Read 2 - MRKOCAKI DOCUMENTO text version

Annals of Microbiology, 51, 145-158 (2001)

Use of Azotobacter chroococcum as potentially useful in agricultural application


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

Abstract ­ Great many papers on this genus have dealt with its significance in plant nutrition and its contribution to soil fertility. Because of this and for better clarity, this paper has been divided into three sections: azotobacteria and their natural habitat (the soil and plant); production of growth substances and their effects on the plant; and possibility of using azotobacteria in agriculture. As soil bacteria, azotobacteria cannot be studied without their natural environment. In Yugoslavia, research on azotobacteria dates back to 1956. The main focus of study was the soil as the natural habitat of these bacteria. Bacteria of the Azotobacter genus synthesizes auxins, cytokinins, and GA­like substances, and these growth materials are the primary substances controlling the enhanced growth. These hormonal substances, which originate from the rhizosphere or root surface, affect the growth of the closely associated higher plants. In order to guarantee the high effectiveness of inoculants and microbiological fertilizers it is necessary to find the compatible partners, i.e. a particular plant genotype and a particular azotobacteria strain that will form a good association. Key words: Azotobacter chroococcum, microbiological fertilizer, plant, soil.

INTRODUCTION The first species of the genus Azotobacter, named Azotobacter chroococcum, was isolated from the soil in Holland in 1901. Ever since, these bacteria have been studied by numerous authors, who have made a number of significant discoveries about this genus. Azotobacteria represent the main group of heterotrophic freeliving nitrogen-fixing bacteria. Azotobacter chroococcum and Azotobacter agilis are studied by Beijerinck (1901). In subsequent years several other types of Azotobacteria group have been found in the soil and rhizosphere such as Azotobacter vinelandii, Lipman (1903); Azotobacter beijernckii, Lipman (1904); Azotobacter nigricans, Krassilnikov (1949); Azotobacter paspali, Döbereiner (1966), Thomp-

* Corresponding author. Phone: 021 421-717; Fax: 621-212; e-mail: [email protected]


son and Skerman (1980); Azotobacter armenicus, Thompson and Skerman (1981); Azotobacter salinestris, Page and Shivprasad (1991). These nitrogen-fixing bacteria are important for ecology and agriculture. Along with nodular bacteria, azotobacteria was considered to be the most extensively studied genus among the saprophytes (Beijerinck, 1908; Winogradsky, 1938; Horner et al., 1942). As far back as the thirties, Winogradsky (1932) discovered that azotobacteria release ammonia into the soil. The first period of research on the genus was marked by studies on its morphological, cytological, and biochemical characteristics (Allison and Gaddy, 1940; Lipman and Mac Lees, 1940; Lee and Burris, 1943). Other areas of research included nutrient media used to grow azotobacteria, their reproduction methods, etc. (Jensen, 1955; Johnson and Magee, 1956; Pr§ 1963). ­a, The results showed that azotobacteria reproduce very well on nitrogen-free nutrient mediums, which marked the beginning of a new phase in azotobacteria research. Many authors tried to find a practical application of this ability, but their results turned out widely different and the conclusion was that the positive effects these bacteria had on the plant were more due to their production of certain growth substances than to their nitrogen-fixing activity (Georgiu and Menuke, 1964; Brown, 1976). At that time, the questions of azotobacteria ability to fix nitrogen and, a little later, of its effectiveness in stimulating and promoting plant growth and yields were re-opened. The significance of Azotobacter chroococcum in plant nutrition and its contribution to soil fertility was thoroughly elaborated in a number of papers. Thus, for better clarity, insight and understanding this paper is divided into three sections: azotobacteria and their natural habitat (the soil and plant); production of growth substances and their effects on the plant; and possibility of using azotobacteria in agriculture.

AZOTOBACTERIA AND THEIR NATURAL HABITAT As soil bacteria, azotobacteria cannot be studied without their natural environment. Thus, ever since the sixties, these microorganisms have been studied in the soil as their natural habitat (Mi§ ­ustin, 1953; Vojinova-Raikova, 1954; Jensen and Petersen, 1955; Vojinoviv, 1956; Blinkov, 1957, 1962; Kirsanina and Volkova, 1960; Kolker and Dahnova, 1960). The Soil - In Yugoslavia, research on azotobacteria dates back to 1956. The main focus of study was the soil as the natural habitat of these bacteria. Thus, Vojinoviv (1956) and Mickovski (1957, 1959, 1960) studied the abundance of azotobacteria in the soils of Serbia and Macedonia, respectively; Sariv and Ra§ ­oviv (1963a, 1963b) investigated azotobacteria dynamics in the soils of the Vojvodina province; Pr§ (1964) studied azotobacteria in the rhizospheres of the Istrian ­a peninsula; Raduloviv and Hauher (1967) and Raduloviv (1969) studied the dynamics of azotobacteria in the rhizosphere of wheat. It is to be pointed out that azotobacteria is not found in all types of soil. Moreover, its abundance varies as per the depth of the soil profile (Vojinoviv, 1961; Sariv, 1969a, 1969b). The abundance of diazotrophs in different types of soil is not always well documented, but information is found for azotobacteria (Rubenchik, 1963; Jensen, 1965; Vancura et al., 1965; Kaputska and Rice, 1976; Balandreau, 1986).

