Amelioration of salinity stress and growth stimulation of mustard (Brassica juncea L.) by salt-tolerant Pseudomonas species

Applied Soil Ecology 149 (2020) 103518

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Amelioration of salinity stress and growth stimulation of mustard (Brassica juncea L.) by salt-tolerant Pseudomonas species

T

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Manisha Phour , Satyavir Singh Sindhu Department of Microbiology, College of Basic Sciences and Humanities, CCS Haryana Agricultural University, Hisar 125004, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Rhizobacterial isolates Biofertilizer Mustard Crop productivity Abiotic stress Soil salinity

Indian mustard (Brassica juncea) is a winter oilseed crop grown across the Northern Indian planes. The growth and yield of the crop have been declining over the recent years due to increase in soil salinity. Applications of plant growth-promoting rhizobacterial strains have been found to enhance crop productivity under salinity conditions. Therefore, the objective of this study was to identify bacterial isolates from the mustard rhizosphere that may help in improving the mustard growth under salt stress conditions. 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. The most promising rhizobacterial isolates HMM57 and JMM15 were identified as Pseudomonas argentinensis and P. azotoformans, respectively by 16SrRNA sequencing. In primary screening, out of ninety-four rhizobacterial isolates tested, only ten isolates HMM6, HMM13, HMM34, HMM39, HMM44, HMM57, HMM65, HMM88, JMM15 and JMM42 showed salt tolerance up to 8% NaCl concentration (0.1–20.0 mm colony size). During secondary screening on agar plates, these rhizobacterial isolates were checked for their potential to enhance seed germination of mustard and growth of seedlings at the high level of salinity (20 dS m−1). Moreover, bacterial isolates HMM57 and JMM15, which exhibited different plant growth-promoting traits, were used as inoculants on the mustard plants grown under high salinity conditions (8 and 12 dS m−1) in a greenhouse. 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−1 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.

1. Introduction Climate change, environmental stresses, water shortage and reduction in soil fertility are the major constraints limiting the growth and productivity of crops and impose a threat to the future of food security for ever-increasing human population (Clair and Lynch, 2010). The abiotic and biotic stresses have been reported to cause 50 and 30% losses, respectively, to agricultural productivity worldwide (Kumar and Verma, 2018). The major abiotic stresses include temperature, drought, salinity, and heavy metal stress (He et al., 2018). Soil salinity affects 800 Mha of land worldwide (Munns and Tester, 2008), and this area is expanding gradually. Salinity stress has a detrimental impact on all aspects of plant growth and development. It limits the productivity of

⁎

crop plants, especially in arid and semi-arid regions, where rainfall is limited (Munns and Gilliham, 2015; Zӧrb et al., 2019). Salt accumulation in soil solution reduces water and nutrient uptake, and this leads to osmotic stress, ion toxicity, nutrient imbalances, and water-deficit. Excessive concentrations of salt ions also affect photosynthesis, injure leaves, and may lead to chlorosis and early leaf senescence (Hanin et al., 2016). Moreover, abiotic stress factors also influence biotic stress, and the major effect of these stresses result in loss of soil microbial diversity, soil fertility and competition for nutrient resources (Chodak et al., 2015). Under conditions of higher salinity (EC 9.6–17.5 dS m−1), average yields of all vital crop plants such as sorghum, barley, wheat, sugarcane, sugarbeet and cotton are reduced by 50% (Panta et al., 2014).

Corresponding author. E-mail address: [email protected] (M. Phour).

https://doi.org/10.1016/j.apsoil.2020.103518 Received 20 September 2019; Received in revised form 3 January 2020; Accepted 19 January 2020 0929-1393/ © 2020 Published by Elsevier B.V.

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M. Phour and S.S. Sindhu

different rhizosphere sites as a composite sample at a depth of 15–30 cm with the help of soil core sampler (Auger). Collected composite samples were transported to the lab in a cold box and stored at 4 °C until processed. The physio-chemical properties of the soil, texture, organic-carbon, available nitrogen, phosphorus, potassium, and total nitrogen were determined as per the described procedures (Hesse, 1971; Jackson, 1973; Brady and Weil, 2002).

