.2%). The other growth and yield parameters like number of branches per plant, plant height, number of opened flowers and number of flower buds were significantly superior in case of Hexaconazole (0.1%) spray treatment followed by Chlorothalonil (0.2%) and Mancozeb (0.2%). Hexaconazole recorded highest yield (76.25 Q/ha) and incremental benefit: cost of 7.16. Hence, it can be recommended to the farmers for the efficient management of Alternaria leaf blight of chrysanthemum.

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* Originals not seen.

Appendix I

Mean monthly meteorological data for the experimental year (2007-08) and the mean of past 57 years (1950 ­ 2006) of Main Agricultural Research Station, University of Agricultural Sciences, Dharwad.

Rainfall (mm) Month 2007-08 April May June July August September October November December January February March Total 86.40 65.00 220.10 211.20 176.00 180.80 74.80 54.00 Trace Trace 00.00 12.80 1081.10 1950-2006 2007-08 48.50 81.06 112.38 151.30 97.17 103.63 127.83 32.62 5.32 0.06 1.09 0.45 761.41 36.70 34.60 29.70 27.00 27.10 27.20 29.70 29.50 29.00 30.40 31.90 35.30

Temperature ( C) Mean maximum 1950-2006 37.35 33.73 28.88 29.09 26.99 28.57 30.07 30.08 29.37 29.62 32.52 36.41 Mean minimum

o

Relative humidity (%) 2007-08 1950-2006 75.52 65.91 80.95 87.00 85.98 81.91 75.84 68.03 62.96 62.81 51.19 55.81

2007-08 21.40 21.30 21.30 21.10 20.50 20.30 19.40 15.10 14.60 14.00 15.70 19.70

1950-2006 19.86 21.39 21.47 20.99 20.29 19.89 18.43 15.90 12.52 14.62 16.42 19.56 55 61 80 85 85 83 68 53 65 72 67 49

Appendix II

List of abbreviations and symbols used % µm Sp.spp Fig. gram ha hrs g

-1

: : : : : : : : : : : : : : : : : : : : : : :

Per cent Micro meter Species (Singular and plural) Figure (S) Gram (S) Hectares Hours Per gram Meter (S) Centimeter Milli meter Milli gram Minute (S) Degree celsius Second (S) Percent disease index Average Critical difference Standard error mean Co-efficient of variation And other co workers Namely Main Agricultural Research Station

m Cm mm mg min

0

C

Sec PDI Avg. CD S. Em CV et al. viz. MARS

Appendix III

Cost of agrochemicals, botanicals, bioagents and labour cost

Particulars Hexaconazole Carbendazim 12% + Mancozeb 63% Propineb Pseudomonas fluorescens Nimbicidin Garlic clove extract Chlorothalonil Mancozeb Carbendazim 25% + Iprodione 25% Labour cost Men labour Women labour Rent for sprayer 100 per head 80 per head 50 each Cost (Rs) 560 per liter 400 per kg 400 per kg 210 per kg 190 per liter 50 per kg 850 per kg 260 per kg 850 per kg

STUDIES ON LEAF BLIGHT OF CHRYSANTHEMUM CAUSED BY Alternaria alternata (FR.) Keissler

ARUN KUMAR G. S.

2008

Dr. B. C. KAMANNA Major Advisor

ABSTRACT

Among the several diseases, Alternaria leaf blight is one of the most destructive foliar disease which causes heavy loss in chrysanthemum. Survey during kharif/rabi 2007 revealed that Alternaria leaf blight was severe in all the four districts viz., Dharwad, Haveri, Gadag and Koppal.. Isolation and morphological studies revealed Alternaria alternata (Fr.) Keissler as causal organism. The weather studies revealed that per cent disease index (PDI) was progressing at linear rate throughout the plant growth and it was negatively correlated with minimum temperature, relative humidity (morning and evening). While, positively correlated with maximum temperature and rainfall. The prediction model developed for PDI was linear i.e., Y = a + b, t, where a = 1.35 and b = -0.17; with high R2 value of 0.96. Out of nine different fungicides tested in vitro, Propiconazole and Hexaconazole at all the concentrations (0.1%, 0.2% and 0.3%) completely inhibited the mycelial growth of A. alternata. Among the six bio-control agents tested against A. alternata under laboratory condition in dual culture, Trichoderma harzianum recorded highest inhibition of radial growth. T. koningii, T. viridae, and T. virens were next in order. Bacterial antagonists proved to be least effective. Out of six plant extracts tested against A. alternata, NSKE followed by neem leaf extract and garlic clove extracts were highly inhibitory to A. alternata, while tulasi leaf extract proved to be least inhibitor. In case of field evaluation of fungicides, botanicals and bio-agent Hexaconazole (0.1%) effectively controlled the disease incidence which recorded very less per cent disease index. followed by Chlorothalonil (0.2%) and Mancozeb (0.2%).

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