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Methane is a greenhouse gas that contributes to global warming and paddy fields are an important source of methane emission. Methanogenic archaea and methane-oxidizing bacteria inhabiting paddy soil ecosystem play crucial roles in the methane emission from paddy fields. This literature review summarizes progress of ecological studies on methanogenic archaea and methane-oxidizing bacteria inhabiting paddy soil ecosystem from a series of investigations conducted in various paddy fields. Emphasis was placed on the isolation and identification of methanogenic archaea and methane-oxidizing bacteria, and also the composition, abundance, and dynamics of these microbial communities in paddy soil ecosystem. Some perspectives for future studies were presented on the robust stability of methanogenic archaeal community structure and the bias of methane-oxidizing bacterial isolates identified at species level, which will provide fundamental information useful for predicting, controlling, and mitigating methane emission from paddy fields., Published online: 20 Jul 2021

Ciliates play important ecological roles as microbial predators in soil. Although arable soils can experience a wide range of temperatures caused by agricultural land management, the effect of temp...

To reveal whether microaerophilic Fe(II)-oxidizing bacteria (FeOB) participate in the Fe(II) oxidation at the oxic-anoxic interface in flooded paddy field soil, distribution of microaerophilic FeOB belonging to Gallionellaceae (Gallionella-related FeOB) in the surface layer of a flooded paddy soil microcosm and O2 conditions for the Fe(II) oxidation by a microaerophilic Fe(II) oxidizer, Ferrigenium kumadai An22, were investigated. Flooded paddy soil microcosms were incubated for 30 days. Five soil layers were sampled at 2-mm intervals from the soil surface after the incubation. The community structure of Gallionella-related FeOB was analyzed with qPCR and DGGE methods. In the culture experiment, O2 and Fe(II) profiles in F. kumadai An22-inoculated and non-inoculated gel-stabilized gradient tubes were analyzed, in which an opposing gradient of O2 and Fe(II) was formed. A thin oxic layer was formed at the soil surface after the incubation. The copy number of 16S rRNA genes of Gallionella-related FeOB was highest at the top 0–2 mm layer of the soil. DGGE analysis showed that several bands derived from Gallionella-related FeOB newly appeared at the top soil layer with high intensity. In the culture experiment, F. kumadai A22 grew at the oxic side of the oxic-anoxic interface with the formation of Fe oxides. The present study indicated that Gallionella-related microaerophilic FeOB proliferated in the surface layer of flooded paddy field soil, presumably by oxidizing Fe(II) in the oxic-anoxic interface.

An evaluation of suppressiveness against soil-borne diseases is important for their control. We herein assessed disease suppression against F. oxysporum f. sp. spinaciae using the Fusarium co-cultivation method in 75 soils collected from croplands around the country. The suppressive effects of soil microbes against F. oxysporum growth were examined by simultaneously culturing soil suspensions and F. oxysporum f. sp. spinaciae on a culture medium. The growth degree of F. oxysporum on the medium varied among the 75 soils tested, and 14 soils showing different degrees of growth were selected to investigate the incidence of spinach wilt by cultivating spinach inoculated with the pathogenic F. oxysporum strain. A correlation (r=0.831, P0.001) was observed between the disease incidence of spinach wilt and the growth degree of F. oxysporum using the co-cultivation method in the 14 selected soils. No correlations were noted between chemical and biological parameters (including pH and the population density of microbes, except for the growth degree of F. oxysporum) and the growth degree of F. oxysporum and incidence of spinach wilt, indicating that it was not possible to predict the degree of growth or disease incidence based on specific chemical and biological characteristics or their combination. The present results suggest that an evaluation of the growth degree of F. oxysporum by the Fusarium co-cultivation will be useful for diagnosing the disease suppressiveness of soil against pathogenic F. oxysporum in croplands.

The community structure of methanogenic archaea is relatively stable, i.e., it is sustained at a high abundance with minimal changes in composition, in paddy field soils irrespective of submergence and drainage. In contrast, the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils. This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments. In the pot experiment, a soil sample obtained from an upland field was incubated under submerged conditions for 275 d. Soil samples periodically collected were subjected to culture-dependent most probable number (MPN) enumeration, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of archaeal 16S rRNA gene, and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene (mcrA) of methanogenic archaea. The abundance of methanogenic archaea increased from 102 to 103 cells g−1 dry soil and 104 to 107 copies of mcrA gene g−1 dry soil after submergence. Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis, members from Methanocellales, Methanosarcinaceae, and Methanosaetaceae proliferated in the soils, and the community structure was relatively stable once established. In the field experiment, the number of viable methanogenic archaea in a rice paddy field converted from meadow (reclaimed paddy field) was monitored by MPN enumeration over five annual cycles of field operations. Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer's paddy field was dressed onto the meadow (dressed paddy field) and an upland crop field converted from the meadow (reclaimed upland field). The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field. Then, the number gradually increased over five years and finally reached 103–104 cells g−1 dry soil, which was comparable to that in the dressed paddy field. These findings showed that the low abundance of autochthonous methanogenic archaea in the non-methanogenic oxic upland field soils steadily proliferated, and the community structure was developed following repeated and long-term submergence. These results suggest that habitats suitable for methanogenic archaea were established in soil following repeated and long-term submergence.

Redox cycle of iron (Fe) is the central process in the biogeochemistry of paddy field soil. Although Fe(II)-oxidizing process is mediated by both abiotic and biotic reactions, microorganisms involv...

