Rhizospheric soil sampling and screening for P-solubilizing bacteria
About 5 g soil from rhizospheres of sorghum, paddy, tobacco, tea and maize crops was collected from local field in the vicinity of Changa village, Dist. Anand, Gujarat (India). Soil sample obtained from about 10 cm depth along the root system was considered as rhizospheric soil. Besides, soil samples were also collected from the rhizosphere in domestic garden as well as non-agricultural surfaces like river bank, lake-side and factory-side locations and stored at 4°C. Soils from these locations were collected across a period of one month and used for bacterial isolation within 3 days of sampling. For isolation of P-solubilizing bacteria, one gram of soil sample was suspended in 9ml sterile distilled water under aseptic condition and was subjected to vigorous shaking (100 rpm, 30°C) for one hour. This soil solution was used to prepare 10^-2 10^-3 10^-4 10^-5 and 10^-6 dilutions serially (Sharma et al., 2011). For primary screening, an aliquot of 0.1 ml of each dilution was spread on Pikovskaya agar plate, and the colonies obtained after 24 to 48 h incubation at 30°C were examined to screen P-solubilizing bacteria. Presence of zone of clearance surrounding the colony was considered as qualitative measure of P-solubilizing ability. Selected P-solubilizing isolates were subsequently sub-cultured on nutrient agar medium and used for further characterization as discussed in following sections. All the isolates were maintained as pure cultures on nutrient agar medium at 4°C, throughout the study.

For quantitation of P-solubilizing ability, bacterial inocula for test isolates were prepared by inoculating single colony of each isolate aseptically in 3 ml nutrient broth and allowing the cultures to grow overnight under shaking conditions in a rotary shaker (Orbitek, Scigenics Biotech Pvt. Ltd., India) maintained at 150 rpm and 30°C. One millilitre of freshly grown culture of each isolate was harvested by centrifuging at 10,000 rpm for 2 minutes. Resulting pellets were washed with 1 ml of sterile normal saline and finally re-suspended in 1 ml of normal saline. A three-microliter aliquot of inoculum for each isolate was aseptically spotted on pre-solidified Pikovskaya agar. The petri plates were incubated at 30°C for 24 to 48 h to allow bacterial growth and formation of clear halo. Normal saline similarly spotted was used as control. P-solubilizing ability was quantified as phosphate solubilising index (PSI), calculated as ratio of diameter of clear halo (mm) to diameter of zone of growth (mm) (Nosrati et al. 2014).

Antibacterial activity
Selected isolates were tested for ability to antagonise E. coli using soft agar overlay technique with slight modification (Hockett and Baltrus 2017). Bacterial inocula of test isolates were prepared as described above, with final re-suspension of cultures in 3 ml of normal saline for further use. To pour the overlay, 24h freshly grown E. coli culture (50µl) was aseptically mixed with sterile warm molten agar (0.6%) and poured onto the already solidified nutrient agar. Plates were carefully tilted in all directions so as to ensure even distribution of overlay, allowed to solidify and used to create 3 mm bores using sterile cork borer. Thereafter, 20µl of fresh test inoculum was transferred into the bore and the plates were incubated at 30°C for 3 days. Clear zone around the zone of growth of test isolate was taken as a measure of antibacterial ability. Antibacterial ability of all the test isolates was recorded qualitatively as positive or negative.

IAA production
Phytohormone IAA was quantitated and detected by method given by Ahmad et al. (2008). Briefly, test cultures were grown in 3 ml Luria broth supplemented with 500µg/ml tryptophan to induce IAA production. Each culture similarly inoculated on Luria broth in absence of tryptophan was used as corresponding control. Cultures grown for 48 h under shaking conditions (150 rpm, 30°C) were harvested and centrifuged 10,000 rpm for 2 minutes at room temperature. Resulting cell free supernatants were used for spectrophotometric determination of IAA at 535 nm using Salkowski reagent (Ahmad et al. 2008). IAA was quantified using a standard curve prepared by similar spectrophotometric measurement of pure IAA in range of 10 to 100µg/ml. IAA quantification for each culture was carried out in triplicates.

Siderophore Production
Siderophore production by test isolates was measured using CAS solution assay (Christina Jenifer et al. 2015). Briefly, the isolates were grown in King’s B medium under shaking conditions (150 rpm, 30°C) for 24 h and centrifuged to derive cell free supernatants. To 0.5 ml of these supernatants, 0.5 ml CAS solution was added and colour developed after 10 minutes of incubation at room temperature was measured spectrophotometrically at 630nm. Un-inoculated medium similarly treated was used as the control. For quantitation, % siderophore units were calculated by following formula [{Ar-As}/Ar]*100; where Ar = absorbance of reference and As = absorbance of sample. Siderophore quantitation for each culture was carried out in triplicates.

HCN production
HCN production by test isolates was measured qualitatively as per method given by Reetha et al. (2014). Test bacteria were streaked on Luria agar plate amended with 4.4 g/L of glycine. Sterile filter paper soaked in 2% sodium carbonate and 0.5% picric acid solution was placed in lid of the petri dishes which were subsequently sealed with parafilm and incubated at 30°C for 48 h to allow bacterial growth. Change in colour of the filter paper from yellow to orange/light brown, brown or reddish-brown indicated weak, moderate or strong production of HCN respectively.

Microscopic and biochemical characterization of bacterial isolates
Test isolates were characterized on preliminary basis using selected microscopic and biochemical tests including Gram staining, catalase, indole production test, nitrate reduction test, Vogues-Proskauer test, Methyl red test and oxidase test, performed according to Bergey’s Manual of Determinative Bacteriology. Besides, fluorescent pigment production on King’s B agar after 24 h of growth at °C was also checked to identify fluorescent Pseudomonas species.

Molecular characterization of bacterial isolates
Identification of selected bacterial isolates was carried out on the basis of 16s rRNA gene sequencing. Genomic DNA of test isolates was isolated using standard procedure as described by Sambrook and Russel (2001) while 16S rRNA gene was amplified using universal primers 8F(5’-AGAGTTTGATCCTGGC-TCAG-3’) and 1492R(5’-ACGGCTACCTTGTTAC-GACTT-3’). The PCR reaction mixture in a total volume of 20µl contained, 20 picomoles of each primer, 0.2mM dNTPs, ~80ng of template DNA, 1X PCR buffer and 1U of Taq DNA polymerase (procured from Takara Bio USA, Inc). The thermal cycler (MJ Mini, Bio-Rad) was programmed as follows: 94°C for 4 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds and primer extension at 72°C for 1 minute; followed by final extension at 72°C for 10 minutes. The PCR amplicons were analysed using 1% agarose gel, stained with ethidium bromide post electrophoresis. PCR products of appropriate sizes were then partially sequenced in single pass reaction using 8F primer (services out sourced from 1^st Base, Singapore). The sequences obtained were subjected to BLAST analysis (http://www.ncbi.nlm.nih.gov) to identify the isolates on the basis of sequence similarities. Moreover, these partial nucleotide sequences were deposited at GenBank and the accession numbers were obtained.

Statistical analysis
Data for quantitative experiments is represented as Mean ± S.D as indicated in the respective figure legends. Correlation analysis between P-solubilization and biocontrol traits was performed using Microsoft Excel, for which absolute values of PSI values were retained while siderophore, IAA and HCN producing abilities were rated on a relative scale of 0-3, based on quantitative and qualitative determinations as described earlier.