The aerial root samples were collected during the blooming period (April-May) from naturally grown epiphytic Rhynchostylis retusa from Jorhat (26.74°NL; 94.14°EL), Assam, India. The climate was humid tropical, with an annual mean temperature of 28.7 °C and 274 mm of rainfall (India Meteorological Department). The roots were rinsed with tap water before sterilization for 1 min with 70% ethanol (v/v) and 5 min with 0.9% sodium hypochlorite (Gangwar et al., 2014). In order to isolate the bacteria, root pieces were taken in cold, sterilized mortar pestles and crushed with saline water (0.85% of NaCl) (Li et al., 2017). An aliquot of 100 µL from each tube up to six-fold serially diluted samples were plated on Jensen’s media and incubated for 48 h at 28°C. Following incubation, morphologically different bacterial colonies were further spotted on nitrogen-fixing bacteria (NFB) media containing DL-Malic acid; 5g/L, NaOH; 3g/L, K₂HPO₄; 0.4g/L, FeSO₄.7H₂O; 0.1g/L, MnSO₄; 0.01g/L, NaCl; 0.02g/L, CaCl₂.2H₂O; 0.01g/L, Na₂MoO₄; 0.002g/L, Agar powder; 15g/L, with pH 6.8 ± 0.1 Bromothymol blue 2 mL of 50 mg in 10 mL 70% alcohol (Dobereiner et al., 1995). The distinctive bacterial colonies were purified with repeated streaking on the nutrient agar (NA). The pure isolates were stored at -80°C in 40% glycerol stock for further study.

The biochemical characterization, as Catalase, Oxidase, Methyl Red, Voges–Proskauer, Carbohydrate utilization, etc., was performed using a biochemical identification test kit (HiMedia, Mumbai, India). Gram staining and morphological characterization using a laboratory microscope, characterization results were compared by Bergey’s manual (Bergey, 1994). The genomic DNA of the bacterial isolates was extracted by the BiOstic Bacteremia DNA kit (Qiagen, Germany). Amplification of the 16S rRNA gene was carried out in a Veriti Thermal Cycler (Thermo Fischer, USA) using universal forward primer 27F (5´-AGAGTTTGATCMTGGCTCAG-3´) and reverse primer 1492 R (5´-GGTTACCTTGTTACGACTT- 3´) (Dos Santos et al., 2019). The PCR products were purified using a Qiagen PCR purification kit, and sequencing was done through Agri Genome Labs India Pvt. Ltd. (Kochi, India). The resulting sequence was compared to the gene bank database using NCBI BLAST. A phylogenetic tree was constructed by MEGA 6.0 software (Tamura et al., 2013).

The amount of IAA produced by the bacterial isolates was determined by the colorimetric technique using Salkowski’s method (Gordon and Weber, 1951). Bacterial strains were cultured in tryptophan-containing broth (750 µg/mL) for 96 h at 28°C, followed by 10 min centrifugation at 10,000 rpm. For quantification of synthesized indole, mainly IAA, the supernatant was mixed (1:2) with Salkowski reagent (1 mL of 0.5 M FeCl₃ with 49 mL of 35% HClO₄) and incubated for 15 min at room temperature. After incubation, the pink color was examined at 530 nm using Biospectrometer (Eppendorf, Germany), along with various concentrations of standard of IAA (Sigma, United States).

Optimization experiments were carried out by cultivating the bacterial strain at different temperatures, pH, incubation times, and substrate concentrations. Further, in order to optimize the process parameters by Microbacterium testaceum Y411, one parameter was altered every time while keeping other parameter constant for IAA production. The temperature was optimized (26, 28, 30, 32, 34, 36, 38, and 40°C) at pH 7, substrate 750 µg/mL for 96 h incubation at 120 rpm shaking condition. Similarly, the pH was optimized at different ranges from 3 to 10 at optimized temperature, substrate 750 µg/mL, and incubation period of 96 h. Subsequently, incubation period (24, 48, 72, 96, and 120 h) and substrate L-tryptophan concentration (250, 500, 750, 1000, 1250, 1500, 1750, and 2000 µg/mL) were optimized for auxin (IAA) production at optimum temperature and pH. All the optimization experiments were performed in triplicates to avoid experimental error.

Microbacterium testaceum Y411, the most active and stable IAA producer, was tested for its ability to stimulate seed germination of mung beans (Vigna radiata). Only healthy seeds (15 numbers) were selected, and abnormally low, discolored, and damaged seeds were removed. The bacterial culture was grown in a tryptophan medium at 30 °C for 48 h. Bacterial cells were removed through centrifugation at 8000 rpm for 10 min. Clear cell-free supernatant (auxin containing) 5 mL was used for seed germination assay in a Petri dish. One-hour sterile water-soaked seeds were placed on a paper towel dampened with water at the bottom of the Petri dish. In control, only sterile media was used over the Mung seed (15 numbers). Three replicates were used for this assay. After 24-48, h seed germination will be recorded from each assay plate as given by Xu et al. (2019).

