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.     

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.     

T4-type bacteriophage communities estimated from the major capsid genes (g23) in manganese nodules in Japanese paddy fields

The present study compared the capsid gene ( g23 ) of T4-type bacteriophages (phages) in Mn nodules with those in the plow layer soil and subsoils of two Japanese paddy fields by applying the primers MZIA1bis and MZIA6 to DNA extracts from the nodules and soils. The deduced amino acid sequences of the g23 genes in the Mn nodules were similar to those in the plow layer soil and in the subsoils. This result indicated that similar T4-type phage communities developed at these sites and that the diversity of T4-type phage communities was wide enough to cover those in the plow layer soil and in the subsoils. The majority of g23 clones formed several clusters with the clones and phages obtained from far-apart paddy fields, and the sequences of two clones were completely identical to a phage and a clone from other paddy fields at the nucleotide or amino acid level, indicating horizontal transfer of g23 genes between those paddy fields. A clone with a long nucleotide residue (686 bp) and a distribution remote from the other clones in the phylogenetic tree indicated that there were many uncharacterized, novel g23 genes in the paddy fields.     

Vertical Changes in Bacterial Communities in Percolating Water of a Japanese Paddy Field as Revealed by PCR-DGGE

Percolating water was sampled from the plow layer and subsoil layer in a Japanese paddy field, and the bacterial communities were compared together with floodwater by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) targeting a partial 16S rRNA gene and subsequent sequencing. The number of DGGE bands ranged from 16 to 28 with no significant differences among the sampling sites and times. Only 2 bands were common for the three sources of water samples. DGGE bands specific for the floodwater samples and percolating water samples from the plow layer were identified, while percolating water samples from the subsoil layer did not show specific bands but displayed common bands to those of the floodwater samples (7 bands) and percolating water samples from the plow layer (1 band). Cluster analysis of the DGGE banding patterns showed a distinct clustering in the samples of percolating water from the plow layer and a closer relationship between the others. These results suggest that the bacterial communities in percolating water changed during downward movement through the plow layer and subsoil layer. Sequences of the DGGE bands specific for the samples of percolating water from the plow layer showed a close relationship with anaerobic bacteria such as iron-reducers or uncultured bacterial DNA isolated from environments that are considered to be less oxic. On the other hand, the sequences of the bands specific for the samples of floodwater and percolating water from the subsoil layer showed a close relationship with uncultured bacterial DNA isolated from freshwater environments.     

Succession and Phylogenetic Profile of Eubacterial Communities in Rice Straw Incorporated into a Rice Field: Estimation by PCR-DGGE Analysis

PCR-DGGE analysis followed by sequencing was conducted to estimate the succession and the phylogenetic profile of the eubacterial communities responsible for the decomposition of rice straw (RS) that was incorporated into a rice field. Leaf sheath and leaf blade parts were separately put in nylon mesh bags, and were placed in the rice field soil under drained conditions during the off-cropping season and under flooded conditions after transplantation of rice. In addition, RS samples that had been placed under drained conditions in the off-cropping season were placed again in flooded rice field soil after transplantation of rice. DGGE patterns of the bacterial communities in the RS samples were classified into two groups, namely leaf sheaths and leaf blades. Principal component analysis of the DGGE patterns revealed the succession along with the duration of placement. These results indicated that the RS part (sheath or blade) mainly determined the structure of the bacterial communities responsible for the RS decomposition, followed by the duration of placement. Sequence analysis of the characteristic DGGE bands indicated that most of the closest relatives associated with the bands belonged to α-, β-, γ-, and δ-Proteobacteria, CFB group, and Spirochaetes. Some bands were closely related to Acidobacteria and Verrucomicrobia. CFB members and α-Proteobacteria predominated commonly in both RS parts, while γ- and δ-Proteobacteria, and Spirochaetes and β-Proteobacteria specifically colonized sheath and blade parts, respectively. In addition, Proteobacteria and CFB members characterized the differences in the bacterial communities under flooded or drained conditions. These results suggest that Proteobacteria, CFB group, and Spirochaetes were responsible for RS decomposition in rice field soil under both flooded and drained conditions.     

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.     

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).     

Comparison of Bacterial Communities Associated with Microcrustaceans from the Floodwater of a Paddy Field Microcosm: Estimation Based on DGGE Pattern and Sequence Analyses

Bacterial communities associated with five kinds of microcrustaceans ( Tanycypris sp., Moina sp., Mesocyclops sp., Cypretta sp. and Heterocypris sp.) from the floodwater of a paddy field microcosm were examined by the application of denaturing gradient gel electrophoresis (DGGE) to PCR-amplified 16S rDNA products with universal bacterial primers and by sequencing of characteristic DGGE bands. The number of DGGE bands of the associated bacteria was small, indicating the association of specific bacterial members with the microcrustaceans studied, among which Tanycypris sp. showed the smallest number of bands. Principal component analysis (PCA) demonstrated that the community structure of the associated bacteria could be divided into three groups: Podocopida ( Tanycypris sp., Cypretta sp. and Heterocypris sp.), Moina sp. and Mesocyclops sp., and further analysis separated Tanycypris sp. and Heterocypris sp. into different clusters. The duration of the incubation period affected the bacteria associated with Tanycypris sp., Moina sp. and Cypretta sp. only. Nearly all of the associated bacteria belonged to Gram-negative bacteria, especially the Cytophaga-Flavobacterium-Bacteroides (CFB) group. Closest relatives of the DGGE bands common to three Podocopida and Mesocyclops sp. belonged to an invertebrate endosymbiont.     

Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter

Abstract

We studied the effects of the application of organic matter (OM) and chemical fertilizer (CF) on soil alkaline phosphatase (ALP) activity and ALP-harboring bacterial communities in the rhizosphere and bulk soil in an experimental lettuce field in Hokkaido, Japan. The ALP activity was higher in soils with OM than in soils with CF, and activity was higher in the rhizosphere for OM than in the bulk soil. Biomass P and available P in the soil were positively related to the ALP activity of the soil. As a result, the P concentration of lettuce was higher in OM soil than in CF soil. We analyzed the ALP-harboring bacterial communities using polymerase chain reaction based denaturing gradient gel electrophoresis (DGGE) on the ALP genes. Numerous ALP genes were detected in the DGGE profile, regardless of sampling time, fertilizer treatment or sampled soil area, which indicated a large diversity in ALP-harboring bacteria in the soil. Several ALP gene fragments were closely related to the ALP genes of Mesorhizobium loti and Pseudomonas fluorescens. The community structures of the ALP-harboring bacteria were assessed using principal component analysis of the DGGE profiles. Fertilizer treatment and sampled soil area significantly affected the community structures of ALP-harboring bacteria. As the DGGE bands contributing to the principal component were different from sampling time, it is suggested that the major bacteria harboring the ALP gene shifted. Furthermore, there was, in part, a significant correlation between ALP activity and the community structure of the ALP-harboring bacteria. These results raise the possibility that different ALP-harboring bacteria release different amounts and/or activity of ALP, and that the structure of ALP-harboring bacterial communities may play a major role in determining overall soil ALP activity.     

Bacterial communities in soybean rhizosphere in response to soil type, soybean genotype, and their growth stage

Three experiments were conducted in this study in order to investigate the impacts of soil type, soybean genotype, and the reproductive growth stage on bacterial communities in the soybean rhizosphere. Communities were evaluated by principal component analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns and sequencing of partial 16S rDNA polymerase chain reaction (PCR) amplicons. A pot experiment analyzing three soybean genotypes grown in two different types of soil (Black soil and Dark Brown soil) indicated that soil type was the major factor in influencing the bacterial communities in the soybean rhizosphere, with a more significant effect observed in the Black soil samples than in the Dark Brown soil samples. A field experiment was conducted in Dark Brown soil using three soybean genotypes, and the results gleaned from both pot and field experiments indicated that bacterial communities in the soybean rhizosphere changed with growth stages, and higher number of DGGE bands observed in early reproductive growth stages, while surprisingly, a significant impact of genotype on the bacterial communities was not observed in these experiments. However, a plate culture experiment targeting the culturable bacterial communities detected a remarkable difference in the community structures of the rhizosphere between the two soybean genotypes, suggesting that a small portion of the total bacteria was influenced by genotype. Sequence analysis of DGGE bands indicated that bacterial phyla of Proteobacteria, Actinobacteria, Bacteroidetes, Nitrospirae, Firmicutes, Verrucomicrobia and Acidobacteria commonly inhabit the soybean rhizosphere.     

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.     

Identification of microbial communities that assimilate substrate from root cap cells in an aerobic soil using a DNA-SIP approach

Although root cap cells are an important substrate for microorganisms in the rhizosphere, little attention has been paid to the decomposition of sloughed root cap cells by microorganisms. This study used rice plant callus cells grown on medium containing ¹³C-labelled glucose as a model material for rice plant root cap cells. Harvested ¹³C-labelled callus cells (78 atom % ¹³C) were subjected to decomposition in an aerobic soil microcosm for 56 days. The low cellulose and lignin levels and the disaggregated nature of the callus cells indicated that these cells were an appropriate model material for root cap cells. DNA was extracted from a soil incubated with ¹²C- and ¹³C-callus cells and subjected to buoyant density gradient centrifugation to identify bacterial species that assimilated carbon from the callus cells. The stability of the total bacterial communities during the incubation was estimated. Many DGGE bands in light fractions of soil incubated with ¹³C-callus cells were weaker in intensity than those from soil incubated with ¹²C-callus cells, and those bands were shifted to heavier fractions after ¹³C-callus treatment. ¹³C-labelled DNA was detected from Day 3 onwards, and the DGGE bands in the heavy fractions were most numerous on Day 21. DGGE bands from heavy and light fractions were sequenced, revealing more than 70% of callus- C incorporating bacteria were Gram-negative, predominantly α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Sphingobacteria and Actinobacteria. These species were phylogenetically distinct from the bacteria reported to be present during plant residue decomposition and resident in rice roots. This study indicates that root cap cells are decomposed by specific bacterial species in the rhizosphere, and that these species augment the diversity of rhizospheric bacterial communities.     

