Api 50ch

The strain YU22 was tested for its growth in Luria–Bertani (LB) agar, tryptic soy agar (TSA) and NA at 32°C. The morphological and biochemical characteristics and Gram staining were performed using routine microbiological methods. Cell growth at pH 5–10 (1.0-unit interval) was examined using the following buffer systems: 0.1 M citric acid/0.1 M trisodium citrate for pH 5.0; 0.2 M Na₂HPO₄/0.2 M NaH₂PO₄ for pH 6.0–8.0 and 0.1 M NaHCO₃/0.1 M Na₂CO₃ for pH 9.0–10.0. To test the oxidation of sole carbons, cell suspension from overnight cultures was prepared in physiological saline and inoculated into Biolog GN2 microplate. Similarly, cell suspensions were also inoculated to API 20 E, API 20 NE, API ZYM and API 50CH (BioMérieux, Marcy-l’Étoile, France) strips for biochemical and enzymatic analysis. All these tests were performed according to manufacturer’s instructions, and the results were recorded within 24 h incubation at 32 °C.

The polar lipids and isoprenoid quinones were performed as described by Minnikin et al. (1984 (link)) and Collins et al. (1977 (link)), respectively. The extraction of cellular fatty acids was performed as described by Kuykendall et al. (1988 (link)) and then analyzed with the Sherlock Microbial Identification System (MIDI) (Sasser, 1990 ). The growth gradients of pH, temperature, and salinity were optimized by the methods described by Li et al. (2016 (link)). Gram staining was carried out as described by Jenkins et al. (2003 (link)). The test of anaerobic growth was performed in an anaerobic jar for a week (Li et al., 2016 (link)). The activities of oxidase and catalase were determined by the methods described by Li et al. (2016 (link)). Enzymatic activity, acid production, and carbon source utilization were performed using API ZYM, API 50CH, and API 20NE (bioMérieux) according to the manufacturer's instructions.

To determine carbohydrate utilization profiles, API 50 CH (bioMérieux) tests were used according to the manufacturer’s instructions. Data for the reference type strain (DSM 20509^T) was taken from the BacDive database at https://bacdive.dsmz.de/strain/6407.

Optimal culture conditions were determined by testing various incubation temperatures (25, 28, 37, 42, and 50 °C), atmospheres (aerobic, anaerobic and microaerophilic), NaCl concentrations (5, 5.5, 7.5, 10, 15, and 20% of NaCl) and pH levels (5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5). The morphology and motility were observed using a new-generation scanning electron microscope (Hitachi High-71 Technologies Corporation, Tokyo, Japan).
Furthermore, three semi-quantitative standardized micro-methods of Analytical Profile Index (API®, bioMérieux®) tests: API® 20A, API® 50 CH, and API® ZYM were used, according to the manufacturer’s instructions¹⁶ , in order to study carbohydrate metabolism and enzymatic activities.
Fatty acid methyl ester (FAME) analysis was explored by Gas Chromatography/Mass Spectrometry, as previously reported^{17 (link), 18} . FAMEs were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500—SQ 8 S, Perkin Elmer®, Courtaboeuf, France). Obtained spectra were compared with those contained in the repertory databases using MS Search 2.0 operated with the Standard Reference Database 1A (National Institute of Standards and Technology-NIST, Gaithersburg, USA), and FAMEs mass spectral database (Wiley, Chichester, UK).

Rhizobacteria were sourced from a commercially available mixture (Tribus^® Original, Impello Biosciences, Fort Collins, Colorado, USA) consisting of B. subtilis, B. pumilis, and B. amyloliquefaciens at a combined cfu count of 10 billion per millilitre. The three species of Gram-positive Bacilli were confirmed in the laboratory by API^® (bioMerieux^® Ltd, Marcy l’Etoile, ARA, France) colour guides. Each Bacilli spp. was extracted from an isolated colony of pure growth samples obtained from serial dilutions and streak plate purification before analysis with API^® 50 CH (bioMerieux^® Ltd, Marcy l’Etoile, ARA, France) in triplicate. The identity was then confirmed with reference to the API^® database (bioMerieux^® Ltd, Marcy l’Etoile, ARA, France). The AM fungal inoculum was cultured to a total mass of 5 g in 200 mL nutrient broth for 1 week and homogenised with an electric blender for 1 min. The species identity was confirmed by Eurofins Scientific^® (Wolverhampton, West Midlands, UK) as R. intraradices.