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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).

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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

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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.

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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.


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