Plant growth promoting characteristics of halophilic and halotolerant bacteria isolated from coastal regions of Saurashtra Gujarat

The study was carried out at “Department of Biotechnology, College of Agriculture, Junagadh Agricultural University, Junagadh” during the academic year 2018–2019. A total of fifteen isolates was used for studying the various plant growth promoting activities with three replication each for each experiment using a completely randomized design (CRD).

Soil sample collection

The soil samples were collected from coastal regions of Saurashtra, Gujarat viz., Junagadh and Porbandar districts located at coordinates 21.52°N 70.47°E and 21°37′48″N 69°36′0″E respectively. The names of the sites within the respective districts from which the soil samples were collected with due consultation and permissions have been shown in Table 11. A total of 15 soil samples (approximately 100 g each) were collected from agricultural fields from a depth of 5 cm around the crop rhizospheres with the help of an agricultural soil sampler.

Preliminary soil analysis

The preliminary analysis of soil samples were carried out to determine the soil chemical properties viz., soil pH by potentiometry and electrical conductivity by conductometry method⁴², and soil organic carbon content by back titration method⁴³, available soil phosphorous by colorimetric method⁴⁴ and potash by flame photometry method⁴².

Isolation of halophilic bacteria from soil samples

Halophilic bacteria were isolated from 15 different soil samples and inoculated by streak plate method on freshly prepared halophilic agar plates containing in grams per litre: 10 g casein acid hydrolysate, 10 g yeast extract, 5 g proteose peptone, 3 g tri-sodium citrate, 2 g potassium chloride, 25 g magnesium sulphate and 20 g agar supplemented with 10% NaCl adjusted to pH 7.2 ± 0.2 (at 25 °C) and incubated at 35 °C for 5 days.

Morphological characterization

Morphological characterization of all isolates were studied by plating on halophilic agar plates and incubating at 35 ± 2 °C for 24 h in BOD incubator. The morphological and cultural characteristics such as size, shape, elevation, margin, texture, opacity and pigment with regard to colonial characters were then observed and recorded from the growth on halophilic agar plates. In addition it also included microscopic characterization by Gram’s staining, motility and scanning electron microscopic observation.

Determination of salt, pH and temperature tolerance test

The salt, pH and temperature tolerance of all isolates were tested separately in halophilic broth tubes supplemented with NaCl concentrations ranging from 5, 10, 15, 20 and 25 per cent for salt tolerance, pH ranging from 2.0, 4.0, and 6.0, 8.0 and 10.0 for pH tolerance, and the temperature ranging from 18, 25, 35 and 45 °C for temperature tolerance. These tubes were inoculated with 0.1 ml (having 10⁷ CFU ml⁻¹) previously grown culture of all isolates. A tube without inoculation served as the negative control for each range of salt, pH and temperature respectively. The isolates were inoculated and incubated at 35 ± 2 °C for 8–10 days. The strain that could grow at particular range of salt, pH and temperature were considered as tolerant by observing the presence or absence of growth and comparing it with negative control respectively for each isolates.

Biochemical characterization

Biochemical characterization of isolates were carried out by using a HiPure Bacterial Identification Kit obtained from HiMedia for biochemical tests viz., glucose, adonitol, lactose, arabinose, sorbitol, citrate, lysine and ornithine utilization, urease detection, phenyl alanine deamination, nitrate reduction and H₂S production tests. Other biochemical tests viz., starch, lipid and gelatin hydrolysis, indole production, methyl red, Voges–Proskauer and catalase tests were carried out manually according to the standard protocol followed by Nezami et al.⁴⁵ while oxidase test was based on the method described by Nyakeri⁴⁶.