146 N. MRKOVA KI and V. MILIv

The presence of azotobacteria in the soils of Japan was studied by Rao et al. (1984), while Kole et al. (1988) investigated how widespread these bacteria are in the soils of eastern Canada. Thompson (1989a, 1989b, 1989c) determined azotobacteria abundance in vertisols, and Reddy and Reddy (1989) studied the competitive saprophytic survival of azotobacteria in black cotton soil. We examined the number of azotobacteria in chernozem soil of Vojvodina province (Mrkova­ki, 1997; Mrkova­ki et al., 1997a, 1998a, 1998b). The Plant - Studies on azotobacteria abundance in the rhizosphere of certain crops, which began almost simultaneously with research on the soil as the habitat of these bacteria, revealed that azotobacteria are much more abundant in the rhizosphere of plants than in the surrounding soil and that this abundance depends on the crop species (Sariv and Ra§ ­oviv, 1963a, 1963b). The rhizospheres of soybean and other legumes contain considerably larger number of these bacteria than the nearby soil, and the abundance also varies from one rhizosphere zone to the other. In the root zone of wheat, in contrast to maize, soybean, sunflower, and sugar beet (Sariv and Ra§ ­oviv, 1963b), no azotobacteria were found during the entire growing season, and their number was higher in the rhizosphere and the adjoining zone than in the nearby soil. Azotobacteria abundance in the soil also depends on the incorporated mineral fertilizer and the plant species grown and it varies during the growing season (Sariv, 1978). The incorporation of maize harvest residues, barnyard manure, and mineral fertilizer inhibits azotobacteria development in both the rhizosphere and the surrounding soil in wheat (Sariv et al., 1983). As pointed out by several authors (Rovira, 1965b; Macura, 1966), azotobacteria populations are not much higher in the rhizosphere compared to soil population. Vancura (quoted by Döberainer, 1974) mentioned high rhizosphere effects on azotobacteria only in the vicinity of Egyptian legume roots (up to 108 per gram of dry rhizosphere soil). Micev (1971), Döberainer (1974) and Barea et al. (1978), report the abundance of this bacterium in chernozem to be 9-37 × 102, while Mrkova­ki (1997) and Mrkova­ki et al. (1997a, 1998a) determined the number of azotobacteria in the rhizosphere of sugar beet to be 3-12 × 103. Previous research prompted the questions of azotobacteria survival in the soil and plant rhizosphere and influence of a particular plant genotype on its interrelationship with azotobacteria. These studies included several species, namely maize, wheat, sunflower, and sugar beet (Sariv et al., 1987, 1988, 1990a, 1990b, 1991; Thilak, 1993; Gururaj and Mallikarjuniah, 1995; Trifkoviv, 1996; Yadav et al., 1996; Mrkova­ki et al., 1996b, 1997b). Field trials carried out in different locations have demonstrated that under certain environmental and soil conditions inoculation with azotobacteria has beneficial effects on plant yields. The effect of Azotobacter chroococcum on vegetative growth and yields of maize has been studied by numerous authors (Hussain et al., 1987; Sariv et al., 1987; Martinez Toledo et al., 1988; Miliv and Sariv, 1988; Nieto and Frankenberger, 1991; Mishra et al., 1995; Pandey et al., 1998; Radwan, 1998), as well as the effect of inoculation with this bacterium on wheat (Emam et al., 1986; Rai and Gaur, 1988; Ga§ et al., 1990; Sariv et al., 1990a; ­iv Badyala and Verma, 1991; Tippanavar and Reddy, 1993, Elshanshoury, 1995; Pati et al., 1995; Fares, 1997). Studies of the effect of Azotobacter chroococcum on soil nitrogen balance also included plant species such as oats (Avena sativa L.) (Shabaev et al., 1991),

Ann. Microbiol., 51, 145-158 (2001) 147

Hordeum vulgare (Martinez Toledo et al., 1990), rice (Kawimandan, 1986), lettuce (Requena et al., 1997), and barley (Troitskaya and Troitskii, 1988; Saha et al., 1991; Belimov et al., 1998). The effects of inoculating sugar beet with strains of Azotobacter chroococcum have been studied by Krstiv et al. (1991), Sariv et al. (1991), Mrkova­ki et al. (1996a), Steinberga et al. (1996); Antipchuk et al. (1997); Mezei et al. (1997). Azotobacter chroococcum cells have been found in maize tissue (Hallberg, 1995; Li et al., 1995; Rai­eviv et al., 1995a, 1995b). The penetration of azotobacteria into plant tissue was studied in vitro using the tissue culture procedure. Callus mass increased in association with azotobacteria (Mezei et al., 1997/98). Mrkova_ki et al. (1995a) confirmed the presence of these bacteria in sugar beet calluses in vertical as well as in horizontal direction.

PRODUCTION OF GROWTH SUBSTANCES AND THEIR EFFECTS ON THE PLANT Growth substances, or plant hormones, are natural substances that are produced by microorganisms and plants alike. They have stimulatory or inhibitory effects on certain physiological-biochemical processes in plants and microorganisms. Brakel and Hilger (1965) showed that azotobacteria produced indol-3-acetic acid (IAA) when tryptophan was added to the medium. Vancura and Macura (1960), Burlingham (1964), and Hennequin and Blachere (1966), on the other hand, found only small amounts of IAA in old cultures of azotobacteria to which no tryptophan was added. Three gibberelin-like substances were detected by Brown and Burlingham (1968) in an Azotobacter chroococcum strain. The amounts found in the 14-dayold cultures ranged between 0.01 and 0.1 µg GA3 equivalent/ml. Bacteria of the genus Azotobacter synthesize auxins, cytokinins, and GA-like substances, and these growth materials are the primary substance controlling the enhanced growth of tomato (Jackson et al., 1964; Barea and Brown, 1974; Azcorn and Barea, 1975). These hormonal substances, which originate from the rhizosphere or root surface, affect the growth of the closely associated higher plants. Brown and Burlingham (1968) and Eklund (1970) have demonstrated in their papers that the presence of Azotobacter chroococcum in the rhizosphere of tomato and cucumber is correlated with increased germination and growth of seedlings. Furthermore, Bagyraj and Menga (1978), Martinez et al. (1997), Barakart and Gabr (1998), Puertas and Gonzales (1999) report that the dry weight of tomato plants inoculated with Azotobacter chroococcum and grown in phosphate-deficient soil was significantly greater than that of noninoculated plants. Reports have also be given of a beneficial effect that phosphate mobilizing organisms can have for other types of microorganisms also beneficial for agriculture, such as azotobacteria (Ramos et al., 1972). The beneficial influence of phosphate-solubilizing bacteria on survival of azotobacteria in the rhizosphere has been observed (Belimov et al., 1995). P uptake was highest folowing inoculation with both microbial species (Azotobacter + Glomus fascilatum) and aplication of 50% of the recommended P rate (Kshirsagar et al., 1994). Phytohormones (auxin, cytokinin, gibberellin) can stimulate root development. They are produced not