Ferrigenium kumadai An22 T (=JCM 30584 T =NBRC 112974 T =ATCC TSD-51 T ) is a microaerophilic iron oxidizer isolated from paddy field soil and belongs to the family Gallionellaceae . Here, we report the complete genome sequence of F. kumadai An22 T , which was obtained from the hybrid data of Oxford Nanopore long-read and Illumina short-read sequencing.

This chapter presents characteristics of soils of the Chubu region, which encompasses between the Sea of Japan and the Pacific Ocean in the middle of Honshu. In the region, there is Mt. Fuji, the active volcano and the highest mountain in Japan. Not only that, the geology of the region is diverse. The region has several mountain ranges, diverse climates and vegetation. So, you can see a lot of different type of soils in the region. In this chapter, we will first give an overview of the soil formation factors and the existing soils in the region. The chapter then describes the agriculture and forestry for the three distinct regions of the Chubu region. The northern part of the Chubu region, called Hokuriku, centers on paddy production on wet and clayey soil by virtue of snow-meltwater and summer high temperature. For the central part of the Chubu region consisting of highland, the sustainable soil management for forestry is described. As for the southern part of the Chubu region, Tokai, characteristics of agriculture for cultivation of vegetables and tea on mainly Red-Yellow soil under mild climate are described, including efforts toward effective use of fertilizers.

Soils are characterized by diverse biotic and abiotic constituents, and this complexity hinders studies on the effects of individual soil components on microorganisms in soil. Although artificial soils have been used to overcome this issue, anoxic soils have not yet been examined. We herein aimed to create artificial soil that reproduces anaerobic methane production by soil from a rice field. Organic materials and mineral particles separated from rice field soil were mixed to prepare an artificial soil matrix; the matrix was added with a small volume of a soil suspension as a microbial inoculum. When the microbial inoculum was added immediately after matrix preparation, anaerobic decomposition was markedly less than that by original soil. When the inoculum was added 9-15 days after soil matrix preparation, anaerobic CO2 and methane production was markedly activated, similar to that by original soil after 40 days of incubation, which suggested that the maturation of the soil matrix was crucial for the reproduction of anaerobic microbial activities. The diversity of the microbial community that developed in artificial soil was markedly less than that in original soil, whereas their predicted functional profiles were similar. Humic substances altered the composition and network patterns of the microbial community. These results suggested that the functional redundancy of soil microorganisms was sustained by different microbial sub-communities. The present study demonstrated that artificial soil is a useful tool for investigating the effects of soil components on microorganisms in anoxic soil.

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.

The conversion factor, kK, for estimation of microbial biomass potassium (K) by the chloroform-fumigation extraction method was determined for some arable soils: upland field soils under different fertilization conditions, an upland field soil under a greenhouse condition, and a paddy field soil under a flooded condition. The kK value varied with land utilization (paddy or upland) or fertilization (chemical or organic fertilizer). Value of kK was different between paddy field soil (0.28–0.38) and upland field soil (0.41–0.73). This study indicates that the value could be useful for the estimation of microbial biomass K in soil by the chloroform-fumigation extraction method and further investigation of the amounts of biomass K in different types of soils under conditions with varied field managements will be necessary.

Preventive measures against soil-borne diseases need to be implemented before cultivation because very few countermeasures are available after the development of diseases. Some soils suppress soil-borne diseases despite the presence of a high population density of pathogens. If the suppressiveness of soil against soil-borne diseases may be predicted and diagnosed for crop fields, it may be possible to reduce the labor and cost associated with excessive disinfection practices. We herein evaluated the suppressiveness of soils in fields with the long-term application of organic amendments by examining the growth of pathogenic Fusarium oxysporum co-cultivated with indigenous soil microorganisms on agar plates. Soils treated with coffee residue compost or rapeseed meal showed suppressiveness against spinach wilt disease by F. oxysporum f. sp. spinaciae or spinach wilt and lettuce root rot diseases by F. oxysporum f. sp. spinaciae and F. oxysporum f. sp. lactucae, respectively, and the growth of pathogenic Fusarium spp. on agar plates was suppressed when co-cultured with microorganisms in a suspension from these soils before crop cultivation. These results indicate the potential of the growth degree of pathogenic F. oxysporum estimated by this method as a diagnostic indicator of the suppressiveness of soil associated with the inhabiting microorganisms. A correlation was found between the incidence of spinach wilt disease in spinach and the growth degree of F. oxysporum f. sp. spinaciae by this co-cultivation method, indicating that suppressiveness induced by organic amendment applications against F. oxysporum f. sp. spinaciae is evaluable by this method. The co-cultivation method may be useful for predicting and diagnosing suppressiveness against soil-borne diseases.

Methanogenic archaea survive under aerated soil conditions in paddy fields, and their community is stable under these conditions. Changes in the abundance and composition of an active community of methanogenic archaea were assessed by analyzing mcrA gene (encoding α subunit of methyl-coenzyme M reductase) and transcripts during a prolonged drained period in a paddy-upland rotational field. Paddy rice (Oryza sativa L.) was planted in the flooded field and rotated with soybean (Glycine max [L.] Merr.) under upland soil conditions. Soil samples were collected from the rotational plot in the first year, with paddy rice, and in the two successive years, with soybean, at six time points, before seeding, during cultivation, and after harvest as well as from a consecutive paddy (control) plot. By the time that soybean was grown in the second year, the methanogenic archaeal community in the rotational plot maintained high mcrA transcript levels, comparable with those of the control plot community, but the levels drastically decreased by over three orders of magnitude after 2 years of upland conversion. The composition of active methanogenic archaeal communities that survived upland conversion in the rotational plot was similar to that of the active community in the control plot. These results revealed that mcrA gene transcription of methanogenic archaeal community in the rotational field was affected by a prolonged non-flooding period, longer than 1 year, indicating that unknown mechanisms maintain the stability of methanogenic archaeal community in paddy fields last up to 1 year after the onset of drainage.