Indian mustard [Brassica juncea (L.)] is a vital oilseed winter crop grown across the Northern Indian plains, which occupies the third place among the various oilseed species due to its considerable economic and nutritional value. The maximum oilseed production area is centered in the North-West agro-climatic zone, where the majorities of soil and groundwater sources are highly saline (1.6–17%) and have sodicity problems (Mandal et al., 2010). To gain the maximum productivity of mustard, irrigation is crucial. When the irrigation water with high salinity level is applied at pre-sowing and flower initiation, it significantly reduces seed germination (Fowler, 1991), seedling growth (Almansouri et al., 2001), plant height (root/shoot elongation), dry matter yield (Mishra and Anju, 1996), branching pattern and pod formation (Shekhawat et al., 2012). It can also reduce the mustard's growth and grain yield by 50% and 70%, respectively. The immensity of the salinity effect varies with the different plant species, their type, and the level of salinity, which may affect the growth, yield, and oil production of mustard. This salt-induced growth reduction is known to be improved by several means including the application of growth-regulating substances (Ashraf and Foolad, 2007; Zhang et al., 2008; Enebe and Babalola, 2018), by improving the nutritional status or by inoculation of salt-tolerant bacteria and mycorrhizal fungi (Plaut et al., 2013). Various plant growth-promoting rhizobacterial (PGPR) strains including Azotobacter, Azospirillum, Acetobacter, Bacillus, Klebsiella, Pseudomonas, and Serratia are widely applied on crops as a means of improving productivity, increasing stress resistance and regulating plant growth (Watanabe et al., 2000; Zhang et al., 2006; Nunkaew et al., 2014; Sharma et al., 2018; Chu et al., 2019). The mechanisms by which PGPR strains enhance the growth of plants under salinity conditions include production of ALA (5-aminolevulinic acid); IAA (indole acetic acid); ethylene and solubilization of potassium and phosphorus (Malik and Sindhu, 2011; Plaut et al., 2013; Egamberdieva and Jabborova, 2015; Phour et al., 2018). Rhizobacteria with these plant growth-promoting traits are known to regulate several key physiological processes such as seed germination, reduced Na+ uptake, enhanced nutritional uptake, altered light reactions, improved scavenging of reactive oxygen species and enhanced photosynthetic assimilation. They are also responsible for the maintenance of nutrients status which leads to less ultra-structural damage in the root tip under stress conditions (Ahmad et al., 2006; Egamberdieva and Jabborova, 2015; Zӧrb et al., 2019). Degradation of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) into 2-oxobutyrate and ammonia by bacterial ACC deaminase lower the ethylene concentration in plant roots, relieves the ethylene repression of auxin response factors synthesis and indirectly increases the plant growth (Glick, 2005; Kang et al., 2010). Thus, application of plant growth-promoting rhizobacterial strains has been found to enhance crop productivity at different levels of salinity. Therefore, there are immense possibilities for developing bacterial biofertilizers using rhizobacterial strains that could make the plant to grow under salinity conditions (Kumar and Verma, 2018). Thus, the objective of the present study was to characterize rhizobacterial isolates for their salt tolerance and plant growth-promoting activities that could improve the mustard growth under salt stress conditions.

2.2. 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. 2.3. 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 (Marsudi et al., 1999). 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. Three replications were used to confirm their salt tolerance activity. 2.4. 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. Three replications were used for quantification in each experiment. 2.4.1. Utilization of 1-aminocyclopropane-1-carboxylate (ACC) by rhizobacteria Rhizobacterial isolates were spotted on medium plates containing minimal medium (Dworkin and Foster, 1958) supplemented with 3 mM ACC (Penrose and Glick, 2003) and minimal medium plates containing ammonium sulphate as nitrogen sources were used as control plate for growth comparison of different bacterial isolates (Khandelwal and Sindhu, 2013). The growth of rhizobacterial strains was recorded after 4–5 days of incubation at 28 ± 2 °C. 2.4.2. Production of δ-aminolevulinic acid (ALA) Rhizobacterial isolates were tested for their ability to produce δaminolevulinic acid by the method as described (Mauzerall and Granick, 1955). 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. A standard curve was used to determine the concentration of ALA in the samples.