148 N. MRKOVA KI and V. MILIv

only by plants but also by rhizosphere bacteria in vitro, e.g. Azotobacter spp., Agrobacterium spp. and by AM fungi. There are many references to effects of phytohormone-producing microorganisms on nutrient uptake by influence on the root surface by an increased phosphatase activity (Höflich et al., 1994). Alkaline phosphatase activity in the peach roots was highest with Azotobacter chroococcum + P fertilizer (Godara et al., 1995). Results of a greenhouse pot experiments with onion showed that application of G. fasciculatum + A. chrooccocum + 50% of the recommended P rate resulted in the greatest root length, plant height, bulb girth, bulb fresh weight, root colonization and P uptake (Mandhare et al., 1998). Inoculation with Azotobacter + Rhizobium + VAM gave the highest increase in straw and grain yield of wheat plants with rock phosphate as a P-fertilizer (Fares, 1997). Elgala et al. (1995). concluded that with microbial inoculation rock phosphate could be used as cheap source of P in alkaline soils and that combined inoculation could reduce the rate of fertilizer required to maintain high productivity. Azotobacter chroococcum produces gibberelins, auxins, and cytokinins, as shown by Reliv et al. (1987), Martinez Toledo et al. (1989), Salmeron et al. (1990), and Gonzales Lopez et al. (1991). Reliv (1989) obtained 9.6-19.8 µg GA eq l/l medium of substance with gibberellic activity. Five cytokinins were identified in an Azotobacter chroococcum culture filtrate (Nieto and Frankenberger, 1989). A study by Govedarica et al. (1993) on the production of growth substances by nine Azotobacter chroococcum strains isolated from a chernozem soil has showed that these strains have the ability to produce auxins, gibberelins, and phenols and, in association with the tomato plant, increase plant length, mass, and nitrogen content. Azotobacter chroococcum strains isolated from the sugar beet rhizosphere (also grown on chernozem) were also shown to produce gibberelins; the growth of pea hypocotyl was equivalent to a GA3 concentration of 0.003-0.1 µg/cm3 culture (Miliv and Mrkova­ki, 1995). Developments in molecular procedures during the last decade, the increased sensitivity and other analytical techniques may allow more precise measurements in the future. Many developmental processes are not regulated by one signal hormone but are under multiple hormonal control (Barendse and Peters, 1995; Voasenek and Blom, 1996).

POSSIBILITY OF USING AZOTOBACTERIA IN AGRICULTURE For a number of years Azotobacter chroococcum was used in the former Soviet Union to inoculate seeds or roots of crop plants (Mishustin and Naumova, 1962). Results from other countries also indicate that inoculation with azotobacteria affects the plant growth, and sometimes the yields as well (Jackson et al., 1964; Rovira, 1965a; Denarié and Blachere, 1966). Azotobacteria is used for studying nitrogen fixation and inoculation of plants due to its rapid growth and high level of nitrogen fixation (Jagnow, 1987; Gouri and Jagasnnatathan, 1995; Maltseva et al., 1995; Mrkova­ki et al., 1996a). However, despite the considerable amount of experimental data concerning azotobacteria stimulation of plant development, the exact mode of action by which azotobacteria enhances plant growth is not yet fully understood.

Ann. Microbiol., 51, 145-158 (2001) 149

Three possible mechanisms have been proposed: N2 fixation; delivering combined nitrogen to the plant; the production of phytohormone-like substances that alter plant growth and morphology, and bacterial nitrate reduction, which increases nitrogen accumulation in inoculated plants. The problem is to identified the cause of the varying efficiency of plant genotypes and microbial strains when free nitrogen-fixing bacteria are applied. According to Sariv et al. (1988), it may be assumed that the relationship between a plant genotype and a strain of microorganism depends on the following characteristics of the partners in the system: ­ the quantitative and qualitative composition of root exudates of the plant genotype; ­ the specificity of the microbial strain's metabolism; ­ the ability of the microbial strain and plant genotype to synthesize phytohormones; ­ the ability of the microbial strain to synthesize inhibitors; ­ influence of the plant genotype-microbial strain system; ­ the microbial strain's ability to colonize root surface and penetrate into plant tissue; ­ influence of the genotype-strain system on the changes of the rhizosphere environmental factors (pH, rH2, pO2, CO2, etc.); ­ the specificity of the plant genotype regarding nitrogen uptake and transport. On the basis of a similar discussion covering the factors in the nitrogen fixation system (Quispel, 1991; Kennedy and Tchan, 1992 ­ who studied nitrogen fixation in cereals ­ Kennedy et al., 1997) gave some new suggestions concerning the future research on the association of diazotrophs and cereals in the interest of more effective nitrogen fixation (the importance of adequate colonization; the oxygen paradox; opposed by the fact that this gas is simultaneously an essential electron acceptor yet is extremely toxic; the effective transfer of nitrogen to the host plant). Triplett (1996) concluded that the development of the diazotrophic endophytic association in maize appears to be the most likely route to success in the development of a corn plant which does not require nitrogen fertilization for optimum growth and yield. In opinion of Kennedy et al. (1997) the need for sustainable nitrogen ­ fixing systems is sufficiently great that there is an obligation on scientists to take some prudent risks in setting research goals. The probability of success of obtaining soon an effective associative system with wheat is low. The progress since 1992 suggests that the research is on schedule to deliver positive outcomes in the medium term of 5-15 years. The ability of a number of azotobacteria strains to colonize sugar beet roots and nitrogenase activity of the studied sugar beet hybrids are correlated with the movement of azotobacteria cells towards the root of the plant (Mrkova­ki et al., 1995a). Pelleted seed mixed with a liquid azotobacteria culture has a variable effect on the reproduction of these microorganisms under laboratory conditions ­ some strains grow well, others somewhat less well, and some even exhibit inhibited growth (Mrkova­ki et al., 1995b, 1999). By evaluating our data from the azotobacteria field inoculation experiments with sugar beet, accumulated over the past 10 years, it can be concluded that this bacterium is capable of improving the yields of agriculturally important crop sugar beet using various Azotobacter chroococcum strains and cultivars. The per150 N. MRKOVA KI and V. MILIv

centage of success due to inoculation with these bacteria was: from 4-26% in increase the sugar beet root yield; from 2.5-5.39% in increase of sugar content and from 7-24% in increase of crystallized sugar yield (Mrkova­ki et al., 2001). The challenge to the research community will be to develop systems to optimize beneficial plant-endophyte bacterial relationships (Sturz et al., 2000). In order to guarantee the high effectiveness of inoculants and microbiological fertilizers it is necessary to find compatible partners, i.e. a particular plant genotype and a particular azotobacteria strain that will form a good association. Ackonowledgements - We are grateful to Prof. Dr Zora Sariv of the Faculty of Agriculture, University of Novi Sad, for her constructive comments on the manuscript. We thank Vojin vupina for technical assistance.


