Phenetic Characterization of Nitrogen Fixing Azotobacter from Rhizospheric Soil of Southern Rajasthan

The present research was conducted to characterize the indigenous plant growth promoting (PGP) Azotobacter strains isolated from plant root interface of semi-arid regions of Rajasthan (India) and to study their potential to be used as bio-fertilizers. A total of 172 Azotobacter strains were isolated, purified and based on the morphological test i.e. gram staining, pigmentation, cyst formation, fluorescence etc, broadly classified as Azotobacter. Further the secluded strains were examined for biochemical analysis and plant growth promoting characters. All the isolates showed different biochemical characteristics and significant PGP traits. IAA activity of the Azotobacter strains ranges from 54.5-6000 μg/mL. Ammonia, HCN and siderophore was produced by 92.4%, 78.4% and 80.23% of the total isolates respectively. Solubilization of phosphate was observed in 97.6% of the total isolates. These strains were also characterized for qualitative and quantitative N2 fixation abilities and the result indicated that 114 strains showed positive results on nitrogen free malate agar medium (NFMM) containing bromothymol blue (BTB) and able to produce 18.93-475.6 N-moles C2h4 mg protein −1 h−1 of acetylene reduced by Azotobacter strains. In vitro pot studies revealed that the selected native Azotobacter strains having high ARA results significantly increase the plant growth characters. Shoot length, root length, root number and chlorophyll content and leaf number increases by 45.62%, 17.60%, 97.49%, 49.69% and 27.83% respectively in pot inoculated with AZO23-3 as compared to control. These effective strains can further be utilized for development of effective microbial formulations.

cytokinins 11 . Therefore considering the above properties, Azotobacter sp. is used as a beneficial biological agent, extensively seek for a various crops like wheat, rice, sorghum, sugarcane, maize, sorghum, and maize 12 . To develop commercial biofertilizer the initial steps are isolation and characterization of native Azotobacter strains. Consequently, this study was designed to isolate and characterize Azotobacter for their various plant growth promoting characteristics and N 2 fixing attributes.

Isolation of Azotobacter sp
The Azotobacter sp. was isolated on Nitrogen free Jensen's medium by serial dilution and spread plate technique 13 . The serial soil dilution of soil samples were done upto 10 -6 and afterwards spread on Jenson medium plates. The inoculated petriplates were kept in invert position for incubation at 28±2°C for about 48 h. The Azotobacter colonies were large, ovoid, pleomorphic in shape and were purified on Azotobacter medium 14 and preserved in glycerol stocks.

Biochemical analysis of Azotobacter sp
Pure strains of Azotobacter sp. were isolated from soil samples and were characterized according to procedure mentioned in Bergey's Manual of Systematic Bacteriology 15 . The cyst formation was induced according to the procedure of Socolofsky and Wyss 16 and the staining of cysts were done by the method of Vela and Wyss 17 . Under biochemical characteristics, starch hydrolysis, H 2 S production, TSI test, Nitrate reduction, Urease, Catalase and Oxidase reaction were studied. Carbon source i.e., glucose, mannitol, lactose and sucrose utilization were also assayed. Antibiotic sensitivity of Azotobacter isolates were determined by disk agar diffusion method 18 using standard concentration of antibiotic discs of Ampicillin, Rifampicin, Kanamycin, Chloramphenicol and Penicillin.

PGP activitiesIAA Production
Quantification measurement of IAA production was followed by the method as described by Gordon and Weber 19 . The produced IAA concentration in the culture was measured by plotting the standard graph of IAA obtained in the range of 10-100μg/ ml.

Nh 3 Production
Ammonia production was tested for Azotobacter strains as described by Cappuccino and Sherman 20 . Overnight grown bacterial culture was inoculated in 10 ml peptone broth and incubated at 35±0.1°C for 48 h in shaking postion. 0.5 mL of Nessler's reagent was added after incubation.

HCN Production
Azotobacter culture was streaked on nutrient agar medium, which containedglycine (4.4g per liter) and in the same petriplate a 0.5% picric acid solution (in 2% sodium carbonate) soaked Whatman filter paper was placed inside the lid. These plates were sealed with parafilm and incubated at 25 ± 2°C for 4 days and the color change of filter paper was observed (Castric, 1977) 21 .

Siderophore Production
Siderophore production was assayed on the Chrome azurol S agar medium as described by Schwyn and Neilands 22 .

Phosphate solubilizing activity
The potential to solubilize insoluble phosphates on the modified Sperber's medium was determined by measuring clear zone around the colonies 23 .

