Ten bacterial isolates procured from National Agriculturally Important Microbial Culture Collection, ICAR-NBAIM, India, five bacterial isolates obtained from Microbial Technology Unit II, ICAR-NBAIM, India, along with one BioNPK formulation were used in this study (Table 1). In this study, BioNPK was utilised as a check which has found effective in stimulating plant growth under drought stress (Saxena et al. 2020).
Growth conditions of microorganisms
All the microorganisms were grown in Nutrient Agar (Himedia Pvt. Limited) except Nesterenkonia which was grown in nutrient broth supplemented with 4% NaCl. Microorganisms were incubated at 30°C for 48 h at 150 rpm in a shaking incubator. Four isolates viz. Bacillus sp. RPB03, Pseudomonas sp. RPB22, Bacillus sp. RPB602 and Pseudomonas sp. RPB609 were incubated at 50°C for 48 h at 150 rpm.
Pot experiment for screening of microorganisms for drought stress alleviation
An initial screening was conducted with all the microorganisms used in the study on the basis of chlorophyll content, proline content and various plant growth parameters. The best isolates obtained from the study would be used later for carrying out detailed investigation. Therefore, one month pot trial was conducted in a glass house at ICAR-National Bureau of Agriculturally Important Microorganisms, Mau, Uttarpradesh, India. Each pot (4” diameter) was filled with 0.5 kg sterilized sand: soil mixture (1:3) which was sterilized in autoclave by tyndallisation technique. The pots were weighed and 100% saturated with water to calculate the field capacity (FC). Thereafter, the weight of each pot (sand: soil and water) was calculated for 50% and 30% FC which was then used to maintain the water levels at FC according to the treatments. Drought stress levels were assessed using 30% (Stressed Control) and 50% (Un-stressed Control) FC. Log phase broth cultures (containing109 CFU/ mL) were mixed with 0.2% carboxymethyl cellulose (CMC) carrier and coated on wheat (HD2967) seeds. Seeds treated with only nutrient broth and CMC were used as uninoculated control. Prior to seed treatment, the wheat seeds were surface sterilised for 1 minute with 70% ethanol and then for 5 minutes with a 1.5% sodium hypochlorite solution (Rudolph et al. 2015). Eight seeds were sown in each pot which were trimmed to four plants after germination. All treatments were taken in triplicates and randomised. Recommended dose of NPK (60:30:20) mg kg-1 of soil was applied in all the treatments. The pots were weighed everyday and water was simply supplied to maintain field capacity (FC) according to drought stress levels of treatments. The experiment was set up using the following treatments –(i) 30% FC- Uninoculated stressed control; (ii) 50% FC- Uninoculated non-stressed control; (iii) 30% FC + microbial inoculation. In our study, a total of 16 microorganisms were used making the total number of treatments as eighteen (Tables 3 and 4).
Similar setup was used for elucidating the impact of selected/efficient bioinoculants on morphological, physiological, biochemical and molecular traits of wheat under drought stress. Bacillus sp. BT-3, Klebsiella sp. HA9 and BioNPK (consortium of Azotobacter sp. , Paenibacilus sp. and Bacillus sp.) were selected for further study. The experiment was designed using the following treatments –
(i) 30% FC- Uninoculated stressed control; (ii) 50% FC- Uninoculated un-stressed control; (iii) 30% FC + . Bacillus sp. BT-3; (iv) 30% FC + Klebsiella sp. HA9; (v) 30%FC + BioNPK (consortium of Azotobacter chroococum, Paenibacilus tylopili and Bacillus decolorationis). The total treatments maintained were five and are presented in Table 5.
Analyses of plant growth and biomass
After 30 days of sowing, three plants were uprooted from each treatment replicate. Root and shoot length were measured by using inch tap. Fresh weight of root and shoot was measured by using weighing balance. To determine the dry weight, wheat roots and shoots were incubated in hot air oven at 80°C for three days.
Analysis of root morphology
Root studies were carried out by collecting plants from three replicates after 30 days of sowing. The adherence of the soil to the roots was detached by the method of Costa et al. (2000). The LA2400 (3rd Gen.) scanner was used to measure root length, surface area, projected area, volume, average diameter, number of root tips, number of forks and number of links. Thereafter, WIN RHIZO Programme V. 2017a software (Regent Instruments Inc. Ltd., Quebec, Canada) was used to analysed actual values of each root parameters.
Relative water content
Weatherly's method was used to analyse the relative water content (RWC) (Weatherly 1950). Leaves were harvested from the plants and weighed. Thereafter, leaves were transferred in distilled water for 24 hrs. Afterthis the leaves were fully turgid and were weighed again. After weighing, the leaves were put in oven at 80°C for 72 h and dry weight was recorded. RWC was calculated using the following formula.
