Phenetic and Molecular Diversity of Nitrogen Fixating Plant Growth Promoting Azotobacter Isolated from Semiarid Regions of India

In the present study, 24 Azotobacter strains were isolated from soils of different areas of southern Rajasthan and characterized at biochemical, functional, and molecular levels. The isolated Azotobacter strains were gram negative and cyst forming when viewed under the microscope. These strains were also screened for their plant growth promoting activities and the ability of these isolates to survive under abiotic stress conditions viz. salt, pH, temperature, and drought stress. All the isolates showed IAA, siderophore, HCN, and ammonia production, whereas seven Azotobacter strains showed phosphate solubilization. Amplified Ribosomal DNA Restriction Analysis (ARDRA) revealed significant diversity among Azotobacter strains and the dendrogram obtained differentiated twenty-four of the strains into two major clusters at a similarity coefficient of 0.64. Qualitative and quantitative N2 fixation abilities of these strains were also detrained, and the amounts of acetylene reduced by Azotobacter strains were in the range of 1.31 to 846.56 nmol C2H4 mg protein−1 h−1. The strains showing high nitrogen fixation ability with multiple PGP activities were selected for further pot studies, and these Azotobacter strains significantly increased the various plant growth parameters of maize plantlets. Furthermore, the best Azotobacter isolates were subjected to 16S rRNA sequencing and confirmed their identities as Azotobacter sp. The indigenous Azotobacter strains with multiple PGP activities could be further used for commercial production.


Introduction
In agriculture, nitrogen deficiency directly influences the yield and profitability of crop plants worldwide and can be overcome by the application of inorganic chemical fertilizers [1]. The increased use of nitrogen based inorganic chemical fertil-izers causes serious adverse effects on the physicochemical properties of soil, i.e., degradation of soil organic carbon (SOC) and soil acidification [2]. Plant growth promoting rhizobacteria (PGPR) provides a promising sustainable solution for increasing agricultural productivity by encouraging plant growth and using plenty of growth-promoting pathways, beneficial plant microbe interactions, etc. and thereby decreases the use of these inorganic chemical based fertilizers [3]. In agriculture, many plant growth promoting microorganisms such as Azotobacter, Rhizobium, and Pseudomonas are used as biofertilizers, which not only provide plant nutrition but also maintain the soil health.
Nitrogen fixing bacteria, especially from the genus Azotobacter, holds an important role in soil fertility [4], since it can provide fixed N 2 to plants and promote plant growth by providing other necessary nutrients to plants. Azotobacter is gram-negative, nonsymbiotic diazotrophic bacteria that belongs to the Azotobacteriaceae family that can fix an average of 20 kg N/ha/per year which can be further increased through inoculation of potent strains [5].
The ecological distribution of Azotobacter spp. is a complicated subject that depends on soil characteristics and climate conditions [6]. There are around six species in the genus Azotobacter and among them Azotobacter chroococcum dominant in Indian soils [7]. Several researchers around the world have successfully used Azotobacter as PGPB in biological N 2 fixation, increased nutrient availability in the rhizosphere, and induced root surface not only yielding attributes but also reduced production costs [8,9].
Biotic and abiotic stress significantly affects crop production and can be overcome by the application of plant growth promoting rhizobacteria, which stimulates plant growth by employing a plethora of growth-promoting mechanisms [10]. Azotobacter not only has the capacity to fix atmospheric nitrogen but also has various PGP activities, which makes Azotobacter the most effective and widespread among the PGPR community [11].
Despite the availability of Azotobacter strains, there is still a need to isolate and characterize native potent indigenous strains adapted to the local environment, which can not only contribute to the formulation of effective bioinoculants but also have a more competitive ability to survive in field conditions [12]. Hence, the aim of this study was to isolate, screen, and characterize the local Azotobacter present in the rhizospheric soil of different districts of Rajasthan for various plant growth promoting attributes and to evaluate their nitrogen-fixing ability by the Acetylene Reduction Assay (ARA).

