Growth stimulation of mustard by Pseudomonas species -

Growth stimulation of mustard by Pseudomonas species

Various plant growth-promoting activities, i.e., production of aminolevulinic acid, indole acetic acid, 1-aminocyclopropane-1-carboxylate deaminase, and solubilization of potassium and phosphorus, were analyzed in isolated salt-tolerant rhizobacterial strains, which may contribute towards salt tolerance and crop productivity in mustard. Rhizobacterial inoculation revealed 139 to 291% increase in root and shoot dry weight even after 80 days of sowing. The results indicated that application of two potential rhizobacterial isolates HMM57, and JMM15, which were able to tolerate high salt concentration (8% NaCl) and having IAA and ALA production along with ACC utilization activities, showed stimulation of the mustard growth even up to 12 dS/m in plates and also under controlled greenhouse conditions. These plant growth-promoting rhizobacterial isolates may be used for the enhancement of mustard crop productivity under salinity stress and could be used as biofertilizer.

Isolation of rhizobacteria and host species

Rhizobacterial colonies were isolated by serial dilution method using King’s B (KB) medium, and ninety-four rhizobacterial isolates were selected based on typical morphological and biochemical characteristics. Seeds of Brassica juncea L. variety 749 were obtained from the Department of Agronomy, C.C.S. Haryana Agricultural University, Hisar, India.

Salt tolerance among rhizobacterial isolates at different salt concentrations

All the rhizobacterial isolates were checked for their ability to grow at different concentrations of NaCl, i.e., 1, 2, 4, 6 and 8% (w/v), on nutrient agar (NA) medium containing 20 mM HEPES (N-2-hydroxyethane-sulphonic acid) buffer. NA plates were spotted with a 20 μl growth suspension of different rhizobacterial isolates and later incubated for 3–4 days at 28 ± 2 °C in an incubator. The susceptibility or tolerance to NaCl by different rhizobacterial was recorded by observing the growth as a positive or negative result, and their effect on colony size was observed at different salt concentrations.

Evaluation of different plant growth-promoting activities

Rhizobacterial isolates were selected on the basis of salt tolerance ability and further studied for the various growth-promoting traits.

Utilization of 1-aminocyclopropane-1-carboxylate (ACC) by rhizobacteria

Rhizobacterial isolates were spotted on medium plates containing minimal medium supplemented with 3 mM ACC and minimal medium plates containing ammonium sulphate as nitrogen sources were used as control plate for growth comparison of different bacterial isolates. The growth of rhizobacterial strains was recorded after 4–5 days of incubation at 28 ± 2 °C.

Production of δ-aminolevulinic acid (ALA)

Rhizobacterial isolates were tested for their ability to produce δ aminolevulinic acid. These cultures were inoculated in triplicates in 10 ml LB (Luria Bertani) broth supplemented with 15 mM glycine and succinate. Then, cultures were incubated at 28 °C for 48 h under stationary conditions of growth, and subsequently, culture samples were withdrawn and centrifuged at 10,000 rpm for 15 min. To 0.5 ml of culture supernatant, 50 μl of acetylacetone and 0.5 ml of 1 M sodium acetate buffer were added, and then tubes were incubated in a water bath for 15 min at boiling temperature. After cooling, 3.5 ml of modified Ehrlich’s reagent were added, and the absorbance of the mixture was measured at 556 nm after 20 min.

Indole acetic acid (IAA) production by rhizobacterial isolates

For quantification of IAA production, rhizobacterial isolates were inoculated in duplicate in 30 ml of LB broth supplemented with DL tryptophan @ 100 μg/ml and were incubated at 28 ± 2 °C for 72 h under stationary conditions of growth. The growth suspension was centrifuged at 10,000 rpm for 15 min. Quantitative estimation of IAA was determined in the culture supernatants by standard method.

Phosphorus solubilization by rhizobacterial isolates

Phosphorus solubilization ability of rhizobacterial isolates was determined by the spot test method on Pikovskaya medium plates containing tricalcium phosphate. Rhizobacterial strains of 48 h old growth were spotted on above-prepared plates and incubated at 28 ± 2 °C for 2–3 days. The formation of solubilization zone around the bacterial colony confirmed the presence of phosphorus solubilization in rhizobacterial isolates.

Potassium solubilization by rhizobacterial isolates

Potassium solubilization by rhizobacterial isolates was also studied by the spot test method on Aleksandrov medium plates having mica powder (an insoluble form of potassium) and acidic dye bromothymol blue. A loopful of 2-days old-growth of the rhizobacterial isolate was spotted on Aleksandrov medium plates and incubated at 28 ± 2 °C for 4–5 days. The presence of potassium solubilization activity in rhizobacterial strains was based upon the ability of solubilization zone formation and change of color from greenish blue to yellow.