Pure colony of the bacterial strain Y411 was cultivated in tryptophan broth for 48h at 30 °C and 120 rpm. The culture was centrifuged at 10,000 rpm, and the supernatant was subjected to extraction with ethyl acetate (1:2) 2 to 3 times to obtain maximum recovery (Mohite, 2013). Further, the extract was dried in a rotary evaporator (Buchi, R215, UK) before dissolved in the optimum amount of methanol.

A silica gel G 60 F 254 pre-coated aluminum plate (Merck, Germany) was used for the solvent-extracted auxin’s in thin-layer chromatography (TLC). The dissolved methanol sample was detected on a silica plate beside an IAA standard of 2mg/mL. After 30 min in the solvent isopropanol-water (60:40 v/v), the plate was air-dried and treated with Salkowski’s reagent. The pink color spot was observed, and the R_f value was calculated about the standard.

The spotted auxin was scrapped from the TLC plate and eluted with a 1:2 v/v mixture of n-hexane and ethyl acetate (Extra pure, EMPLURA) by SPE (solid phase extraction) using a glass column (1.0 x 12cm) packed with silica gel of 60-120 mesh as a stationary phase. Before adding the sample, the column was pre-washed with n-hexane. The eluted product was thoroughly dried and analyzed by HPLC, FT-IR, and HRMS.

The FT-IR (Perkin Elmer, Spectrum 100 UK) analysis of the purified sample has been carried out using transmission mode with a wavenumber ranging from 400 to 4000 cm^-1. HPLC (Waters, USA) analysis was carried out using a C18 column (5μm×4.6×150mm). The absorbance of IAA was measured at 280 nm using a mobile phase of water and acetonitrile (40:60 v/v ratio) for 30 min at a flow rate of 1mL/min. A 20 μL sample was injected, and the retention time of the compound was compared to the IAA standard (Sigma) (Aswathy et al., 2013).

The UHPLC-HRMS (Xevo XS QT, Waters, USA) instrument (mass range 20-4000 amu) was coupled with Xevo XS QT mass spectrometer through the ESI interface. The program was used as ESI voltage 4.5 kV; capillary voltage 10V; tube lens 50V N₂ gas was used as an auxiliary, sweep, sheath at 5,2,10 arbitrary units, respectively. The MS ions spectra were acquired using Xevo XS QT mass spectrometer in the scan range of 50 to 300 m/z. For HRMS analysis, 20 µL of control IAA and purified bacterial extracted sample were used to detect the MS spectra of IAA and its metabolites.

The surface-sterilized capsule containing seeds of Cymbidium aloifolium (epiphytic orchid) was slit in half longitudinally (Arditti, 2008). The exposed seeds were inoculated on MS medium that contained 0.8% agar and was supplemented with a range of bacterial auxin and synthetic auxin concentrations, including 0.5, 1, 1.5, and 2.0 mg/L, respectively. BAP (1 mg/L) was used as a cytokinin by Murashige and Skoog (1962), while NAA was employed as a positive control. The flasks were incubated for 16 h under photoperiod conditions (white fluorescent light) at 25 ± 1°C in an experimental tissue culture chamber. The physical observations such as seed germination, callus formation, root length, and shoot length were investigated regularly every week for 120 days. The % germination was calculated using the following formula:

Where, X represents the number of seeds inoculated

Y represents the number of seeds germinated

One-way ANOVA was conducted, followed by Tukey’s post-test for each treatment using the SPSS software (ver. 10.1, SPSS Inc., www.spss.com). The significance level was p=0.05 for the effect of culture media on various parameters such as seed germination, callus formation, root number, root length, shoot length, and survival duration.

All the bacterial isolates were tested for IAA synthesis; the potential bacterial species were shown prolific growth and IAA production in the presence of Tryptophan broth growth media at 28°C for 96 h of incubation. The production range of IAA was between 9 to 158.62 µg/mL, and Y411 produced the highest IAA (158.62 µg/mL), followed by Bacillus aryabhattai Y-715 (115.82 µg/mL) the details are given in Figure 1 .

The genera of isolated bacterial species are identified as Microbacterium, Pseudomonas, Achromobacter, Brevibacterium, Arthrobacter, Stenotrophomonas, Staphylococcus, and Bacillus. A highly efficient auxin-producing bacterial isolate, Microbacterium testaceum Y411, was identified based on morphological, biochemical, and molecular characterization. The isolate was morphologically rod-shaped and non-motile. The biochemical characterization revealed that the isolate was yellowish-orange pigmented, gram-positive with catalase-positive, and oxidase-negative. The strain can use glucose, sucrose, arabinose, mannitol, and rhamnose and grow in malic acid media ( Table 1 ). The 16S rRNA gene sequence (1459 bp) was compared with the NCBI database, and a phylogenetic tree was constructed with a total branch length of 10.92302698 using the neighbor-joining method to see them closely related to homologous sequences of GenBank ( Figure 2 ) (Saitou and Nei, 1987). The strain was identified as Microbacterium testaceum from NCBI data base (Pisarska and Pietr, 2015). A phylogenetic tree was constructed with a bootstrap value of 1000 replicates shown next to the branches. The resulting dataset had 1400 locations of 10 nucleotide sequences, with codon positions 1^st+2^nd+3^rd+Noncoding included (Tamura et al., 2013). The sequence was deposited to NCBI GenBank with accession number MZ367021.