Materials and Methods
2.1. Isolation of Azotobacter. Isolation of Azotobacter sp. from rhizospheric soils was done by the procedure described by Upadhyay et al. [13]. The rhizospheric soil samples collected from the districts of Rajasthan viz. Udaipur, Banswara, Dungarpur, Rajsamand, and Chittorgarh and were stored at 4°C until processed (Supplementary data sheet Table: S1). Rhizosphere soil was diluted up to 10 -7 dilutions under aseptic conditions, and Azotobacter strains were isolated on Nitrogen free Jensen's medium [14].

Screening of Plant Growth Promoting (PGP) Attributes
and Abiotic Stress Tolerance of Azotobacter. All Azotobacter strains were further screened for multiple plant growth promoting activities viz. IAA production, siderophore production, ammonia production, HCN production, phosphate solubilization, and different abiotic stress tolerance viz. pH tolerance, temperature tolerance, salinity tolerance, and drought tolerance according to the methodologies published in our previous research [17,18].

Molecular Characterization
Using ARDRA of the 16S rDNA Region. The total genomic DNA of Azotobacter strains was carried out following the method outlined by Jain et al. [19]. The DNA patterns of the restriction digested 16S rDNA region were amplified and analyzed on agarose gel for all Azotobacter isolates as outlined by Jain et al. [19] and Kour et al. [17]. The 16S rDNA amplicons were digested with HinfI, AluI, and TaqI restriction endonucleases [20]. Data analysis was performed using the NTSYS-PC (Numerical Taxonomy and Multivariate Analysis System) software and SIMUQUAL Jaccards similarity coefficient [21].

Qualitative and Quantitative Estimation of Nitrogen
Fixation. The qualitative nitrogen fixing ability of Azotobacter was detected in petri plates containing nitrogen-free malate media (NFM) with bromothymol blue (BTB) dye, and the positive strains produced a blue color zone around the inoculated strains. The amount of total nitrogen produced by Azotobacter in Jensen's broth was evaluated by micro-Kjeldahl method as described by Jackson [22]. Furthermore, Azotobacter isolates were screened for acetylene reduction ability (ARA) according to the methodologies of Marag and Suman [23], and the ARA results were expressed in nanomoles of ethylene produced per mg protein per hour.
2.6. In Vitro Studies on the Effect of Azotobacter on the Growth and Yield of Maize Seedlings. To study the effect of Azotobacter on maize (variety FEM-2), the pot experiments with selected Azotobacter strains with multiple PGP and high ARA activities were conducted in triplicate in a complete randomized design (CRD) as per our previous research [17]. The seeds treated with Azotobacter inoculant were sown whereas uninoculated control was also maintained. Different plant growth parameters, i.e., shoot length, root length, root number, leaf number, and chlorophyll content, were recorded after 30 days of germination.

Sequencing and Phylogenetic
Analysis of 16S rDNA of Potent Azotobacter Strains. The PCR amplified 16S rDNA regions of efficient Azotobacter were sequenced using an automated DNA Sequencer (ABI model 377, Applied Biosystems) as per the method described by Jain et al. [18]. The 16S rDNA sequences were edited before the BLAST using the BioEdit software package. The sequences obtained in the study were compared with previously submitted sequences of the nucleotide database GenBank at National Centre for Biotechnology (NCBI) using the nucleotide BLAST (blastn) 2 BioMed Research International [24]. The 16S rDNA consensus sequences were aligned using an online tool CLUSTAL-W [25]. This alignment was further used for phylogenetic tree construction through the MEGA 6.06 software using the Maximum Likelihood method [26].

