The soil pH was determined in 1:2.5 soil:water suspension using the method described by Jackson (1973). The soil was mixed with distilled water in a 1:2.5 ratio to prepare a suspension. A calibrated pH meter was used to measure the pH, ensuring that the electrode remained submerged without touching the beaker walls. The pH of the diluted soil mixture was recorded.

Organic carbon was determined using the chromic acid titration method of Walkley and Black (1934). In this method, 1.00 g of processed soil was weighed, and 10 mL of potassium dichromate and 20 mL of sulfuric acid were added. After 5 min, the mixture was diluted with deionized water to 125 mL, mixed, and allowed to cool. Five to six drops of ferroin indicator were added, and then the mixture was titrated with ferrous sulfate, recording the volume to the nearest 0.1 mL. Blank samples were titrated before unknown samples, ensuring that the titrant volume was within 0.2 mL.

Available nitrogen was determined using the alkaline permanganate method of Subbiah (1956). A 20-g soil subsample (<2 mm) was placed in an 800-mL Kjeldahl digestion flask with 100 mL of 0.32% KMnO₄, 100 mL of 2.5% NaOH, and 20 mL of water. The flask was connected to a macro-Kjeldahl distillation unit, and 75 mL of distillate was collected in 25 mL of boric acid indicator mixture. The ammonia was titrated with 0.05 N H₂SO₄ to determine the available nitrogen content.

The P content of soil was determined according to the method of Olsen (1954). Briefly, 0.5 N sodium bicarbonate (NaHCO₃) at pH 8.5 was used to extract available phosphorus, and the resulting solution was subjected to P estimation using a spectrophotometer.

Available potassium was extracted using neutral normal ammonium acetate (Merwin and Peech, 1951) and determined on a flame photometer. Plant-available potassium was measured by analyzing the filtered extract on an atomic absorption spectrometer set to emission mode at 766.5 nm. The results were reported as parts per million (ppm) of potassium (K) in the soil.

The soil samples were analyzed for viable bacterial count before the experiment. One gram of soil in 9 mL of sterilized water blank and the soil suspension was diluted in a 10-fold series, and then bacterial count was determined using the standard pour plate technique on different media as described by Subba Rao (1999). The count was expressed as colony-forming units per gram of soil (cfu/g soil).

The biofilm-producing bacteria were isolated from different soil samples by serial dilution and spread plate techniques. The serially diluted suspensions of samples were spread on a pre-poured nutrient agar medium. The plates were incubated for 24 h at 30°C ± 2°C. The pure culture of bacteria was obtained by repeated sub-culturing. The isolates were further screened for plant growth-promoting (PGP) traits such as phosphate solubilization, nitrogen-fixing ability, IAA production, HCN production, ammonia production, and siderophore production.

The phosphorus-solubilizing activity of isolates was determined qualitatively. The isolates were spot-inoculated on Pikovskaya’s Medium (PVK) medium as described by Pikovskaya (1948) and incubated at 30°C ± 2°C for 72 h. The yellow-colored halo zones around the bacterial colony indicate the phosphate-solubilizing activity of the bacterial isolates. The halo zone diameter around the colony was calculated by subtracting the colony size from the total halo zone size. The phosphate solubilization index (PSI) was measured using the following formula (Edi-Premono et al., 1996):

Bacterial isolates were spot-inoculated on Jensen’s medium and incubated at 28°C ± 2°C for 96 h. The plates that showed growth in the form of a colony were selected (Jensen, 1954).

Bacterial isolates were inoculated in a nutrient broth enriched with tryptophan (1%–2%) and incubated for 24 h at 28°C ± 2°C under shaking conditions. Cultures were centrifuged at 15,000 rpm for 20 min. Following the centrifugation, 2 mL of Salkowski reagent was added to the supernatant, incubated for 30 min at 28°C ± 2°C, and observed for the development of pink color as an indication of IAA production. Quantitative estimation of IAA from supernatant was performed using the method of Gordon and Paleg (1957).

Bacterial isolates were screened out for the production of hydrogen cyanide (HCN) as per the method described by Bakker and Schippers (1987). Bacterial cultures were streaked on pre-poured plates of King’s B medium amended with 1.4 g/L glycine. Whatman No. 1 filter paper strips were soaked in 0.5% picric acid in 2% sodium carbonate and placed in the lid of each Petri plate. The plates were sealed with parafilm and incubated at 30°C ± 2°C for 1–4 days. Uninoculated control was kept for comparison of results. Plates were observed for change in color of filter paper from yellow to orange-brown.

