Bacterial Communities Responsible for the Decomposition of Rice Straw Compost in a Japanese Rice Paddy Field Estimated by DGGE Analysis of Amplified 16S rDNA and 16S rRNA Fragments

To estimate the succession and phylogenetic composition of the bacterial communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analyses targeting 16S rDNA and 16S rRNA, followed by sequencing were conducted in a Japanese paddy field. The DGGE bands of the bacterial communities in the rice straw compost were significantly more numerous in the DNA samples than in the RNA samples. Although the band number of the DNA samples was almost constant throughout the period, RNA samples showed fewer DGGE bands after mid-season drainage than before it. Thus, about 81% of the bacteria present in rice straw compost were considered to be metabolically "active" before mid-season drainage and about 62% after it. The changes in the DGGE patterns of bacterial DNA and RNA before and after mid-season drainage, respectively, were also revealed by cluster analysis and principal component analysis of the DGGE patterns. These results indicated that the bacterial communities of rice straw compost incorporated into flooded paddy fields changed gradually along with the decomposition, except for the period of mid-season drainage, but that they were influenced by mid-season drainage. Members of β-, γ- and δ-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Chlorobia, Verrucomicrobia, Chloroflexi, Spirochaetes, Firmicutes (clostridia) and Actinobacteria were present during the decomposition of rice straw compost. Characteristic "active" bacteria among them were as follows: Clostridium, Acinetobacter (γ-Proteobacteria) and β-Proteobacteria before mid-season drainage, Flavobacterium, Chondromyces , Chlorflexi and δ-Proteobacteria after mid-season drainage, and Spirochaeta and myxobacteria throughout the period.     

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.     

Succession and phylogenetic profile of eukaryotic communities in rice straw incorporated into a rice field: Estimation by PCR-DGGE and sequence analyses

Abstract

Succession and the phylogenetic profile of eukaryotic communities associated with rice straw decomposition in a rice field were studied using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by 18S rDNA sequencing. Nylon mesh bags containing leaf sheaths or blades were buried in the plow layer of a rice field under flooded conditions after transplanting (Experiment 1) and under drained conditions during the off-crop season (Experiment 2). In addition, rice straw samples in Experiment 2 were taken out before plowing in spring and re-placed in the rice field under flooded conditions at transplanting. Statistical analyses based on DGGE patterns showed that eukaryotic communities were divided into two groups, namely group A before the placement in soil, after the mid-season drainage in Experiment 1 and under the drained conditions in Experiment 2 and group B before the mid-season drainage in Experiment 1 and under the flooded conditions in Experiment 2. Based on the sequence analysis of DGGE bands, which characterized the eukaryotic communities, succession of the communities was revealed, that is, most of the bands in group A were closely related to fungi, whereas the bands in group B were closely related to protozoa. These results indicated that eukaryotic communities associated with rice straw decomposition in the rice field are mainly affected by soil conditions, such as oxic or reduced conditions, irrespective of rice straw parts (leaf sheaths and blades).     

Molecular characterization of methane-oxidizing bacteria associated with rice straw decomposition in a rice field

Abstract

Methane-oxidizing bacteria (MOB) are crucial to the reduction of CH₄ emitted to the atmosphere. However, it is unclear how MOB in rice straw are affected by straw decomposition processes. In a Japanese rice field, a year-round experiment was set up to study the effects of agricultural practice (rice cultivation/winter fallow), straw parts (leaf sheath/blade) and the site of straw placement (plow layer/soil surface) on MOB communities in rice straw using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing analyses of key MOB functional genes (pmoA and amoA). Thirty-eight different DGGE bands were observed over the entire investigation period. Principal component analysis of DGGE pattern suggested that agricultural practice is the key factor regulating the MOB communities. Sequencing of dominant DGGE bands showed that: (1) during the rice cultivation period, methanotrophs (particularly type I methanotrophs) dominated the MOB community, (2) during the winter fallow season both type I and type II methanotrophs were dominant in sheath segments placed both on the soil surface and in the plow layer, whereas ammonia oxidizers seemed to dominate blade segments placed in the plow layer. Alignment of diagnostic amino acid sequences of MOB suggested the presence of novel ammonia oxidizers in rice straw in rice fields.     