Solarization makes a great impact on the abundance of ammonia oxidizers and nitrifying activity in soil. To elucidate fluctuations in the abundance of ammonia oxidizers and nitrification in solarized soil, copy numbers of amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA), viable number of ammonia oxidizers and inorganic nitrogen contents were investigated in greenhouse experiments. The copy number of amoA gene and the viable number of ammonia oxidizers were determined by the quantitative polymerase chain reaction and most probable number methods, respectively. Abundance of AOB based on the estimation of amoA gene copy numbers and viable counts of ammonia oxidizers was decreased by the solarization treatment and increased during the tomato (Solanum lycopersicum L.) cultivation period following the solarization. Effect of solarization on the copy number of amoA gene of AOA was less evident than that on AOB. The proportion of nitrate in inorganic nitrogen contents was declined by the solarization and increased during the tomato cultivation period following the solarization. Positive correlations were found between the proportion of nitrate in inorganic nitrogen content and the copy number of bacterial or archaeal amoA gene or the viable number of ammonia oxidizers; the copy number of bacterial amoA gene showed a strong correlation with the viable number of ammonia oxidizers. The present study revealed influences of solarization on the fluctuation in the abundance of ammonia oxidizers and dynamics of inorganic nitrogen contents in soil and the results indicate that the determination of amoA gene of AOB is possibly a quick and useful diagnostic technique for evaluating suppression and restoration of nitrification following solarization., ファイル公開日: 2018/08/18

Changes in the relative abundances of the transcripts of hydA gene paralogs for [FeFe]-hydrogenase in Clostridium sp. strain H2 and Desulfovibrio sp. strain A1 isolated from paddy field soil were analyzed during H2 production. Strains H2 and A1 had at least five and two phylogenetically different hydA genes, respectively. The relative abundances of their hydA transcripts differed among the paralogs and H2 production activity changed in a manner that depended on the growth phase and conditions. Increases or decreases in the relative abundances of the transcripts of two out of five hydA genes in strain H2 correlated with changes in H2 production rates, whereas those of the others remained unchanged or decreased. In strain A1, the relative abundances of the transcripts of two hydA genes differed between monoculture, sulfate-reducing, and syntrophic, methanogenic conditions. The relative abundance of the transcripts of one hydA gene, predicted to encode a cytosolic [FeFe]-hydrogenase, was higher under syntrophic, methanogenic conditions than sulfate-reducing conditions, while that of the transcripts of the other hydA gene decreased with time under both conditions. This study showed that the transcription of the hydA gene during growth with active H2 production was differently regulated among the paralogs in H2 producers isolated from paddy field soil.

Plant residues are one of the main sources of soil organic matter in paddy fields, and elucidation of the bacterial communities decomposing plant residues was important to understand their function and roles, as the microbial decomposition of plant residues is linked to soil fertility. We conducted a DNA stable isotope probing (SIP) experiment to elucidate the bacterial community assimilating 13-carbon (13C) derived from plant residue under an anoxic soil condition. In addition, we compared the bacterial community with that under the oxic soil condition, which was elucidated in our previous study (Lee et al. in Soil Biol Biochem 43:814–822, 2011). We used the 13C-labeled dried rice callus cells as a model of rice plant residue. A paddy field soil was incubated with unlabeled and 13C-labeled callus cells. DNA extracted from the soils was subjected to buoyant density gradient centrifugation to fractionate 13C-enriched DNA. Then, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rDNA band patterns and band sequencing method were used to evaluate bacterial community. DGGE analysis showed that the band patterns in the 13C-enriched fractions were distinctly changed over time, while the changes in the community structure before fractionation were minor. Sequencing of the 13C-labeled DGGE bands revealed that Clostridia were a major group in the bacterial communities incorporating the callus-derived carbon although Gram-negative bacteria, and Actinobacteria also participated in the carbon flow from the callus under the anoxic condition. The proportion of Gram-negative bacteria and Actinobacteria increased on 14 days after the onset of incubation, suggesting that the callus was decomposed by diverse bacterial members on this phase. When the bacterial groups incorporating the 13C were compared between under anoxic and oxic soil conditions, the composition was largely different under the two opposite conditions. However, some members of Gram-negative bacteria were commonly found under the anoxic and oxic soil conditions. The majority of bacterial members assimilating the callus carbon was Clostridia in the soil under anoxic conditions. However, several Gram-negative bacterial members, such as Acidobacteria, Bacteroidetes, and Proteobacteria, also participated in the decomposition of callus under anoxic soil conditions. Our study showed that carbon flow into the diverse bacterial members during the callus decomposition and the distinctiveness of the bacterial communities was formed under the anoxic and oxic soil conditions.