IAA production

In vitro IAA production of isolates were determined following the protocol described by Khalid et al.⁴⁷ on a freshly prepared 10 ml Glucose Phosphate Broth (GPB) medium prepared in 100 ml Erlenmeyer flasks. l-Tryptophan was added at desired concentration to the liquid medium by filter sterilization and passing through 0.2 μm membrane filter. 1.0 ml of 3-days old isolates broth (10⁷ CFU ml⁻¹) were then inoculated into each flasks and incubated at 30 ± 2 °C for 48 h. A flask containing the same broth medium without inoculation was kept as negative control for comparison. Following incubation, the isolates’ broth culture contents were filtered through Whattman filter paper No.2 and 3.0 ml of culture filtrate were taken in test tubes and 2.0 ml of Salkowski’s reagent was added for measuring IAA production. The test tubes containing the contents were then allowed to stand for 1/2 h for color development. The colour development were observed in both standard solutions and culture filtrate of IAA and the intensity of color developed were measured by spectrophotometer at 530 nm and recorded. The concentration of IAA produced by the bacterial isolates were then deduced by calculation from the IAA standard curve.

Nitrogen fixation capacity

The nitrogen fixing capacity of the selected halophilic bacterial isolates were studied on a nitrogen free Jensen’s agar medium. The plates were inoculated by streaking the isolate culture onto the plates and incubated at 35 ± 2 °C for 5 days. The nitrogen fixation capacity of the bacterial isolates were then determined by micro Kjeldahl digestion and distillation method⁴⁸. The uninoculated plate of the same media was used as negative control. The nitrogen content in the nitrogen free media which is equivalent to the amount of nitrogen fixed by the isolates were then determined by calculating with the following formula:

where, 0.05 = Normal concentration of H₂SO₄ used; 0.014 = Conversion factor.

Qualitative phosphate solubilization test in solid medium

Qualitative phosphate solubilization test in Sperber’s medium was carried out as per standard protocol followed by Nosrati et al.⁴⁹. To examine Pi (Inorganic phosphorus) solubilization capabilities, 10 μl of the bacterial suspensions (~ 10⁴ CFU ml⁻¹) was spotted with the help of sterile inoculating needle onto the center of Sperber medium plate containing insoluble Pi. The inoculated plates were incubated at 28 °C. A Sperber agar plate spotted with E. coli was kept as the negative control. The zone of solubilization was recorded at 3, 5, 7 and 10 days after inoculation. The Solubilization Index (SI) was determined by measuring the ratio of halo (clear zone) diameter (mm) and the colony diameter as per the formula followed by Nautiyal⁵⁰. And the Solubilization Efficiency (SE) was calculated by the formula followed by Nguyen et al.⁵¹.

Quantitative phosphate solubilization test in liquid medium

Quantitative phosphate solubilization test in liquid medium was carried out in phosphate solubilization medium as per standard protocol followed by Dahale⁵². Erlenmeyer flasks (250 ml) containing 50 ml of phosphate solubilization estimation medium (containing per litre: 0.5 g yeast extract, 10 g dextrose, 5 g CaCl₂, 0.5 g (NH₄)₂SO₄, 5 g Ca₃(PO₄)₂, 0.2 g KCl, 0.1 g MgSO₄, 0.0001 g MnSO₄ and 0.0001 g FeSO₄, pH 7.0) were inoculated with 100 μl of bacterial suspension (approx. 10⁷ CFU/ml) in triplicates and incubated on rotary shaker (180 rpm) at 28 °C. After an intervals of 3, 5, 7 and 10 days, samples were drawn aseptically and centrifuged at 5000×g for 10 min to pellet the cell biomass and insoluble phosphate and the supernatants were used for the measurement of pH using pH meter for the determination of acidity and liberated Pi following phosphomolybdic blue color method⁴². The amount of Pi released in respective broths were estimated from three flasks each after incubation (DAI) in comparison with a set of uninoculated control. The concentration of Pi released were then calculated by plotting a graph of OD versus concentration of phosphates released in μg for standard and samples followed by their comparisons.

Estimation of pH change in broth culture

The supernatant obtained after centrifugation of each isolate culture at 3, 5, 7 and 10 DAI were examined for change in pH for determining acidity using a digital pH meter. The drop in pH in supernatants of each respective culture broth were recorded by comparing with the initial pH kept at 7.0.

