The partial sequences from the 16S rRNA genes of 531 bacteria

The partial sequences from the 16S rRNA genes of 531 bacteria isolated from the main root of the sugar beet (L. reported in the sweet potato (18). The diversity and community composition of culturable bacteria from the main root of the sugar beet were assessed in the present study using 16S rRNA gene sequencing in order to provide basic ecological information and construct a resource for an efficient survey and utilization of plant-growth-promoting rhizobacteria (PGPR). The seeds of the sugar beet (cultivar Rycka) were sown on 27 April 2011 in an experimental field of the Hokkaido Agricultural Research Center (Memuro, Hokkaido, Japan, 4289.2 N/1430.7.7 E). The width of the rows and spacing between plants were 60 cm and 22.5 cm, respectively. The field was dressed with S014 (150, 315, and 210 kg/ha for N, P2O5, and K2O, respectively; Hokuren Fertilizer Co., Sapporo, Japan) as a basal fertilization. Based on a visual inspection, three healthy sugar beets were randomly sampled from the experimental field on 11 August 2011. The main roots were carefully washed with tap water to remove loosely adhering soil and debris and then with sterilized water. The lateral roots on the 186544-26-3 main root were manually removed, and the main root was then separated into surface (SU) and core (CO) parts. SU represented a right part of the main root with a 10-mm thickness from the root surface, and CO symbolized a core area of the primary root without the main surface area. Around 200 g of every area of the tissue produced from three plant life was put into 500 186544-26-3 mL of 67 mM phosphate buffer (pH 7.0) and homogenized within a blender. The homogenate was filtered through a bit of Miracloth (Calbiochem, Darmstadt, Germany), as well as the supernatant was after that stored being a 15% glycerol share at C80C until afterwards bacterial isolation. Garden soil samples were gathered from three sampling sites by an auger (between a depth of 5 cm to 15 cm) after getting rid of surface area garden 186544-26-3 soil on 12 Oct 2011, and had been 186544-26-3 combined being a amalgamated garden soil sample. The chemical substance characteristics from the garden soil sample were dependant on the Tokachi Nokyoren Agricultural Analysis Institute (Obihiro, Hokkaido, Japan) (Desk S1). Four bacterial isolate choices were made of the SU and CO elements of the main base of the sugar beet by R2A (BD, Franklin Lakes, NJ, USA) and HM media. The HM medium was altered Coles HM medium (3) by adding 0.1% L-arabinose and 0.03% yeast extract. The pH was adjusted to 6.8 with 2N NaOH prior to autoclaving. Homogenates of the SU and CO parts of the main root were serially diluted with 67 mM phosphate buffer (pH 7.0), and 100 l of each dilution was inoculated on 1.5% agar plates of R2A medium containing 50 mg L?1 cycloheximide and HM medium containing 50 mg L?1 polymyxin B. After an incubation at 24C in the dark for 7 d, colonies were randomly collected and subjected to single colony isolation twice by streaking them onto new medium. The purified bacterial strains were stored as a 15% glycerol stock at C25C. Regarding DNA extraction, strains were cultured on an agar plate of the R2A or HM medium for any few days at 24C. An aliquot of bacterial cells was collected with an inoculation loop and total DNA was extracted from your cells using a previously explained DNA extraction method (7). PCR amplification for 16S rRNA gene sequencing, and the editing and analyses of sequences for the strains isolated in the present study were conducted as previously explained (17). A total of 531 strains were isolated from the surface and core parts of the main root of the sugar beet using two media (Table 1). Clustering analysis (99% identity) was used to group 531 strains into 155 OTUs, and library protection was 83.1%. Statistical analysis revealed that the number of OTUs and both Shannon and Simpson diversity indexes were higher for the surface tissue collection than for the core tissue collection in R2A and HM Rabbit Polyclonal to HSF1 media (Table 1). However, these differences were small, which was consistent with the findings reported by Lilley (12). Differences were also attributed to the thickness of the surface tissue (10 mm), which may have.