A survey of metabolic changes in potato leaves by NMR-based metabolic profiling in relation to resistance to late blight disease under field conditions.

Non-targeted nuclear magnetic resonance (NMR)-based metabolic profiling was applied to potato leaves to survey metabolic changes associated with late blight resistance under field conditions. Potato plants were grown in an experimental field, and the compound leaves with no visible symptoms were collected from 20 cultivars/lines at two sampling time points: (i) the time of initial presentation of symptoms in susceptible cultivars and (ii) 12 days before this initiation. 1 H NMR spectra of the foliar metabolites soluble in deuterium oxide- or methanol-d4 -based buffers were measured and used for multivariate analysis. Principal component analysis for six cultivars at symptom initiation showed a class separation corresponding to their levels of late blight resistance. This separation was primarily explained by higher levels of malic acid, methanol, and rutin and a lower level of sucrose in the resistant cultivars than in the susceptible ones. Partial least squares regression revealed that the levels of these metabolites were strongly associated with the disease severity measured in this study under field conditions. These associations were observed only for the leaves harvested at the symptom initiation stage, but not for those collected 12 days beforehand. Subsequently, a simple, alternative enzymatic assay for l-malic acid was used to estimate late blight resistance, as a model for applying the potential metabolic marker obtained. This study demonstrated the potential of metabolomics for field-grown plants in combination with targeted methods for quantifying marker levels, moving towards marker-assisted screening of new cultivars with durable late blight resistance. Copyright © 2016 John Wiley & Sons, Ltd.

Rice Bran Amendment Suppresses Potato Common Scab by Increasing Antagonistic Bacterial Community Levels in the Rhizosphere.

Potato common scab (PCS), caused by pathogenic Streptomyces spp., is a serious disease in potato production worldwide. Cultural practices, such as optimizing the soil pH and irrigation, are recommended but it is often difficult to establish stable disease reductions using these methods. Traditionally, local farmers in southwest Japan have amended soils with rice bran (RB) to suppress PCS. However, the scientific mechanism underlying disease suppression by RB has not been elucidated. The present study showed that RB amendment reduced PCS by repressing the pathogenic Streptomyces population in young tubers. Amplicon sequencing analyses of 16S ribosomal RNA genes from the rhizosphere microbiome revealed that RB amendment dramatically changed bacterial composition and led to an increase in the relative abundance of gram-positive bacteria such as Streptomyces spp., and this was negatively correlated with PCS disease severity. Most actinomycete isolates derived from the RB-amended soil showed antagonistic activity against pathogenic Streptomyces scabiei and S. turgidiscabies on R2A medium. Some of the Streptomyces isolates suppressed PCS when they were inoculated onto potato plants in a field experiment. These results suggest that RB amendment increases the levels of antagonistic bacteria against PCS pathogens in the potato rhizosphere.

Metaproteomic identification of diazotrophic methanotrophs and their localization in root tissues of field-grown rice plants.

In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.

A rice gene for microbial symbiosis, Oryza sativa CCaMK, reduces CH4 flux in a paddy field with low nitrogen input.

Plants have mutualistic symbiotic relationships with rhizobia and fungi by the common symbiosis pathway, of which Ca(2+)/calmodulin-dependent protein kinase (encoded by CCaMK) is a central component. Although Oryza sativa CCaMK (OsCCaMK) is required for fungal accommodation in rice roots, little is known about the role of OsCCaMK in rice symbiosis with bacteria. Here, we report the effect of a Tos17-induced OsCCaMK mutant (NE1115) on CH4 flux in low-nitrogen (LN) and standard-nitrogen (SN) paddy fields compared with wild-type (WT) Nipponbare. The growth of NE1115 was significantly decreased compared with that of the WT, especially in the LN field. The CH4 flux of NE1115 in the LN field was significantly greater (156 to 407% in 2011 and 170 to 816% in 2012) than that of the WT, although no difference was observed in the SN field. The copy number of pmoA (encodes methane monooxygenase in methanotrophs) was significantly higher in the roots and rhizosphere soil of the WT than in those of NE1115. However, the mcrA (encodes methyl coenzyme M reductase in methanogens) copy number did not differ between the WT and NE1115. These results were supported by a (13)C-labeled CH4-feeding experiment. In addition, the natural abundance of (15)N in WT shoots (3.05‰) was significantly lower than in NE1115 shoots (3.45‰), suggesting greater N2 fixation in the WT because of dilution with atmospheric N2 (0.00‰). Thus, CH4 oxidation and N2 fixation were simultaneously activated in the root zone of WT rice in the LN field and both processes are likely controlled by OsCCaMK.