Chlorophyll and carotenoid content
Chlorophyll a, b, total chlorophyll and carotenoid were analysed following the method described by Arnon (1949). One gram fresh leaves were grounded in liquid nitrogen followed by homogenization in 80% acetone. A spectrophotometer was used to measure the absorbance of the supernatant at 663, 645, and 480 nm (Analytik Jena).
Quantitative determination of osmoprotectants
The acid-ninhydrin approach established by Bates et al. (1973) was used to determine the proline content of root and leaf tissues using spectrophotometry. 0.5 g plant sample was crushed with liquid nitrogen and homogenized in10 ml of 3.0% sulfosalicylic acid. Then, it was centrifuged at 10,000 rpm for 20 min. 2.0 ml of supernatant was mixed with equal amounts of acid ninhydrin and glacial acetic acid followed by heating at 100°C for 1 hr. Reaction was stopped by putting the tubes in ice. 4.0 ml of toluene was added and mixed thoroughly by vortexing for 15 to 20 min. Absorbance of the upper layer was recorded at 520 nm by using a spectrophotometer (Analytik Jena). A standard curve was prepared using L-proline (10-100 µg/ml) as a standard.
Dubois et al (1951) method was used to calculate sugar content. 200 mg plant samples (root and leaves) were cooked for one hour with 10 ml of 80% ethanol. The extract was filtered through Whatman no. 1 filter paper after cooling. One ml of filtrate was added with 1.0 ml of 5% phenol and 5 ml concentrated H2SO4 and vortexed well. Absorbance was recorded at 490 nm and the concentration of sugar was calculated with reference to standard curve made from glucose (0-100 µg/ml).
The Bradford assay was used to analyse the protein content of plant roots and leaves (Bradford 1976). Plant materials weighing 0.5 g were crushed in liquid nitrogen and homogenised in 5 mL sodium phosphate buffer (pH 7.0). The suspension was then centrifuged for 20 minutes at 12000 rpm, and 0.5 mL of clear supernatant was combined with 3 mL of Bradford reagent. A spectrophotometer was used to measure the absorbance at 595 nm (Analytik Jena). The concentration of protein in unknown sample was calculated with reference to standard curve made from Bovine serum albumin (100-1000 µg ml-1).
Grieve and Grattan (1983) method was applied to analyse the glycine betaine in plant tissues.
Lipid peroxidation was assayed following the methods described by Heath and Packer (1968). Plant samples were finely ground in liquid nitrogen and homogenised in 10.0 ml of 0.1% trichloro-acetic acid. The homogenate was centrifuged at 1500 g for 15 min. 1.0 ml of supernatant was added with 4.0 ml of 0.5% thiobarbituric acid in 20% trichloro-acetic acid. The mixture was then heated for 30 minutes at 95°C before being chilled in an ice bath. The mixture was centrifuged for 10 minutes at 10,000 g after cooling. At 532 nm and 600 nm, the absorbance of the supernatant was measured. The extinction coefficient for thiobarbituric acid reactive substance is 155 mM-1 cm-1. The results were represented as nmol malondialdehyde (MDA) equivalents per gram of fresh weight.
Quantitative determination of Antioxidant enzymes
Superoxide dismutase (SOD)
The methodology reported by Dhindsa et al. (1981) was used to determine SOD activity (1981). Plant samples (100 mg) were crushed and centrifuged (15000 g, 20 min.) in 0.1 M phosphate buffer (pH 7.5). 0.2 ml 200 mM methionine, 0.1 ml 2.25 mM nitroblue tetrazolium chloride (NBT), 0.1 ml 3 mM EDTA, 1.5 ml 100 mM phosphate buffer (pH 7.8), 0.1 ml 1.5 M sodium carbonate, and 0.1 ml enzyme extract were used to make 3 ml reaction mixtures. Water was used to make up the final volume (3 ml). Thereafter, 0.4 mL of 2μmol l−1 riboflavin was added and exposed to light (15 W fluorescent lamp, 15 min). After deactivating the enzyme activity in the dark, the absorbance was measured at 560 nm. One unit of SOD was represented by a 50% decrease in absorbance when compared to the control, which lacked enzyme extract.
POD activity was determined by using the method reported by Castillo et al. (1984). Plant samples (100 mg) were ground, homogenised, and centrifuged (15000 g, 20 min.) in 0.1 M phosphate buffer (pH 7.5). 0.5 ml of 96 mM guaiacol, 1.0 ml of 100 mM phosphate buffer (pH 6.1), 0.5 ml of H2O2 (12 mM), and 0.1 ml enzyme extract were combined to make a 3.0 ml reaction mixture. The change at 470 nm was recorded at every 30s interval and the enzyme activity was calculated as Units (U) (tetra guaiacol) min−1 g−1fresh weight. Tetra guaiacol has an extinction coefficient of 26.6 mM-1 cm-1.