Comparison of community structures of microbiota at main habitats in rice field ecosystems based on phospholipid fatty acid analysis

The present study compares the community structures of microbiota at different habitats in Japanese rice fields by comparing their phospholipid fatty acid (PLFA) compositions to understand the contribution of different habitats to microbiological diversity. The data were collected from four neighboring rice fields. Comparison was made for the PLFA compositions extracted from the floodwater, percolating water, rice soils under flooded and drained conditions, rice straw (RS) placed in flooded and drained rice soils, RS in the composting process, and RS compost placed in a flooded rice field. Average amounts of PLFAs were 33 μg L⁻¹ in the floodwater, 17.1 μg L⁻¹ in the percolating water from plow layers, 34.6 μg L⁻¹ in the percolating water from subsoil layers, 108 μg g⁻¹ dry weight basis (dw) in flooded rice soils, 382 μg g⁻¹ dw in RS materials, 2,510 μg g⁻¹ dw in RS composts, 2,850 μg g⁻¹ dw in RS composts after application to a flooded rice soil, 222 μg g⁻¹ wet weight basis (ww) in RS in drained rice soils, and 284 μg g⁻¹ ww in RS in flooded rice soils. The total amount of PLFAs to the soil depth of 10 cm was estimated to be about 12 g m⁻². The PLFA compositions were different from each other depending on the habitats. Rice soils were characterized by the predominance of actinomycetes and Gram-positive bacteria in comparison with the other habitats. In contrast, the microbial communities in the floodwater and percolating water were characterized by the predominance of Gram-negative bacteria and eukaryotes (presumably algae), and Gram-negative bacteria, respectively. The microbial community of RS materials was dominated by fungi. Gram-positive bacteria became predominant in RS after application to flooded rice soils, while RS placed in a drained rice field after harvesting rice was characterized by the predominance of Gram-negative bacteria and fungi. The community structures at respective habitats were stable and specific, irrespective of the season of sampling and the duration of decomposition of RS.     

Community structure of bacteria and fungi responsible for rice straw decomposition in a paddy field estimated by PCR-RFLP analysis

Rice straw including leaf sheaths and blades put in nylon mesh bags was placed in the plow layer of a Japanese paddy field after harvest under upland conditions and after transplanting of rice seedlings under flooded conditions. In addition, rice straw that was decomposed under the upland conditions during the off-crop season in winter was placed again in soil at the time of transplanting. The materials were collected periodically to analyze the community structure of the bacteria and fungi responsible for rice straw decomposition by PCR-RFLP analysis. The PCR products with 27f and 1492r primers designed for bacterial 16S rDNA and with EF3 and EF4 primers designed for fungal 18S rDNA were digested with four restriction endonucleases (Hinf I, Sau3A I, Hae III, EeoR I). Bacterial communities in the decomposing rice straw were different from each other between upland and flooded conditions, between leaf sheaths and blades, and between straw samples with and without decomposition under upland conditions during the off-crop season. Fungal communities in the decomposing rice straw were also different between the leaf sheaths and blades under upland soil conditions. Score plots of bacterial and fungal communities in the principal component analysis were separated from the plot of the straw materials along with the duration of the placement, indicating the succession of bacterial and fungal communities in decomposing rice straw with time.     

Comparison of bacterial community structures at main habitats in paddy field ecosystem based on DGGE analysis