Estimation of Nitrogen production and fixation Qualitative estimation
For qualitative estimation of nitrogen production Nitrogen free Malate media, containing with bromothymol blue (BTB) as an indicator, was used for detection of nitrogen fixing activity. Plates were inoculated with purified isolates and incubated at 37ºC up to 24 h. Those isolates producing blue colored zone on Nitrogen free Malate media were considered nitrogen fixers and they were further tested by quantification of nitrogen fixation by Kjeldahl and acetylene reduction activity (ARA) method.

Nitrogen Production
Total nitrogen fixation by the Azotobacter isolates in the growing medium was quantified by the method 24 .
Total Nitrogen was calculated and expressed as % (percentage) using the formula: Quantitative estimation by ARA Quantification of nitrogen fixation was carried out by ARA assay following the method of Stewart et al. 25 Briefly, five ml of modified Ashby's medium 26 were inoculated with Azotobacter and incubated at 30°C for 24 h. Further, the cotton plugs of the tubes were replaced with rubber stopper and 5 ml head space was injected with 10% (v/v) acetylene and incubated at 30°C for 2 h. The C 2 H 4 production was measured using gas chromatography and the ARA values were recorded as N moles C 2 H 4 mg protein −1 h −1 . In vitro studies on the effect of selected Azotobacter on growth of maize seedling The pot experiment with selected Azotobacter strains having high nitrogenase activities were conducted in plastic pots filled with sterile perlite/vermiculite/coco-peat mixture in triplicate as per CRD design on maize (FEM-2 variety) 27 . Various plant growth parameters like leaf number, chlorophyll content of leaf, average shoot length, root length and root numbers, were analyzed.

Isolation and characterization of the Azotobacter strains
Total of 172 Azotobacter strains were isolated from different rhizospheric soil samples on Azotobacter selected media. The phenetic characteristics of Azotobacter strains are summarized (supplementary sheet Table  1). Most of the bacterial colonies isolated were circular (even) in shape, whitish in colour and the size ranges between 1.0-4.0 mm in size Jimenez et al. 28 , reported that most Azotobacter isolates showed circular form, entire margin with whitish (cream color), smooth, irregular, shining, 3-8mm diameter colonies. All the bacterial isolates were gram negative. Out of 172 isolates, 22 produced water soluble yellow pigment, 10 produced brown pigment, 2 isolates produced red pigment. Out of 172 isolates, 27 showed a green fluorescence, 3 presented a yellow fluorescence and 5 displayed a red fluorescence. Only two isolates did not form cyst, remaining 170 isolates formed cyst. Shaikh and Mohd. Shakir 29 performed cyst formation test on 39 Azotobacter vinelandii, and found that 38 out of 39 Azotobacter produced cyst.
A l l t h e b a c te r i a l i s o l ate s we re characterized by detecting their biochemical characteristics. Results of the investigation results indicated that out of 172 isolates, 129 isolates were found to hydrolyze starch; 146 isolates were Multiple antibiotic resistances were seen in a majority of the isolates. Out of 172 strain 105 isolates showed sensitivity and 87 shows resistance against Ampicillin. Only 3 strains showed resistance and 169 were sensitive for Rifampicin. 122 isolates were sensitive and 50 are resistant for Kanamycin. Only one strain out of 172 shows resistance for Chloramphenicol. 85 Azotobacter strains were found resistant to penicillin while 87 were sensitive to penicillin. Bhaduri et al. 32 studied among 18 isolates of Asymbiotic N 2fixers (Azotobacter) and symbiotic N 2 -fixers (Rhizobium spp for the antibiotic resistance and susceptibility of against different antibiotics including Chloramphenicol and Rifampicin. The antibiotics ampicillin, chloramphenicol, kanamycin, penicillin and rifampicin resistance tested on 117 isolates of A. chroococcum were tested by the antibiotic disc method by Sindhu et al. 33 .
In present study the obtained ARA ranges were significantly higher than other observations and corroborated with Rodelas et al. 42 who reported the ARA results of pure cultures of strains were between 9.70 to 257.73 nmol C 2 H 4 h −1 vial −1 . According to Tejera et al. 28 A. chroococcum isolates from soil showed ARA production within ranged between 79.6 to 329.5 nmol C 2 H 4 h −1 culture.
Nitrogen fixation potentiality of the selected isolates of Azotobacter sp was estimated by Akhter et al. 43 , ranging from 04.95 to 10.55 mg N/g substrate. Kizilkya 44 reported that the Azotobacter fix nitrogen in the range of 3.50 to 29.35 μg N mL -1 for medium culture. Variation in efficiency of Azotobacter may be due to difference in strains and different growth conditions being used in different studies 45 .

