Keywords

1 Introduction

Biofertilizers also called as bio-inoculants, the organic preparations containing microorganisms are beneficial to agricultural production in terms of nutrient supply particularly with respect to N and P. When applied as seed treatment or seedling root dip or as soil application, they multiply rapidly and develop a thick population in rhizosphere. Biofertilizers can fix atmospheric N through the process of biological nitrogen fixation (BNF), solubilize plant nutrients like phosphates and stimulate plant growth through synthesis of growth promoting substances. They have C: N ratio of 20:1 indicating the capacity of the biofertilizer to release nutrients. Being eco-friendly, non hazardous and non-toxic products, biofertilizers are nowadays gaining the importance in agriculture (Sharma et al. 2007; Hakeem et al. 2016). They are cheaper and low capital intensive.

Biofertilizers benefiting the crop production include Azotobacter, Azospirillum, Blue green algae, Azolla, P-solubilizing microorganisms, mycorrhizae and sinorhizobium (Selvakumar et al. 2009). Azotobacter chroococcum and Azotobacter agilis were first of all studied by Beijerinck (1901). The first species of the genus Azotobacter, named Azotobacter chroococcum family Azotobacteriaceae, was isolated from the soil in Holland in 1901. In subsequent years several other types of Azotobacter 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, Dobereiner (1966), Azotobacter armenicus, Thompson and Skerman (1981); Azotobacter salinestris, Page and Shivprasad (1991).

2 Taxonomy, Morphology and Distribution of Azotobacter

The genus Azotobacter includes 6 species, with A. chroococcum most commonly inhabiting many soils all over the world (Mahato et al. 2009). Among the saprophytes along with nodular bacteria, genus Azotobacter was considered to be the most extensively studied (Horner et al. 1942). Aerobic bacteria belonging to the genus Azotobacter represent a diverse group of free-living diazotrophic (with the ability to use N2 as the sole nitrogen source) microorganisms commonly inhibiting the soil. The taxonomic classification of Azotobacter is shown below.

Domain

:

 Bacteria

Kingdom

:

 Bacteria

Phylum 

:

 Proteobacteria

Class 

:

 Gammproteobacteria

Order 

:

 Pseudomonas

Family 

:

 Pseudomonadaceae/Azotobaceriaceae

Genus 

:

 Azotobacter

Azotobacter represents the main group of heterotrophic, non-symbiotic free living nitrogen-fixing bacteria principally inhabiting the neutral or alkaline soils. These bacteria are Gram negative and vary in shape. They are generally large ovoid pleomorphic cells of 1.5–2.0 um or more in diameter ranging from rods to coccoid cells. The cells can be dispersed or form irregular clusters or occasionally chains of varying lengths in microscopic preparations. In fresh cultures, the cells are mobile due to the numerous flagella present on their body surface but later the cells lose their mobility, become almost spherical and produce a thick layer of mucus, forming the cell capsule. The shape of the cell is affected by the amino acid glycine which is present in the nutrient medium peptone. Their distribution of existence is diverse and occurs either singly, in paired or irregular clumps and sometime in chains of varying length. Fig. 1 shows different stained Azotobacter species cells. Azotobacter possesses some unique features among the biofertilizers. They possess more than one type of nitrogenase enzymes (Joerger and Bishop 1988). They do not produce endospores but form cysts, oval or spherical bacteria that form thick-walled cysts (means of asexual reproduction under favorable condition) (Salhia 2013). The formation of cysts is induced by changes in the concentration of nutrients in the medium and addition of some organic substances such as ethanol, n-butanol, or β-hydroxybutyrate. The formation of cysts is also induced by chemical factors and is accompanied by metabolic shifts, changes in catabolism, respiration and biosynthesis of macromolecules (Sadoff 1975). The cysts of Azotobacter are spherical and consist of the so-called ‘central body’a reduced copy of vegetative cells with several vacuoles and the ‘two-layer shell’. The inner part of the shell has a fibrous structure and is called intine and outer part has a hexagonal crystalline structure called as exine (Page and Sadoff 1975). The central body can be isolated in a viable state by some chelation agents (Parker and Socolofsky 1968). The main constituents of the outer shell are alkyl resorcinol composed of long aliphatic chains and aromatic rings.