Each of the bacterial isolates was cultured in peptone water at 30°C ± 2°C for 4 days. One milliliter of Nessler’s reagent was added to each tube. The development of a yellow-to-orange color indicated the production of ammonium by the bacterial isolates (Ahmad et al., 2008).

Siderophore production was detected using the chrome azurol S (CAS) plate assay method (Patel et al., 2018; Schwyn and Neilands, 1987). Sterilized blue agar was prepared by mixing CAS (60.5 mg/50 mL distilled water) with 10 mL of iron solution (1 mM FeCl₃·6H₂O in 10 mM HCl). This solution was slowly added to a hexadecyltrimethyl ammonium bromide (HDTMA) solution prepared by dissolving 72.9 mg HDTMA in 40 mL distilled water. Thus, 100 mL of CAS dye was prepared; 750 mL of nutrient agar was mixed with 1,4-piperazine diethane sulfonic acid (30.24 g), and pH 6.8 was adjusted with 0.1 N NaOH. It was autoclaved separately and then mixed with CAS (100 mL) under aseptic conditions, and then the plates were prepared for further experiments. A bit of 72-h-old culture of each test bacterium was placed on pre-poured blue-colored CAS agar plates. Plates were incubated at 30°C ± 2°C for 24 h and observed for the production of an orange halo around the bit.

where SE is the siderophore efficiency (%), Z is the halo zone diameter (mm), and C is the colony diameter (mm).

The selected PGP isolates were then tested for biofilm production by inoculating bacteria freshly isolated from agar plates into 10 mL of trypticase soy broth (TSB) with 1% glucose (Christensen et al., 1985). The broths were incubated at 37°C ± 2°C for 24 h and then diluted with 1:100 fresh medium; 200 µL of diluted cultures was placed in each well of a sterile 96-well flat-bottom polystyrene tissue culture plate. The negative control wells contained uninoculated sterile broth. The plates were incubated at 37°C ± 2°C for another 24 h. After incubation, the contents of each well were gently tapped out and washed four times with 0.2 mL of phosphate-buffered saline (PBS; pH 7.2) to remove any free-floating bacteria. Biofilms formed by bacteria adhering to the well walls were fixed with 2% sodium acetate and stained with 0.1% crystal violet. The excess stain was removed by washing with deionized water, and the plates were left to dry. After drying, the wells were filled with 99.9% isopropyl alcohol (IPA) to dissolve the crystal violet. The optical density (OD) of the stained adherent biofilm was measured at 570 nm using a micro-ELISA auto reader. The experiment was conducted in triplicate and repeated three times. Biofilm production was assessed using the criteria established by Stepanovic et al. (2007).

Standard biochemical assays were used to evaluate the phenotypic characteristics of rhizobacterial isolates. Catalase activity was measured by observing the formation of oxygen bubbles in pure cultures after adding 3% H₂O₂ (Graham and Parker, 1964). Gram staining was conducted on log-phase cultures and viewed using a phase-contrast microscope, as described by Coico (2006). Citrate utilization was assessed on Simmons citrate agar, with the appearance of a transparent zone indicating positive utilization (Koser, 1924). Casein hydrolysis was examined on skim milk agar plates, and clear zones formed, indicating protease activity (Wehr and Frank, 2004; Jadhav et al., 2020). All tests included uninoculated controls.

Bacterial genomic DNA was isolated by taking a 24-h-old broth culture (10 mL) of bacterial isolates. The broth culture was centrifuged at 2,000 rpm for 10 min, and the supernatant was discarded. One milliliter of freshly prepared extraction buffer was added to the pellet, the resulting solution was transferred to a 2-mL Eppendorf tube and incubated at 65°C in a water bath for 30 min, and an equal volume of an ice-cold solution of phenol:chloroform:isoamyl alcohol (25:24:1) was added to it. The solution was mixed well and centrifuged at 10,000 rpm for 10 min at 4°C, and the upper aqueous layer was transferred to a new Eppendorf tube. An equal volume of an ice-cold solution of phenol:chloroform:isoamyl alcohol (25:24:1) was added to this aqueous layer, and the step was repeated three to four times. In the final aqueous layer, the ice-cold absolute alcohol was added in excess to precipitate the bacterial genomic DNA, and the solution was centrifuged at 10,000 rpm at 4°C for 10 min. The supernatant was discarded, the pellet was washed with 100 µL of 70% (v/v) ethanol and centrifuged at 10,000 rpm for 15 min at 4°C, the supernatant was discarded, and the pellet was dissolved in 50 µL of Tris-EDTA (TE) buffer (pH 8.0) and stored at −20°C for further studies (Sambrook et al., 1989).