Seasonal Shifts in Bacterial Community Structures in the Lateral Root of Sugar Beet Grown in an Andosol Field in Japan.

To investigate functional plant growth-promoting rhizobacteria in sugar beet, seasonal shifts in bacterial community structures in the lateral roots of sugar beet were examined using amplicon sequencing ana-lyses of the 16S rRNA gene. Shannon and Simpson indexes significantly increased between June and July, but did not significantly differ between July and subsequent months (August and September). A weighted UniFrac principal coordinate ana-lysis grouped bacterial samples into four clusters along with PC1 (43.8%), corresponding to the four sampling months in the order of sampling dates. Taxonomic ana-lyses revealed that bacterial diversity in the lateral roots was exclusively dominated by three phyla (Actinobacteria, Bacteroidetes, and Proteobacteria) in all samples examined. At the lower taxonomic levels, the dominant taxa were roughly classified into three groups. Therefore, the relative abundances of seven dominant genera (Janthinobacterium, Kribbella, Pedobacter, Rhodanobacter, Sphingobium, Sphingopyxis, and Streptomyces) were the highest in June and gradually decreased as sugar beet grew. The relative abundances of eight taxa (Bradyrhizobiaceae, Caulobacteraceae, Chitinophagaceae, Novosphingobium, Phyllobacteriaceae, Pseudomonas, Rhizobiaceae, and Sphingomonas) were mainly high in July and/or August. The relative abundances of six taxa (unclassified Comamonadaceae, Cytophagaceae, unclassified Gammaproteobacteria, Haliangiaceae, unclassified Myxococcales, and Sinobacteraceae) were the highest in September. Among the dominant taxa, 12 genera (Amycolatopsis, Bradyrhizobium, Caulobacter, Devosia, Flavobacterium, Janthinobacterium, Kribbella, Kutzneria, Pedobacter, Rhizobium, Rhodanobacter, and Steroidobacter) were considered to be candidate groups of plant growth-promoting bacteria based on their previously reported beneficial traits as biopesticides and/or biofertilizers.

Effects of Different Types of Additional Fertilizers on Root-associated Microbes of Napa Cabbage Grown in an Andosol Field in Japan.

The effects of different types of additional fertilizations (a compound fertilizer and Chiyoda-kasei) on the root-associated microbes of napa cabbage grown in an Andosol field were investigated by molecular community ana-lyses. Most of the closest known species of the bacterial sequences whose relative abundance significantly differed among fertilizers were sensitive to nitrogen fertilization and/or related to the geochemical cycles of nitrogen. The fungal community on the roots of napa cabbage was dominated by two genera, Bipolaris and Olpidium. The relative abundance of these two genera was affected by the types of fertilizers to some extent and showed a strong negative correlation.

Autoregulation of nodulation interferes with impacts of nitrogen fertilization levels on the leaf-associated bacterial community in soybeans.

