Azotobacter vinellandii SINAz1 Increases Growth and Productivity in Rice under Salinity Stress

The world population is increasing very fast and it will reach around 9 billion by 2050 (Godfrey et al., 2010). To feed this huge population, food supplies has to increased upto 70-100% (Tillman et al., 2011). Rice is a very important crop, which is cultivated and consumed by maximum number of world population than other crop (Huang et al., 2018). Besides that, rice cultivation provides much of the raw materials needed by today’s manufacturing industry (Kenmore, 2003). Thus, rice production directly affects food security as well as the economy of the people. Therefore, the production of rice must be increased to fulfil the requirements of growing world population.

Rice production depends mainly on nitrogen (N) fertilizer. The use of chemical fertilizers for supply of nitrogen intrinsically degrade soil environment and environmental health through emission of nitrogen oxides, (especially N2O) from the anaerobic (flooded) rice ecologies (Bhattacharjee et al., 2008). Therefore, biological (especially associative) nitrogen fixation (BNF) should be exploited to supplement N for rice production which would supplement 20-25% of the total N i.e. about 80000 tones atmospheric N/ha (Subba Rao, 2007). Azotobacter is generally regarded as a free-living aerobic nitrogen-fixer (Saharan and Nehra, 2011). Besides, nitrogen fixation, Azotobacter also promote the plant growth by producing thiamine, riboflavin, indole acetic acid (IAA) and Gibberellins (GA) (Sahoo et al., 2012).

In this study, we report the discovery of nitrogen fixing free living bacteria Azotobacter vinellandii strain SINAz1 among 20 rhizospheric bacteria from different locations of Odisha, India. Along with nitrogen fixation, it secretes plant growth promoting hormones like IAA, GA3, ABA and zeatin significantly compared to other. It also protects rice plants from the toxic effect of salinity stress (200 mM NaCl) up to 15 days in pot experiments. This strain notably increased the growth and yield of rice plant without application of any chemical fertilizer. So our bacterium is novel and bears unique characteristics collectively nitrogen fixation, hormone secretion and salinity stress tolerance.

Isolation of nitrogen fixing (Azotobacter spp.) organisms

For isolation Azotobacter species, soil samples were collected from four different rice fields situated in different locations of Odisha, India, viz. OUAT experimental field 1 and 2, Experimental field of Mahanga and Sindhupur, Cuttack, Odisha, India, where rice is cultivated at least for last 20 years. The rice plants (Oryza sativa L. var. IR64) were up rooted and the soil was scrapped off from the root and used for isolation. The isolates were phenotyped (Kennedy et al., 2005; Sahoo et al., 2014) grouped on the basis of phenotypic characters and one representative of each group of isolates were used for the remainder experiments.

Morphological and staining characteristics of the bacteria

Morphological characteristics (shape, size, motility) of the bacteria were checked under a phase contrast light microscope (100X objective). Gram’s stain and spore stain (malachite green) of the isolates were done following standard microbial methods (Collee and Miles, 1989).
 
Physiological and biochemical characterization of isolates
 
Physiological and biochemical tests (oxidase, catalase, urease, indole production, methyl red, acetoin production, nitrate reduction, citrate utilization, hydrogen sulphide (H₂S) production, carbohydrate fermentation, arginine dihydrolase, starch hydrolysis, lipase, tributyrin and vegetable oil hydrolysis, cholesterol hydrolysis, protein hydrolysis, gelatine and casein hydrolysis, pectin and chitin hydrolysis, lecithin hydrolysis) were done for identification of all the bacterial isolates. The detailed procedure of each test was performed according to the method described by Sahoo et al., (2014).
 
Acetylene reduction assay (ARA), quantification of IAA, GA3, ABA and zeatin produced by bacterial isolates
 
Nitrogen fixation efficiency in culture by the four Azotobacter isolates were assessed by ARA in the laboratory (Hardy et al., 1968) cultivated on N-free Jensen agar (Jensen, 1954). The extraction and quantification of IAA, GA3, ABA and zeatin were done according to the method described earlier (Sahoo et al., 2014).
 
Amplification of nifK, nifD, nifH gene
 
The genomic DNA was amplified using full length nif K primers (forward 5'- ATGAGCCAGCAAGTCGATAA-3' and reverse 5'- TGGTGCTGGACCATGCGATT-3'), nif D primers (forward 5'- ATGACCGGTATGTCGCGCCA-3' and reverse 5'- CGGCGGTCGCGGACT-3’) and nifH primers (forward 5'- ATGGCTATGCGTCAATGCGC-3' and reverse 5'- TCAGACTTCTTCGGCGGTTT-3') designed by using primer-3 software (http://bioinfo.ut.ee/primer3-0.4.0/). These primers were synthesized and supplied by Eurofins Amar Immunodiagnostics, Hyderabad, A.P., India.
 