Allison F.E., Gaddy V.L. (1940). Synthesis of coenzyme R. by certain rhizobia and by Azotobacter chroococcum. J. Bacteriol., 39: 273. Antipchuk A.F., Rangelova V.M., Tatsyurenko O.V., Shevcenko A.I. (1997). Effect of Azotobacter on the yield and quality of sugar beet. Mikrobiol. Zhurnal., 59: 90-94. Azcorn R., Barea J.M. (1975). Synthesis of auxins, gibberellins and cytokinins by Azotobacter vinelandi and Azotobacter beijerinckii related to effects produced on tomato plants. Plant Soil, 43: 609-619. Badiyala D., Verma S.P. (1991). Effect of Fym and biofertilizers on wheat equivalent yield, diversity index and land utilization index of maize + soybean-wheat cropping sequence under mid hills of Hemichal Predesh. Farming Systems, 7: 122-125. Bagyraj D.J., Menga J.A. (1978). Interaction between a VA mychoriza and Azotobacter and their effects on rhizosphere microflora and plant growth. New Phytol., 80: 567573. Balandreau J. (1986). Ecological factors and adaptive processes in N2-fixing bacterial population of the plant environment. Plant Soil, 90: 73-92. Barakart M.A.S., Gabr S.M. (1998). Effects of different biofertilizer types and nitrogen fertilizer levels on tomato plants. Alexandria J. Agricultural Research, 43: 149-160. Barea J.M., Brown M.E. (1974). Effect on plant growth produced by Azotobacter paspali related to synthesis of plant growth regulating substance. J. Appl. Bacteriol., 37: 583593. Barea J.M., Ocampo J.A., Azcon R., Olivares J., Montoya (1978). Effects of ecological factors on the establishment of Azotobacter in the rhizosphere. Ecol. Bull., 26: 325330. Barendse G.W.M., Peters T.J.M. (1995). Multiple hormonal control in plants. Acta Bot. Neer., 44: 3-17. Beijerinck M.W. (1901). Über ologonitrophile mikroben. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. II Abt., /: 561-582. Beijerinck M.W. (1908). Fixation of Free Atmospheric Nitrogen by Azotobacter in Pure Culture. Koninel. Ned. Acad. Weteucchapp, Prac. 11. Belimov A.A., Kojemiakov A.P., Chuvarliyeva C.V. (1995). Interaction between barley and mixed cultures of nitrogen fixing and phosphate-solubilizing bacteria. Plant Soil, 173: 29-37. Belimov A.A., Ivanchikov A.Y., Vorobev N.I. (1998). The effect of predominant flora of

Ann. Microbiol., 51, 145-158 (2001)


the barley rhizoplane on the interaction between introduced diazotrophs and the plant. Microbiology, 67: 340-345. Blinkov G.H. (1957). Geograficeskoje rasprostranenie azotobaktera v po­vah Sibirii. AN. ´ SSSR, Referativni ­urnal, Biologija, 1, Moskva. Blinkov G.H. (1962). O rasprostranenii i osobenostjah Azotobacter chroococcum. Mikrobiologija, 3. Brakel J., Hilger F. (1965). Etude qualitative et quantitative de la synthese de substances de nature auxinique par Azotobacter chroococcum in vitro. Bull. Inst. Agron. Stns. Rech. Gembloux, 33: 469-487. Brown M.E., Burlingham S.K. (1968). Production of plant growth substances by Azotobacter chroococcum. J. Gen. Microbiol., 53: 135-144. Brown M. (1976). Role of Azotobacter paspali in association with Paspalum notatum. J. Appl. Bacteriol., 40: 341-348. Burlingham S.K. (1964). Growth regulators produced by Azotobacter in culture media. Ann. Rep. Rothamsted. Exp. Stat., p. 92. Denarié J., Blachere H. (1966). Inoculation de graines de vegetaux cultives a l' aide de souches bacteriennes. Ann. Inst. Pasteur, Suppl. to 3: 57-74. Döberainer J. (1966). Azotobacter paspali sp. nov., uma bacteria fixadora de nitrogenio na rhizosfera de Paspalum. Pesquisa Agropecuaria Brasileira, 1: 357-365. Döberainer J. (1974). Nitrogen fixing bacteria in the rhizosphere. Biological Nitrogen Fixation, 33: 86-120. Eklund E. (1970). Secondary effects of some Pseudomonads in the rhizosphere of peat grown cucumber plant. In: Pharis R.P., Reid D.M., eds, Hormonal Regulation of Development. Vol 3, Springer-Verlag, N.Y., p. 613. Elgala A.M., Ishac Y.Z., Abdel Monem M., ElGhandour I.A.I. (1995). Effect of single and combined inoculation with Azotobacter and VA micorrhizal fungi on growth and mineral nutrient contents of maize and wheat plants. In: Huang P.M., Berthelin J., Bollag J.M., Mcgill W.B., Page A.L.,eds, Environmental Impact of Soil Component Interaction, Vol. 2: Metals, Other Inorganics, and Microbial Activities, PP. 109-116. Elshanshoury A.R. (1995). Interaction of Azotobacter chroococcum, Azospirillum brasilense and Streptomyces mutabilis, in relation to their effect on wheat development. J. Agronomy Crop Sci., 175: 119-127. Emam N.F., Fayez M., Makboul H.E. (1986). Wheat growth as affected by inoculation with Azotobacter isolated from different soils. Zentralbl. Microbiol., 141: 17-23. Fares C.N. (1997). Growth and yield of wheat plant as affected by biofertilisation with associative, symbiotic N2-fixers and endomycorrhizae in the presence of the different P-fertilizers. Ann. Agr. Sci., 42: 51-60. Ga§v O., Hong. Z., Popoviv M., Lukiv V., Sariv M., Sariv Z. (1990). Activity of nitroge­ Ø nase and nitrogen assimilation enzymes in wheat inoculated with Azotobacter chroococcum. Biochem. Physiol. Pflanzen, 186: 169-178. Georgiu V., Menuke L. (1964). Azotobacter chroococcum (§ ­tam 100) kak producent auksinopodobnih ve§ ­ vestv. V. sb. Bakterijalne udobrenija, 166-176. Godara R.K., Awasthi R.P., Kaith N.S. (1995). Effect of biofertilizer and fetilizer on Azotobacter population, crown gall infection and alkaline phosphatase activity in peach. Indian J. Plant Physiol., 38: 334-336. Gonzales Lopez, J., Martinez Toledo M.V., Reina S., Salmeron V. (1991). Root exudates of maize on production of auxins, gibberellins, cytokinins, amino acids and vitamins by Azotobacter chroococcum chemically defined media and dialysed soil media. Toxicol. Environ. Chem., 33: 69-78. Gouri P.S.V.M., Jagasnnatathan R. (1995). Biotechnology in organic farming. Biotechnol. Dev. Review, 5: 34-47.