Estimation of Pi released from culture supernatant

The available phosphorous content in the broth supernatant of each respective isolates were estimated by following phosphomolybdic blue color method⁴². A known varying concentration of potassium dihydrogen phosphate was used to prepare the standard curve. One ml supernatant of each isolates and control were then taken in 50 ml volumetric flask and 10 ml of chloromolybdic acid was added to it and the contents were mixed thoroughly. The volume was adjusted to three fourth with distilled water and 0.25 ml chlorostannous acid was added followed by immediate adjustment of volume to 50 ml with distilled water. After 15 min, the intensity of blue color developed were measured in UV spectrophotometer at 610 nm using reagent blank.

Potash solubilization capacity

The potash solubilization capacity of the selected halophilic bacterial isolates were tested qualitatively on an aleksandrow agar plates. A loopful of bacterial cells were picked from the respective fresh isolate culture broth and spotted in the middle of the plates containing solidified medium. The inoculated plates were then incubated in the incubator by placing them upside down at 30 °C for 4 days. An Aleksandrow agar plate spotted with an E. coli culture in the centre served as the negative control. The different bacterial isolates showing ability to solubilize potash by forming zone around their colony growth were considered as positive potash solubilizing bacteria. The diameter of zone of clearance or solubilization (halo) observed around the bacterial colony and the diameter of colony were measured after 3, 5, 7 and 10 days of inoculation in triplicates. Potash solubilization index (KSI) was then calculated as the ratio of diameter of halo (mm)/diameter of colony (mm) as per the formula followed by Nautiyal⁵⁰. And the Solubilization Efficiency (SE) was calculated by the formula followed by Nguyen et al.⁵¹.

Siderophore production potentiality

The siderophore production potentiality of isolates were determined by following qualitative plate assay on Chrome Azurol S blue agar medium (CAS) to detect the siderophore production by isolates as per method described by Bhatt⁵³. The CAS agar media was prepared according to the step-by-step procedure described by Louden et al.⁵⁴. The individual CAS plates were then spot inoculated by overnight grown cultures of respective isolates and incubated at 30 ± 2 °C for 24 h. The isolates showing yellow to orange colored ring around the colonies were then considered as positive siderophore producing strains. The siderophore types produced were further identified as hydroxymate, catechol or carboxylate type by Tetrazolium, Arnow’s, and Carboxylate test respectively as per the standard protocol described by Bhatt⁵³.

Qualitative ACC deaminase production

Qualitative ACC deaminase production test was carried out using a simple plate assay following the standard protocol³⁸ in Dworkin and Foster (DF)⁵⁵ minimal salts medium containing per litre of distilled water: 4.0 g KH₂PO₄, 6.0 g Na₂HPO₄, 0.2 g MgSO₄.7H₂O, 2.0 g glucose, 2.0 g gluconic acid and 2.0 g citric acid with trace elements: 1 mg FeSO₄.7H₂O, 10 μg H₃BO₃, 11.19 μg MnSO₄.H₂O, 124.6 μg ZnSO₄.7H₂O, 78.22 μg CuSO₄.5H₂O, 10 μg MoO₃, adjusted to pH 7 and then supplemented with 1.8% (w/v) Bacto-Agar (Difco Laboratories, Detroit, MI, USA) which has a very low nitrogen content. After sterilizing the media by autoclaving at 15 psi 121 °C for 20 min, the heat-labile ACC was filter-sterilized through a 0.2-mm membrane at a concentration of 3 mM and the filtrate was added to the DF salts minimal medium as a sole nitrogen source and poured onto each plates marked as DF-ACC. After solidification, the plates were spot inoculated with fresh isolates inoculum and incubated for 72 h at 32 ± 2 °C but not higher than 35 °C to avoid inhibition of ACC deaminase activity of the isolates. Plates containing only DF minimal salts medium without ACC marked as DF plates were used as the negative control and those with (NH₄)₂SO₄ (0.2% w/v) in place of ACC marked as ACC-NH₃ plates were used as the positive control⁵⁶. Isolates showing growth on ACC-supplemented plates were compared to the negative and positive controls and considered as positive for ACC deaminase production as indicated by their ability to break down ACC and utilize it as the nitrogen source for growth.