2. Materials and methods 2.1. Site description and soil sampling Soil samples used for isolation of rhizobacterial isolates, were collected randomly from various locations of mustard fields grown in Choudhary Charan Singh (CCS) Haryana Agricultural University, Hisar, Haryana, India (29°15′19.26″N; 75°68′83.53″E). The farm soil was a coarse loamy mixed, hyperthermic, Typic Haplustepts (Soil Survey Staff, 2014). From each location, samples were collected from six

2.4.3. 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 DLtryptophan @ 100 μg ml−1 (Hartman et al., 1983; Malik and Sindhu, 2

Applied Soil Ecology 149 (2020) 103518

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Nine treatments (T1–T9) were used for this experiment. Uninoculated mustard seeds were sown as a control (T1 to T3), whereas T4–T6 were inoculated with bacterial isolate JMM15, and T7–T9 were inoculated with bacterial isolate HMM57. In treatments T2, T5, and T8, soil salinity was maintained at 8 dS m−1 EC, whereas 12 dS m−1 EC was maintained in T3, T6, and T8. The mustard plants were grown in the pot-house under daylight conditions. The plants were irrigated with water (0.4 dS m−1 EC and pH 8.4), as and when required.

2011) 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 (Remi Instruments, Mumbai, India). Quantitative estimation of IAA was determined in the culture supernatants by the method as described by Gordon and Weber (1951). 2.4.4. 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 (Pikovskaya, 1948; Sindhu et al., 1999). 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.

2.5.3. Tolerance index The tolerance index (TI) as describe by Cano et al. (1998) was calculated from the following relation: Tolerance Shoot + root fresh weight of stressed plants index = Shoot + Root fresh weight of control plants × 100 . Tolerance index was calculated for bacterial inoculated pots (T5, T6, T8 and T9) with respect to control pots (T2 and T3), which do not receive any bacterial treatments.

2.4.5. 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 (Hu et al., 2006; Parmar and Sindhu, 2019). 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.

2.6. Molecular identification of selected rhizobacterial isolates The 16S rRNA sequence analysis was used for the identification of selected rhizobacterial isolates HMM57 and JMM15. The extracted genomic DNA of these rhizobacterial isolates was amplified by PCR using the universal primers 8-27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) (Pitcher et al., 1989). Purification of the product was done as described previously (Pandey et al., 2002). The amplified 16S rRNA gene was sequenced by the dideoxy chain terminator method using a Big Dye terminator kit followed by capillary electrophoresis on an ABI 310 Genetic Analyzer (Applied Biosystems). The partial 16S rRNA gene sequence of the selected bacterial isolates was then used as a query to search for similar sequences in the GenBank database.

2.5. Efficacy of inoculated rhizobacteria on the growth of mustard under salinity conditions 2.5.1. Agar plate experimental design For evaluating the effect of salt concentration on seed germination of mustard, selected rhizobacterial isolates (107–108 cells ml−1 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−1. Inoculated seeds were grown at 28 ± 2 °C in the plant incubator, and the growth of seedlings was recorded on the 5th and 10th days after sowing. The observations for retardation or stimulation of root and shoot growth of mustard seedlings (with or without salt) were recorded. Five replications were used for the agar plate experiment.

2.7. Data analysis In all experiments, the values have been expressed as the mean ± standard deviation of experiments with average values of five plants in three replications each. The results were statistically analyzed using ANOVA, F test, t-test, and Holm-Šídák method using a Sigma Plot (Systat Software, San Jose, CA, USA). The F–statistic shows the mean between two variables are significantly different, and the t-statistic is the ratio of the departure of the estimated value of a parameter from its hypothesized value to its standard error. It is used in hypothesis testing via Student's t-test.

2.5.2. Experimental design in greenhouse and description of treatments Efficient rhizobacterial isolates selected based on different plant growth-promoting activities, their salt tolerance ability and a stimulatory effect on mustard seedling growth, were used for inoculation of mustard under greenhouse conditions. The experiment was conducted in triplicate during the month of October to January, and each treatment had an average of five plants in three replications each. Each replication was used to record different plant growth parameters after 40 and 80 days of sowing. Seeds were inoculated with 20 ml culture suspension (107–108 cells ml−1 of growth suspension) of selected bacterial isolates according to the treatment schedule and grown in the respective earthen pots containing saline soil of varying salt concentrations. The earthen pots of 10–15 kg soil capacity were filled with sandy loam soil. The physio-chemical properties of the soil used were sand (67%), silt (17.4%), clay (15.6%), pH (8.4), EC (0.54 dS m−1), texture (sandy loam), organic-carbon (0.32%), available nitrogen (170 kg ha−1), P (22 kg ha−1), K (278 kg ha−1) and total nitrogen (0.056%) (Hesse, 1971; Jackson, 1973; Brady and Weil, 2002; Phour and Sindhu, 2019). The soil was supplemented with required doses of nitrogen (80 kg ha−1) and phosphorus (40 kg ha−1) fertilizers. The same volume of bacterial suspension was added after one week of sowing in respective pots according to treatment schedule to check the inoculation effect on mustard's plant growth. The mustard seeds were sown in the second week of October at a distance of 15 cm × 5 cm.