Fig. 1
figure 1

Azotobacter species cells, stained with Heidenhain’s iron hematoxylin, ×1000

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The population of Azotobacter is generally low in the rhizosphere of the crop plants in uncultivated soils. Jensen’s N-free medium is frequently used for the mass multiplication of Azotobacter. Azotobacter grows well at an optimum temperature range between 20 and 30 °C and grows best in neutral to alkaline soil (pH of 6.5–7.5), but does not thrive when the pH is below 6 and hence not present in acidic soil. This organism has been reported to occur in the rhizosphere of a number of crop plants such as rice, maize, sugarcane, bajra, vegetables and plantation crops (Arun 2007) hence called rhizobacteria and or occurs endophytically (Hecht-Buchholz 1998). They work better in the root region of crop non-symbiotically when sufficient organic matter is present. They are reported to occur also in parenchymatous cells of root cortex and leaf sheath. Azotobacter is generally used in any non-legume crop (Singh and Dutta 2006). They can exhibit a variety of characteristics responsible for influencing the overall plant growth (Tippannavar and Reddy 1989). The Azotobatcteria also categorised as Plant growth Promoting Rhizobacteria (PGPB) are considered to promote plant growth directly or indirectly. These rhizobacteria derive their food and energy from the organic matter present in the soil and root exudates and fix atmospheric N (Maryenko 1964) depending on the amount of carbohydrates utilized by them. These non-specific associative nitrogen-fixing rhizobacteria are important for ecology and play a great role in soil fertility in agriculture.

3 Mode of Action of Azotobacter on Plant Growth

Despite the considerable amount of experimental data available concerning Azotobacter stimulation of overall plant development, however the exact mode of action by which Azotobacter enhances plant growth is not yet fully understood (Wani et al. 2013). Three possible mechanisms have been proposed to explain the action: N2 fixation; delivering combined nitrogen to the plant; the production of phytohormone – like substances that change the plant growth and morphology and bacterial nitrate reduction, thereby increasing nitrogen accumulation in inoculated plants.

3.1 Nitrogen Fixation

Nitrogen fixation is considered as one of the most important biological processes and interesting microbial activity on the surface of earth after photosynthesis as it makes the recycling of nitrogen and gives a fundamental contribution to nitrogen homeostasis in the biosphere. Biological nitrogen fixation plays an important role in maintaining soil fertility (Vance and Graham 1995). Azotobacteria is used for studying nitrogen fixation and inoculation of plants due to its rapid growth and high level of nitrogen fixation. They are extremely tolerant to oxygen while fixing nitrogen and this is due to respiration protection of nitrogenase (Robson and Postgate 1980; Hakeem et al. 2016). They have respiratory protection, uptake of hydrogenases and switch on-off mechanisms for protection of nitrogenase enzyme from oxygen (Chhonkar et al. 2009). Azotobacter chroococcum is shown to have uptake hydrogenase which metabolises hydrogen (H2) evolved during nitrogen fixation (Partridge et al. 1980). Azotobacter is capable of converting nitrogen to ammonia, which in turn is taken up by the plants (Kamil, et al 2008). Iswaran and Sen (1960a) reported that the presence of optimum levels of calcium nutrient is essential for better growth of Azotobacter and its nitrogen fixation. However, the efficiency of Azotobacter was found to decrease with increased N level as reported by Soleimanzadeh (2013). Azotobacter spp. are non-symbiotic heterotrophic bacteria and capable of fixing about 20 kg N/ha/per year (Kizilkaya 2009) and it may be used in crop production as a substitute for a portion of mineral nitrogen fertilizers (Hajnal et al. 2004). According to Soliman et al. (1995) inoculation with Azotobacter replaced up to 50 % of urea-N for wheat grown in a greenhouse trial under aseptic conditions. The isolated culture of Azotobacter can fix about 10 mg nitrogen g−1 of carbon source under in-vitro conditions. The schematic representation of nitrogen fixation involved in nitrogen cycle in the biosphere by diazotrophs (Azotobacteria) is shown in Fig. 2.

Fig. 2
figure 2

Nitrogen fixation by diazotrophs (Azotobacter spp.)