Isolation and Characterization of the Azotobacter Strains.
In the present study, 24 Azotobacter strains were isolated from different rhizospheric soil samples of Southern Rajasthan on Jensen's medium. The morphological characteristics of Azotobacter strains are summarized in Table S2 (Supplementary data sheet). Based on cell morphology and pigment production, these isolates were preliminary characterized as Azotobacter. All these isolates were able to grow in nitrogenfree medium without producing water-soluble pigments.
Microscopic studies confirmed that all these strains are cyst forming gram-negative rods when examined under the microscope. The present findings were corroborated with those of Khosravi and Dolatabad [27] who isolated Azotobacter from rhizospheric soils from arid and semiarid regions of Iran.

Screening of Plant Growth Promoting (PGP) Attributes and Abiotic Stress Tolerance of Azotobacter Strains. All 24
Azotobacter strains were subjected to various plant growth promoting activities, and the results are summarized in Table 1. All 24 Azotobacter strains were able to produce IAA ranging from 16.5 to 34 μg ml -1 in media supplemented with tryptophan. The maximum IAA production was observed by Azo4 (34 μg ml -1 ) followed by Azo7 (33.5 μg ml -1 ) whereas the least by Azo16 (16.5 μg ml -1 ). In the present study, IAA production by Azotobacter strains were slightly higher as compared with an earlier report from Pandey et al. [11] where 23 μg/ml of IAA production was recorded in Azotobacter strain AU1. Out of the 24 strains, only seven were phosphate solubilizers, and the phosphate solubilization index (PSI) ranged between 1.14 and 3.61, and the strain Azo15 showed the highest solubilization index, i.e., 3.61. Nosrati et al. [28] studied the phosphate solubilization potentials of twenty Azotobacter isolates and reported the PSI for these strains between 3:5 ± 0:1 and 1:4 ± 0:1 which was also observed in the present investigation.
In the present study, all 24 strains were found to be positive for siderophore production, which was determined by their ability to produce the distinct orange halo on the chrome azurol S (CAS) agar media. The highest siderophore production index was shown by Azo21 (1.81) whereas the least by Azo6 (1.08). Ferreira et al. [29] reported that A. vinelandii produced the highest levels of siderophore (80-140 μmol l −1 ) after 72 h incubation. All Azotobacter strains were able to produce HCN, and out of 24 isolates, 16 isolates exhibited strong (+++) production, 5 isolates as moderate (++), and 3 isolates as weak (+). Whereas all Azotobacter strains were ammonia producers and 10 were recorded as strong (+++) ammonia producers, 12 as moderate (++) ammonia producers, and 2 showed poor (+) ammonia production. Chennappa et al. [30] studied the PGP activities of ten Azotobacter strains and reported that all strains were able to produce HCN and ammonia, which also supports finding of the present investigation.
The ability of Azotobacter strains to tolerate abiotic stress was also evaluated, and the results are summarized in supplementary data sheet (Table S3-S6). Among the 24 strains of Azotobacter, 6 strains exhibited growth at pH 4.0; all the strains were able to grow at pH 6.0, whereas 4 strains were able to grow at pH 8.0 and one strain at pH 10.0. Similar results were reported by Jimenez et al. [31] where pH 6.0-7.0 were determined as optimum for Azotobacter strains isolated from different crop plant soil samples.
