Colonization of rice plants was evaluated in vivo using a rifampicin-resistant mutant of strain REICA_142T, denoted REICA_142TR. The mutant was selected on R2A agar medium amended with 25 μg ml-1 rifampicin (Sigma-Aldrich, St. Louis, MO) and streaked to purity. One-day-old germinated rice seeds
were incubated for 1 h with 8.4 log cells of REICA_142TR CFU ml-1 (REICA_142TR treatment) or with sterile phosphate buffer solution (pH 6.5; control treatment) . For each treatment, four replicate rice seedlings were grown in autoclaved as GSK3235025 well as natural V soil  for up to 4 weeks at 70% water holding capacity. Water lost from the pots was replaced daily using sterile demineralized water. Following growth, all rice plants were surface-sterilized , rice tissue was treated with mortar and pestle, after which serial dilutions of the resulting mTOR inhibitor homogenates were made and plated onto selective agar (R2A supplemented with Rif). Following plate incubations at 28°C for 72 h, the bacterial communities obtained from the plant tissue were enumerated. The ability of strain REICA_142TR to invade rice plants from the V soil was thus confirmed by isolating colonies from the relevant plates (at least one per replicate) and HMPL-504 price performing BOX-A1R
PCR on these . Availability of supporting data The accession numbers for the 16S rRNA gene sequences of Enterobacter oryziphilus strains REICA_084, REICA_142T and REICA_191 are [GenBank:JF795012, JF795013, JF795014], and of Enterobacter oryzendophyticus strains REICA_032, REICA_082T and REICA_211 are [GenBank:JF795010, JF795011, JF795015], Selleckchem Rapamycin respectively. The accession numbers for the rpoB gene sequences of strains REICA_084, REICA_142T
and REICA_191 are JF795018, JF795019 and JF795020, and of Enterobacter oryzendophyticus strains REICA_032, REICA_082T and REICA_211 are JF795016, JF795017 and JF795021, respectively. The generated phylogenetic trees from the 16S rRNA and rpoB genes were deposited in the publicly-accessible TreeBASE data repository with the project number 14166. Acknowledgments We thank Dr. Darshan Brar at IRRI for providing the rice material, Dr. Peter Kämpfer and Dr. Roger Stephan for providing the type strains of Enterobacter radicincitans, Enterobacter turicensis, Enterobacter helveticus and Enterobacter pulveris, and Dr. Jiří Jirout for assistance in the fatty acid analyses (BC ASCR, ISB). This study was supported by the joint RUG-WUR initiative on rice endophytes in the context of a DOE-JGI project on the rice endophyte metagenome and by a grant provided by the FWF (National Science Foundation, grant no. P 21261-B03) to A.S. P.R.H. was supported by the Soil Biotechnology Foundation. Electronic supplementary material Additional file 2: Figure S2: Maximum-likelihood tree based on rpoB gene sequences showing the phylogenetic position of Enterobacter oryziphilus sp. nov.