Sites for pot experiments
 
The pot experiments was conducted in the green house of Department of Soil Science and Agricultural Chemistry, Orissa University of Agriculture and Technology, Odisha, India, in the year 2018-2019, to unveil the native efficient Azotobacter strain for formulation and production of potent indigenous biofertilizer for commercial exploitation in salinity soil.
 
Formulation of biofertilizers
 
Biofertilizers were formulated aseptically under a laminar
air flow using the following composition. Sterile (autoclaved) charcoal powder 700 g/kg, CaCO₃ 100 g/kg, gum acacia 20 g/kg and liquid culture 180 g/kg (180 ml containing 10⁹ cfu/ml) i.e. final population 2 × 10⁸ cfu/g formulation (according to Bureau of Indian Standards (BIS).
 
Treatment of seedlings and design of pot experiments
Healthy, 21d old rice (Oryza sativa L. var. IR 64, a salt sensitive variety) seedling were dipped separately in biofertilizer suspensions (10% w/v i.e. 2 × 10⁸ cfu/ml) for 2 h as recommended for commercial formulations by Bureau of Indian Standards (BIS) and transplanted in different pots with three replications each viz. Control (C) without any fertilizer; Treatment 1 (T1) with Azotobacter vinelandii. isolated from OUAT experimental field 1; Treatment 2 (T2) with Azotobacter vinelandii. isolated from OUAT experimental field 2; Treatment 3 (T3) with Azotobacter vinelandii. isolated from experimental field Sindhupur, Cuttack.
 
Growth parameters
 
Growth parameters like plant height (cm), tiller/hill (no), effective tiller/hill (no), panicle length (cm), leaf area (sq. cm) and panicle length (cm) were measure prior to harvest. The crop was harvested after 90 d and the post harvest observations like root length (cm), root dr. wt. (g), root volume (ml), panicle weight (g), grain yield/plant (g), filled grain/panicle (no.) and 1000 grain wt. (g) were recorded.
 
Salinity stress tolerance study
 
The above pot experiments were repeated for salinity stress tolerance assay. The 3 selected Azotobacter vinellandii (Based on the ARA assay) strains along with control were used for these studies. The treatments (C, T1, T2 and T3) were used for this salinity tolerance assay. Rice plants after 6 weeks in soil were subjected to salinity (200 mM NaCl) stress. All the pots (C, T1, T2 and T3) were kept in one big tank filled with 200 mM NaCl solution. The plants were grown in the green house and the white light was provided (16 h photo period) by white fluorescent tubes (36 W Philips TLD) with a photon flux density of 52 μ /m2s (PAR).
 
16S rRNA gene sequencing
 
The 16S rRNA gene was PCR amplified by using the forward (5'- AGAGTTTGATCMTGGCTCAG-3') and reverse primer 5'- GTTACCTTGTTACGACTTAAGTCGTAACAAGGTAACC-3' using the genomic DNA isolated from the most efficient Azotobacter vinellandii The amplified products were sequenced. The sequencing was done on ABI 3130xl analyser based on Sangers dideoxy termination method and submitted to NCBI gene bank (ncbi.nlm.nih.gov).
 
Statistical analysis
 
All statistical analysis were performed using the graph and prism software. The experimental data values were mean values from three independent series, each done with three replicates and the results presented as means ± standard error (SE), based on three replications. The statistical significance at P<0.05 has been calculated.

Morphological, colony and biochemical characteristics of tentative Azotobacter on Jensen’s media
 
Five types of soil bacteria isolated from each experimental rice fields using Jensen’s medium. The organisms were studied by their colony characters (Table 1). The colonies of the bacteria of Jensen’s medium produced convex, circular, fluorescent, brown, off white or white, low convex, flat, plicate, size ranges from 0.60-1.00 mm, gummy, not gummy, mucoid (Table 1). The characteristics viz., shape, size (length and breadth) motility, Gram’s stain of the bacteria were checked under a phase contrast microscope (100X objective). The morphological characteristics of all 20 colonies were presented in Table 2.




 
Biochemical characterization
 
The biochemical tests such as oxidase test, phosphatase test, nitrate reduction test catalase, carbohydrate utilization, carbohydrate fermentation, nitrate reduction, citrate utilization etc. were carried out for identification of isolates. The isolates were examined for catalase, oxidase and for urease test. In citrate utilization test, the bacteria of Jensen’s medium showed positive some of them showed negative. Biochemical characterization of all isolates were given in the Table 3.


 
Identification of Azotobacter spp.
After biochemical tests some of them are identified as Azotobacter vinellandii, Azotobacter chroococcum, Klebsiella spp. Beijerinckia spp. Pseudomonas spp. from the 20 number of isolates (Table 4). The identified Azotobacter vinelandii were used for further assays.


 
Acetylene reduction assay (ARA) of isolated Azotobacter spp.
 
In vitro nitrogen fixing efficiency of identified Azotobacter spp. were studied and among the 11 isolates, the Azotobacter vinelandii isolated and identified from the different fields showed higher nitrogen fixation efficiency (Table 5). All the 3 Azotobacter vinelandii isolates (Az1a, Az2b and Az4a) identified from different fields were used in 3 treatments (T1, T2 and T3) respectively along with control (C) for further studies. Because these three Azotobacter vinelandii isolates have higher nitrogenise activity than others.