The diversities leaf-associated bacteria on nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S rRNA gene. To analyze the impact of nitrogen fertilization on the bacterial leaf community, soybeans were treated with standard nitrogen (SN) (15 kg N ha(-1)) or heavy nitrogen (HN) (615 kg N ha(-1)) fertilization. Under SN fertilization, the relative abundance of Alphaproteobacteria was significantly higher in Nod(-) and Nod(++) soybeans (82% to 96%) than in Nod(+) soybeans (54%). The community structure of leaf-associated bacteria in Nod(+) soybeans was almost unaffected by the levels of nitrogen fertilization. However, differences were visible in Nod(-) and Nod(++) soybeans. HN fertilization drastically decreased the relative abundance of Alphaproteobacteria in Nod(-) and Nod(++) soybeans (46% to 76%) and, conversely, increased those of Gammaproteobacteria and Firmicutes in these mutant soybeans. In the Alphaproteobacteria, cluster analyses identified two operational taxonomic units (OTUs) (Aurantimonas sp. and Methylobacterium sp.) that were especially sensitive to nodulation phenotypes under SN fertilization and to nitrogen fertilization levels. Arbuscular mycorrhizal infection was not observed on the root tissues examined, presumably due to the rotation of paddy and upland fields. These results suggest that a subpopulation of leaf-associated bacteria in wild-type Nod(+) soybeans is controlled in similar ways through the systemic regulation of autoregulation of nodulation, which interferes with the impacts of N levels on the bacterial community of soybean leaves.

The genotype of the calcium/calmodulin-dependent protein kinase gene (CCaMK) determines bacterial community diversity in rice roots under paddy and upland field conditions.

The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria, including endophytes and epiphytes, were examined by using a Tos17 rice mutant line under paddy and upland field conditions. Roots were sampled at the flowering stage and were subjected to clone library analyses. The relative abundance of Alphaproteobacteria was noticeably decreased in R plants under both paddy and upland conditions (0.8% and 3.0%, respectively) relative to those in D plants (10.3% and 17.4%, respectively). Population shifts of the Sphingomonadales and Rhizobiales were mainly responsible for this low abundance in R plants. The abundance of Anaerolineae (Chloroflexi) and Clostridia (Firmicutes) was increased in R plants under paddy conditions. The abundance of a subpopulation of Actinobacteria (Saccharothrix spp. and unclassified Actinosynnemataceae) was increased in R plants under upland conditions. Principal coordinate analysis revealed unidirectional community shifts in relation to OsCCaMK gene dosage under both conditions. In addition, shoot length, tiller number, and plant weight decreased as the OsCCaMK gene dosage decreased under upland conditions. These results suggest significant impacts of OsCCaMK on both the diversity of root-associated bacteria and rice plant growth under both paddy and upland field conditions.

Community Analysis-based Screening of Plant Growth-promoting Bacteria for Sugar Beet.

Clone libraries of bacterial 16S rRNA genes (a total of 1,980 clones) were constructed from the leaf blades, petioles, taproots, and lateral roots of sugar beet (Beta vulgaris L.) grown under different fertilization conditions. A principal coordinate analysis revealed that the structures of bacterial communities in above- and underground tissues were largely separated by PC1 (44.5%). The bacterial communities of above-ground tissues (leaf blades and petioles) were more tightly clustered regardless of differences in the tissue types and fertilization conditions than those of below-ground tissues (taproots and lateral roots). The bacterial communities of below-ground tissues were largely separated by PC2 (26.0%). To survey plant growth-promoting bacteria (PGPBs), isolate collections (a total of 665 isolates) were constructed from the lateral roots. As candidate PGPBs, 44 isolates were selected via clustering analyses with the combined 16S rRNA gene sequence data of clone libraries and isolate collections. The results of inoculation tests using sugar beet seedlings showed that eight isolates exhibited growth-promoting effects on the seedlings. Among them, seven isolates belonging to seven genera (Asticcacaulis, Mesorhizobium, Nocardioides, Sphingobium, Sphingomonas, Sphingopyxis, and Polaromonas) were newly identified as PGPBs for sugar beet at the genus level, and two isolates belonging to two genera (Asticcacaulis and Polaromonas) were revealed to exert growth-promoting effects on the plant at the genus level for the first time. These results suggest that a community analysis-based selection strategy will facilitate the isolation of novel PGPBs and extend the potential for the development of novel biofertilizers.

Community- and genome-based views of plant-associated bacteria: plant-bacterial interactions in soybean and rice.

