Monday, 10 July 2006

Comparison of Bacterial Community Structures at Main Habitats in Paddy Field Ecosystem Based on DGGE Analysis.

Susumu Asakawa and Makoto Kimura. Graduate School of Bioagricultural Sciences, Nagoya University, Furocho, Chikusa, Nagoya, Japan

Paddy field is a unique agro-ecosystem, where the field is flooded during the most period of rice cultivation and left under drained conditions during the off-crop season. The paddy field ecosystem, therefore, consists of diverse habitats for microorganisms such as floodwater and reduced soil layer caused by flooding as well as rice root, rice straw, composted materials, percolating water, etc. Our group has analyzed bacterial communities in these various habitats in Japanese paddy fields by the Denaturing Gradient Gel Electrophoresis (DGGE) method and elucidated the predominant members in bacterial communities and relative successions at the respective habitats. However, direct comparison has not been conducted among bacterial communities at various habitats to understand the “whole community structures of bacteria in paddy field ecosystem”, although it is requisite to understand the diversity of bacterial world in the paddy field ecosystem. The present study compared the bacterial community structures at different habitats, i.e., floodwater, percolating water, microcrustaceans inhabiting in floodwater, plow layer soil, rice root, rice straw and rice straw compost incorporated into soil, rice straw placed on the soil surface, plant residue in paddy fields and rice straw under composting process, by analyzing data of the DGGE banding patterns and the sequenced DGGE bands. The study fields were located in the Aichi-ken Anjo Research and Extension Center, Central Japan (latitude 34.8º N, longitude 137.5º E) and the soil was an Anthraquic Yellow Soil (Oxyaquic Dystrudept). The samples mentioned above except rice root, microcrustacean and composting rice straw were collected from the paddy fields. Rice straw samples under composting process were taken from a storehouse in the same center. Rice root and microcrustacean samples were collected from the pot and microcosm experiments, respectively, using soil samples and/or 5 kinds of microcrustaceans taken from the paddy fields in the center. DNA was extracted from these samples and subjected to PCR-DGGE pattern and sequence analyses targeting bacterial 16S rDNA. The richness, diversity and variability of bacterial communities at the respective habitats were estimated based on the number and the relative intensity of the DGGE bands. The total different number of DGGE bands were largest for the samples of plant residue, rice straw buried in soil and rice straw placed on soil surface (97, 78 and 61, respectively), indicating that these habitats had high phylotype (band) richness. The samples of plow layer soil and rice straw compost buried in soil showed the highest Shannon-Wiener's (H') diversity index (2.59 and 2.16, respectively) and exhibited the lowest variability for the band pattern, which suggests that the bacterial communities in these habitats were very diverse and stable. In contrast, the samples of rice straw placed on soil surface and microcrustacean had the lowest values of the diversity index (0.83 and 0.52, respectively) and the highest variability of the patterns. The bacterial communities at these habitats seemed to be less diverse and variable though the community in rice straw placed on soil surface had high phylotype richness. Sequences of totally 250 DGGE bands were assigned to phylogenetic groups (phyla or classes for Proteobacteria) and their distribution in different phylogenetic groups was determined among the DGGE clones of the respective habitats. Aquatic habitats (floodwater and percolating water) were predominated with the Betaproteobacteria, Bacteroidetes (CFB group) and Actinobacteria clones. Betaproteobacteria and Bacteroidetes were also dominant groups in the DGGE clones of rice root and microcrustacean, respectively. Proteobacteria clones were more abundant in the DGGE bands of rice straw and rice straw compost incorporated into soil, rice straw placed on soil surface and plant residue than of other habitats. DGGE clones affiliated to Firmicutes and Actinobacteria dominated the DGGE bands of composting rice straw, whereas abundant groups belonged to Chloroflexi and Actinobacteria in the bands of plow layer soil. “Comprehensive mapping” of these DGGE clones was conducted according to Garrity et al. (Bergey's Manual of Systematic Bacteriology 2nd ed. vol. 2, 2005) by using principal component analysis of evolutionary distances for each of the sequences of bands to 202 reference sequences of type strains of type families, where possible, in the domain Bacteria. Bacterial communities at the respective habitats were differently scattered on the principal component plots, indicating that the communities were distinct to each other and specific.

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