Bacterial communities at different habitats in a Japanese paddy field ecosystem were compared to understand the bacterial world in the ecosystem as a whole by analyzing data of the denaturing gradient gel electrophoresis (DGGE) band patterns and the sequenced DGGE bands. The habitats were floodwater, percolating water, microcrustacean inhabiting in floodwater, plow layer soil, rice roots, rice straw and rice straw compost incorporated in soil, rice straw placed on the soil surface, plant residues in paddy fields, and rice straw under composting process. Phylotype (band) richness, diversity, evenness, and stability of the bacterial communities at the respective habitats were evaluated based on the DGGE profile data. Phylotype richness was greater near plant residues, rice straw buried in soil and rice straw placed on soil surface, while it was smaller at microcrustacean and rice straw compost buried in soil. The samples from plow layer soil and rice straw compost buried in soil showed considerably higher index values for diversity, evenness, and stability, while those from rice straw placed on soil surface and microcrustacean had lower values of the indices than other habitats. Sequences of totally 250 DGGE bands were assigned to phyla or classes. Distribution of bacterial members to phylogenetic taxa was different among the respective habitats. Inhabitants in plow layer soil were most widely distributed among the groups (nine phyla: Proteobacteria, Chloroflexi, Chlorobi, Verrucomicrobia, Acidobacteria, Nitrospira, candidate division OP10, Cyanobacteria, and Actinobacteria), while those in floodwater and microcrustacean were restricted to only three phyla (Proteobacteria, Bacteroidetes, and Actinobacteria). Proteobacteria and Bacteroidetes were found at all the habitats and the habitats except for plow layer soil, respectively, whereas abundant members belonged to Chloroflexi and Actinobacteria in plow layer soil. “Comprehensive mapping” of DGGE fragments was conducted by principal component analysis based on evolutionary distances of the fragments to 202 reference bacterial strains to overview phylogenetic relationships of bacterial members among the respective habitats. The score plots with the first and second principal components distinctly characterized bacterial members at the respective habitats, and the similarity between the respective communities was clearly demonstrated. Overall, bacterial communities at the respective habitats were distinct and different in the diversity and stability to each other, which may have contributed to the diversity of overall bacterial communities in the paddy field ecosystem.     

Effect of Free-Air CO2 Enrichment (FACE) on Methanogenic Archaeal Communities Inhabiting Rice Roots in a Japanese Rice Field

The effect of free-air CO₂ enrichment (FACE) on the methanogenic archaeal communities inhabiting rice roots was studied in a Japanese rice field by separately collecting rice roots three times (at mid-tillering, panicle initiation, and heading stages) according to their nodal number, extracting DNA from the roots and subjecting it to polymerase-chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and sequence analyses. Appearance of roots indicated that aging and senescence occurred faster under the FACE conditions than under the ambient conditions. The number of DGGE bands of methanogenic archaeal communities tended to increase with the growth stages. Cluster analysis showed that the succession of methanogenic archaeal communities in the ambient plot preceded that in the FACE plot, while the trend of the appearance of rice roots was opposite. All the closest relatives associated with the DGGE bands belonged to Methanomicrobiales and Rice cluster I, and FACE did not affect the phylogenetic position of the closest relatives associated with the characteristic DGGE bands. Faster succession of methanogenic archaeal communities in the ambient plot and similar phylogenetic members between the plots were observed in rice roots in years with both warmer (1999) and cooler (2003) weather during the rice cultivation period than in average years.     

Bacterial community in plant residues in a Japanese paddy field estimated by RFLP and DGGE analyses

Plant residues (PRs) are “hot spots” of microbial activities in soil. PRs with the size more than 0.5 mm were collected from a Japanese paddy field during rice cultivation period (from May to September) and fractionated into four categories by size (>4, 2-4, 1-2, and 0.5-1 mm) using sieves. Restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) patterns were compared among the fractions after DNA extraction from the PRs and PCR amplification. The total amount of PRs with the size over 0.5 mm decreased in the field with the first-order kinetics (r²=0.810, p<0.01) with time from rice transplanting to harvest. RFLP analysis showed that the bacterial community structure in PRs with the 0.5-2 mm fraction was different from that in PRs with the >2 mm fraction and the latter community structure changed after the midseason drainage. In contrast, the DGGE patterns of the bacterial community in the PRs indicated the succession from June to September during rice cultivation forming three major groups irrespective of the fraction size. Sequence analysis of DGGE bands showed that Firmicutes (clostridia), α-, γ-, δ-Proteobacteria (myxobacteria), Nitrospira, Acidobacteria, Bacteroidetes, Verrucomicrobia and Spirochaetes were predominant members in the PRs irrespective of fraction size.     

Effect of Application of Rice Straw and Compost on the Bacterial Communities Associated with Moina sp. in the Floodwater of a Paddy Soil Microcosm: Estimation Based on DGGE Pattern and Sequence Analyses

Bacterial communities associated with Moina sp. in the floodwater of a paddy field microcosm were examined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA. Eighteen out of 20 eubacterial DGGE bands were sequenced. The associated eubacterial communities mainly consisted of the Cytophaga-Flavobacterium-Bacteroides group and α-, β-, and γ-Proteobacterial groups, irrespective of the application of rice straw and rice straw compost. The effect of the application of rice straw and compost on the communities was not appreciable, compared with host specificity. An uncultured Cytophagales bacterium was estimated to be specifically associated with Moina sp. Presence of bacteria that are specific to rice straw treatment was also estimated.     