Efficacy of inoculated rhizobacteria on the growth of mustard under salinity conditions

Agar plate experimental design

For evaluating the effect of salt concentration on seed germination of mustard, selected rhizobacterial isolates (107–108 cells/ml of growth suspension) were studied for germination of mustard seeds on water agar (0.8%) plates having different levels of NaCl to obtain an electrical conductivity (EC) ranging from 0 to 20 dS/m. Inoculated seeds were grown at 28 ± 2 °C in the plant incubator and the growth of seedlings was recorded on the 5^th and 10^th days after sowing. The observations for retardation or stimulation of root and shoot growth of mustard seedlings (with or without salt) were recorded.

Results

Salt tolerance of rhizobacterial isolates

Screening of rhizobacterial isolates for their salt tolerance capacity at different NaCl concentrations showed that out of ninety-four rhizobacterial isolates, only ten isolates, i.e., HMM6, HMM13, HMM34, HMM39, HMM44, HMM57, HMM65, HMM88, JMM15, and JMM42 revealed larger colony size ranging from 10.1 to 20.0 mm at 8% NaCl concentration.

Rhizobacterial isolates, i.e., HMM39, HMM57, HMM88 and JMM15 revealed significant growth on ACC supplemented plates, and two bacterial isolates HMM65 and HMM13 showed moderate growth. Observations were taken for the ALA producing ability of rhizobacterial isolates in the culture supernatant and maximum ALA production was observed in bacterial isolate JMM15 (17.45 μg/ml). Rhizobacterial isolate JMM15 also produced maximum 12.24 μg/ml IAA and four bacterial isolates HMM39 (1.27 μg/ml), HMM57 (4.62 μg/ml), HMM88 (3.34 μg/ml) and JMM42 (2.86 μg/ml) produced IAA in the range of 1.0–5.0 μg/ml. The promising isolate JMM15 also showed phosphate solubilization zone varying between 1.51 and 2.0 mm on tricalcium phosphate containing medium plates after 2 days of growth. Other rhizobacterial isolates, i.e., HMM13 (1.43 mm), HMM39 (1.03 mm), JMM42 (1.4 mm) showed significant phosphate solubilization zone, whereas two bacterial isolates HMM57 and HMM65 showed an insignificant zone (0.2 mm) of phosphorus solubilization. Four bacterial isolates lacked phosphate solubilization ability. Out of the eleven rhizobacterial strains tested for potassium solubilization, only four isolates revealed a significant zone of K solubilization on mica containing modified Aleksandrov medium plates. Rhizobacterial isolate JMM15 (5.28) showed the highest potassium-solubilizing index (K-SI) followed by isolate HMM57 (4.50).

Effect of inoculated rhizobacteria on the growth of mustard under salinity conditions

An increase in germination percentage was observed by inoculation of all selected bacterial isolates up to 20 dS/m as compared to untreated control. Seeds inoculated with rhizobacterial isolate HMM39, HMM57, or JMM15 showed a more stimulatory effect on the growth of seedlings even at 16 and 20 dS/m salt concentrations in comparison to un-inoculated control. Rhizobacterial isolates HMM57 and JMM15, which possessed most of the plant growth-promoting attributes, were finally selected assuming that these cultures could help mustard plants to withstand varied biotic and abiotic stresses because of their plant growth-promoting activities. A significant increase in tolerance index was observed with the inoculation of bacterial isolates HMM57 and JMM15. Therefore, inoculation of rhizobacterial isolates HMM57 and JMM15 could ameliorate the effect of salt stress and may stimulate the seed germination and plant growth under salt stress conditions.

Conclusion:

There are immense possibilities for developing biofertilizer using rhizobacterial strains that could enhance crop growth under salt stress. In this study, two potential rhizobacterial strains, i.e., Pseudomonas argentinensis strain HMM57 and P. azotoformans strain JMM15, were found to tolerate high salt concentration up to 8% NaCl. The seed inoculation with these bacterial strains showed stimulation of mustard growth up to 12 dS/m in plates as well as in pots under greenhouse controlled conditions. The results suggested that the production of ALA and IAA along with an expression of ACC deaminase activity by the rhizobacterial isolates contribute towards the growth stimulation of seedlings and mustard plants under different salt concentrations. Therefore, these rhizobacterial Pseudomonas species having different plant growth activities may be exploited for ameliorating the adverse effect of salt stress and for promoting the growth of mustard crop under field conditions.

Reference:

Phour, M. and Sindhu, S.S., 2020. Amelioration of salinity stress and growth stimulation of mustard (Brassica juncea L.) by salt-tolerant Pseudomonas species. Applied Soil Ecology, 149: 103518.