Yasunori Nakajima

Succession and phylogenetic composition of eubacterial communities in rice straw during decomposition on the surface of paddy field soil

Abstract To estimate the bacterial communities in rice straw left on the soil surface of paddy fields, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis was conducted. Rice straw samples were placed on the soil surface in a Japanese paddy field under drained conditions after harvest and under flooded conditions after the transplanting of rice. The residual samples on the soil surface under upland conditions were collected just before spring plowing and were replaced again on the soil surface after transplanting, under flooded conditions. The DGGE patterns of the bacterial communities in rice straw on the surface of paddy field soil were divided into three groups, namely rice straw samples before placement, under drained conditions and under flooded conditions. Sequence analysis of DGGE bands indicated that most of the bacterial members in rice straw during decomposition on the soil surface in the paddy field belonged to Gram-negative bacteria. The bands that commonly existed throughout the periods under flooded and upland conditions were closely related to α-Proteobacteria. The groups of Spirochaetes and δ-Proteobacteria were often observed during flooded periods, although the members of Bacteroidetes, α-Proteobacteria, Gemmatimonadetes and Actinobacteria were also detected under flooded conditions. In contrast, the bands characteristic to the rice straw left on the soil surface of the paddy field under drained conditions belonged to Bacteroidetes and γ-Proteobacteria. This finding clearly indicates that the bacterial communities responsible for rice straw decomposition were determined by the water regime in the paddy field, and various members of Gram-negative bacteria have contributed to the decomposition of rice straw left on the soil surface in a paddy field.

Molecular analysis of the ammonia oxidizing bacterial community in the surface soil layer of a Japanese paddy field

Abstract Ammonia oxidation to nitrite is the key part in the nitrification–denitrification process that occurs in the oxic–anoxic interface of paddy fields and is, therefore, an important component of the nitrogen cycle. Ammonia oxidation is carried out by autotrophic ammonia oxidizing bacteria (AOB). In this study, we investigated the AOB community in the surface paddy soil layer using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing methods using amoA-specific primers targeting a partial stretch of the gene that encodes ammonia monooxygenase A (amoA). DGGE bands with eight different levels of mobility were obtained from the surface soil. The DNA sequences of five bands were identified. All DNA sequences were closely related to Nitrosospira spp. and no Nitrosomonas-like AOB were identified. The deduced amino acid sequences of the five retrieved DGGE bands were closely related (99–100%) to previously published amoA amino acid sequences of the cultured strains Nitrosospira Ka3 and Nitrosospira CT2F. A phylogenetic tree based on the amoA amino acid sequence revealed that all five AOB sequences were grouped within the amoA cluster 1.

Community structure of the microbiota in the floodwater of a Japanese paddy field estimated by restriction fragment length polymorphism and denaturing gradient gel electrophoresis pattern analyses

Abstract. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-denaturing gradient gel electrophoresis (DGGE) patterns of 16S rDNA were studied to elucidate the effects of the type of fertilization and the growth stage of rice plants on the community structure of the microbiota in the floodwater of a Japanese paddy field under a long-term fertilizer trial. From the mid tillering stage, a higher pH and temperature were observed in the plot without fertilization (NoF plot) than in the plots supplied with chemical fertilizers (CF plot) and with compost (CM plot). DNA fragments specific to the respective plots and common to every plot were detected after the digestion of PCR products by restriction enzymes. Cluster analysis separated the RFLP and DGGE patterns of the microbiota in the floodwater into four clusters; the microbiota in (1) the NoF plot, (2) the CF plot, (3) the CM plot, and (4) the CF and CM plots in the early growing stage. The effect of fertilizer application on the community structure was more conspicuous than that of seasonal variation.

Cyanobacterial communities of rice straw left on the soil surface of a paddy field

To estimate diversity, seasonal variation, and phylogeny of the cyanobacterial communities in rice straw placed in nylon mesh bags and left on the soil surface of a paddy field, total DNA was extracted from straw, amplified by polymerase chain reaction targeting 16S rRNA genes of cyanobacteria, and the amplicons were separated by denaturing gradient gel electrophoresis (DGGE). These DGGE bands were sequenced. The paddy field was under flooded condition after transplanting of rice (Experiment 1) and under drained conditions after harvest (Experiment 2). The residual samples on the soil surface under upland conditions were collected just before spring plowing and were placed again on the soil surface after transplanting under flooded conditions. DGGE band patterns of cyanobacterial communities of rice straw were different under drained conditions, under flooded conditions when fresh rice straw samples were placed (Experiment 1), and under flooded conditions when residual rice straw samples were replaced (Experiment 2), indicating that the communities were influenced by both water regime of the paddy field and the degree of the rice straw decomposition. Sequence analysis of DGGE bands indicated that most of the cyanobacteria in rice straw on the soil surface in the paddy field were filamentous members belonging to Subsections III and IV. Filamentous cyanobacterial cells were observed in rice straw under flooded conditions by epifluorescence microscopy.