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3.2 Growth Promoting and Other Substances Produced by Azotobacter

Although most specifically noted for their nitrogen fixing ability, Azotobacter spp. have also been noted for their ability to produce different growth hormones (IAA and other auxins, such as gibberllins and cytokinins) (Barea and Brown 1974), vitamins, antibacterial and antifungal compounds and siderophores (Pandey and Kumar 1989b) which directly or indirectly effect the plant growth and microbial activity. 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 and biochemical processes in plants and microorganisms. These hormonal substances which originate from the rhizosphere or root surface affect the growth of the closely associated higher plants. Brakel and Hilger (1965) showed that Azotobacter produced indol-3-acetic acid (IAA) when tryptophan was added to the medium while as Hennequin and Blachere (1966) found only small amounts of IAA in old cultures of Azotobacter to which no tryptophan was added. 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 (Azcorn and Barea 1975). Azotobacter spp. can also produce antifungal compounds to fight against many plant pathogens (Jen-Hshuan 2006). Many strains of Azotobacter have been reported to produces pigments which are involved in the metabolism of microbial organisms. For example, A. chroococcum forms a dark-brown water-soluble pigment melanin. This process occurs at high levels of metabolism during the fixation of nitrogen and is thought to protect the nitrogenase system from oxygen (Shivprasad and Page 1989). Other Azotobacter species produce pigments from yellow-green to purple colours (Jensen 1954) including a green pigment which fluoresces with a yellow-green light and a pigment with blue-white fluorescence. Azotobacter acts as indicator hence enhances the microbial activity in soils. Jafari et al. (2012) reported that a more realistic indicator of total microbiological activity of soil is dehydrogenase activity which points to the intensity of oxidation reduction, i.e. the intensity of metabolic activity of microorganisms. In this research, dehydrogenase activity increased in all the variants where Azotobacter was applied (Table 1). Mutant A. vinelandi is more suitable for the biosynthesis of alginate in view of its latent utilization as a food stabilizer has better qualitative properties (Chen et al. 1985). Alginate the polymers are linear polysaccharides, which are composed of variable amounts of (1–4)-a-D-mannuronic acid and its epimer, a-L-guluronic acid. Several strains of Azotobacter are capable of producing amino acids when grown in culture media amended with different carbon and nitrogen sources (Lopez et al. 2005). Substances like amino acid produced by these rhizobacteria are involved in many processes that explain plant-grown promotion. Biochemical analysis of chlorophyll, nitrogen, phosphorous, potassium and protein content was higher in Azotobacter inoculated plants as compared to non-inoculated control plants (Naseri et al. 2013).

Table 1 Effect of inoculation on dehydrogenase activity (μg TPF/10 g of soil)
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3.3 Response of Crops to Growth Promoting Substances

Large number of field trials and various experiments carried throughout India and whole world have convincingly established the importance of Azotobacter as microbial inoculant. Various crops like wheat, barley, maize, sugar beet, carrot, cabbage, potato were inoculated with Azotobacterin during its preparation. In India many crops like wheat, rice, onion, brinjal, tomato and cabbage were tested during experiment to study the effects of Azotobacter. Eklund (1970) demonstrated that the presence of A. chroococcum in the rhizosphere of tomato and cucumber is correlated with increased germination and growth of seedlings. 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. A study conducted by Govedarica et al. (1993) on the production of growth substances by nine A. 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. Puertas and Gonzales (1999) reported that dry weight of tomato plants inoculated with A. chroococcum and grown in phosphate-deficient soil was significantly greater than that of non inoculated plants. Phytohormones (auxin, cytokinin, gibberellin) can stimulate root development. A. chroococcum produces an antibiotic which inhibits the growth of several pathogenic fungi in rhizosphere thereby seedling mortality (Subba Rao 2001). Incidence of some diseases of mustard and rapeseeds could be reduced by inoculating with Azotobacter (Singh and Dutta 2006) Vijayan et al. (2007) observed that foliar application of A. chroococcum to mulberry grown under saline soil conditions showed significant level of improvement in biochemical and morphological parameters of leaf. Under greenhouse conditions inoculation of A. chroococcum recorded a significant N and P uptake in both seed and stover in brown sarson (Brasssica campestris L.) over the control (Wani 2012). Dual inoculation of Azotobacter and Azospirillum showed synergistic effects by improving growth prompting hormones, controlling pathogenesis and growth reducing agents due to producing fungicide antibiotics and compounds (antagonistic effect) and also air molecular N fixing and also producing growth prompting hormones such as oxine, cytokenine and gibberellins and solving mineral compound (Naseri et al. 2013).