3. Results 3.1. 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. Whereas, six bacterial isolates HMM15, HMM18, HMM40, HMM46, HMM69 and HMM81 did not grow at 8% NaCl concentration. Other isolates showed colony size ranging from 0.5 to 10.0 mm up to 6% salt concentration (Fig. 1). Rhizobacterial isolates were further tested in LB broth containing 8% NaCl to confirm the tolerance level, and all the ten isolates showed luxuriant growth, which was observed on the basis of turbidity level with respect to control. Therefore, these rhizobacterial isolates were classified as salt-tolerant (Willey et al., 2009). Further, NaCl tolerance of bacterial isolates JMM15 and HMM57 were also tested spectrophotometrically, and revealed the OD (optical density) values of 0.049 and 0.072, respectively at 8% NaCl concentration. Subsequently, CFU 3

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No. of rhizobacterial isolates

M. Phour and S.S. Sindhu

45 40

Other rhizobacterial isolates did not cause K solubilization on mica containing plates.

39

35 30 22

25 20

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

16

15

11

10

Six rhizobacterial isolates HMM39, HMM44, HMM57, HMM65, HMM88, and JMM15 were tested for seed germination percentage at different salt concentrations, i.e., 0, 4, 8, 16 and 20 dS m−1. An increase in germination percentage was observed by inoculation of all selected bacterial isolates up to 20 dS m−1 as compared to untreated control (Fig. 2). However, with an increase in salt concentration, germination declined. All the bacterial isolates also exhibited stimulation of root and shoot growth at both 5 and 10 days of growth seedlings (Tables 2, 3; Supplementary Figs. S2 and S3). Seeds inoculated with rhizobacterial isolates HMM39, HMM57, or JMM15 showed a more stimulatory effect on the growth of seedlings even at 16 and 20 dS m−1 salt concentrations in comparison to un-inoculated control (Table 2). More specifically, 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. These two rhizobacterial isolates were tested further for their effect on the growth of the mustard crop in the soil at different EC 0, 8, and 12 dS m−1 levels in earthen pots. The results showed that inoculation of rhizobacterial isolates HMM57 and JMM15 stimulated the growth of mustard at 0, 8, and 12 dS m−1 salinity level under pot house conditions (Supplementary Fig. S4). However, an increase in salinity, varying from 0 to 12 dS m−1, caused progressive and significant reduction in the growth of mustard plants in comparison to un-inoculated control. After 40 days of sowing, inoculation of bacterial isolate HMM57 showed 131 (F = 220.50, t = 14.64, p < 0.001),100 (F = 197.27, t = 7.181, p < 0.001) and 58% (F = 93.48, t = 13.66, p < 0.001) increase in root dry weight and 53 (F = 554.24, t = 14.678, p < 0.001), 254 (F = 4354.79, t = 77.837, p < 0.001) and 8% (F = 44.41, t = 5.27, p < 0.001) increase in shoot dry weight of mustard at 0, 8 and 12 dS m−1, respectively (Fig. 3a, b, c) as compared to control. Similarly, inoculation of bacterial isolate JMM15 showed 94 (F = 220.50, t = 20.36, p < 0.001), 130 (F = 197.27, t = 19.629, p < 0.001) and 27% (F = 93.48, t = 6.31, p < 0.001) increase in root dry weight and 23 (F = 554.24, t = 33.219, p < 0.001), 301 (F = 4354.79, t = 83.508, p < 0.001) and 14% (F = 44.41, t = 9.401, p < 0.001) increase in shoot dry weight of mustard at 0, 8 and 12 dS m−1, respectively (Fig. 3a, b, c). After 80 days of sowing, inoculation of bacterial isolate HMM57 showed 119.7 (F = 3492.45, t = 72.81, p < 0.001), 135 (F = 18,108.50, t = 189.63, p < 0.001) and 139% (F = 2806.13, t = 58.15, p < 0.001) increase in root dry weight and 40.4

6

5 0 0.5-2.5mm

2.51-5.0mm

5.1-10.0mm

10.1-20.0mm

No growth

Colony size Fig. 1. Salt tolerance of rhizobacterial isolates in nutrient agar medium containing 8% NaCl concentration.