Mung seeds and rooting assays was performed to investigate the effects of Microbacterium testaceum Y411 supernant (containing bacterial auxin) supported 100% seed germination with healthy radicle with an average length of 2.2 ± 0.13 cm. By contrast, in the control plate, seed germination with bent radicle with an average length of 0.9 ± 0.15 cm (see Figure 3 ).

The effect of temperature on IAA synthesis has been investigated with a range of 26 to 40°C. As the temperature was raised from 26 to 30 °C, the IAA production increased from 15 to 158 µg/mL; however, as the temperature was increased further, as shown in Figure 4A , the IAA production dropped from 158 to 17 µg/mL. The production of IAA was maximum at 30 °C when it produced 158 µg/mL. As pH increased from 3 to 7, IAA production increased from 10 to 150 µg/mL, decreasing as pH rose again from 7 onward ( Figure 4B ). Further, the influence of the incubation period from 0 to 120 h with 24 h intervals on IAA biosynthesis was investigated. At 48 h of incubation, IAA biosynthesis was reached 159.5 µg/mL, and at 72 h of incubation, no change in the IAA production was observed ( Figure 4C ). In the case of tryptophan concentration optimization, 1000 µg/L was the optimum concentration for IAA production by Microbacterium testaceum Y411 ( Figure 4D ). Finally, the maximum IAA production (170 µg/mL) was recorded at optimum temperature (30°C), pH (7.0), incubation time (48h), and tryptophan concentration (1000 µg/L) at 150 rpm by Microbacterium testaceum Y411.

TLC analysis was confirmed by observing the collected fraction’s pink spot after spraying with the Salkowski reagent. It was found that solvent-extracted bacterial auxin and standard IAA chromatographs had the same R_f value of ~0.53. For maximum recovery, the bio-extract was purified in a silica gel glass column using solvent (n-hexane: ethyl acetate ratio 20:80). The TLC purified fraction was further analyzed through HPLC and compared with standard IAA. It was observed that purified bacterial IAA had a strong peak with a retention time of 6.36 min, which was similar to the retention time of 6.33 min of standard IAA ( Figures 5A, B ).

The FT-IR spectra of purified bacterial auxins were analyzed. The broad absorption band at 3385.50 cm^-1 corresponds to the carboxylic -OH group, -C=O stretching matches the peak at 1688.31 cm^-1 (generally obtained at 1670 cm^-1), and -NH bends at 1455.90 cm^-1. Furthermore, the peaks at 1302-1405 cm^-1 were associated with the aromatic ring’s -C-H vibration, while the wave number at 1098.55 cm^-1 was indicated for –C-H bending. The band represents -CH₂ at 736.05 cm^-1 ( Figure 6 ).

High-resolution mass spectrometric (HR-MS) techniques using time-of-flight mass spectrum (TOF- MS ES+) provide high selectivity, mass accuracy, and structural information, allowing forging high selectivity. The target compounds were identified to confirm the identification of auxin metabolites. HRMS was used to detect the MS spectra of IAA and its metabolites in purified bacterial extracted materials. The mass-to-charge ratio of control IAA was found at m/z 176.06 ( Figure 7A ). The chlorinated derivative of the IAA (4-Chloro indole acetic acid) compound is composed of fragmented ions C₁₀H₈ClNO₂ and C₁₀H₉ClNO₂ were found at m/z 209.16 and 212.06, indicating notable auxin activity ( Figure 7B ). The m/z ratios of IAA in the bacterial sample match those of commercially available synthetic chemicals, indicating that metabolites exist.

Orchid seed germination and tissue development were investigated using pure form of bacterial auxin extracted from Microbacterium testaceum Y411. A combination of auxin and MS media was used to observe seed germination. In this study within 45 days, the combination of MS medium, 6-benzyl amino purine (BAP) (1 mg/L), and bacterial auxin (1 mg/L) produced the maximum seed germination ( Table 2 and Figure 8 ). Using this medium combination, the number of roots (4 Nos.), root length (4 cm), and shoot length (6.5 cm) were found to be higher. On the other hand, in the control (synthetic) media combination of MS+BAP at 1 mg/L and 1 mg/L of NAA seed germination was observed in 55 days. Plant growth metrics were revealed to be less affected by MS media with 2 mg/L of bacterium auxin and synthetic auxin (NAA) ( Table 2 ). It was revealed that the culture medium with bacterial auxin had a significant effect (p<0.05) on seed germination, callus formation, root length, shoot length, and survival period ( Table 2 ; Figure 8 ).