Biofertilizer is a wide term which includes a diverse category of bioinoculants such as nitrogen fixers, phosphate solubilizers, phosphate mobilizers and plant growth promoting rhizobacteria. Biofertilizers are the organisms which are naturally present in all types of soils. Applications of these biofertilizers are environment friendly, means to supplement nutrient to the plants. Important biofertilizers are nitrogen fixers, phosphate solubilizers and phosphate mobilizers [1]. However, the concept of biofertilizers was developed with the discovery of nitrogen fixing Azospirillum. Azospirillum was first reported by Beijerinck [2] and it was named as Spirillum lipoferum by Schroeder [3]. It gained the reputation of being the most studied plant associative bacterium only after it was rediscovered by Dobereiner J [4] in the roots of Digitaria decumbens. The meaning of Azospirillum was from ‘azite’ a French word meaning nitrogen and ‘spira’ a Greek word meaning spiral. Azospirillum means a small nitrogen spiral. The name lipoferum means fat bearing; brasilense from brasiliensis pertaining to the country of Brazil, South America. They also explored the high potentiality of Azospirillum as microbial inoculant with tropical grasses and other crop plants. It has been enlarged to encompass other possible bacterial associations by adopting the terminology “Diazotropic biocoenosis”.

Azospirillum is a gram negative, motile, curved rod of variable size, ranging from 0.5 – 1μm in length, exhibits spirillar movement and polymorphism, containing poly-β- hydroxy butyrate (PHB) granules and fat droplets [4,5] They contain peritrichous flagella and polar flagellum used for swarming [6]. It was associated with the root system of plants making use of the nutrients exuded by them. Azospirillum is colonized the root region of crop plants in large numbers and fixes substantial amount of nitrogen [7] and they exerted beneficial effects on plant growth and yield many crops of economic importance [8].It is used extensively in rice and other cereal crops as biofertilizers [8]. Tarrand et al. [9] and Magalhaes et al. [10] proposed as Azospirillum, the genus distinguished into two species based on physiological and morphological differences between various strains on DNA homology experiments like Azospirillum brasilense and A. lipoferum [9,10] Later, four additional Azospirillum species were described, A. amazonense. Isolated from many grasses in the Amazonian area of Brazil [11], the salt tolerant species A. halopraeferans, associated exclusively with roots of kallar grass [12], A. irakense [13] and A. dodereinerae [14].

Azospirillum is grown in N-free medium, it behaves as microaerophilic, fixes nitrogen and when supplemented with nitrogen it grows as an aerobe [4]. The population levels of Azospirillum were reported from 10⁴ to 10⁶ cells per gram of dry soil or root by Magalhaes et al. [10] and 7 x 10⁴ per gram of fresh roots in Stenocerus pruinosus and 1.1 x 10⁴ in Opuntia ficus - indica by Mascarua-Esparza et al. [15]. Maximum Azospirillum numbers were detected in a laterite soil and the minimum in an extremely acid sulphate saline kari soil [16]. The present study aims to isolate, identify, screen and study the biology of Azospirillum spp from various soils of Srivilliputtur Taluk, Virudhunagar District, Tamil Nadu, India.

Study area

Tamil Nadu is situated in Southern end of India, towards east of Kerala north of Andhra Pradesh and Karnataka. Several folds or parts of Western Ghats separate the states of Tamil Nadu and Kerala. The area of investigation, Srivilliputtur is located in the Southern side of Virudhunagar District; this area is a boundary of Theni and Madurai Districts in North, Tirunelveli District in South and Kerala in Southwest.

Collection of soil and root samples

Soil and Root samples were collected from different crop soils in various places of Srivilliputtur Taluk, Virudhunagar District. The soil samples (Rhizosphere soil) were air dried under shade and used for isolation and enumeration of Azospirillum.

Isolation and enumeration Azospirillum

For the isolation of Azospirillum, the rhizosphere soil samples were serially diluted from 10⁴ to 10⁶ using sterile water. One mille liter of the soil diluents from each dilution was transferred to the tubes containing 10 mille liter of nitrogen free malic acid semi solid medium and kept it in incubation for three days at 37±2ºC.

Enumeration of Azospirillum in soil samples were carried out by Most Probable number method (MPN). One mille liter successive dilutions of 10⁴, 10⁵ and 10⁶ soil samples were transferred to test tubes containing nitrogen free malic acid semi solid medium. Then the tubes were incubated at room temperature for 3 days. The positive tubes were counted and the population was calculated and expressed as number of Azospirillum per gram dry weight of soil samples.

Identification and screening of Azospirillum

The selected bacterial strains were identified using standard biochemical tests as listed in the Bergey’s Manual of Determinative Bacteriology. The following tests/methods were used for isolation and identification of Azospirillum from the soils.

Cell morphology: To study the cell morphology, log phase cultures of Azospirillum were properly diluted with sterile distilled water. Smear was prepared on clean glass slides by using a loopful of culture dilution. The smears were air-dried, heat fixed and stained with crystal violet. The cells were observed under light microscope with oil immersion objective to see the size and shape.

Motility: Motility was tested by hanging drop method. Slides were prepared with cultures and motility was observed under oil immersion.

Biotin requirement: The biotin requirements of the bacterial isolates are tested using semisolid nitrogen free malic acid medium prepared in two sets of tubes, one set of medium prepared with the addition of biotin (100 μg l^-1) and other without biotin. The growth was observed by the change in colour from yellowish green to blue.

Catalase test: Selected strains were inoculated on LB agar plates and incubated at 28°C for 24 h. About 3 to 4 drops of 3 percent H₂O₂ solution was allowed to flow over the culture. Formation of bubbles or effervescence indicated a positive result.