The redox cycle of iron (Fe) is a key process in the biogeochemistry of paddy field soil. It has been recently remarked that Fe(II)-oxidizing bacteria belonging to the family Gallionellaceae (Gallionella-related FeOB) are one of the key players in the Fe(II) oxidation in the soil. The present study aimed to investigate the influence of alternate wetting and drying (AWD) irrigation for water-saving rice cultivation on the dynamics of the community of Gallionella-related FeOB in the paddy field soil over three seasons (wet-dry-wet seasons) of rice cultivation. The AWD irrigation brought about the reduction of Fe(II) contents in the soil, compared with the continuously flooding (CF) irrigation, especially in the last half of the rice cultivation period in the dry season. In contrast, a trend of higher copy number of 16S rRNA genes of Gallionella-related FeOB was observed in the last half of the rice cultivation period of the AWD paddy soil. As a result, a significant negative correlation was observed between the Fe(II) contents and the copy numbers of 16S rRNA genes. PCR-DGGE analysis of 16S rRNA genes of Gallionella-related FeOB showed that the band patterns were different between the CF and AWD paddy soils. Meanwhile, the results of clone library analysis indicated that differences in the taxonomic composition of Gallionella-related FeOB were not observed between the CF and AWD paddy soils, suggesting that Gallionella-related FeOB responded to the AWD water-saving irrigation on the individual strain levels. The potential of Fe(II)-oxidizing activity and its apparent contribution of the activity of Gallionella-related FeOB to the Fe(II) oxidation in the soil of the investigated field were estimated to be 2.4 × 10−3 to 1.7 × 10−1 mg Fe(II) g−1 dry soil day−1 and 4.6%, respectively, based on the reported Fe(II) oxidation rates of cells. The present study showed that the fluctuation of the redox status of Fe caused by the repeated cycle of wetting and drying of the field brought about the changes in the community structure of Gallionella-related FeOB in the soil. Furthermore, it was suggested that Gallionella-related FeOB were certainly involved in the Fe(II)-oxidizing process in the AWD paddy field soil.

Rice is one of the staple crops in Burkina Faso. However, the local production covers only 47% of the population demands. One of the main reasons of the poor productivity in Burkina Faso is iron toxicity which is related mainly to the activity of Iron Reducing Bacteria in the rice field’s ecosystems. In order to control the harmful effects of Iron Reducing Bacterial populations and to improve rice productivity, a pots experiment was conducted at the experimental site of the University Ouaga I Pr. Joseph KI-ZERBO. An iron toxic soil from Kou Valley (West of Burkina Faso) and two rice varieties, BOUAKE-189 and ROK-5, sensitive and tolerant to iron toxicity, respectively, were used for the experiment. The pots were drained for 14 days (D2) and amended with chemical fertilizers (NPK + Urea and NPK + Urea + Ca + Mg + Zn complexes). Control pots without drainage and fertilization (D0/NF) were prepared similarly. The kinetics of Iron Reducing Bacterial populations and ferrous iron content in soil near rice roots were monitored throughout the cultural cycle using MPN and colorimetric methods, respectively. The total iron content was evaluated in rice plant using a spectrometric method. Data obtained were analyzed in relation to drainage and fertilization mode, rice growth stage and rice yield using the Student’s t-test and XLSTAT 2014 statistical software. The experiment showed that the combined application of subsurface drainage and NPK + Urea + Ca + Mg + Zn fertilization, reduced significantly the number of IRB in the soil near rice roots for both rice varieties (p = 0.050 and p = 0.020) increased the leaf tissue tolerance to excess amounts of Fe, and rice yield.

The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil.

Soil-borne diseases caused by pathogenic microorganisms are one of the main factors responsible for the decline in crop yields in farmlands. Pathogenic Fusarium oxysporum causes serious damage to various crops, and, thus, a feasible diagnostic method for soil-borne diseases is required. We herein examined a simple method to evaluate the suppressiveness of soil microorganisms against a pathogen by co-cultivating indigenous soil microorganisms and a pathogenic fungus (F. oxysporum f. sp. spinaciae). We inoculated F. oxysporum onto the center of agar medium plates mixed with a dilution series of a suspension of organic fertilizers or soil. After an approximately one-week cultivation, the growth degree of F. oxysporum was estimated based on the size of the colonies that formed on the plates. The growth degree of F. oxysporum significantly differed among the organic fertilizers tested, indicating the usefulness of the method for evaluating suppressiveness by organic fertilizers. Differences in the growth degrees of F. oxysporum were associated with the incidence of disease in spinach on soil treated with organic fertilizers and inoculated with a pathogenic F. oxysporum strain. These results suggested that this method provides some useful information on the suppressiveness of organic fertilizers and soil against Fusarium wilt.

Iron toxicity is a major stress to rice caused by a high concentration of reduced iron, in the soil in many lowlands worldwide. To reduce iron toxicity in the West African lowlands, an investigation was performed at the site of the University of Ouagadougou, in pots containing an iron toxic soil from the Kou Valley (West of Burkina Faso). The experiment objective was to study the effect of mineral fertilizer on Iron Reducing Bacteria (IRB) dynamics and activity during rice cultivation, iron accumulation in rice plant and rice biomass yield under iron toxicity conditions. BOUAKE-189 and ROK-5 rice varieties, sensitive and tolerant to iron toxicity, respectively, were used for the experiment. The pots were amended with chemical fertilizers (NPK + Urea and NPK + Urea + Ca + Mg + Zn complex). Control pots without fertilization were prepared similarly. The kinetics of IRB and ferrous iron content in soil near rice roots were monitored throughout the cultural cycle using MPN and colorimetric methods, respectively. The total iron content was evaluated in rice plant using spectrometric method. Data obtained were analyzed in relation to fertilization mode, rice growth stage and rice yield using the student’s t-test and XLSTAT 2014 statistical software. The experiment revealed that NPK + Urea and NPK + Urea + Ca + Mg + Zn fertilization, decreased significantly (p

