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.
Table 1: Colony characteristics of the isolated tentative Azotobacter spp.
Table 2: Morphological characteristics of the tentative Azotobacter isolated on Jensen’s media.
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.
Table 3: Biochemical characteristics of the Azotobacter spp. isolated.
After biochemical tests some of them are identified as Azotobacter vinellandii
, Azotobacter chroococcum
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.
Table 4: Identification of tentative Azotobacter spp.
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
Table 5: Nitrogen fixation efficiency (Acetylene reduction assay) by 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
Fig 1: (a) Rice plants of different treatments (C, T1, T2 and T3) inoculated with Azotobacter vinellandii exposed to salinity stress (200 mM). (b) PCR conformation of the nif H gene showing amplification of 0.87kb. (c) nif D gene (1.4 kb) (d) nif K gene (1.5 kb).
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
Fig 2: Endogenous hormone content of rice plants (C, T1, T2, T3) inoculated with Azotobacter vinellandii under 200 mM NaCl stress. (a) Endogenous content of IAA; (b) Content of ABA; (c) Content of zeatin; (d) Content of GA3.
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
Table 6: Phenotypic growth characteristics (plant height, root length, root dry weight, leaf area); photosynthetic characteristics (chlorophyll content, net photosynthetic rate, stomatal conductance and internal CO2 concentration, total protein); nutrient content (nitrogen, phosphorus, potassium, sodium) of rice plants at different treatments (Az1, Az2, Az3) and control (C) after 15 days salinity (200 mM) stress.
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 C2
/mg bact./h. The Azotobacter vinelandii
SINAz1 (isolate no. Az4a) of experimental field Sindhupur, Cuttack which produced 128.57 nmole C2
/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 C2
/h/culture or 57-686 nmole C2
/mg protein/h (Barua et al., 2012).
In rice, ARA of heterotrophic or endophytic Azotobacter
spp. was 12.10-53.40 nmol C2
/mg bact./h (Barua et al., 2012)
which conformed with the present study. The 16S rDNA of the most potent A. vinelandii (
(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
inoculation increases the growth and yield of chickpea under saline (5.8 dS m-1
) arid condition (Abdiev et al., 2019).