(colony forming unit) count of 4.44 and 4.51 log10 CFU ml−1 were observed in bacterial isolates HMM57 and JMM15, respectively (Supplementary Fig. S1). 3.2. Determination of different plant growth-promoting activities In addition to salt-tolerant capability, the other plant growth-promoting traits of selected rhizobacterial isolates were also studied (Table 1) in which the production of exopolysaccharides may also contribute towards amelioration of salt stress. ACC utilization was observed after 2 days of incubation on the minimal medium (Dworkin and Foster, 1958) plates supplemented with 3 mM ACC. 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 (Mauzerall and Granick, 1955), and maximum ALA production was observed in bacterial isolate JMM15 (17.45 μg ml−1). Rhizobacterial isolate JMM15 also produced maximum 12.24 μg ml−1 IAA and four bacterial isolates HMM39 (1.27 μg ml−1), HMM57 (4.62 μg ml−1), HMM88 (3.34 μg ml−1) and JMM42 (2.86 μg ml−1) produced IAA in the range of 1.0–5.0 μg ml−1. 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). Table 1 Different plant growth-promoting activities of rhizobacterial isolates. Characteristics

ALA production (μg ml−1) Salt tolerance ACC utilizationa IAA production (μg ml−1) P solubilizing activity (solubilizing zone)b K solubilizing activity (K-SI)c

Rhizobacterial isolates HMM6

HMM13

HMM34

HMM39

HMM44

HMM57

HMM65

HMM88

JMM15

JMM42

13.66 8% +++ − − −

12.74 8% + − 1.43 −

11.24 8% − − − 1.2

11.41 8% ++++ 1.27 1.03 −

11.99 8% − − 0.3 2.9

11.74 8% ++++ 4.62 0.2 4.50

11.12 8% +++ − 0.2 0.9

12.33 8% + 3.34 0.4 −

17.45 8% ++++ 12.24 2.0 5.28

11.62 8% − 2.86 1.4 −

Growth characteristics of rhizobacterial isolates on minimal medium ++: less growth, +++: moderate growth, ++++: significant growth, −: no growth. P solubilization ability of bacterial isolates were scored on the basis of the zone of solubilization formed on TCP containing Pikovskaya medium plates. c K solubilization ability of bacterial isolates was scored on the basis of potassium-solubilizing index. Potassium-solubilizing index = Colony diameter + Clear zone diameter / Colony diameter. The values are average values of three replications. a

b

4

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M. Phour and S.S. Sindhu

Fig. 2. Germination percentage of mustard after inoculation of selected rhizobacterial isolates at different salinity levels on 0.8% plain water agar plates. Error bars represent SE. All the values differ from the control significantly by Holm Šídák test with p < 0.001.

120

Seed gemination (%)

100 Control HMM39 HMM44 HMM57 HMM65 HMM88 JMM15

80

60

40

20

0 0 dSm-1

4 dSm-1

8 dSm-1

16 dSm-1

20 dSm-1

Salt concentration (EC)

Table 2 Effect of various rhizobacterial isolates at different salinity levels at 5th day of seedling growth. Sr. no.

1. 2. 3. 4. 5. 6. 7.