4 Interaction of Azotobacter with Other Microorganisms

4.1 Interaction with Rhizobium

A synergistic relation of Azotobacter with Rhizobium interaction as co-inoculants have been observed in a majority of studies conducted under conditions like laboratory, greenhouse or field crops. Combined inoculation of Azotobacter and Rhizobium spp. has observed a positive response from crops. By significantly increasing nodulation Azotobacter spp. greatly influence Rhizobium activity. Increasing N2 content within roots and shoots of respiring/metabolizing plant cells improves conditions within the rhizosphere and enhances synergistic interactions between the host and Azotobacter sp. in an open field conditions. Associative effect of A. chroococcum on Bradyrhizobium strains (BM 42 and BM 43) specific to moong bean (Vigna radiata) was also observed (Yadav and Vashishat 1991). The effect was more pronounced when A. chroococcum was co-inoculated with both the strains of Bradyrhizobium.

4.2 Interaction with Azospirillum

The beneficial effects of Azotobacter and Azospirillum interaction on plants are mainly attributed to improvements in root development, an increase in the rate of water and mineral uptake by roots, the displacement of fungi and plant pathogenic bacteria and to a lesser extent, biological nitrogen fixation (Okon and Itzigsohn 1995). Associative effect of Azospirillum lipoferum and Azotobacter chroococcum with Rhizobium spp. improved the growth of chick pea grown on both loamy sand and sandy soils (El-Mokadem et al. 1989). Both Azotobacter and Azospirillum have been shown to improve growth yields in various soil mineral compositions. This suggests that a mutualistic relationship exists between Azotobacter and Azospirillum where both interact with the Rhizobium to improve Cicer arietinum (chick pea) yields (Parmar and Dadarwal 1997). However, maximum values were obtained with Azospirillum application. Similarly, positive reports on application of Azotobacter and Azospirillum on the yield of mustard (Brassica juncea) are available (Tilak and Sharma 2007). Yasari et al. (2009) reported that inoculation of seed with Azotobacter chroococcum, Azospirillium brasilense and Azospirillium lipoferum recorded 1000 seed weight of 4.10 g, pods plant −1 of 125.10 and seed yield of 1668 kg ha−1 in rapeseed (Brassica napus. L) at maturity in a field experiment conducted at Gharakheil Agricultural Research Station in Mazandaran province (Iran) during rabi season.

Some of the studies have shown that a relationship exists between chemotactic behaviour and Azotobacter’s influence on plant growth such as cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) (Kumar et al. 2007). In the areas of soil where plant root exudates or secretions such as sugars, glucose, amino acids and organic acids have been deposited, bacteria mobilize towards these exudates through chemotactic attraction. Increased yields and enhanced growth using A. chroococcum indicate a positive response attributed to nitrogen fixation, phosphorus mobilization, bacterial production and the release of phytohormones (Kumar et al. 2007).

5 Possibility of Using Azotobacter in Crop Production

Azotobacter has beneficial effects on crop growth and yield through biosynthesis of biologically active substances, stimulation of rhizospheric microbes, producing phyopathogenic inhibitors (Lenart 2012). Azotobacter makes availability of certain nutrients like carbon, nitrogen, phosphorus and sulphur through accelerating the mineralization of organic residues in soil and avoid uptake of heavy metals (Levai et al. 2008). Azotobacter can be an important alternative of chemical fertilizer because it provides nitrogen in the form of ammonia, nitrate and amino acids without situation of over dosage, which might be one of the possible alternatives of inorganic nitrogen source (eg. Urea). Azotobacter as nitrogen biofertilizer increases the growth and yield of various crops under field conditions (Table 2).

Table 2 Effect Of Azotobacter On Crop Yield
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5.1 Effects of Azotobacter on Growth and Yield of Crops