Quantitative ACC deaminase production

The ACC deaminase activity of isolates were estimated by performing quantitative test as described hereafter. The bacterial isolates were cultured first in rich medium and then transferred to minimal medium with ACC as the sole nitrogen source in order to create culture conditions that favors the induction of isolates ACC deaminase activity. The isolates exhibiting positive ACC deaminase activity in the qualitative test were cultured first in a 15 ml fresh TSB broth and incubated at 35 ± 2 °C for 24–48 h on a shaker incubator at 150–200 rpm. After incubation, the accumulated biomass of the respective isolate cultures were harvested by centrifugation at 8000 rpm for 10 min at 4 °C and supernatants were discarded. The cell pellet of each isolates were then washed with 5 ml of either 0.1 M Tris–HCl (pH 7.6) or fresh DF salts minimal medium and centrifuged again and pellets resuspended in 15 ml DF minimal salt medium containing ACC at a final concentration of 3 mM in triplicates and incubated at 32 °C for another 36–72 h on a shaker incubator. The bacterial cells were then harvested again by centrifugation at 8000 rpm for 10 min at 4 °C and the supernatants were discarded and the cell pellet of each isolates were washed twice with 5 ml of 0.1 M Tris–HCl (pH 7.6) to ensure the pellets were free of the bacterial growth medium³⁸ and re-suspended in 1 ml of 0.1 M Tris–HCl (pH 7.6) in new 1.5 ml microcentrifuge tubes. The contents of the 1.5 ml microcentrifuge tubes were centrifuged at 16,000×g for 5 min and the supernatants were removed and the pellets were re-suspended in 600 μl of 0.1 M Tris–HCl (pH 8.5). Thirty microliter of 5% toluene (v/v) were added to the respective cell suspensions and vortexed for 30 s to labilize the cells. Then, 200 μl of labilized cell suspensions were transferred to a clean 1.5 ml microcentrifuge tubes and 20 μl of 0.5 M ACC was added and briefly vortexed for 5 s and then incubated at 30 °C for 15 min. About 200 μl of labilized cell suspension without ACC was kept as negative control and 0.1 M Tris–HCl (pH 8.5) with 20 μl of 0.5 M ACC was kept as blank. The samples were then mixed thoroughly with 1 ml of 0.56 N HCl by vortexing and the cell debris removed by centrifugation at 12,000 rpm for 5 min at room temperature and 1 ml of supernatants were transferred to a glass test tube and mixed with 800 μl of 0.56 N HCl and 300 μl of DNP solution i.e. 2,4-dinitrophenylhydrazine reagent (0.2% 2,4-dinitrophenylhydrazine in 2 M HCl). The content mixture were then vortexed and incubated at 30 °C for 30 min. Then 2 ml of 2 N NaOH were added to the sample mixture prepared above and the absorbance were measured at 540 nm in spectrophotometer⁵⁷. Finally, the ACC deaminase activity of isolates were determined by measuring the amount of α-ketobutyrate formed by the cleavage of ACC by ACC deaminase in each samples by comparing their absorbance at 540 nm to a standard curve generated by α-ketobutyrate.

Molecular identification by 16S rRNA gene amplification

The DNA for 16S rRNA gene amplification were isolated using Qiagen’s DNA extraction DNeasy^® Blood and Tissue kit (250) and the 16S rRNA gene amplification were carried out on capillary sequencer (Applied Bio Systems 3130) with the help of a pair of universal oligonucleotide primers viz., forward and reverse primers designated as 005F and 907R with sequences 5′-TGGAGAGTTTGATCCTGGCTCAG-3′ and 5′-CCGTCAATTCMTTTRAGTTT-3′ respectively. The partial 16S rRNA gene sequence obtained from studied bacteria were analyzed and identified with nucleotide BLAST search in Gene Bank of NCBI.

Consent to participate/publish

I, as the main corresponding author of this paper hereby state that the entire research work was carried out with the appropriate consent of all the concerned authors and that all the concerned authors in relation to this work described has given their approval for participation into this publication process.