Community structure of methanogenic archaea in paddy field soil under double cropping (rice-wheat)

Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.     

Eukaryotic communities associated with the decomposition of rice straw compost in a Japanese rice paddy field estimated by DGGE analysis

To estimate the succession and phylogenetic composition of the eukaryotic communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analysis targeting 18S rDNA followed by sequencing was conducted in a Japanese paddy field. The eukaryotic communities in rice straw compost incorporated into the flooded paddy field were influenced by the mid-season drainage and mainly composed of fungi (Ascomycota, Zygomycota, and Chytridiomycota) and protozoa (Ciliophora, Euglyphida, and Dactylopodida), most of which existed continuously during the cultivation period of paddy rice. The results indicated that these eukaryotic members were associated with the decomposition of rice straw compost in paddy field soil directly or indirectly.     

Microbial community responsible for the decomposition of rice straw in a paddy field: estimation by phospholipid fatty acid analysis

To estimate the microbial communities responsible for rice straw decomposition in paddy field, phospholipid fatty acid (PLFA) composition of leaf sheaths and blades was analyzed during the decomposition of both residues under upland conditions after harvest and under flooded conditions at the time of transplanting of rice plants. In addition, rice straw that had been placed in the field under upland conditions (November to April) was taken out in spring, and placed again in the same field under flooded conditions at the time of transplanting. High proportions of the branched-chain PLFAs were observed under flooded conditions (June to September); the proportions of straight mono-unsaturated and straight poly-unsaturated PLFAs were high under upland conditions in the winter season for 4 months. The dominant PLFAs in straight mono-unsaturated, straight poly-unsaturated and branched-chain PLFA groups were 18:19, 18:17 and 16:17c, 18:26c and i15:0, i17:0 and ai15:0, respectively, under both upland and flooded conditions. These findings indicated the important roles of Gram-negative bacteria and fungi under upland conditions and of Gram-positive bacteria and anaerobic Gram-negative bacteria under flooded conditions. Cluster analysis of PLFA composition showed the difference of community structure of microbiota in rice straw between upland and flooded conditions. In addition principal component analysis revealed the difference between leaf sheaths and blades under upland conditions and indicated that the content of straight unsaturated PLFAs (sheaths > blades) characterized their community structures.     

Bacterial communities in manganese nodules in rice field subsoils: Estimation using PCR-DGGE and sequencing analyses

Abstract

The phylogenetic positions of bacterial communities in manganese (Mn) nodules from subsoils of two Japanese rice fields were estimated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by sequencing of 16S rDNA. The DGGE band patterns and sequencing analysis of characteristic DGGE bands revealed that the bacterial communities in Mn nodules were markedly different from those in the plow layer and subsoils. Three out of four common bands found in Mn nodules from two sites corresponded to Deltaproteobacteria and were characterized as sulfate-reducing and iron-reducing bacteria. The other DGGE bands of Mn nodules corresponded to sulfate and iron reducers (Deltaproteobacteria), methane-oxidizing bacteria (Gamma and Alphaproteobacteria), nitrite-oxidizing bacteria (Nitrospirae) and Actinobacteria. In addition, some DGGE bands of Mn nodules showed no clear affiliation to any known bacteria. The present study indicates that members involved in the reduction of Mn nodules dominate the bacterial communities in Mn nodules in rice field subsoils.     