There is increment in dry matter accumulation in Azotobacter inoculated plants; it stimulates development of foliage, roots, branching, flowering and fruiting which is triggered by fixed nitrogen and plant growth regulator like substance produced. It also increases plant tolerance to lack of water under adverse condition (Zena and Peru 1986). The rate of increase in the leaf area determines the photosynthetic capacity of plant, which leads to better assimilation of produce and towards yield. Using Azotobacter spp. potato yield has been increased by 33.3 % and 38.3 % (Zena and Peru 1986). 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. Yield increased ranges from 2 to 45% in vegetables, 9 to 24% in sugarcane, 0 to 31% in maize, sorghum, mustard etc., on Azotobacter inoculation (Pandey and Kumar 1989a, b). Tandon (1991) estimated the fertilizer equivalent of important biofertilizers. According to the estimate, fertilizer equivalent of 19–22 kg ha−1 for rhizobium 20 kg N ha−1 for Azotobacter and Azospirillium, 20–30 kg N ha−1 for blue green algae (BGA) and 3 to 4 kg N ha−1 of Azolla. Results of pot experiments a under greenhouse conditions 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). Laxminarayan (2001) reported that seed inoculation with Azotobacter produced higher grain and stover yield compared to uninoculated treatments. Singh and Dutta (2006) reported a significant in seed yield (7.86q ha−1) in rapeseed and mustard (var. yella) due to inoculation with Azotobacter. Sharma (2002) reported the effect of biofertilizers and nitrogen on growth and yield of cabbage cv. Pride of India. Biofertilizer application significantly increased the leaf number, weight of non-wrapper leaves per plant, head length and width, gross and net weight of head per plant and yield per hectare over no biofertilizer application. Azotobacter in balanced nutrient condition results in 3.5 % increment in LAI at rosette stage of canola crop and additional application of Azotobacter shot up the yield by 21.17 % over the control (chemical fertilizers) (Yasari and Patwardhan 2007). According to Das and Saha (2007) combined inoculation of Azotobacter, Azospirillium along with diazotrophs increased grain and straw yield of rice by 4.5 and 8.5 kg ha−1, respectively. The dual inoculation of A. chroococcum and P. indica had beneficiary response on shoot length, root length, fresh shoot and root weight, dry shoot and root weight, and panicle number that affect growth of rice plant (Kamil et al. 2008). Similar result put forwarded by Sandeep et al. (2011) which revealed that there is better growth response of Azotobacter inoculated plants as compared to non-inoculated control plants. Jafari et al. (2011) reported that the use of azotobacter had a positive effect on the grain yield of maize. In the variants where Azotobacter was applied, the grain yield increased in three maize hybrids (Table 3). In ZP 555 su, the yield increased by 1000 kg/ha, in NS 6030 by 280 kg/ha and in 620 k by 450 kg/ha. In NS 609b hybrid, the inoculation did not have any effect. An investigation was conducted under field conditions Milosevic et al. (2012), on a chernozem soil to study the effect of wheat seed inoculation (the cultivars Renesansa and Zlatka) with A. chroococcum, strain 86 (2–5 × CFU 108 ml−1) reported that inoculation increased the energy of germination by 1 to 9 % and seed viability by 2 to 8 %. The largest increase in 1000 seed weight was obtained in the case of the cultivar Renesansa (16 %). A. chroococcum inoculation increased the seed yield of both cultivars and highest yield increase (74 %) was registered in the case of the cultivar Zlatka. According to Salhia (2013) azotobacter inoculants have a significant promoting effect on growth parameters like root, shoot length and dry mass of bamboo and maize seedlings in vitro and in pot experiments. Under green house conditions plant height, leaf number/plant, number of primary and secondary branches/plant, fresh and dry weight of whole plant, number of siliqua/plant, seeds/siliqua of brown sarson increased significantly with Azotobacter inoculation than no inoculation with seed and stover yield of 10.107 g pot −1 and 22.400 g pot−1 respectively (Wani 2012). Naseri et al. (2013) while studying the effect of A. chroococcum and Azospirillum brasilense on grain yield, yield components of maize (S.C.704) as a second cropping in western Iran indicated that the dual inoculation with Azotobacter and Azospirillum on plant height, number of grain per row, 1000-grain weight, grain yield, biological yield and protein content was significant. Estiyar et al. (2014) reported that, number of branches, pod per plant and 1000 grain weight also increased with Azotobacter application.

Table 3 Effect of inoculation on the grain yield of maize (t/ha)
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6 Conclusion

Azotobacter spp. of bacteria, regarded as Plant Growth Promoting Rhizobacteria (PGPR) synthesize growth substances that greatly enhance plant growth and development and inhibit phytopathogenic growth by secreting inhibitors. There is a great significance of A. chroococcum in plant nutrition and its contribution to soil fertility. It is thus an important component of integrated nutrient management system due to its significant role in soil fertility. More research is necessary in future to explore the potentiality of Azotobacter in soil fertility using modern technology of soil genomics etc. The challenge to the research community will be to develop systems to optimize beneficial plant-endophyte bacterial relationships (Sturz et al. 2000) for long term effective role. 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 especially adapted to local edaphic and climatic conditions.