Azotobacter growth was also determined for salt tolerance using varying concentration of NaCl in Jensen broth. At 1-4% NaCl concentration, all 24 strains showed profuse growth, and at 5% NaCl concentration, 20 strains showed profuse growth, and 4 strains showed moderate growth, whereas at 6% NaCl concentration, 7 strains showed profuse growth, and the remaining 17 strains showed moderate growth. At 7% NaCl concentration, 3 strains showed moderate, and 13 strains showed week growth, and at 8% NaCl concentration, 3 strains showed moderate, and 1 strain showed weak growth. At 9%NaCl concentration, 2 strains showed moderate, and 2 showed weak growth, whereas at 10% salt concentration, only two strains showed weak growth. The above result agreed with the findings of Chennappa et al. [30] who studied four Azotobacter species for salt tolerance and reported that A. chroococcum, A. vinelandii, and A. salinestris can tolerate up to 8% NaCl concentration; however, the cell counts were reduced with high concentration of NaCl.
All 24 strains were subjected to temperature stress, and at 20°C, poor growth was noted in 8 Azotobacter strains whereas moderate growth was observed in 16 isolates. At 30°C, all 24 Azotobacter strains showed strong growth whereas at 35°C and 40°C only 2 strains and 1 strain, respectively, showed strong growth. At 45°C, 20 Azotobacter strains showed poor growth, and 4 isolates failed to grow at this temperature. Similar results are corroborated by Chennapa et al. [30] where they studied temperature tolerance in Azotobacter and reported that the strains of A. chroococcum were found to be tolerant to the temperature range of 35-45°C.
Azotobacter growth was determined for drought stress using varying concentration of PEG in Jensen broth. At 10%, 20%, 30%, and 40% PEG concentration, Azo18, Azo11, Azo10, and Azo7 strains showed moderate to high growth, respectively. Reports are available on PGP bacteria tolerance to drought stress (20-40%) induced by PEG by Marulanda et al. [32]. Ali et al. [33] reported drought tolerance among thirty-two selected PGP bacterial isolates by PEG (6000) and hence could withstand under water stress conditions.   (Figure 1(a)). On the basis of restriction digestion by HinfI, 21 different patterns were obtained, whereas digestion by TaqI and AluI produced 16 and 20 different patterns, respectively. Based on UPGMA clustering analysis, the Azotobacter strains showed significant molecular diversity, and the dendrogram obtained differentiated 24 strains into 2 major clusters A and B comprising 17 and 7 strains, respectively, at the similarly coefficient of 0.10 (Figure 1(b)). Similarity indices established on the basis 57 bands of the three restriction enzymes ranged from 0.04 to 0.45. Genetic relationship among 24 Azotobacter strains was also visualized by performing PCA, and the Azotobacter strains occupied the same position in two-dimensional and three-dimensional scaling as observed in the dendrogram. Therefore, different fingerprinting profiles were obtained which showed significant genetic diversity of Azotobacter strains selected from different districts of southern Rajasthan. Rubio et al. [34] identified genetic diversity among A. chroococcum, A. salinestris, and A. Armeniacus by PCR-RFLP of 16S rDNA sequences using the enzymes RsaI and HhaI. Mazinani and Asgharzadeh [35] also reported the molecular identification of three Azotobacter sp., i.e., A. chroococcum, A. vinelandii, and A. beijernckii using RsaI, HpaII, and HhaI. Khosravi and Dolatabad [27] studied the molecular differentiation and diversity analysis of Azotobacter species and reported that the ARDRA technique with HpaII, BOX, and REP PCR based markers was able to differentiate between A. chroococcum and A. salinestris.