Diverse microorganisms are living as endophytes in plant tissues and as epiphytes on plant surfaces in nature. Questions about driving forces shaping the microbial community associated with plants remain unanswered. Because legumes developed systems to attain endosymbioses with rhizobia as well as mycorrhizae during their evolution, the above questions can be addressed using legume mutants relevant to genes for symbiosis. Analytical methods for the microbial community have recently been advanced by enrichment procedures of plant-associated microbes and culture-independent analyses targeting the small subunit of rRNA in microbial ecology. In this review, we first deal with interdisciplinary works on the global diversity of bacteria associated with field-grown soybeans with different nodulation genotypes and nitrogen application. A subpopulation of Proteobacteria in aerial parts of soybean shoots was likely to be regulated through both the autoregulation system for plant-rhizobium symbiosis and the nitrogen signaling pathway, suggesting that legumes accommodate a taxonomically characteristic microbial community through unknown plant-microbe communications. In addition to the community views, we then show multiphasic analysis of a beneficial rice endophyte for comparative bacterial genomics and plant responses. The significance and perspectives of community- and genome-based approaches are discussed to achieve a better understanding of plant-microbe interactions.

Thiosulfate-dependent chemolithoautotrophic growth of Bradyrhizobium japonicum.

Thiosulfate-oxidizing sox gene homologues were found at four loci (I, II, III, and IV) on the genome of Bradyrhizobium japonicum USDA110, a symbiotic nitrogen-fixing bacterium in soil. In fact, B. japonicum USDA110 can oxidize thiosulfate and grow under a chemolithotrophic condition. The deletion mutation of the soxY(1) gene at the sox locus I, homologous to the sulfur-oxidizing (Sox) system in Alphaproteobacteria, left B. japonicum unable to oxidize thiosulfate and grow under chemolithotrophic conditions, whereas the deletion mutation of the soxY(2) gene at sox locus II, homologous to the Sox system in green sulfur bacteria, produced phenotypes similar to those of wild-type USDA110. Thiosulfate-dependent O(2) respiration was observed only in USDA110 and the soxY(2) mutant and not in the soxY(1) mutant. In the cells, 1 mol of thiosulfate was stoichiometrically converted to approximately 2 mol of sulfate and consumed approximately 2 mol of O(2). B. japonicum USDA110 showed (14)CO(2) fixation under chemolithotrophic growth conditions. The CO(2) fixation of resting cells was significantly dependent on thiosulfate addition. These results show that USDA110 is able to grow chemolithoautotrophically using thiosulfate as an electron donor, oxygen as an electron acceptor, and carbon dioxide as a carbon source, which likely depends on sox locus I including the soxY(1) gene on USDA110 genome. Thiosulfate oxidation capability is frequently found in members of the Bradyrhizobiaceae, which phylogenetic analysis showed to be associated with the presence of sox locus I homologues, including the soxY(1) gene of B. japonicum USDA110.

Development of a bacterial cell enrichment method and its application to the community analysis in soybean stems.

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.

Variovorax sp. Has an Optimum Cell Density to Fully Function as a Plant Growth Promoter.

Utilization of plant growth-promoting bacteria colonizing roots is environmentally friendly technology instead of using chemicals in agriculture, and understanding of the effects of their colonization modes in promoting plant growth is important for sustainable agriculture. We herein screened the six potential plant growth-promoting bacteria isolated from Beta vulgaris L. (Rhizobium sp. HRRK 005, Polaromonas sp. HRRK 103, Variovorax sp. HRRK 170, Mesorhizobium sp. HRRK 190, Streptomyces sp. HRTK 192, and Novosphingobium sp. HRRK 193) using a series of biochemical tests. Among all strains screened, HRRK 170 had the highest potential for plant growth promotion, given its ability to produce plant growth substances and enzymes such as siderophores and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, respectively, concomitantly with active growth in a wider range of temperatures (10⁻30 °C) and pH (5.0⁻10.0). HRRK 170 colonized either as spots or widely on the root surface of all vegetable seedlings tested, but significant growth promotion occurred only in two vegetables (Chinese cabbage and green pepper) within a certain cell density range localized in the plant roots. The results indicate that HRRK 170 could function as a plant growth promoter, but has an optimum cell density for efficient use.