Elevated concentrations of atmospheric CO2 ([CO2]) enhance the production and emission of methane in paddy fields. In the present study, the effects of elevated [CO2], elevated temperature (ET), and no nitrogen fertilization (LN) on methanogenic archaeal and methane-oxidizing bacterial community structures in a free-air CO2 enrichment (FACE) experimental paddy field were investigated by PCR-DGGE and real-time quantitative PCR. Soil samples were collected from the upper and lower soil layers at the rice panicle initiation (PI) and mid-ripening (MR) stages. The composition of the methanogenic archaeal community in the upper and lower soil layers was not markedly affected by the elevated [CO2], ET, or LN condition. The abundance of the methanogenic archaeal community in the upper and lower soil layers was also not affected by elevated [CO2] or ET, but was significantly increased at the rice PI stage and significantly decreased by LN in the lower soil layer. In contrast, the composition of the methane-oxidizing bacterial community was affected by rice-growing stages in the upper soil layer. The abundance of methane-oxidizing bacteria was significantly decreased by elevated [CO2] and LN in both soil layers at the rice MR stage and by ET in the upper soil layer. The ratio of mcrA/pmoA genes correlated with methane emission from ambient and FACE paddy plots at the PI stage. These results indicate that the decrease observed in the abundance of methane-oxidizing bacteria was related to increased methane emission from the paddy field under the elevated [CO2], ET, and LN conditions.

Soil solarization is an ecologically friendly method of controlling various plant pathogens and pests, but also affects non-pathogenic members of the soil biota. Here, we studied the impact of soil solarization on the community structure of soil ciliates using a culture-independent molecular approach, namely denaturing gradient gel electrophoresis (DGGE) of targeted 18S rRNA gene fragments. Greenhouse soil with added organic fertilizers was solarized for 33 days at an average temperature of 47–48°C. Solarization caused a drastic change in the ciliate community. The variation between replicates was large, which suggested that the distribution of ciliates was spatially heterogeneous in the soil, probably due to their decreased numbers. In contrast, non-solarized soil had a stable and homogeneous ciliate community during the experimental period. In solarized soil, most of the original ciliate community recovered 76 days after solarization. Sequence analysis of DGGE fragments indicated that both r-selected and K-selected species of ciliates were affected by solarization but recovered with time after solarization. Our results demonstrated both the vulnerability and resilience of the ciliate community to soil solarization and also the utility of using molecular-based analysis of ciliate communities as bioindicators of soil stress caused by solarization.

An aerobic, methane-oxidizing bacterium (strain RS11D-PrT) was isolated from rice rhizosphere. Cells of strain RS11D-PrT were Gram-stain-negative, motile rods with a single polar flagellum and contained an intracytoplasmic membrane system typical of type I methanotrophs. The strain utilized methane and methanol as sole carbon and energy sources. It could grow at 20–37 °C (optimum 31–33 °C), at pH 6.8–7.4 (range 5.5–9.0) and with 0–0.2 % (w/v) NaCl (there was no growth at above 0.5 % NaCl). pmoA and mmoX genes were present. The ribulose monophosphate and/or ribulose bisphosphate pathways were used for carbon assimilation. Results of sequence analysis of 16S rRNA genes showed that strain RS11D-PrT is related closely to the genera Methylococcus, Methylocaldum, Methyloparacoccus and Methylogaea in the family Methylococcaceae. The similarity was low (94.6 %) between strain RS11D-PrT and the most closely related type strain (Methyloparacoccus murrellii R-49797T). The DNA G+C content was 64.1 mol%. Results of phylogenetic analysis of the pmoA gene and chemotaxonomic data regarding the major cellular fatty acids (C16 : 1ω7c, C16 : 0 and C14 : 0) and the major respiratory quinone (MQ-8) also indicated the affiliation of strain RS11D-PrT to the Methylococcus–Methylocaldum–Methyloparacoccus–Methylogaea clade. On the basis of phenotypic, genotypic and phylogenetic characteristics, strain RS11D-PrT is considered to represent a novel genus and species within the family Methylococcaceae, for which the name Methylomagnum ishizawai gen. nov., sp. nov. is proposed. The type strain is RS11D-PrT ( = JCM 18894T = NBRC 109438T = DSM 29768T = KCTC 4681T).

Effects of solarization on microbial communities in soil were investigated with an incubation experiment and in a greenhouse experiment with isolated bed culture of tomato (Solanum lycopersicum L.) by molecular techniques. Microbial biomass carbon in soil was decreased by about half in the incubation experiment treated with 45°C for 14 days and the greenhouse experiment with solarization for 45 days. Bacterial and fungal communities in soil were affected by the heat and solarization treatments in both the experiments and greatly different from the communities in the unheated soil or the soil before solarization. Copy number of amoA gene of ammonia-oxidizing bacteria in soil was decreased to one-tenth or less by the treatments in both the experiments. This study revealed that the solarization made a great impact on the abundance and composition of microbial communities in soil.

Oral administration of hydrogen water ameliorates Parkinson's disease (PD) in rats, mice, and humans. We previously reported that the number of putative hydrogen-producing bacteria in intestinal microbiota is low in PD compared to controls. We also reported that the amount of hydrogen produced by ingestion of lactulose is low in PD patients. The decreased hydrogen production by intestinal microbiota may be associated with the development and progression of PD. We measured the amount of hydrogen production using gas chromatography by seven bacterial strains, which represented seven major intestinal bacterial groups/genera/species. Blautia coccoides and Clostridium leptum produced the largest amount of hydrogen. Escherichia coli and Bacteroides fragilis constituted the second group that produced hydrogen 34- to 93-fold lower than B. coccoides. Bifidobacterium pseudocatenulatum and Atopobium parvulum constituted the third group that produced hydrogen 559- to 2164-fold lower than B. coccoides. Lactobacillus casei produced no detectable hydrogen. Assuming that taxonomically neighboring strains have similar hydrogen production, we simulated hydrogen production using intestinal microbiota that we previously reported, and found that PD patients produce a 2.2-fold lower amount of intestinal hydrogen compared to controls. The lower amount of intestinal hydrogen production in PD was also simulated in cohorts of two other countries. The number of hydrogen-producing intestinal bacteria may be associated with the development and progression of PD. Further studies are required to prove its beneficial effect.