Bacterial isolates

Control HMM39 HMM44 HMM57 HMM65 HMM88 JMM15

0 dS m−1

4 dS m−1

6 dS m−1

8 dS m−1

16 dS m−1

20 dS m−1

R

S

R

S

R

S

R

S

R

S

R

S

7.24 8.40 6.14 7.86 9.60⁎ 6.62 7.90

2.30 4.32⁎ 2.08 2.66 3.52⁎ 3.70⁎ 4.56⁎

7.80 8.66 7.62 10.08⁎ 9.30⁎ 8.24 9.78⁎

2.46 5.30⁎ 4.50 2.72 4.38⁎ 4.40 4.94⁎

6.34 7.34 6.9 8.64⁎ 6.04 8.24⁎ 8.00

2.16 2.46 3.24⁎ 2.30 3.58⁎ 3.22⁎ 3.52⁎

4.46 7.22⁎ 6.24 7.16⁎ 6.06 6.24 7.42⁎

1.24 2.62⁎ 2.68⁎ 1.50 2.44⁎ 2.45⁎ 2.82⁎

2.00 4.38⁎ 4.58⁎ 4.90⁎ 3.52⁎ 2.06 4.46⁎

1.46 2.24⁎ 1.64 2.10⁎ 1.90⁎ 1.58 2.04⁎

0.92 1.90⁎ 1.32 1.78⁎ 1.14 1.30 1.42

0.50 1.50⁎ 1.04⁎ 1.32⁎ 1.06⁎ 1.65⁎ 1.24⁎

The values are the average values of five replications. R and S represents root and shoot in cm, respectively, and the values with * superscript differ significantly from control with p < 0.001. Table 3 Effect of various rhizobacterial isolates at different salinity levels at 10th day of seedling growth. Sr. no.

1. 2. 3. 4. 5. 6. 7.

Bacterial isolates

Control HMM39 HMM44 HMM57 HMM65 HMM88 JMM15

0 dS m−1

4 dS m−1

6 dS m−1

8 dS m−1

16 dS m−1

20 dS m−1

R

S

R

S

R

S

R

S

R

S

R

S

16.12 16.46 14.58 20.40⁎ 7.78⁎ 15.98 19.36⁎

4.26 5.46⁎ 4.26 4.30 3.84 6.16⁎ 4.84

14.82 15.40 13.44 19.70 18.94 12.98 17.40

4.86 5.00 5.20 4.10 6.82⁎ 5.54 5.96

14.76 16.70 11.86 19.12⁎ 12.40 11.98 17.36

3.42 3.38 3.40 4.26 5.96⁎ 4.52⁎ 5.38⁎

10.58 14.62⁎ 10.26 17.32⁎ 4.34⁎ 8.68 16.20⁎

2.20 3.24⁎ 3.08 4.28⁎ 2.72 3.16⁎ 4.08⁎

2.38 5.08⁎ 4.84⁎ 6.28⁎ 6.00⁎ 3.65 6.52⁎

1.78 2.32 1.68 2.22 2.10 1.34 2.5

0.80 2.56⁎ 1.90⁎ 2.12⁎ 1.00 1.78⁎ 2.14⁎

1.20 2.14⁎ 1.20 1.70⁎ 1.30 1.64⁎ 1.50

The values are the average values of five replications. R and S represents root and shoot in cm respectively, and the values with * superscript differ significantly from control with p < 0.001.

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.

(F = 3341.49, t = 39.83, p < 0.001), 22.5 (F = 97,449.49, t = 236.26, p < 0.001) and 200% (F = 24,664.69, t = 177.41, p < 0.001) increase in shoot dry weight of mustard at 0, 8 and 12 dS m−1, respectively (Fig. 3d, e, f) in comparison of control. Similarly, inoculation of bacterial isolate JMM15 showed 118 (F = 3492.45, t = 71.94, p < 0.001), 77.6 (F = 18,108.50, t = 108.68, p < 0.001) and 168% (F = 2806.13, t = 69.98, p < 0.001) increase in root dry weight and 83 (F = 3341.49, t = 81.74, p < 0.001), 42 (F = 97,449.49, t = 441.10, p < 0.001) and 290.5% (F = 24,664.69, t = 204.43, p < 0.001) increase in shoot dry weight of mustard at 0, 8 and 12 dS m−1, respectively (Fig. 3d, e, f). Tolerance index (Ti) of stressed mustard plants was calculated at 8, and 12 dS m−1 with respect to un-inoculated control (Fig. 4). A significant increase in tolerance index was observed with the inoculation of bacterial isolates HMM57 and JMM15. Therefore, inoculation of

3.4. Identification of rhizobacterial isolates The rhizobacterial isolates HMM57 and JMM15 were identified as Pseudomonas argentinensis (GeneBank accession number: MK355573) and Pseudomonas azotoformans (GeneBank accession number: MK355571), respectively (Fig. 5) by using universal primers 8-27F ( 5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The query sequence of the salt-tolerant bacterial strains HMM57 and JMM15 showed 100% and 99.21% match with the Pseudomonas argentinensis (GeneBank accession number: NR043115), Pseudomonas azotoformans (GeneBank accession number: NR037092), 5

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Fig. 3. Efficacy of two rhizobacterial isolates HMM57 and JMM15 on plant biomass (mg/plant) of mustard at different level of salinity at (a) 0 dS m−1 (b) 8 dS m−1 (c) 12 dS m−1 after 40 days of sowing; (d) 0 dS m−1 (e) 8 dS m−1 (f) 12 dS m−1 after 80 days of sowing. Values given as average values of five plants in three replication, RDW and SDW represent as root and shoot dry weight. Error bars represent SE. All the values differ from the control significantly by Holm Šídák test with p < 0.001.

