Isolation of fluorescent Pseudomonas
Wheat plants cultivated locally in the vicinity of Aligarh city, India were uprooted carefully and rhizospheric soil was collected. Briefly, soil tightly adhered to roots were collected by vortexing the roots in 0.85% NaCl solution to prepare stock suspension. Hundred microlitre of appropriately diluted sample was spread on King’s B agar plates and incubated for 48 h at 28°C. Typical colonies producing yellow-green fluorescence detected under UV light were selected and purified. The cultures were given a strain number and maintained in the laboratory by subculturing. One set of culture was preserved at -80°C in glycerol broth. The fluorescent Pseudomonas strains were biochemically characterized and tentatively identified using standard methods^9-10.

Assay for production of plant growth promoting activities
Plant growth promoting activities of isolated bacteria was determined using standard methods as described previously⁶ with little modifications. A brief description is given here. Production of Indole-3-acetic acid (IAA) was analyzed using the standard method of Loper and Scroth¹¹ with the amendment of media by tryptophan (100 µg mL^-1). Siderophore production was studied by growing the cultures in Modi medium. Catechol-type phenolates were measured as described by Reeves et al.¹². Phosphate solubilization was studied by using standard method¹³.

The exopolysaccharides (EPS) production was done by the method of Mody et al¹⁴.  1-aminocyclopropane-1-carboxylate (ACC)  deaminase activity of the strains was checked by growing them onto DF (Dworkin and Foster’s) salts minimal agar medium¹⁵ amended with 3.0 mM ACC (Sigma-Aldrich, USA) instead of (NH₄)₂SO₄ as nitrogen source¹⁶. Bacterial strains were also evaluated for their antifungal activity against Fusarium oxysporum (MTCC-3930), Fusarium ciceri (ITCC-3636), Fusarium solani (ITCC- 6953), Aspergillus niger (ITCC-6370), Alernaria alternata (ITCC-6778) by dual culture assay¹⁷.

Assay for biofilm development
The biofilm formation was determined in 96 well polystyrene microtitre plates as described by O’Toole and Kolter¹⁸. Briefly, bacteria were grown in the tryptic soy broth (TSB, HiMedia, India) at 28 °C for 24 h. Each well was filled with 200 µL (≈10⁷ CFU mL^-1) of culture in fresh medium and allowed to grow for 24 h at 28 °C. The wells were washed with phosphate buffer saline (PBS), followed by staining with 0.1% crystal violet for 20 min. Unbound strain was removed by washing each well with PBS. In each well ethanol (95%) was added to solubilize the bound dye and absorbance was recorded at 590 nm using micro plate reader (ThermoScientific Multiskan EX, UK). Based on absorbance value of dye, the strains were distinguished as strong and moderate biofilm formers according to the criteria adopted by Christensen et al¹⁹.

Identification of MW4 strain based on 16S rRNA gene sequence analysis
The methods adopted at Macrogen (South Korea) for molecular characterization is briefly described below. Genomic DNA was extracted from MW4 and used as template in a 30 µL reaction mixture by using an EF-TaqDNA polymerase (SolGent, South Korea). The primers 518F (5’-CCAGCAGCCGCGGTAATACG-3´) and 800R (5´-TACCAGGGTATCTAAT CC-3´) were used for the PCR (Polymerase chain reaction). The PCR reaction was performed with 20 ng of genomic DNA as the template Activation of Taq polymerase at 95°C for 2 min, 35 cycles of 95°C for 1 min, 55°C, and 72°C for 1 min each were performed, finishing with a 10 min step at 72°C. The amplified PCR products were purified with a multi screen filter plate (Millipore Corp., USA). Sequencing reaction was performed using a PRISM BigDye Terminator v3.1 Cycle Sequencing Kit. The DNA samples containing the extension products were added to Hi-Di formamide (Applied Biosystems, CA, USA). The mixture was incubated at 95°C for 5 min, followed by 5 min on ice and thenanalyzed by ABI Prism 3730XL DNA analyzer (Applied Biosystems, Macrogen Inc, Seoul, South Korea). The resulting nucleotide sequence measuring 997 bp in size was submitted to GenBank sequence database (accession number KF555376). The sequence obtained were analysed using Basic Local Alignment Search Tool (BLASTN) and matched against known bacterial sequences present in NCBI GenBank data base (http: /www.ncbi.nlm.nih.gov). Related sequences obtained from the database were used to construct a phylogenetic tree using MEGA5 software.

Rhizosphere colonization assay
For colonization study under non sterile soil condition, rifampicin resistant mutant of the two parent strains MW4 and MW3 was selected based on sequential exposure of rifampicin and selecting the mutant on rifampicin amended Kings B agar plate and checked their stability for several generations. Triticum aestivum L. (var. PBW-343) surface sterilized seeds were treated with rifampicin resistant mutants MW3 and MW4 (≈ 2.8×10⁷ CFU seed^-1) separately and sown in natural loam soil. Collection of rhizosphere and rhizoplane samples (five plants) was made at 15, 30, and 45 days after sowing. Serial dilutions from rhizosphere and rhizoplane samples were prepared separately in normal saline solution (NSS) and plated onto King’s B agar medium containing 800µg mL^-1 rifampicin. Untreated plants maintained in similar condition were also subjected to enumerate presence of naturally occurring rifampicin resistant bacteria capable of growing on King’s B agar medium at 800µg mL^-1 rifampicin. Only fluorescent colonies grown on rifampicin amended plates were counted.

Biofilm formation on glass cover slips by MW3 and MW4
Crystal violet assay with little modifications as described by O’Toole and Kolter¹⁸ was performed to establish the biofilm development on glass coverslips. Briefly, bacterial strains were grown in tryptic soy broth (TSB) as described earlier. A 12 well tissue culture plate with flat bottom containing sterile coverslips were filled with TSB and 100 µL of inoculum (≈10⁷ CFU mL^-1) per well and incubated statically at 28 °C for 48 h. After growth coverslips were removed and rinsed in PBS followed by staining with 0.1% crystal violet for 20 min. After staining, coverslips were washed with PBS to remove excess dye and examined under a light microscope (Olympus BX60, Japan) at 100X.

Confocal laser scanning microscopy of biofilms by MW3 and MW4
The biofilms formed on coverslips as mentioned above were stained with acridine orange (Sigma-Aldrich, USA). Microscopic observations and image acquisitions were performed using confocal laser scanning microscopy (CLSM) (Olympus Fluoview FV1000) using standard protocol.

Scanning electron microscopy (SEM) and CLSM and of biofilms formed on the roots of T. aestivum
Triticum aestivum L. (var. PBW 343) surface sterilized seeds were sown in sterile sand-soil mixture for raising seedlings. After 15 days of growth, the plant was uprooted, and roots were washed with sterile distilled water and treated with bacterial culture by dipping the roots in 10 mL diluted culture of fluorescent Pseudomonas MW4 and MW3 (≈10⁵ CFU mL^-1) prepared in TSB. In the control plants, sterile distilled water (SDW) was used instead of culture. After 24 hours the root samples was fixed in 2.5% glutaraldehyde and washed in the same buffer followed by fixation in osmium tetraoxide. The root were dehydrated by treating with graded concentrations of ethanol and dried in CO₂ with a critical point-dryer, mounted on stubs, and shadowed with gold, (Sputter Coater-Polaron SC7640 UK) for viewing under an scanning electron microscopy (SEM) (Carl ZeissEVO 40, Germany). For CLSM, 24 h-treated root samples were first stained with acridine orange and images were acquired as mentioned above.