Soil-borne diseases caused by pathogenic microorganisms are one of the main factors responsible for the decline in crop yields in farmlands. Pathogenic Fusarium oxysporum causes serious damage to various crops, and, thus, a feasible diagnostic method for soil-borne diseases is required. We herein examined a simple method to evaluate the suppressiveness of soil microorganisms against a pathogen by co-cultivating indigenous soil microorganisms and a pathogenic fungus (F. oxysporum f. sp. spinaciae). We inoculated F. oxysporum onto the center of agar medium plates mixed with a dilution series of a suspension of organic fertilizers or soil. After an approximately one-week cultivation, the growth degree of F. oxysporum was estimated based on the size of the colonies that formed on the plates. The growth degree of F. oxysporum significantly differed among the organic fertilizers tested, indicating the usefulness of the method for evaluating suppressiveness by organic fertilizers. Differences in the growth degrees of F. oxysporum were associated with the incidence of disease in spinach on soil treated with organic fertilizers and inoculated with a pathogenic F. oxysporum strain. These results suggested that this method provides some useful information on the suppressiveness of organic fertilizers and soil against Fusarium wilt.

Potassium (K) in microbial cells, microbial biomass K, in soil has been recently recognized as a K pool for plant growth. We determined soil microbial biomass K in paddy fields to reveal its importance as a K pool in paddy field soil for the first time. Microbial biomass K ranged from 5 to 21 mg K kg−1 in the soil samples periodically collected from a paddy field and the value corresponded to 41% of the exchangeable K on average. Both microbial biomass K and exchangeable K increased conspicuously due to the long-term application of livestock manure compost or rice straw compost. Biomass K was higher than exchangeable K under K-deficient conditions in the long-term experimental plots without K application. The present study revealed that the microbial biomass contained considerable amounts of K in paddy field soil, indicating the need for evaluation of the microbial biomass K as a source and a stock of K in soil that has been overlooked.

Nitrification is a key process in the global nitrogen cycle, of which the first and rate-limiting step is catalyzed by ammonia monooxygenase. Root cap cells are one of substrates for microorganisms that thrive in the rhizosphere. The degradation of root cap cells brings about nitrification following ammonification of organic nitrogen derived from the root cap cells. This study was designed to gain insights into the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to mineralized N from root cap cells and the composition of active bacterial and archaeal ammonia oxidizers in rice soil. Rice callus cells were used as a model for root cap cells, and unlabelled (12C) and 13C-labelled callus cells were allowed to decompose in aerobic soil microcosms. Real-time quantitative polymerase chain reaction (PCR), DNA-based stable isotope probing (SIP), and denaturing gradient gel electrophoresis (DGGE) were applied to determine the copy number of bacterial and archaeal amoA genes and the composition of active AOB and AOA. The growth of AOB was significantly stimulated by the addition of callus cells compared with the growth of AOA with a much lesser extent. AOB communities assimilated 13C derived from the callus cells, whereas no AOA communities grew on 13C-callus. Sequencing of the DGGE bands in the SIP experiments revealed that the AOB communities belonging to Nitrosospira spp. dominated microbial ammonia oxidation with rice callus amendment in soil. The present study suggests that root cap cells of rice significantly stimulated the growth of AOB, and the active members dominating microbial ammonia oxidation belonged to Nitrosospira spp. in rice rhizosphere.

Reduced fertilizer usage is one of the objectives of field management in the pursuit of sustainable agriculture. Here, we report on shifts of bacterial communities in paddy rice ecosystems with low (LN), standard (SN), and high (HN) levels of N fertilizer application (0, 30, and 300 kg N ha(-1), respectively). The LN field had received no N fertilizer for 5 years prior to the experiment. The LN and HN plants showed a 50% decrease and a 60% increase in biomass compared with the SN plant biomass, respectively. Analyses of 16S rRNA genes suggested shifts of bacterial communities between the LN and SN root microbiomes, which were statistically confirmed by metagenome analyses. The relative abundances of Burkholderia, Bradyrhizobium and Methylosinus were significantly increased in root microbiome of the LN field relative to the SN field. Conversely, the abundance of methanogenic archaea was reduced in the LN field relative to the SN field. The functional genes for methane oxidation (pmo and mmo) and plant association (acdS and iaaMH) were significantly abundant in the LN root microbiome. Quantitative PCR of pmoA/mcrA genes and a (13)C methane experiment provided evidence of more active methane oxidation in the rice roots of the LN field. In addition, functional genes for the metabolism of N, S, Fe, and aromatic compounds were more abundant in the LN root microbiome. These results suggest that low-N-fertilizer management is an important factor in shaping the microbial community structure containing key microbes for plant associations and biogeochemical processes in paddy rice ecosystems.