Studies of In vitro effect of Azotobacter on growth and yield of maize seedling
The experiment of pot culture was conducted in plastic pots filled with sterile planting mixture. Plant growth promoting activity of Azotobacter isolates were studied on FEM-2 variety of Maize. Cultivable seeds were treated with Azotobacter inoculant thorough seed bacterization method and sown. Five Azotobacter strains with higher ARA values were selected for pot studies. Pot experiment data were recorded after 30 days of germination were summarized in Table 2.
O Maize seedlings raised from bacterized seeds with selected Azotobacter isolates, observed higher growth in treated ones as compared with absolute control. This indicates the positive effect mL. Kannapiran and Ramkumar 36 have reported that the amount of IAA produced by Azotobacter chroococcum and Azotobacter beijerinckii were 23.6 and 17.6 μg/mL respectively. Deshmukh and Vidhale 37 isolated four Azotobacter species from the rhizospheric soil in the alluvial valley of Purna river and its tributaries occupy parts of Amravati and found to produce high level (9.2 to 40.0mg/ mL) of IAA. According to Mali and Bodhankar 38 , 25 isolates of Azotobacter chroococcum isolated from the rhizosphere soil of groundnut were tested for production of IAA in 55 μg/mL.
The production of ammonia is an essential PGPR trait that can influence the plants growth indirectly. Estimation of ammonia production examined by the development of faint yellow to dark brown color after addition of Nessler's reagent to overnight grown bacterial culture in peptone broth indicated 92.4% ammonia production by the bacterial isolates the. These results are similar to those of Ahmad et al. 39 , who revealed the production of ammonia commonly detected in the Azotobacter isolates. HCN production was indicated by the change in yellow to brown to red color of the filter paper strips. Out of 172 isolates, 78.4% isolates gave positive reaction for HCN production. For siderophore production, formation of yellow orange halo around the colony on CAS agar medium showed that 80.2% of 172 isolate produce siderophore. Altaf and Malik 40 examined the 15 Azotobacter sp. for various PGPR activity and found that 33% and 80% of the isolates showed HCN and siderophore production respectively. Solubilization of phosphorous was indicated by observing for the zone of clearance around the bacterial colony in the plates. Out of 172 isolates, 97.6% isolates solubilize phosphate producing clear zone around the colony. The results observed were in close agreement with the previous works of Farajzadeh et al. 41 .

Estimation of Nitrogen production and fixation
Azotobacter strains were preliminary screened for nitrogen fixation on nitrogen free Malate agar medium containing Bromothymol Blue (BTB) as an indicator. Out of 172 bacterial isolates, 114 isolates produced blue color zone on medium indicating fixation of nitrogen by them. Further, these 114 Azotobacter isolates were quantified for nitrogen production by Kjeldahl of Azotobacter strains on maize plantlet. Leaf number, total chlorophyll content, Shoot length, root number, root length, have been significantly increased in the maize plantlet inoculated with the selected Azotobacter strains as compared to uninoculated control.
Shoot length, root length, root number and chlorophyll content increases by 45.62%, 17.60%, 97.49% and 49.69% respectively in pot inoculated with AZO23-3 followed by AZO2-12 (36.86%, 14.74%, 90.06% and 44.29%, respectively). Leaf number increases by 27.83% in pot inoculated with AZO23-3 followed by AZO2-12 and AZO8.2 (11.17%). All the Azotobacter strains significantly influenced the observed parameters as compared to uninoculated control and contributed to plant growth. Similar finding was observed by Mahato and Neupane 46 who reported Azotobacter seed bacterization in maize promote the growth of treated plants and find increased growth in root and shoot lengths. Perdomo et al. 47 reported Azotobacter bacterial inoculation in cotton positively influenced plant growth parameters and reduced 50% nitrogen fertilization doses. These findings advocated the application of Azotobacter for improvement of plant growth due to their intrinsic abilities of fixing atmospheric nitrogen and expressing plant growth-promoting substances.
The plant growth promoting traits and the nitrogen fixation by the local Azotobacter strains is a very critical for the selection of such strains for biofertilizer formulations in order to replace the ineffective strains. The results indicated that inoculation by multi PGP Azotobacter strains significantly improved the plant growth under in vitro condition and may be used for commercial production. Hence, the dedicated field studies are required to confirm the efficacy of these Azotobacter strains.