Govedarica M., Miliv V., Gvozdenoviv Dj. (1993). Efficiency of the association between Azotobacter chroococcum and some tomato varieties. Soil plant, 42: 113-120. Guraraj R., Mallikarjuniah R.R. (1995). Interactions among Azotobacter chroococcum, Penicillium glaucum and Glomus fasciculatum and their effect on the growth and yield of sunflower (Helianthus annus L.). Helia, 18: 73-84. Hallberg T.B. (1995). Nitrogen fixing bacteria associated with maizes native of Oaxaca. In: Proceedings of the 10th International Congress on Nitrogen Fixation, St Petersburg, Russia, No 638. Hennequin J.R., Blachere H. (1966). Recherches sur la synthese de phytohormones et de composes phenoliques par Azotobacter et des bacteries de la rhizosphere. Ann. Inst. Pasteur, 3: 89-102. Höflich G., Wichc W., Kühn G. (1994). Plant growth stimulation by inoculation with symbiotic and associative rhizosphere microorganisms. Experientia, 50: 897-905. Horner C.K., Burk D., Allison F.E., Sherman M.S. (1942). Nitrogen fixation by Azotobacter as influenced by molybdenum and vanadium. J. Agric. Res., 65: 173-193. Hussain A., Arshad M., Hussain F. (1987). Response of maize (Zea Mays) to Azotobacter inoculation. Biol. Fert. Soils, 4: 73-77. Jackson R.M., Brown M.E., Burlingham S.K. (1964). Similar effects on tomato plants of Azotobacter inoculation and application of gibberellins. Nature, 203: 851-852. Jagnow G. (1987). Inoculation of cereal crops and forage grasses with nitrogen­fixing rhizosphere bacteria: possible causes of success and failure with regard to yield response. A review ­ Z. Pflanzenernahr Bodenkd., 150: 361-368. Jensen H. (1955). The Azotobacter-flora of some Danish water courses. Dansk bot. Tidsskr., 52: 143-157. Jensen H., Petersen E. (1955). Studies on the Occurence of Azotobacter in Danish Forest Soils. Royal Veterinary and Agricultural College Yearbook, pp.107-126. Jensen H.L. (1965). Nonsymbiotic nitrogen fixation. In: Bartholomew W.V., Clark F.E., eds, Soil Nitrogen. Amer. Soc. Agron. Inc. Publisher, Madison, pp. 436-480. Johnson M.K., Magee L.A. (1956). Some factors affecting the respiratory response of Azotobacter to 24-D, and related compounds. Appl. Microbiol. 4:169-178. Kaputska L.A., Rice F.L. (1976). Acetylene reduction (N2 fixation) in soil and old field succession in Central Oklahoma. Soil Biol. Biochem., 8: 497-503. Kavimandan S.K. (1986). Influence of Rhizobia, Azotobacter and blue green algae on Ncontent and yield of rice. Plant Soil, 96: 133-135. Kennedy I.R., Tchan Y.T. (1992). Biological nitrogen fixation in nonlegumes field crops: recent advances. Plant Soil, 141: 93-118. Kennedy I.R., Pereq-Gerk L.L., Wood C., Deaker R., Gilchrist K., Katupitiya S. (1997). Biological nitrogen fixation in non-leguminous field crops: Facilitating the evolution of an effective association between Azospirillum and wheat. Plant Soil, 194: 65-79. Kirsanina E.F., Volkova V.A. (1960). Nekotorie danne o rasprostanenii azotobaktera v po­vah Gorno - Altaiskoj avtonomnoi oblasti. Mikrobiologija, 29: 551-554. Kole M.M., Altasaar J., Page W.J. (1988). Distribution of Azotobacter in eastern Canadian soils and in association with plant rhizospheres. Can. J. Microbiol., 34: 815-817. Kolker I.I., Dahnova E.N. (1960). Rasprostanenie azotobaktera v po­vah Krima. Mikrobiologija, 29: 555-562. Krassilnikov N.A. (1949). Opredeliteli Bakterii i Aktinomicetov. Izd. AN sssr.M. Krstiv B., Sariv M., Sariv Z. (1991). Efficiency of Azotobacter strains depending on nitrogen level and sugar beet genotypes: In: Polsinelli M., Materassi R., Vincenzini, eds,

Ann. Microbiol., 51, 145-158 (2001)