利用PCR-DGGE技术研究长期自然恢复、种植作物和裸地条件下黑土土壤细菌群落结构变化

Soil microbial biomass and community structures are commonly used as indicators for soil quality and fertility. A investigation was performed to study the effects of long-term natural restoration, cropping, and bare fallow managements on the soil microbial biomass and bacterial community structures in depths of 0--10, 20--30, and 40--50 cm in a black soil (Mollisol). Microbial biomass was estimated from chloroform fumigation-extraction, and bacterial community structures were determined by analysis of 16S rDNA using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Experimental results showed that microbial biomass significantly declined with soil depth in the managements of restoration and cropping, but not in the bare fallow. DGGE profiles indicated that the band number in top 0--10 cm soils was less than that in depth of 20--30 or 40--50 cm. These suggested that the microbial population was high but the bacterial community structure was simple in the topsoil. Cluster and principle component analysis based on DGGE banding patterns showed that the bacterial community structure was affected by soil depth more primarily than by managements, and the succession of bacterial community as increase of soil depth has a similar tendency in the three managements. Fourteen predominating DGGE bands were excised and sequenced, in which 6 bands were identified as the taxa of Verrucomicrobia, 2 bands as Actinobacteria, 2 bands as α-Proteobacteria, and the other 4 bands as δ-Proteobacteria, Acidobacteria, Nitrospira, and unclassified bacteria. In addition, the sequences of 11 DGGE bands were closely related to uncultured bacteria. Thus, the bacterial community structure in black soil was stable, and the predominating bacterial groups were uncultured.     

Microbial communities responsible for the decomposition of rice straw compost in a Japanese rice paddy field determined by phospholipid fatty acid (PLFA) analysis

To identify the microbial communities responsible for the decomposition of rice straw compost in soil during the rice cultivation period, phospholipid fatty acid (PLFA) composition of rice straw compost was determined by periodically sampling the compost from a Japanese rice field under flooded conditions. About 21% of the compost was decomposed within a period of 3 months. The total amount of PLFAs, as an indicator of microbial biomass, was significantly lower under drained conditions than under flooded conditions and was relatively constant during the flooding period. This indicates that the microbial biomass in the compost samples did not increase during the gradual decomposition of rice straw compost under flooded conditions. The proportion of branched-chain PLFAs (biomarker of Grampositive and anaerobic Gram-negative bacteria) slightly decreased during the early period after placement, and increased gradually afterwards. Among the branched-chain PLFAs, i15:0, ail5:0, i16:0 and i17:0 PLFAs predominated and their proportions increased gradually except for i16:0. The proportion of straight mono-unsaturated PLFAs (biomarker of Gramnegative bacteria) was almost constant throughout the period, and 18:1ω9 and 18:1ω7 PLFAs predominated. The proportion of straight poly-unsaturated PLFAs as a biomarker of eukaryotes including fungi was also constant throughout the period, except for a decrease under drained conditions. Straight poly-unsaturated PLFAs consisted mainly of 18:2ω6c PLFA. Therefore, these results suggest that the proportions of Gram-positive and anaerobic Gram-negative bacteria increased during the decomposition of rice straw compost in flooded paddy field. Statistical analyses enabled to divide PLFA patterns of microbiota in the rice straw compost into two groups, one group consisting of rice straw compost samples collected before mid-season drainage and the other of samples collected after mid-season drainage. Small squared distances among samples in cluster analysis indicated that the community structure of microbiota was similar to each other as a whole. These results suggest that the microbial communities changed gradually during the period of placement, and that mid-season drainage may have affected the community structure of microbiota. Principal component analysis of the PLFA composition suggested that the succession of microbiota along with the decomposition in flooded soil was similar between rice straw compost and rice straw and that the changes in the community structure during the decomposition in flooded soil were more conspicuous for rice straw than for rice straw compost.     

Molecular identification of methane oxidizing bacteria in a Japanese rice field soil

By using cultivation-independent techniques, community changes of methane-oxidizing bacteria (MOB) in rice bulk soils were investigated under field conditions in a Japanese rice field. The representative soil samples were collected during the typical rice growing season and nonrice growing period all year round. Statistical characterization of denaturing gradient gel electrophoresis (DGGE) community patterns of MOB pmoA/amoA functional gene fragments showed that MOB community structures in the rice bulk soils remained largely unchanged throughout the investigated period. The total intensity of six common DGGE bands that appeared consistently throughout the investigated period accounted for 64% of the total intensity of all 18 different DGGE bands detected. The low squared distance of the Ward cluster analysis of the DGGE pattern and the high Sorensen similarity coefficient (81%) also implied the high similarity of the MOB community structures. The stable MOB community structure did not couple well with the wide variation of soil water contents all year round. Sequencing analysis of the nine characteristic bands including six common bands revealed the presence of Type I, Type II methanotrophs, and β-proteobacterial ammonia oxidizers in rice bulk soils. In comparison with MOB type species, three DGGE bands showed a wide variation of the highly conserved amino acid residues, implying the presence of novel MOB bacteria inhabiting the rice bulk soil. The high diversity of MOB composition suggested that rice bulk soils might serve as an ideal reservoir for the dynamic changes of MOB in a rice field ecosystem in response to environment changes.     

