Poster: Biology & Disease Mgmt: Bacteriology
Improvement of the environmental DNA extraction method for profiling the culturable microbiome of bacterial wilt-conducive and non-conducive soils
R. GICANA (1), W. Deng (1), J. Wu (1) (1) National Chung Hsing University, Taiwan
The culturable microbiome profile of the bacterial wilt-conducive and nonconducive soils were compared using the PCR-DGGE profile of the V4-V5 hypervariable region of the 16S rRNA, and using the high throughput sequencing (HTS) analysis of the V1-V3 region of the 16S rRNA. PCR-DGGE results showed DNA fingerprints unique only to either the conducive or the nonconducive soils, which was confirmed by HTS analysis with 63 culturable taxa unique only to the nonconducive soil compared to 94 in the conducive soil. Exclusively cultured taxa in the nonconducive soil belong to the Phylum Planctomycete, Nitrospirae and Gemmatimonadetes while those enriched in the conducive soil belong to the Phylum Verrucomicrobia, Alphaproteobacteria and Gammaproteobacteria. While studying the culturable microbiome of the two soil types, we have improved a previous DNA extraction method to generate high quality DNA used in this study. The modified method needs no further purification steps, yields more DNA which are unsheared and with high molecular weight, with low humic acid content, and can extract DNA from bacteria of different cell wall compositions. The microbiome was cultured using low nutrient medium and with an improved DNA isolation technique we were able to capture some taxa previously reported uncultured or were recalcitrant to culturing under traditional laboratory methods such as Nitrospira calida, Planctomyces sp., and several genera belonging to the classes Gemm-3 and Gemm-5.