Soil characteristics regulate various belowground microbial processes including methanogenesis and, consequently, affect the structure and function of methanogenic archaeal communities due to change in soil type which in turn influences the CH4 production potential of soils. Thus, five different soil orders (Alfisol, Entisol, Inceptisol, Podzol and Vertisol) were studied to assess their CH4 production potential and also the methanogenic archaeal community structure in dryland irrigated Indian paddy soils. Soil incubation experiments revealed CH4 production to range from 178.4 to 431.2 μg CH4 g-1 dws in all soil orders as: Vertisol

Flooded paddy fields are the major anthropogenic sources of methane (CH4) emission, and organic materials of rice plant origin were estimated to be important as its source. This study used rice (Oryza sativa L. cv, Yukihikari) callus cells as a model material for slough-off root cap cells, and carbon-13 (13C)-labelled callus cells were subjected to decomposition in aerobic and anaerobic soil microcosms for 56 days. DNA was extracted from a soil incubated with carbon-12 (12C)- and 13C-callus cells and subjected to buoyant density gradient centrifugation to identify methanogenic archaeal species that assimilated carbon from the callus cells. 13C-labelled 16S rRNA gene (16S rDNA) fragments from methanogenic archaea were not polymerase chain reaction (PCR)-amplified in heavy fractions under aerobic soil conditions, while they were successfully done from day 3 onwards under anaerobic soil conditions. Eighty-four denaturing gradient gel electrophoresis (DGGE) bands in heavy fractions were sequenced, re...

As the most abundant biological entities, viruses are increasingly recognised as a major driving force of global biogeochemical nutrient cycles. This study demonstrated that T4-type bacteriophages drive the microbial loop of carbon from root cap cells in rice rhizospheres based on an analysis of the major capsid gene ( g23 ). Rice callus cells were used as a model for root cap cells, and 13 C-labelled callus cells were allowed to decompose. DNA extracted from the soils after incubation under aerobic and anaerobic soil conditions was subjected to PCR–DGGE after density gradient centrifugation. Although 13 C-labelled g23 fragments were not detected in soil incubated under anaerobic conditions, many 13 C-labelled g23 fragments belonging to Paddy Groups V, VIII and IX were obtained from the soil incubated under aerobic conditions. Some g23 fragments were detected throughout the incubation period, and others were obtained only during the early or late incubation period. This study demonstrated that the level of infection of soil bacteria by T4-type phages was very different between aerobic and anaerobic soil conditions. Based on the results of this study, the roles of phages in the decomposition of the root cap cells of rice plants in rice fields are discussed.

The present study observed bacterial consortia in iron-deposited colonies formed on the surface of paddy soils. Plow layer soils under reduced condition, which were collected from a paddy field in Japan and paddy fields located at two sites in Mekong Delta, Vietnam, were incubated at 25 and 30°C in test tubes under a nitrogen (N2) atmosphere with sleeve stoppers made from a synthetic rubber. The sleeve stopper enabled the establishment of microaerobic conditions because of gradual diffusion of oxygen (O2) into the tube (29.2 nmol mL−1 day−1). Many colonies were observed on the soil surface in the test tubes after 2–3 weeks of incubation from both Japanese and Vietnam paddy soils. Bright orange and blood red colonies, which had 0.5–1-mm diameters and circular shapes, were observed. Micro X-ray fluorescence analysis of the colonies showed deposition of iron (Fe) in the colonies, indicating bacteria were enriched in the Fe oxidation. Microscopic observation showed acicular, floc and concentric cryst...

The frequency of visibly infected bacterial cells (FVIC) by phages was reported so far to be larger in soil environments than in aquatic environments. However, it was evaluated with a transmission electron microscope (TEM) and might be biased depending on the criterion of judgment of phage infection by researchers. This study directly compared FVIC with a TEM in soils and in the overlying floodwaters in two Japanese rice fields (Oryza sativa L. Japonica). Soil and floodwater samples were collected three times from three sites in each field during the rice cultivation period. Proportions of bacterial morphology were long-bacteria > short-rod bacteria > coccoid bacteria in this order in both floodwater and soil samples. FVIC was higher in soil samples than in floodwater samples with a range from 4.2 to 11.4% (average 7.4%) and from non-detectable to 5.2% (average 1.2%), respectively. The frequency of phage-infected bacterial cells calculated from FVIC ranged from 32 to 52%, which indicates that the...

Changes in the community structure of methanogenic archaea in a paddy field soil brought about by an alternate wetting and drying (AWD) water-saving practice were shown by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR analyses targeting archaeal 16S rRNA gene and methanogenic archaeal mcrA gene. Soil samples were collected four times from the field plots under the treatments of continuous flooding (CF) and AWD water-saving, where irrigation was conducted when soil water potential at 15 cm depth decreased to −20 kPa. The community analysis was performed over three rice cultivation periods in the wet season in 2008 (2008WS) and the dry and wet seasons in 2009 (2009DS and 2009WS, respectively). PCR-DGGE analysis showed that the archaeal 16S rRNA genes differed depending on water management. During the three rice cultivation periods, the community of the CF plots gradually changed with time, while the community of the AWD plots possessed a relatively large variation. Principal component analysis and sequencing analysis of selected 16S rRNA genes indicated that members of Methanosarcinales dominated in the CF plots while members of Methanocellales and uncultured group of “ Thaumarchaeota ” characterized the AWD plots. Numbers of archaeal 16S rRNA and methanogenic archaeal mcrA genes were significantly different between the CF and AWD plots in 2008WS, but not in 2009DS and 2009WS. Thus, the AWD water-saving practice did not reduce populations of the methanogenic archaea but moderately influenced the community structure in the paddy field soil.

The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil.