4. Discussion

respectively. The ideal tree with the sum of branch length (0.07868118) appeared and the level of duplicate trees, in which the related taxa grouped in the bootstrap test (1000 replicates), also revealed next to the branches (Felsenstein, 1985). The evolutionary separations were figured out by utilizing the Kimura-2 parameter strategy, and investigations were led in MEGA7 software (Kimura, 1980; Kumar et al., 2016).

Under conditions of moderate salinity (EC 4–8 dS m−1), the yields of major crop plant species were reduced by 50–80% (Panta et al., 2014). For improving crop performance under saline conditions, the agriculture scientists have to identify the physiological, biochemical, molecular, and genetic basis of salt tolerance. A wide range of 6

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200 180 160

180

40 DAS 80 DAS

160 140

Tolerance Index

140 Tolerance Index

40 DAS 80 DAS

120 100 80 60

120 100 80 60 40

40

20

20 0

0

Control

HMM57

JMM15

Control

Rhizobacterial Isolates

HMM57

JMM15

Rhizobacterial Isolates

(a)

(b)

Fig. 4. Tolerance index (Ti) by inoculation of different rhizobacterial isolates at salinity level of 12 dS m−1 after (a) 40 days and (b) 80 days growth of mustard growth. Error bars represent SE. All the values differ from the control significantly by Holm Šídák test with p < 0.001.

biotic stresses (Siddikee et al., 2010; Enebe and Babalola, 2018; Kumar and Verma, 2018). Therefore, it is a challenging task for scientists to develop biofertilizers with the microbial strains, which could ameliorate the effect of salt stress and other abiotic stresses. In the present investigation, screening of the cultures for salt

adaptations and mitigation strategies are required to cope with salinity stress for producing large amounts of food to match the ever-increasing human population growth. One of the possible alternatives is the inoculation with PGPR strains and mycorrhizal fungi, which helps the plant growth and development under different types of abiotic and

Fig. 5. Phylogenetic tree of rhizobacterial isolates HMM57 and JMM15 with other closely related species of Pseudomonas. The tree is drawn to scale which shown branch lengths measured in the number of substitutions per site. 7

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and 12 dS m−1 with respect to uninoculated control (Fig. 4). Similar stimulation effects of bacterial inoculation were also observed in commercially important other crops resulting in increased seed germination rate, drought tolerance, biomass, and yield through enhanced nutrient absorption under stress environment (Raju et al., 1999; Damodaran et al., 2013). These studies emphasized that the survival of rhizospheric bacteria in saline soil and its beneficial interaction with the plant are responsible for mobilization and availability of nutrients through the production of IAA and ALA, phosphorus and potassium solubilization and siderophore production (Duffy, 1994; Gyaneshwar et al., 1998; Mantelin and Touraine, 2004). Two most promising salt-tolerant and plant growth-promoting bacterial strains were identified as Pseudomonas argentinensis and Pseudomonas azotoformans (Fig. 5). The plant growth-promoting ability of salt-tolerant rhizobacterial isolates HMM57 and JMM15 suggested their application as biofertilizer for mitigation of salt stress effect on mustard under field conditions.