Detection of Nitrogen Fixing Activity.
To identify the free nitrogen fixers among these isolates, they were preliminary screened on nitrogen-free malate agar medium (NFMM) containing bromothymol blue (BTB) as an indicator. Out of 24 isolates, twenty Azotobacter strains produced a blue color zone on the medium indicating the fixation of nitrogen by them. The studies by Suleiman et al. [36] and Gothwal et al. [37] also reported a similar result. Furthermore, the amounts of total N 2 fixed by Azotobacter strains ranged between 3.17 and 24.19 mg g -1 sucrose using micro-Kjeldhal method ( Table 2). The highest amount of N 2 was fixed by Azo1 while the lowest by Azo22. Upadhyay et al. [13] reported the amounts of N 2 fixed by different Azotobacter strains ranged from 18.88 to 6.04 mg g -1 sucrose and is in close agreement with the results of N 2 fixation in the present study.

In Vitro Studies on the Effect of Azotobacter on the
Growth and Yield of Maize Seedlings. The pot culture experiment was conducted in plastic pots filled with sterile planting mixture. To study the plant growth promoting activity of Azotobacter isolates, maize cultivable variety (FEM-2) seeds were treated with Azotobacter inoculant (seed bacterization method) and sown. Fourteen Azotobacter strains were selected for pot studies based on their higher ARA values. Pot experiment data recorded after 30 days of germination are summarized in Table 3.
Higher plant growth was observed in maize seedlings raised from bacterized seeds with selected Azotobacter isolates compared with absolute control. This indicates the Value ± standard deviation. * Positive Azotobacter strains produced a blue color zone on the medium indicating the nitrogen fixation; * * using micro-Kjeldhal method; * * * acetylene reduction assay.  Shoot length, root length, root number, and total chlorophyll content have been significantly increased in the maize plantlets inoculated with the selected Azotobacter strains as compared to uninoculated control. Shoot length increases by 51.20% in pots inoculated with Azo1 followed by Azo20 (47.92%). Root length increases by 57.39% in pots inoculated with Azo20 followed by Azo13 (49.94%). Root number increases by 89.75% in pots inoculated with Azo1 and Azo13. Leaf number increases by 33.4% in pot inoculated with Azo20 followed by Azo1 and Azo13 (26.0%). Chlorophyll content increased by 80.46% in pots inoculated with Azo20 followed by Azo1 (75.28%). All 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 [39] who reported Azotobacter seed bacterization in maize stimulated the growth of treated plants as characterized by the increase of root and shoot length. Romero-Perdomo et al. [40] reported Azotobacter bacterial inoculation in cotton positively influenced plant growth parameters reducing 50% nitrogen fertilization dose. These findings advocated the application of Azotobacter for the improvement of plant growth due to their intrinsic ability of fixing atmospheric nitrogen and expressing plant growth-promoting substances.
3.6. Molecular Identification of the Isolates. The full length sequence of 16S rDNA gene of the most promising Azotobacter strains based on the multiple PGP activities, nitrogen fixation ability, and in vitro performance viz. Azo1, Azo7, Azo13, Azo15, and Azo20 were sequenced and analyzed using the nucleotide BLAST tool. Based on the BLAST results, these strains showed the greatest sequence identity with the previously reported type strains of Azotobacter chroococcum and Azotobacter vinelandii (Figure 2). Indian soils are previously reported to have both chroococcum and vinelandii species of Azotobacter [11,41] also supporting the present study. The molecular identities and NCBI Gen-Bank accession number assigned to these strains are presented in Table 4.
In the present study, biochemical and molecular characterization of Azotobacter strains was reported from the soils of Southern Rajasthan, and based on 16S rDNA sequencing, the potent strain was characterized as Azotobacter chroococcum and Azotobacter vinelandii. In this research, additional molecular techniques including ARDRA revealed significant genetic diversity among Azotobacter strains studied in this study. The plant growth promoting traits and nitrogen fixation by local Azotobacter strains is very critical for the selection of such strains for biofertilizer formulations in order to replace the ineffective strains. The results indicated that inoculation with multi-PGP Azotobacter strains significantly improved the plant growth under in vitro conditions and may be used for commercial production. Hence, dedicated field studies are required to confirm the efficacy of these Azotobacter strains.

Data Availability
The data used to support the findings of this study are included within the supplementary information file(s).

Conflicts of Interest
No potential conflict of interest was reported by the authors.

Acknowledgments
The financial assistance from the All India Network Project on soil biodiversity and biofertilizers and the Rastriya Krishi VikasYojana (RKVY) research project is highly acknowledged. Table S1: location of different isolated Azotobacter strains from Rajasthan. Table S2: morphological characterization of Azotobacter isolates. Table S3: pH tolerance in Azotobacter isolates. Table S4: temperature stress tolerance in Azotobacter isolates. Table S5: salinity tolerance in Azotobacter isolates.