Paddy field is a major emission source of methane. Methane is the terminal product of anaerobic decomposition of organic matter and generated by methanogenic archaea under flooded conditions in paddy fields. This study aimed to reveal the effect of winter flooding on methanogenic archaeal community structure in paddy fields of Andosols under organic farming. Soil samples were collected from experimental paddy fields in the Field Science Center, Tohoku University, for two years. They were under flooding conditions during winter with organic farming, under non-flooding conditions during winter with organic farming and under non-flooding conditions during winter with conventional farming (non-organic farming). Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of methanogenic archaeal 16S rRNA gene revealed that the DGGE patterns were nearly the same irrespective of the treatment and sampling times. Twenty-three bands were observed from each treatment and 4, 13 and 6 sequen...

A novel methane-oxidizing bacterium, strain Fw12E-YT, was isolated from floodwater of a rice paddy field in Japan. Cells of strain Fw12E-YT were Gram-negative, motile rods with a single polar flagellum and type I intracytoplasmic membrane arrangement. The strain grew only on methane or methanol as sole carbon and energy source. It was able to grow at 10–40 °C (optimum 30 °C), at pH 5.5–7.0 (optimum 6.5) and with 0–0.1 % (w/w) NaCl (no growth above 0.5 % NaCl). 16S rRNA gene sequence analysis showed that strain Fw12E-YT is related most closely to members of the genus Methylomonas , but at low levels of similarity (95.0–95.4 %). Phylogenetic analysis of pmoA and mxaF genes indicated that the strain belongs to the genus Methylomonas (97 and 92 % deduced amino acid sequence identities to Methylomonas methanica S1T, respectively). The DNA G+C content of strain Fw12E-YT was 57.1 mol%. Chemotaxonomic data regarding the major quinone (MQ-8) and major fatty acids (C16 : 1 and C14 : 0) also supported its affiliation to the genus Methylomonas . Based on phenotypic, genomic and phylogenetic data, strain Fw12E-YT is considered to represent a novel species of the genus Methylomonas , for which the name Methylomonas koyamae sp. nov. is proposed. The type strain is Fw12E-YT ( = JCM 16701T = NBRC 105905T = NCIMB 14606T).

Methanogens are not expect to grow in oxic soil because they are obligate anaerobes that require high concentrations of H2/acetate and low oxidation–reduction potentials. However, we verified their growth in an oxic soil microcosm by a DNA-SIP (stable isotope probing) experiment using 13C-labeled rice callus cells (78 at.%) as a substrate. Soils with 13C-labeled and unlabeled dried rice callus were sampled six times during oxic incubation for 56 days. After isopycnic centrifugation of the extracted DNA and PCR amplification of fractionated DNA with methanogen-specific primers, we compared DGGE band patterns of respective fractions between 13C-labeled and unlabeled treatments. The shift of many bands to the fractions of heavier buoyant density (BD) was observed in the 13C-labeled treatment. DNA in the heaviest fractions of the 13C-labeled treatment with a BD from 1.754 to 1.759 g ml−1 was subjected to population analysis of methanogens (13C-assimilating populations). Partial 16S rRNA genes from methanogens were successfully PCR-amplified from day 3 onwards, and 35 DGGE bands sequenced were closely related to Methanosaeta, Methaosarcina, Methanobacterium and Methanocella. Because the estimated BD of non-labeled (12C) DNA of the genera Methanosarcina, Methanosaeta and Methanobacterium was less than 1.72 g ml−1, these results verified the growth of methanogens in the oxic soil microcosm. Detection of 13C-labeled acetoclastic Methanosaeta and Methaosarcina and hydrogenotrophic Methanobacterium and Methanocella indicates the growth of methanogens on acetate and H2/CO2 in the soil microcosm. Based on these results, the growth of acetoclastic and hydrogenotrophic methanogens was discussed in dried callus under oxic soil conditions.

Root cap cells are an important substrate for rhizospheric microorganisms. In this study carbon (13C)-labelled callus cells of rice (Oryza sativa L. cv, Yukihikari) (84 atom % 13C) were used as a model material for root cap cells and subjected to decomposition in an anaerobic soil microcosm for 56 days. DNA was extracted from soils incubated with 13C-labelled and unlabelled callus cells and subjected to buoyant density (BD) gradient centrifugation to identify bacterial species that assimilated callus cells. Many denaturing gradient gel electrophoresis (DGGE) bands were detected in the heavy BD fractions in the soil with 13C-callus cells from day 3 onwards. Sequencing 13C-labelled DGGE bands revealed that they were mainly derived from Gram-negative bacteria and that Acidobacteria, δ-Proteobacteria and Firmicutes were predominant bacteria assimilating rice callus cells. Strictly anaerobic bacteria such as Clostridium spp., Geothrix spp. and Holophaga spp. were predominant decomposers of callus cells. These ...

Cyanobacteria play an important role in maintaining soil fertility in rice fields. Some cyanobacterial members can grow heterotrophically under dark conditions, which might be an important trait for the survival of cyanobacteria during the drained period after harvest. This study aimed to elucidate the heterotrophic growth of cyanobacteria and the microbial loop mediated by cyanophages (cyanobacteria-cyanophages-dissolved organic carbon) in soil using carbon-13 (13C)-labeled dried callus cells as a model material of plant residues. This study used the stable isotope probing (SIP) method coupled with denaturing gradient gel electrophoresis (DGGE). Although heterotrophic growth of cyanobacteria in soil was not observed, the phage-mediated microbial loop in the transformation of callus carbon was elucidated from the detection of 13C-labeled g20 genes in the heaviest fractions (buoyant density: 1.754 g mL−1). The closest relatives of eleven sequenced DGGE bands from the heaviest fractions were uncultured cyan...