Bacterial communities associated with nodal roots of rice plants along with the growth stages: Estimation by PCR-DGGE and sequence analyses

Bacterial communities in rice roots that developed from different nodes and at different growth stages were compared by using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA. Rice root samples were collected at three stages, namely tillering (July 2), maximum tillering (July 21), and ripening (September 12). The bacterial diversity in rice roots was found to increase along with the growth stages of the rice plants as well as the root age from the numbers of DGGE bands. The community structure of the bacteria was also found to change with the growth stages and root age from cluster analysis. Sequence analysis of the DGGE bands indicated that the dominant bacteria associated with rice roots were Gram-negative bacteria, especially β-Proteobacteria irrespective of the growth stages and root age. DGGE bands related to Janthinobacterium agaricidamnosum W1r3T and Clostridium sp. FCB90-3 were ubiquitous in many roots irrespective to the sampling date. Principal component analysis enabled to characterize the DGGE bands related to nitrogen-fixing Azoarcus spp., and Azovibrio sp. BS20-3 in the samples collected on July 2 and on July 21, and the myxobacteria collected on September 12, respectively, as representative bacteria in the bacterial communities. The habitat around older rice roots at every sampling date was more reductive than that around younger rice roots, and the DGGE bands related to Spirochaeta spp. were specific in older roots at every sampling date. Some specific bacteria that were most closely related to the DGGE bands were found from principal component analysis to characterize young and old. roots at each growth stage as follows: aerobes Flavobacterium sp. 90 clone 2 and Janthinobacterium agaricidamnosus W1r3T in young roots and facultative anaerobes Dechloromonas sp. MissR and Anaeromyxobacter dehalogenans 2CP-3 in old nodal roots on July 2, strict anaerobe Geobacter pelophilus Dfr2 and aerobes Nitrosospira sp. Nsp17 and uncultured Nitrospira sp. clone 4-1 in old roots on July 21, and different Clostridium spp. in both young and old roots and Desulfovibrio magneticus RS-1 in old roots on September 12, respectively. A larger number of the closest relatives of anaerobic bacteria grew at the late stage than at the early stages, and in old roots than in younger roots. Thus, the environment of paddy roots was remarkably heterogeneous as a bacterial habitat, where not only the whole root system but also a root may create oxic and anoxic environments.     

Diversity of methanogenic archaeal communities in Japanese paddy field ecosystem, estimated by denaturing gradient gel electrophoresis

Diversity of methanogenic archaeal communities in Japanese paddy field ecosystem was evaluated by the denaturing gradient gel electrophoresis (DGGE) after PCR amplification of the 16S rRNA genes (16S rDNAs), sequencing analysis and data evaluation by principal component analysis. Data were obtained from samples collected from the plowed soil layer, rice roots, rice straws incorporated in soil, plant residues (mixture of weeds, rice litters, rice roots, and rice stubbles) in soil, and composing rice straw. The number of bands of DGGE profiles ranged from 12 to 26 with the highest numbers in rice roots and rice straws incorporated in soil. However, the diversity indices based on both the numbers and intensity of bands indicated that the community of the plowed soil layer was the most diverse, even, and stable. Sequencing of the main DGGE bands showed the presence of Methanomicrobiales, Methanosarcinales, Methanobacteriaceae, and Methanocellales. The plowed soil layer included all phylogenetic groups of the methanogenic archaea of the other studied habitats, with prevalence of the members of Methanomicrobiales and Methanocellales. The phylogenetic diversity was compared with that of paddy soils collected in Italy, China, and the Philippines and that of 12 anaerobic environments (fen, waste, coast, permafrost, natural gas field, bovine rumen, riparian soil, termite, ciliate endosymboints, lake sediment, landfill, and seep rumen). The phylogenetic diversity was more similar among paddy soils than with the other anaerobic environments. Probably, the methanogenic archaeal communities of the paddy field soils were characterized by indigenous members and some of the members of the community of the plowed soil layer colonized rice roots, rice straws, and plant residues.