Ascorbate peroxidase (APX)
APX was analysed by using Nakano and Asada method (Nakano and Asada 1981). Plant samples (100 mg) were ground, homogenised, and centrifuged (15000 g, 20 min.) in 0.1 M phosphate buffer (pH 7.5) containing 1mM ascorbic acid and 0.5 mM EDTA. 0.1 ml of EDTA (3 mM), 1.5 ml of 100 mM phosphate buffer (pH 7.0), 0.1 ml of 0.1 mM H2O2, 0.5 ml of 3.0 mM ascorbic acid, and 0.1 ml enzyme extract were used to make a 3.0 ml reaction mixture. After 60 seconds, absorbance was taken at 290 nm, and activity was represented as U min−1 g−1fresh weight.
CAT was assayed by Aebi method (Aebi 1983). Plant samples (100 mg) were ground in 0.1 M phosphate buffer (pH 7.5), homogenized and centrifuged (15000 g, 20 min.). 1.5 ml of 100 mM (pH 7.0) buffer, 0.5 ml of 75 mM H2O2, and 50 l enzyme extract were combined to make a 3.0 ml reaction mixture. The final volume of the reaction mixture was made up by adding water. At 30s intervals, the change at 240 nm was measured, and the enzyme activity was expressed as Umin−1 g−1fresh weight.
Glutathione reductase (GR)
Smith et al. (1988) method was used for measuring glutathione reductase activity. Plant samples (100 mg) were ground, homogenised, and centrifuged (15000 g, 20 min.) in 10 ml of 0.1 M phosphate buffer (pH 7.5). 1.0 ml of 0.2 M phosphate buffer containing 1 mM EDTA, 0.5 ml of 3.0 mM 5, 5-dithiobis [2-nitrobenzoic acid] (DTNB), 0.1 ml of 2.00 mM NADPH, 0.1 ml of 20 mM glutathione disulphide (GSSG), and 0.1 ml of enzyme extract were used to make a 3.0 ml reaction mixture. Spectrophotometric measurements were taken to determine the increase in absorbance at 412 nm. The extinction coefficient of NADPH is 6.22 mM-1 cm-1. The activity was expressed as U min−1 g−1fresh weight.
Histo-chemical detection of peroxide and superoxide radicals
Method described by Fryer et al. (2002) was followed for staining of superoxide and peroxide radicals in plant leaves. Plant leaves were placed in tubes and immersed in nitroblue tetrazolium (NBT) (0.2%) and 3, 3′-Diaminobenzidine (DAB) (1.0 mg/ ml, pH 3.8) staining solution for staining of superoxide and peroxide radicals respectively. The tubes were placed in the desiccator and attached to a vacuum pump for increasing the infiltration of staining solution. Tubes were rolled up in aluminium foil and left overnight at room temperature. After incubation period, staining solution was drained off. Stained leaves were thoroughly washed for 5 minutes in an acetic acid-glycerol-ethanol (1:1:3) solution at 100°C. Leaves were transferred onto a paper towel saturated with 60% glycerol. Superoxide radicals were visualised as a dark blue due to NBT precipitation and peroxide radicals were visualised as reddish brown due to DAB polymerization.
Analyses of expression of genes associated with drought response in root and shoot of wheat
Thirty-day-old root and shoot samples were collected, and total RNA was extracted using the Trizol method (Rio et al. 2010). qPCR was used to validate the expression of genes (DHN, DREB, L15, and TaABA-8OH) with potential roles in drought stress response. Three independent samples of each were used. TOPscriptTM cDNA synthesis kit (Enzynomics, Republic of Korea) was used synthesize cDNA from 2 g of total RNA, according to the manufacturer's protocol. Table 2 lists the gene-specific primers used for qPCR. The Agilent Mx3000P™ PCR platform and Maxima SYBR Green qPCR kit Master Mix (2X) Universal (Thermo Fisher Scientific Baltics, UAB) were used for the qPCR, which was carried out according to the manufacturer's instructions. The 2−ΔΔCt method was used to calculate the relative expression levels of the selected genes normalised to the expression level of actin from cycle threshold values. Three independent biological replicates with three technical replicates were used in the experiment.
The results of the experiment were provided as the average of three replications. The results of each experiment were statistically analysed using MiniTab 17's one-way analysis of variance (ANOVA). Tukey's test was used to compare mean values of acquired data between treatments (P≤ 0.05).