Proceedings of the Fifth International Symposium on Nitrogen Fixation. with Nonlegumes. Kluwer Academic Publishers, Dordrecht, Boston, London, pp. 329-331. Kshirsagar C.R., Mandhare V.K., Kalbhor H.B., Patil P.L. (1994). Response of onion to Azotobacter and VA-mycorrhizal inoculation along with phosphorus levels. J. Maharashtra Agricultural Universities, 19: 476-477. Lee S.B., Burris R.H. (1943). Large scale production of Azotobacter. Industr. Ind. Eng. Chem., 35: 112-121. Li F., Liu R., Wang C.C., Wand Y.Z. (1995). Research on distribution of 32P labelled nitrogen fixing bacteria in plant. In: Proceedings of the 10th International Congress on Nitrogen Fixation, St Petersburg, Russia, No 623. Lipman J.G. (1903). Report on the New Jersey Agricultural Experiment Station, 24: 217285. Lipman J.G. (1904). Report on the New Jersey Agricultural Experiment Station, 25: 237289. Lipman C.B., Mac Lees E. (1940). Dissociation of Azotobacter chroococcum (Beijerinck). Soil Sci., 50: 75-82. Macura J. (1966). Interactions nutritionnells plantesbacteries et bases experimentales de la bacterisation des graines. Ann. Inst. Pasteur, 3: 9-38. Maltseva N.N., Nadkernichnaya E.V., Kanivets N.A. (1995). Associations of nitrogen-fixing bacteria with winter rye. In: Proceedings of the10th International Congress on Nitrogen Fixation., St. Petersburg, Russia, N. 614. Mandhare V.K., Patil P.L., Gadekar D.A. (1998). Phosphorus uptake of onion as influenced by Glomus fasciculatum, Azotobacter and phosphorus levels. Agricultural Science Digest, 18: 228-230. Martinez R., Dibut B., Casanova I., Ortega M. (1997). Accion estimuladora de Azotobacter chroococcum sobre el cultivo del tomate en suelo ferracitico rojo. I. Efecto sobre los semilleros. Agrotecnia de Cuba, 27: 23-26. Martinez Toledo M.V., Gozalez-Lopez J., De la Rubia T., Moreno J., Ramos-Cormenzana A. (1988). Effect of inoculation with Azotobacter chroococcum on nitrogenase activity of Zea mays roots grown in agricultural soils under aseptic and non-sterile conditions. Biol. Fert. Soils, 6: 170-173. Martinez Toledo M.V., Moreno J., De la Rubia T., Gozalezlopez J. (1989). Root exudates of pea mays and production of auxins, gibberellins and cytokinins by Azotobacter chroococcum. Plant Soil, 110: 149-152. Martinez Toledo M.V., Salmeron V., Gozalezlopez J. (1990). Effect of Azotobacter inoculation on nitrogenase activity of Hordeum vulgare. Chemosphere, 21: 243-250. Mezei S., Popoviv M., Kova­ev L., Mrkova­ki N., Nagl N., Kova­ev L., cacØ N. (1997). v In vitro inoculation of sugar beet calli with Azotobacter chroococcum. In: Proceedings of the 60th IIRB Congress, Cambridge, pp. 521-525. Mezei S., Popoviv M., Kova­ev L., Mrkova­ki N., Nagl N., Malen ­ D. (1997/98). Effect Ø v of Azotobacter strains on sugar beet callus proliferation and nitrogen metabolism enzymes. Biol. Plantarum, 40: 277-283. Micev N. (1971). Results of investigation of free nitrogen fixation (Azotobacterium) in the rhizosphere of some plants and soil types of Macedonia. Agrohemija, 7-8: 309-317. Mickovski M. (1957). Vlijanie na nikotinot vrz porastot i razvitokot na azotobakterot. Socijalisti­ko zemjodelstvo, Skopje, 9: 9-15. Mickovski M. (1959). Pridones kon poznavanjeto ra§ ­irenosta na azotobakterot vo atiskite po­vi na NR Makedonija. Socijalist­ko zemjodelstvo, Skopje, 11: 1-77. Mickovski M. (1960). Zastupenost na azotobakterot i uslovite za negoviot razvitak vo nekoi po­vi na NR Makedonija. Godi§ Zbornik na Zemljodelsko­umarskiot fakul­en § tet, Skopje, 13: 59-108.



Miliv V., Sariv M. (1988). Efficacy of Azotobacter in dependence of maize genotype and the content of nitrogen in nutritive solution. Mikrobiologija, 25: 45-56. Miliv, V., Mrkova­ki N. (1995). Production of growth substances gibberellin type in strains of Azotobacter chroococcum. In: Proceedings of VII Congres Microbiologists of Yugoslavia, Herceg Novi, p. 28. Mishra O.R., Tomar U.S., Sharama R.A., Rajput A.M. (1995). Response of maize to chemicals and biofertilizers. Crop Research, 9: 233-237. Mi§ ­ustin E.N., (1953). O rasprostranenii azotobaktera v po­vah. Mikrobiologija, XXII, 4, 1953. Mishustin E.N., Naumova A.N. (1962). Bacterial fertilizers, their effectiveness and mechanism of action. Mikrobiologija, 31: 543-545 . Mrkova­ki N., Mezei S., Kova­ev L. (1995a). Specific relationship between Azotobacter strains and sugar beet plants. Soil plant, 44: 9-17. Mrkova­ki N., Kova­ev L., Mezei S., ­ v N. (1995b). The growth of Azotobacter strains Ø with pelleted sugar beet seeds. In: Abstracts of XI Symposium JSPP, Novi Sad, p.110. Mrkova­ki N., Mezei S., Kova­ev L., Miliv V., Popoviv M. (1996a). Interrelationship of Azotobacter with sugar beet. In: Abstract of Nitrogen Fixation Conference, Poznan, Poland, p.186. Mrkova­ki N., Mezei S., Kova­ev L.(1996b) Effect of Azotobacter inoculation on dry matter mass and nitrogen content in the hybrid varieties of sugar beet. A Periodical of Scientific Research on Field and Vegetable Crops, 25: 107-113. Mrkova­ki N.(1997). Microbial abundance of the soil and rhizosphere during the growing season as effected by sugar beet genotype. Acta Agriculturae Serbica, 4: 89-95. Mrkova­ki N., Mezei S., Kova­ev L., Sklenar P., Miliv V. (1997a). Number of Azotobacter chroococcum in soil and rhizophere of inoculated sugar beet plants. In: Papers of the IX Congress of the Yugoslav Society of Soil Science, Novi Sad, pp 443-448. Mrkova­ki N., Mezei S., Verepbaranji I., Popoviv M., Sariv Z. and Kova­ev L. (1997b). Associations of sugar beet and nitrogen fixing bacteria in vitro. Biologia Plantarum, 39: 419-425. Mrkova­ki N., Mezei S., Kova­ev L., Sklenar P. (1998a). The effect of inoculation of sugar beet with Azotobacter chroococcum on the bacteria's number on the root and in the rhizosphere. Archiv. Biol. Sci., 50: 189-193. Mrkova­ki N., ­ v N., Mezei S., Miliv V. (1998b). Effect of inoculation on the number Ø of Azotobacters in soil and rhizosphere during sugarbeet growing season. Acta Agriculturae Serbica, 5: 53-59. Mrkova­ki N., Kova­ev L., ­ v N., Mezei S. (1999). Compatibility of Azotobacter Ø chrooccocum with pelleted sugar beet seed. In: Proceedings of the 62nd IIRB Congress, Sevilla, pp. 183-187. Mrkova­ki N., Kova­ev L., ­ v N., Mezei S. (2001).Application of microbiological Ø preparation in sugarbeet production. A Periodical of Scientific Research on Field and Vegetable Crops, 35: 67-73. Nieto K.F., Frankenberger W.T. (1989). Biosynthesis of cytokinins by Azotobacter chroococcum. Soil Biol. Biochem., 21: 967-972. Nieto K.F., Frankenberger W.T. (1991). Influence of adenine, isopentyl alcohol and Azotobacter chroococcum on the vegetative growth of Zea mays. Plant Soil, 135: 213221. Page W.J., Shivprasad S. (1991). Azotobacter salinestris sp. nov., a sodium-dependent, microaerophili, and aeroadaptive nitrogen-fixing bacterium. Int. J. Syst. Bacteriol., 41: 369-376.