tolerance revealed that the growth of the rhizobacterial isolates was adversely affected by an increase in salt concentrations from 1 to 8% (Fig. 1). Out of 94 rhizobacterial isolates, only ten (10.06%) isolates, i.e., HMM6, HMM13, HMM34, HMM39, HMM44, HMM57, HMM65, HMM88, JMM15, and JMM42 showed salt tolerance capacity up to 8% NaCl concentration. In earlier studies, it has been reported that 33% of bacterial isolates survived up to 8% NaCl (w/v) and only 19% isolates showed PGP attributes at higher NaCl concentrations (Upadhyay et al., 2012). Various salt-tolerant root-colonizing rhizobacteria were identified as Klebsiella, Pseudomonas putida, P. extremorientalis, P. chlororaphis, and P. aurantiaca, which showed significant increase in the shoot, root and dry matter of wheat and barley, and were able to survive in saline soil (Tilak et al., 2005; Egamberdieva and Kucharova, 2009; Sapre et al., 2018). In the present study, the inoculation effect of eight rhizobacterial isolates on the germination of mustard seeds and growth of seedlings was evaluated up to 20 dS m−1 salt concentration. It was observed that seed germination was reduced at high salt concentration, but inoculation of rhizobacterial isolates improved seed germination (Fig. 2). Similarly, the growth of mustard seedlings was adversely affected by increasing NaCl concentration both at 5 and 10 days of growth, and variable effects on the growth of seedlings were observed by inoculation of different rhizobacterial isolates (Tables 2, 3). Three salt-tolerant rhizobacterial isolates HMM39, HMM44, HMM57, and JMM15 effectively stimulated the growth of mustard up to 20 dS m−1 salt concentration in comparison to un-inoculated control. Interestingly, rhizobacterial strains HMM39, HMM57, and JMM15 also showed significant ACC utilization ability, suggesting that lowering of ethylene synthesis due to ACC deaminase activity stimulated the growth of mustard seedlings even up to 20 dS m−1 salt level. Similar variations in the growth of seedlings in other crops at different salt concentrations have been reported. For example, stimulation in root and shoot length of maize (Zea mays) was observed under 6 dS m−1 NaCl stress after the inoculation of PGPR (Nadeem et al., 2006). Application of ALA improved the relative growth rate of root and shoot, and leaf water relations (osmotic potential and relative water content) of Brassica napus plants under different NaCl (100, 200 mM) concentrations (Naeem et al., 2011). Isolates JMM15, HMM39 and HMM57 were found to have other plant growth-promoting activities rather than the production of aminolevulinic acid, ACC utilization, i.e., IAA production, P and K solubilization, which could help the mustard plants to withstand extreme salt stress conditions. It has been earlier reported that PGPR having a potential of solubilizing phosphorus, potassium, and calcium could protect the plants against salt toxicity (Grichko and Glick, 2001). ALA production by these rhizobacterial isolates (Table 1) may also partially contribute along with ACC utilization towards growth stimulation of seedlings under different salt levels (Tables 2, 3). Similar stimulation of plant growth under salt stress conditions by inoculation of bacterial strains having different plant growth-promoting activities, especially production of ALA and ACC utilization, were reported in different crops (Almansouri et al., 2001; Kausar and Shahzad, 2006; Fu et al., 2014; Abd Allah et al., 2018; Gupta and Pandey, 2019). In the present study, inoculation of rhizobacterial isolates HMM57 and JMM15 caused 8 to 53% increase in root and shoot dry weight of mustard even at 12 dS m−1 salt concentrations at 40 days after sowing (Supplementary Fig. S4a, b; Fig. 3a, b, c). At later stages of growth, a more pronounced increase in root and shoot dry weight of mustard, i.e., 139 to 291% was observed by the inoculation of rhizobacterial isolates HMM57 and JMM15 even at 12 dS m−1 salt concentrations at 80 days after sowing (Supplementary Fig. S4c, d; Fig. 3d, e, f). In similar studies, the application of the 14 bacterial cultures on canola plants showed an increase in the root length ranging from 5.2–47.8%, and also enhanced the dry weight varying from 16.2–43% (Siddikee et al., 2010). A significant increase in tolerance index was observed with inoculation of bacterial isolates HMM57 and JMM15 in stressed mustard plants at 8

5. 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−1 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. Abbreviations ACC ALA CFU EC IAA HEPES K KB P K-SI NA OD PGPR T

1-aminocyclopropane-1-carboxylate 5-aminolevulinic acid colony forming unit electrical conductivity indole acetic acid N-2-hydroxyethane-sulphonic acid potassium King's B phosphorus potassium-solubilizing index nutrient agar optical density plant growth-promoting rhizobacteria treatment

Acknowledgments The authors thank Head of Microbiology Department for the kind provision of needful facilities. Funding The authors gratefully acknowledge funding from the Department of Microbiology, CCS Haryana Agricultural University, Hisar, India. Availability of data and materials All the data supporting our findings is contained within the 8

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manuscript.

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