The DDBs were characterized by Gram staining and the 16S rRNA genes were analysed as described by Ikunaga et al. (2011). This yielded approximately 1200 bp of useful 16S rRNA gene sequence. Sequences similar to the 16S rRNA gene of isolated strains were identified using blast searches (National Center AZD2281 for Biotechnology Information, http://www.ncbi.nlm.nih.gov). The

16S rRNA gene sequences determined in this study have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers AB627753 to AB627765. Culture samples were filtered through 0.45-μm membranes (Advantec, Tokyo, Japan) and 10 μL was directly injected on to an HPLC system. The HPLC system (Waters, Milford, MA) consisted of a 600E pump, a 2487 dual absorbance detector, a Waters Symmetry C18 column (3.9 mm ID × 150 mm; Waters) and empower 2 software. The mobile phase contained methanol and water (15:85, v/v) at a flow rate of 1.0 mL min−1. A wavelength of 220 nm was used. The column was heated to 40 °C. Authentic DON and 3-epi-DON were detected at retention

times of 6.5 and 4.5 min, respectively. The DDBs were cultured on the agar plates at 28 °C for 5 days. These bacteria (OD600 nm of 0.2) were then preincubated in DON mineral medium (DMM, MM containing 20 μg mL−1 DON), 1/3R2A and 1/3LB liquid media at 28 °C for 24 h. After incubation, bacterial cells were recovered by centrifugation at 6000 g for 10 min, washed twice with 50 mM potassium phosphate buffer (pH 7.0), and resuspended

Cyclopamine cost in the same buffer containing 100 μg mL−1 DON to achieve an OD600 nm of 0.8 (equivalent to 1.3 mg dry weight mL−1). Samples were incubated at 25 °C, collected at various time points, filtered and subjected to HPLC. Initial DON degradation rates were measured within the period of linear DON degradation. Three buffers, noninoculated cells containing DON, inoculated cells without DON and inoculated autoclaved cells (121 °C, 20 min) containing DON, were also analysed as controls. Cells were inoculated into MM with or without 100 μg mL−1 DON and incubated at 120 r.p.m. and 28 °C after precultivating on the agar plates at 28 °C for 5 days. Culture media were RG7420 datasheet collected every other day, appropriately diluted and spread onto 1/3LB agar plates for strains SS5 and RS1, and onto 1/3R2A agar plates for the other strains. These agar plates were incubated at 28 °C for 7 days, and the numbers of colonies were counted. Statistical analysis of the data was carried out using jmp software (version 5.01J; SAS Institute Japan, Tokyo, Japan). Significant differences between the means were determined by using a t-test and Tukey test variance analysis (P < 0.05). A total of 169 environmental samples (61 soil, 78 wheat leaf and 30 wheat spikelet samples) were used for enrichment culture.