Phylogenetic study on a bacterial community in the floodwater of a Japanese paddy field estimated by sequencing 16S rDNA fragments after denaturing gradient gel electrophoresis

Phylogenetic positions of characteristic bands of 16S rDNA that were obtained from the floodwater of a Japanese paddy field by denaturing gradient gel electrophoresis (DGGE) analysis in a previous work (Biol Fertil Soils 36:306–312, 2002) were determined to identify dominant bacterial members in the floodwater. Sequences of DGGE bands were affiliated with the Cytophaga–Flavobacterium–Bacteroides group, β-Proteobacteria, and Actinobacteria and showed phylogenetically close relationships with species inhabiting other aquatic environments, although most of their closest relatives were uncultured bacterial clones.

Comparison of Phospholipid Fatty Acid Composition in Percolating Water, Floodwater, and the Plow Layer Soil during the Rice Cultivation Period in a Japanese Paddy Field

Abstract The composition of phospholipid fatty acids (PLFAs) in the percolating water (PW), the floodwater (FW), and the plow layer soil (PLS) in a paddy field was compared during the period of rice cultivation. The amounts of PLFAs in PW, FW, and PLS ranged from 19.2 to 40.3 μg L−1, from 41.6 to 72.3 μg L−1, and from 56.4 to 75.3 μg g−1 dry-soil, respectively. There was no appreciable seasonal variation in the amount of PLFAs in PW and PLS. However, the amount of PLFAs in FW tended to decrease after the middle stage of rice cultivation. Dominant PLFAs in PW and FW were 16 : 1ω7c, 18 : 1ω7, and 16 : 0 PLFAs, and those in PLS were 16 : 1ω7c, 18 : 1ω7, 16 : 0, 18 : 1ω9, i15 : 0, ai15 : 0, and 10Me17 : 0 PLFAs. The proportion of straight, mono-nnsaturated PLFAs and branched-chain PLFAs ranged from 36.6 to 64.8% and from 9.8 to 23,8% in PW, from 49.2 to 63.7% and from 12.6 to 27.5% in FW, and from 23.4 to 28.1% and 41.7 to 47.7% in PLS, respectively. Principal component analysis of the PLFA composition enabled to clearly differentiate the community structure of the microbiota in PW and FW from that in PLS. A potential indicator of the environmental stress imposed upon the microbiota expressed by the trans vs. cis ratio of 16 : 1ω7 PLFA in the PLFA composition was consistently lower in PW and FW than in PLS, suggesting that the microbial community in PW was not appreciably stressed from the percolation through PLS.

Effect of fungicide thiram on microbial communities of rice seed surface estimated by culture-dependent and culture-independent (PCR-RFLP) methods

Abstract Coating of rice seeds with fungicide Thiram improved the seed germination capability over a long period of time (11 weeks) under low temperature conditions (4 and 8°C), which simulated the sowing of rice seeds in the winter season (the farmers slack season). To analyze the effect of Thiram on the community structure of microorganisms on the rice seed surface, culture-dependent and culture-independent (PCR-RFLP) methods were applied. PCR-RFLP patterns of 16S rDNA showed that the bacterial communities on the rice seed surface were different between coated and uncoated treatments under 8°C conditions, but that they were very similar under 4°C conditions. PCR-RFLP patterns of 18S rDNA revealed the remarkable effect of Thiram on the fungal community structure under both 4 and 8°C conditions. Although the fungal communities were quite different between coated and uncoated seeds at the beginning of incubation, the fungal communities on the coated seed surface became similar to those of uncoated seeds along with the duration of the incubation period. As the dominance percentage of Fusarium spp. among the isolates increased with the duration of the incubation period for both coated and uncoated seeds, Fusarium was considered to be a responsible for the poor germination of rice seeds that were sown in the winter season.