Ann. Microbiol., 51, 145-158 (2001)


Pandey A., Sharma E., Palni L.M.S. (1998). Influence of bacterial inoculation on maize in upland farming system of the Sikkim Himalaya. Soil Biol. Biochem., 30: 379-384. Pati B.R., Sengupta S., Chjandra A.K. (1995). Impact of selected phyllospheric diazotrophs on the growth of wheat seedlings and assay of the growth substances produced by the diazotrophs. Microbiological Research, 150: 121-127. Pr§ M. (1963). Synthese des acides amines dans les varietes de Azotobacter chroococ­a cum. Ann. Sci. Agronomiques, 54: 51-57. Pr§ M.(1964). Azotobacter in the rhizosphere of some agronomic cultures, in red soil of ­a the Istrian peninsula. Scientific Agriculture, 19. Puertas A., Gonzales L.M. (1999). Aislamiento de cepas nativas de Azotobacter chroococcum en la provincia Granma y evaluacion de su actividad estimuladora en plantulas de tomate. Cell. Mol. Life Sci., 20: 5-7. Quispel A. (1991). A critical evaluation of the prospects for nitrogen fixation with nonlegumes. Plant Soil, 137: 1-11. Raduloviv V., Hauher O. (1967). Relationship between Azotobacter chroococcum and microorganisms in wheat rhizosphere. Soil Plant, 16: 215-221. Raduloviv V. (1969). Effect of agrotechnic on dynamics of Azotobacter chroococcum in wheat rhizosphere. Mikrobiologija, 6: 51-63. Radwan F.I. (1998). Response of some maize cultivars to VA ­ mycorrhizal inoculation, biofertilization and soil nitrogen application. Alexandria Journal of Agricultural Research, 43: 43-56. Rai S.N., Gaur A.C. (1988). Chraracterization of Azotobacter spp. and effect of Azotobacter and Azospirillum as inoculant on the yield and N ­ uptake of wheat crop. Plant Soil, 109: 131-134. Rai­eviv V., Sariv M., Sariv Z., Bogdanoviv V. (1995a). Azotobacter movement in maize root. In: Proceedings of the the 10th International Congress of nitrogen Fixation, St. Petersburg, Russia, No 607. Rai­eviv V., Sariv M., Sariv Z., Bogdanoviv V. (1995b). Colonization and adsorption of some Azotobacter strains in maize root. In: Proceedings of the 10th International Congress of nitrogen Fixation, St. Petersburg, Russia, No 608. Ramos A., Barea J.M., Callao V. (1972). A phosphate dissolving and nitrogen fixing microorganism and its possible influence on soil fertility. Agrochimica, 16: 345-350. Rao N.S., Tsuru S., Singh C.S. (1984). A study on the nature of Rhizobium, Azotobacter and Azospirillum in a Japanese soil amended with organic and inorganic manures. Zentralblatt fur Mikrobiologie, 139: 607-613. Reddy M.V.R., Reddy T.K.R. (1989). Competititive saprophytic survival of Azotobacter chroococcum in black cotton soil. Current Sci., 58: 139-140. Reliv B., Govedarica M., Neskovis M. (1987). Plant hormone activity in Azotobacter cul ture. In: Book of Abstracts of VIII Simposum of Yugoslav Society of Plant Phisiology, Tuheljske Toplice, p.79. Reliv B. (1989). Plant horomone activity in Azotobacter culture and effct on wheat. Master thesis, Faculty of Natural Sciences, University Novi Sad. Requena N., Baca T.M., Azcon R. (1997). Evolution of humic substances from unripe compost during incubation with lignolytic or cellulolytic microorganisms and effects on the lettuce growth promotion mediated by Azotobacter chroococcum. Biol. Fert. Soils, 24: 59-65. Rovira A.D. (1965a). Effects of Azotobacter, Bacillus and Clostridium on the growth of wheat. Plant Microbe Ralationships. In: Symposium on Relationships between Soil Microorganisms and Plant Roots. Prague, 1963, p. 193. Rovira A.D. (1965b) Interactions between plant roots and soil microorganisms. Ann. Rev. Microbiol., 19: 241-266.