Utilization of carbon, nitrogen, amino acid and vitamin sources

The utilization of different carbon, nitrogen, amino acid and vitamin sources of different Azospirillum isolates were estimated in LB broth. Filter sterilized carbon, nitrogen, amino acid and vitamin sources were inoculated aseptically into the sterile medium at 1 percent level. The Azospirillum cultures were inoculated at the rate of 1.0 mille liter and incubated at room temperature. The growth was observed by the turbidity of the broth read at 560 nm.

Totally fifteen Azospirillum strains were isolated from the soil samples collected from different crop plants at various places of Srivilliputtur Taluk, Virudhunagar district. The isolated strains were brought to pure culture by several subcultures. The purified Azospirillum strains were maintained in nutrient agar slants and stored at 4ºC for future use (Table 1). The result showed that the population level of Azospirillum was higher in cotton followed by tomato. Among different crop plants, Azospirillum population was least in soil samples collected from bhendi (Table 1). According to Haahtela et al. [17] and Eckert et al. [14] Azospirilla were isolated from a wide variety of plants including many grasses and cereals from all over the world, in tropical, temperate and cold climates. In addition to isolation of Azospirillum, enumerates the population level in soil samples were also collected from different crop plants. Azospirillum population was higher in cotton and least in soil samples collected from bhendi. The association of these organisms with the roots of non-Graminae family plants was also reported by [18]. Seasonal variation in MPN counts, which showed a similar pattern of decrease or increase with the variable climatic conditions, confirm that all types of microorganisms were influenced by the temperature fluctuations in a similar fashion in tea soils Govindan and Purushothaman [19]. Plantation crops like areca nut, cashew, cocoa, rubber, cardamom and sapota grown in acid soils colonized by Azospirillum in their root system [20].

Table 1: Isolation of Total Number of Azospirillum Strains and their Population level in different Crop Plants.

The results indicated that the Azospirillum strains were differed in their ability to change colour intensity of the medium. The difference between the regions in nitrogen fixing ability was related to geographic variables, including soil type [21]. In this study, the isolated strains were screened under in vitro condition. The isolated Azospirillum strains were screened by noting the changes of colour and pH in the Azospirillum growing medium. After selection the Azospirillum strains were screened by noting the colour and pH change of the medium. These two observations were used for the screening of Azospirillum in the laboratory conditions. In the nitrogen free malate broth, Azospirillum strains were able to change colour. The initial green colour changed into blue colour. The ability of colour change was differed between the strains. The result revealed that all the strains were able to change the colour and pH of the medium on 3rd day. But some strains started to change the colour even in the first day after inoculation. Among fifteen strains, the T1, T3, B1, B3, C1, CT1 and CT3 strains were superior in colour change than others (Table 2), however, among fifteen strains, the ability to pH changes is differed between the strains. In strains T3, C1 and CT3 were increased in pH than other strains (Table 2).

Table 2: Results of colour changes, pH and Gram staining in the medium used for the growth of various Azospirillum strains.

Identification of Azospirillum

Based on the present study, the in vitro experiment shows that the totals of 15 Azospirillum strains were screened for their efficiency using various biochemical tests. In Gram stain all the fifteen strains were Gram negative because all the strains were stained with safranin rather than crystal violet. Based on the staining, fifteen strains were revealed Gram negative (Table 2), but the size of the selected strains were ranged from 1-2.0μm in diameter and 2.0-3.5μm in length. Microscopic examination of the isolates revealed that they were vibroid in shape and in the motility levels of all the fifteen strains were showed spiral movement. In catalase and biotin content of Azospirillum, all the strains except T1, T2 were negative to catalase test. (Table 3). The selected fifteen strains were cultured in nitrogen free semisolid medium with or without biotin. Biotin test is one of the important test to differentiate the Azospirillum species mainly A. lipoferum and A. brasilense (Table 3). According to Okon and Itzigsohn R [22], Azospirilla was gram negative, the cured rods of variable size exhibits spirillar movement and contain PHB as reserve food material.

Table 3: Identification, catalase and biotin contents of various Azospirillum strains in different crop plants.

Utilization of carbon sources

The Azospirillum strains utilized different types of chemicals as carbon sources. The utilization of these different types of carbon sources varied from strain to strain. There was a marked difference between the Azospirillum species in the pattern of carbohydrate utilization. The preferential carbon sources varied from strain to strain. Most of the strains preferred fructose and maltose as carbon source but sucrose was moderately utilized while lactose was found to be a poor source (Table 4). The disaccharides except lactose, starch and mannitol supported little growth of isolates belonging to both species, while trisaccharides, polysaccharides did not support their growth. This suggests that sugars are supposed to be very poor substrates of carbon and energy for A. brasilense but better source for A. lipoferum [23].

Table 4: Carbon utilization by Azospirillum strains.

Utilization of vitamin source

All Azospirillum strains were utilized for various vitamin sources. The preferential vitamin sources varied from strain to strain. Most of the strains preferred myoinosital, as vitamin source but nicotinic acid was also moderately utilized while, thiamine, pyridoxine was found to be a poor source (Table 5).

Table 5: Utilization of Vitamins by Azospirillum strains.

Our results demonstrate remarkably different strains of Azospirillum from various crop soils in Srivilliputtur Taluk.

The isolated strains were contains different features in their morphology, carbon utilization and their vitamin contents. This type of results which would be very useful in the field of agriculture to develop organic/bio fertilizer.

Authors are thankful to the University Grants Commission for funding the financial support by minor research project to Dr. N.T. Balaiah.