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.     

稻秸对土壤细菌群落分子多态性的影响

模拟稻秸原位还田条件,分别在水稻土和红壤中添加水稻秸秆培养70d ,第0、5、2 5、4 5、70天采集土样。采用非机械破壁法直接提取水稻土和红壤细菌总DNA ,水稻土细菌总DNA经过二次纯化;红壤细菌总DNA经过一次纯化后,PCR扩增其16SrDNAV3可变区,均可获得清晰的目的条带,对扩增产物进行DGGE分析,结果显示:水稻土和红壤样品的DGGE条带增加,说明稻秸能够增加土壤细菌群落分子多态性的丰富度,随着培养期的延长,施有稻秸的处理中土壤细菌群落多态性的变化远远复杂于空白对照土壤中的细菌群落变化;同时发现在稻秸刺激下不同土壤细菌群落多态性高峰期出现时间不同     

DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.     

Response of soil microbial abundance and diversity in Sacha Inchi (Plukenetia volubilis L.) farms with different land-use histories in a tropical area of Southwestern China

Land-use conversion affects the soil community and microbial abundance, which are essential dynamic indicators of soil quality and sustainability. However, little to no work has been performed to analyse the impact from different land-use histories (i.e. fallow, tea, rice, banana, and maize) on the microbial abundance and diversity in the soil of sacha inchi (Plukenetia volubilis L.). Real-time quantitative PCR (qPCR) was performed to quantify soil bacterial and fungal abundance. Denaturing gradient gel electrophoresis (DGGE) combined with cloning and sequencing was used to assess the microbial communities. Our results showed that the bacterial and fungal abundance in fallow land-use conversion soils was significantly lower than that in the other four land-use conversion soils (tea, rice, banana, and maize). Moreover, the highest abundance of bacteria and fungi was detected in the soils converted from maize to sacha inchicultivation. In addition, canonical correspondence analysis (CCA) showed that the total N and pH were significantly related to bacterial and fungal community structures. These results suggest that land-use conversion from maize fields to sacha inchi farms is an effective way to maintain the soil microbial quantity and hence the sustainability of the soil.     

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.     

Azospirillum inoculation and nitrogen fertilization effect on grain yield and on the diversity of endophytic bacteria in the phyllosphere of rice rainfed crop

We assessed the Azospirillum inoculation and N-fertilization effect on grain yield and on the phyllosphere endophytic diversity of nitrogen-fixing bacteria in a rice rainfed crop. We used cultivation-based techniques and cultivation-independent methods involving PCR-16S rRNA and denaturing gradient gel electrophoresis (DGGE). In general, we observed that grain yield was improved when inoculated with Azospirillum (depending on the genotype) and/or fertilized with urea. A similar behavior was observed in total N-content in grain and the MPN determination, as the highest values occurred when seeds were inoculated with A. brasilense REC3 (S1) than with A brasilense 13-2C (S2). A positive nitrogenase activity and PCR-nifH amplification suggests that the bacteria associated to inner tissues of rice phyllosphere could have contributed to the different N-contents detected. The bacterial diversity, observed in the number and intensity of DGGE profiles, showed a higher number of bands when total DNA was obtained using only CTAB than with CTAB + PVP. The DGGE profiles revealed great stability in the dominating bands, which presumably represent numerically dominant species. Application of A. brasilense strains as inoculants did not influence the dominant members of the endophytic microbial communities in the phyllosphere, but improved N-content and production of rainfed rice crop.     

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.     

Microbial DNA extraction and analyses of soil iron–manganese nodules

Iron–manganese (Fe–Mn) nodules and concretions are soil new growth, reflecting soil environmental conditions during their formation. Bacteria play a dominant role in the oxidation of dissolved Mn(II) in aqueous systems and the formation of marine and freshwater Fe–Mn nodules. However, the role and significance of bacteria in soil Fe–Mn nodule formation have not been well recognized. In this paper, microbial DNA was directly extracted from two Fe–Mn nodule samples collected from Wuhan and Guiyang in central China. The extracted DNA was amplified by polymerase chain reaction (PCR) and cloned. The clones were then screened by amplified ribosomal DNA restriction analysis (ARDRA). Twenty patterns were obtained for Wuhan sample and Guiyang sample, respectively. DNA sequencing and phylogenetic analyses revealed that the bacterial compositions of the Fe–Mn nodules were mainly belonged to Firmicutes, β-proteobacteria, γ-proteobacteria branches of the domain bacteria. These divisions had close relativeness with Mn(II)-oxidizing bacteria identified from marine Fe–Mn nodules, implying the possible contributions of these bacteria to soil Fe–Mn nodule formation.