 
PGP functions of the Azotobacter isolates
 
Plant growth promotion (PGP) functions of the Azotobacter vinelandii isolates (Az1a, Az2b and Az4a) were presented in Fig 1. The activities were highly variable. The isolate Az4a possess higher IAA, ABA, GA3 and zeatin content than other two (Az1a and Az2b).


 
Amplification of nif gene clusters
 
The gene expected size (1.4kb) was obtained in gel picture of nif D gene, size of (1.5 kb) band was obtained in case of nif K gene and size of 0.87 kb was obtained in case of nif H gene for all the Azotobacter isolates (Fig 2).



16S rRNA sequencing of Azotobacter vinelandii isolate
 
The amplified fragment of 16s rRNA of Azotobacter vinellandii (Az4a) was sequenced and the sequence was submitted to NCBI gene bank and catalogued the accession number as MN135308.1.
 
Growth observations in pot experiments under salinity stress
 
Effects of the the 3 Azotobacter vinelandii isolates (Az1a, Az2b and Az4a) on the growth and productivity of the rice plant along with control (C) experiment were studied. The control plants (C) were died and the plants of other treatments (T1, T2 and T3) were grew well and showed better phenotypic growth characteristics. Among them more tiller number, more plant height were observed in case of T3 (Table 6).


 
Azotobacter vinellandii provide salinity (200 mM) tolerance to plants
 
There was a significant difference in survival and agronomic parameters of rice plants of 3 different treatments (T3-T6) when compared with the plants of C. Better agronomic characteristics were observed in all the treatments under 200 mM salinity stress except C (Table 1). The rice plants of C pot died due to toxic stress of chromium. But other treatment (T1, T2 and T3) plants survived up to maturity.

All together 20 tentative Azotobacter spp. were isolated from Jensen’s N-free medium and they were phenotyped by morpho-physiological and biochemical characters (Kennedy et al., 2005; Sahoo et al., 2014). The morphological, physiological and biochemical characters identified the isolates viz. Az1a, Az2b, Az4a as Azotobacter vinelandii, Az1b, Az2c, Az2d, Az3b, Az3d, Az3e, Az4e as Azotobacter chroococcum and Az3a as Azotobacter spp. (Hill and Sawers, 2000) and the species of other isolates remained unknown. The results proved that the population of Azotobacter spp. of rice rhizosphere was diverse. Similarly, diverse species of Azotobacter i.e. A. vinelandii, chroococcum etc. were identified from rice rhizosphere elsewhere (Saharan and Nehra, 2011). Nitrogen fixation efficiency (acetylene reduction assay, ARA) of the Azotobacter spp. varied between 26.16-128.57 nmole C₂H₄/mg bact./h. The Azotobacter vinelandii SINAz1 (isolate no. Az4a) of experimental field Sindhupur, Cuttack which produced 128.57 nmole C₂H₄/mg bact./h, was more efficient nitrogen fixing organism in culture than the other isolated bacteria. The results proved that nitrogen fixation efficiency of the A. vinelandii SINAz1 was superior to other indigenous BNFs viz. acetylene reduction by heterotrophic or endophytic Azotobacter spp. which fixed 79.6-329.50 nmol C₂H₄/h/culture or 57-686 nmole C₂H₄/mg protein/h (Barua et al., 2012). In rice, ARA of heterotrophic or endophytic Azotobacter spp. was 12.10-53.40 nmol C₂H₄/mg bact./h (Barua et al., 2012) which conformed with the present study. The 16S rDNA of the most potent A. vinelandii (Az3) (other isolates were not done) produced an amplicon of 1.4 kbp size which conformed to that of the other Azotobacter spp. (Sahoo et al., 2014). Plant hormones control plant growth and developmental and played a role in adaptation to different stresses (Peleg and Blumwald, 2011). The gibberellic acids (GA3) mitigate plant from the negative effects of salinity (Qin et al., 2011). The stress-induced production of cytokinin in plants confers tolerance to transgenic plants to stress (Ha et al., 2012). In the present study, we reported higher GA3, zeatin and IAA in rice plants inoculated with A. vinellandii SinAz1. It has been reported that the root and shoot biomass was increased with improved tolerance to salinity in the presence of growth promoting microorganisms (Fan et al., 2011). Our study agree with the similar report on Azotobacter inoculation on chickpea (Cicer arietinum L.). Azotobacter inoculation increases the growth and yield of chickpea under saline (5.8 dS m^-1) arid condition (Abdiev et al., 2019). 

Thus, the conformity of the phenotypic and genetic pattern profiles confirmed the identity of the re-isolates as introduced Azotobacter vinelandii SINAz1. The results also proved that the introduced Azotobacter SINAz1 established in the experimental pots under salinity conditions and substantially survived up to harvest of the crop with improved yield compared to uninoculated plants.