Rubenchik L.I. (1963). Azotobacter and its use in agriculture. Israel Program for Scientific Translations, Jerusalem, p. 278. Saha J., Chowdhury A., Chaudhuri S. (1991). Stimulation of heterotrophic dinitrogen fixation in barley root association by the herbicide pendimethalin and its metabolic transformation by Azotobacter spp. Soil Biol. Biochem., 23: 569-573. Salmeron V., Martinez Toledo M V., Gonzalezlopez J. (1990). Nitrogen fixation and production of auxins, gibberellins and cytokinin by an Azotobacter chroococcum strain isolated from root of zea mays in presence of insoluble phosphate. Chemosphere, 20: 417-422. Sariv Z., Ra§ ­oviv B. (1963a). The influence of the maize on the dynamic of Azotobacter in the soil. Soil Plant, 13: 273-277. Sariv Z., Ra§ ­oviv B. (1963b). The effect of some plants on the dynamics of Azotobacter in the soil. Annals of Scientific Work at the Faculty of Agriculture, Novi Sad, 7: 1-11. Sariv Z. (1969a). Biogenic levels of the horizons of calcerous chernozem in Vojvodina. Contemporary Agriculture, 17: 819-825. Sariv Z. (1969b). Biogenity of limeless chernozem inVojvodina. Annals of Scientific Work at the Institute for Research in Agriculture, Novi Sad, 7: 145-151. Sariv Z. (1978). The influence of mineral fertilizers on the population of Azotobacter and oligonitrophilic bacteria in chernozem. Mikrobiology, 15: 153-166. Sariv Z., Miliv V., Jarak M., Govedarica M. (1983). Effect of organic residues on microbiological changes and fertility of soil. Annals of Scientific Work at the Faculty of Agriculture, Novi Sad, 13: 147-165. Saric Z., Saric M., Govedarica M. Stankoviv Z. (1987). The effect of maize genotype and ´ ´ nitrogen level on the efficiency of different Azotobacter strains. J. Plant. Nutr., 10, 916, 1774-1786. Sariv M.R., Sariv Z., Govedarica M. (1988). Efficiency of strain combination of different genera of nitrogen fixing bacteria on sunflower genotypes. In: 12th Intern. Sunflower Conference, Novi Sad, pp. 187-191. Sariv M.R., Sariv Z., Govedarica M. (1990a) Variability of molecular nitrogen fixation and its dependence on plant genotype and diazotroph strains. In: El Bassam N. et al., eds, Genetic Aspects of Plant Mineral Nutrition. Kluwer Academic Publ., Netherlands, pp. 373-379. Saric Z., Micanovic D., Trifkovic V., Saric R.M. (1990b) Effect of Azotobacter on wheat. ´ ´ ´ ´ ´ Microbiology, 27: 139-146. Sariv R.M., Sariv Z., Krstiv B. (1991). Specific responses of Azotobacter strains and sugar beet genotypes. In: Polsinelli M., Materassi R., Vincenzini, eds, Proceedings of the Fifth International Symposium on Nitrogen Fixation. with Non-legumes. Kluwer Academic Publishers, Dordrecht, Boston, London, pp. 333-335. Shabaev V.P., Smolin V.Y., Strekozova V.I. (1991). The effect of Azospirillum brasilense Sp. 7 and Azotobacter chroococcum on nitrogen - balance in soil under cropping with oats (Avena sativa L.). Biol. Fert. Soils, 10: 290-292. Steinberga V., Apsite A., Bicevskis J., Strikanska V., Viesturs V. (1996). The effect of Azotobacterium on the crop yield and biological activity of the soil. In: Wojtovich A., Stepkowska J., Szlagowska A., eds, Proceedings of 2nd European Nitrogen Fixation Conference. Poznan, pp.191. Sturz A.V., Christie B.R., Nowak J. (2000). Bacterial Endophytes: Potential Role in Developing Sustainable Systems of Crop Production. Critical Reviews in Plant Sciences, 19: 1-30. Thilak K.V.B.R. (1993). Associative effects of versicular ­ arbuscular mycorrizae with nitrogen fixers. In: Proceedings of the Indian National Science Academy, Part B, Biological Sciences, 59: 325-331.

Ann. Microbiol., 51, 145-158 (2001)


Thompson J.P., Skerman V.B.D. (1980). Azotobacteraceae: the taxonomy and ecology of the aerobic nitrogen ­ fixing bacteria. Academic Press, London. Thompson J.P., Skerman V.B.D. (1981).Validation list No6. Int. J. Syst. Bacteriol., 31: 215-218. Thompson J.P. (1989a). Counting viable Azotobacter chroococcum in vertisoils 1. Comparison of media. Plant Soil, 117: 9-16. Thompson J.P. (1989b). Counting viable Azotobacter chroococcum in vertisoils 2. The non proportionality phenomenon. Plant Soil, 117: 17-29. Thompson J.P. (1989c). Counting viable Azotobacter chroococcum in vertisoils 3. Methods for preparation of soil suspensions. Plant Soil, 117: 31-40. Tippannavar C.M., Reddy T.K.R. (1993). Seed treatment of wheat (Triticum aesativum L.) on the survival of seed borne Azotobacter chroococcum. Karnataka Journal of Agricultural Sciences, 6: 310-312. Trifkoviv V. (1996). Associative capability of genus Azotobacter with maize. Ph. D. Thesis. Faculty of Agriculture, Zemun. Tripllet E.W. (1996). Diazotrophic endophytes: progress and prospects for nitrogen fixation in monocots. Plant Soil, 186: 29-38. Troitskaya T.M., Troitskii N.A. (1988). Nitrogen fixation by Azotobacter chroococcum in association with barley. Microbiology, 57: 234-237. Vancura V., Macura J. (1960). Indole derivatives in Azotobacter cultures. Folia Microbiol., 5: 293 Vancura V., Abdel Malek Y., Zayed M.N. (1965). Azotobacter and Beijerinckia in the soils and rhizospheres of plants in Egypt.. Folia Microbiol., 10: 224-229. Voesenek L.J., Blom P.M. (1996). Plants and hormones - An Ecophysiological View on Timing and Plasticity. J. Ecol., 84: 11-119. Vojinova-Raikova Z. (1954). Rasprostranenie azotobaktera v po­vah Bulgarii. Mikrobiologija, XXVI, 4. Vojinovi v . (1956). The widespread Azotobacter in Serbian soils. Journal for Scientific Z Agricultural Research, 26: 97-112. Vojinoviv Z. (1961). Mikrobiological properties of main types soils in Serbia for nitrogen cycling. Journal for Scientific Agricultural Research, 43: 3-25. Winogradsky S.N. (1932). Sur la synthese de l' ammoniaq par les Azotobacters du sol. Ann Inst. Pasteur, 48: 269-300. Winogradsky S. (1938). Etudes sur la microbiologie du sol et des eaux. sur la morphologie et l'oecologie des Azotobacter. Ann. Inst. Pasteur, 60: 351-400. Yadav K.S., Sunita Suneja, Sharma H.R. (1996). Seed bacterization studies with Azotobacter chroococcum in sunflower (Helianthus anuus L.) Crop Research, 11: 239-243.





14 pages

Report File (DMCA)

Our content is added by our users. We aim to remove reported files within 1 working day. Please use this link to notify us:

Report this file as copyright or inappropriate


You might also be interested in