Phylogenetic study on microbial communities at various habitats in paddy field ecosystem

为了解稻田生态系统微生物群落结构，从洪水、稻草、表层氧化和还原土层等不同生境中提取DNA和RNA，并用真细菌、真核生物、产甲烷菌、甲烷氧化菌和氨氧化菌的特异引物进行PCR扩增。通过对聚合酶链式反应产物进行变性梯度凝胶电泳法(DGGE)分析，确定了不同生境的季节变化和专一性，并测定了DGGE谱带的DNA序列。对各生境特征DGGE条带的序列分析表明，存在代表该群落的特有微生物，微生物群落可分为洪水和渗漏水(水生生境)、稻草、稻草堆肥和植物废弃物(植物残渣生境)、水稻根和微型甲壳动物(共生生境)。对提取的DNA进行DGGE分析时，还检测到耕层土壤中的真细菌和产甲烷群落。所有的真细菌和产甲烷菌全年都是活跃的。有趣的是，根据环境DNA的PCR-DGGE分析估计的微生物群落与通过磷脂脂肪酸(PLFA)分析估计的微生物群落相似。

For elucidating the community structure of microorganisms in the paddy field ecosystem, DNA and RNA were extracted from the samples collected from various habitats such as floodwater, rice straw on and in the plow layer, and surface oxidized and reduced soil layers, and they were PCR-amplified with primers specific to eubacteria, eukaryotes, methanogens, methanotrophs, and ammonia oxidizers. PCR products were subjected to denaturing gradient gel electrophoresis (DGGE) to estimate the seasonal variations and the specificity of respective habitats with the subsequent determination of DNA sequence for characteristic DGGE bands.In general, microbial communities estimated from PCR-DGGE patterns were phylogenetically endemic at respective habitats with relatively small seasonal variations. Sequence analysis of characteristic DGGE bands at each habitat indicated the presence of endemic microorganisms representing the community, and the microbial habitats were grouped into floodwater and percolating water (aquatic habitat), rice straw, rice straw compost and plant debris (plant residue habitat), rice roots and microcrustaceans (symbiotic habitat).As eubacterial and methanogenic communities in the plow layer soil that were observed in DGGE analysis of extracted DNA were also detected in DGGE analysis of extracted RNA, all the eubacteria and methanogens were estimated to be active all the year round.Interesting was the finding that microbial communities estimated from the PCR-DGGE analysis of environmental DNA was similar to those estimated from phospholipid fatty acid (PLFA) analysis.