Universal primers were applied to amplify the 16S rDNA sequences of the biofilm-producing PGP isolates. To amplify the bacterial 16S rDNA gene, a conserved ~1.5-kb region was targeted with the following primer pair:

Forward primer (16S-F): 5′-GGATGAGCCCGCGGCCTA-3′.

Reverse primer (16S-R): 5′-CGGTGTGTACAAGGCCCGG-3′.

These primers were chosen for their high specificity and Guanine-Cytosine (GC) content (72.22% for forward and 68.42% for reverse), and they were designed to assure efficient and accurate amplification with high-fidelity DNA polymerase. The primer pair amplifies a region that is useful for taxonomic identification and phylogenetic analysis of bacterial isolates.

The PCR was carried out in a 20-µL reaction containing ~50 ng of template DNA, 20 pM of each primer, 0.2 mM dNTPs, and 1 U Taq polymerase (Genei, Bangalore, India) in 1× PCR buffer. Amplification was performed using a thermal cycler (multigene PCR system, Labnet, Edison, NJ, USA). Reactions were cycled 35 times at 94°C for 3 min, 50°C for 30 sec, and 72°C for 1.5 min followed by a final extension at 72°C for 10 min. The PCR products were analyzed on 1% agarose gel in 1× Tris-Acetate-EDTA (TAE) buffer and ran at 100 V for 1 h, and the size was determined using a 1-kb DNA ladder (Thermo Scientific, Waltham, MA, USA). The amplified product was purified and sequenced by Biokart Genomics Lab, Bengaluru, India. The sequences were aligned using ClustalW 1.74 followed by the construction of a neighbor-joining phylogenetic tree using MEGA11 (Tamura et al., 2013). The sequences were aligned using ClustalW 1.74, and a neighbor-joining phylogenetic tree was constructed using MEGA11 (Tamura et al., 2013).

The seeds of the tomato (cultivar ‘Solan Lalima’) were procured from the Department of Seed Science and Technology, Dr. YS Parmar University of Horticulture and Forestry, Nauni-Solan, India. This cultivar was chosen for the study, as it is a local and commonly grown tomato variety in the area and is also very responsive to the inoculation of biofertilizers.

For the preparation of the inoculum, biofilm-producing PGP bacterial isolates were cultured in a liquid medium. The medium was incubated at 28°C for 48 h. The population density (1.5 OD at 540 nm) that resulted in the formation of 10⁸ cfu/mL of the bacterial isolate was used for the preparation of inoculum formulation.

The selected seeds of the cultivar ‘Solan Lalima’ were immersed in a sterile beaker for 1 h in a liquid culture of various bacterial formulations. The seeds were inoculated with selected bacterial isolates, and the nursery was produced by transferring the seeds into disposable cups. Following germination, the seedlings were planted into pots. One uninoculated control treatment was kept for comparison. The pots were arranged in triplicate, with each treatment having three replications, and placed under net house conditions.

The soil was obtained from a furrow slice (0- to 15-cm depth) of the soil science field, UHF Nauni Solan. The soil was passed through a 2-mm sieve and used for a pot culture experiment. The potting mixture was prepared by mixing sand, soil, and farm yard manure (FYM) in a ratio of 1:2:1. The mixture was sterilized for three successive autoclave cycles of 1 h each at 121°C. Five kilograms of potting mixture was filled in each pot. Moisture in the potting mixture was maintained near field capacity.

Plant height was measured from the soil base to the tip of the fully expanded leaf, and values were recorded in centimeters.

The shoot of the plants was weighed separately immediately after harvesting, and values were expressed in grams.

The length of roots was measured using a scale for individual plants and expressed in centimeters.