Microbial community analysis of field-grown soybeans with different nodulation phenotypes.

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.

NMR-Based Metabolic Profiling of Field-Grown Leaves from Sugar Beet Plants Harbouring Different Levels of Resistance to Cercospora Leaf Spot Disease.

Cercospora leaf spot (CLS) is one of the most serious leaf diseases for sugar beet (Beta vulgaris L.) worldwide. The breeding of sugar beet cultivars with both high CLS resistance and high yield is a major challenge for breeders. In this study, we report the nuclear magnetic resonance (NMR)-based metabolic profiling of field-grown leaves for a subset of sugar beet genotypes harbouring different levels of CLS resistance. Leaves were collected from 12 sugar beet genotypes at four time points: seedling, early growth, root enlargement, and disease development stages. ¹H-NMR spectra of foliar metabolites soluble in a deuterium-oxide (D2O)-based buffer were acquired and subjected to multivariate analyses. A principal component analysis (PCA) of the NMR data from the sugar beet leaves shows clear differences among the growth stages. At the later time points, the sugar and glycine betaine contents were increased, whereas the choline content was decreased. The relationship between the foliar metabolite profiles and resistance level to CLS was examined by combining partial least squares projection to latent structure (PLS) or orthogonal PLS (OPLS) analysis and univariate analyses. It was difficult to build a robust model for predicting precisely the disease severity indices (DSIs) of each genotype; however, GABA and Gln differentiated susceptible genotypes (genotypes with weak resistance) from resistant genotypes (genotypes with resistance greater than a moderate level) before inoculation tests. The results suggested that breeders might exclude susceptible genotypes from breeding programs based on foliar metabolites profiled without inoculation tests, which require an enormous amount of time and effort.

Bacterial Compatibility in Combined Inoculations Enhances the Growth of Potato Seedlings.

The compatibility of strains is crucial for formulating bioinoculants that promote plant growth. We herein assessed the compatibility of four potential bioinoculants isolated from potato roots and tubers (Sphingomonas sp. T168, Streptomyces sp. R170, Streptomyces sp. R181, and Methylibium sp. R182) that were co-inoculated in order to improve plant growth. We screened these strains using biochemical tests, and the results obtained showed that R170 had the highest potential as a bioinoculant, as indicated by its significant ability to produce plant growth-promoting substances, its higher tolerance against NaCl (2%) and AlCl3 (0.01%), and growth in a wider range of pH values (5.0-10.0) than the other three strains. Therefore, the compatibility of R170 with other strains was tested in combined inoculations, and the results showed that the co-inoculation of R170 with T168 or R182 synergistically increased plant weight over un-inoculated controls, indicating the compatibility of strains based on the increased production of plant growth promoters such as indole-3-acetic acid (IAA) and siderophores as well as co-localization on roots. However, a parallel test using strain R181, which is the same Streptomyces genus as R170, showed incompatibility with T168 and R182, as revealed by weaker plant growth promotion and a lack of co-localization. Collectively, our results suggest that compatibility among bacterial inoculants is important for efficient plant growth promotion, and that R170 has potential as a useful bioinoculant, particularly in combined inoculations that contain compatible bacteria.

Molecular cloning and expression analysis of the replacement histone H3 gene of Italian ryegrass (Lolium multiflorum).

The replacement histone H3 gene and its 5'-flanking sequence were isolated from Italian ryegrass by polymerase chain reaction and inverse polymerase chain reaction, respectively. Expression analysis showed that this gene is constitutively expressed in the entire plant. The expression level in leaves was found to be significantly low when compared with that in other tissues. However, the gene expression level in leaves was increased by the treatment with abscisic acid and abiotic stresses such as cold, heat and high-salinity (NaCl). The motif search of the 5'-flanking sequence of the replacement histone H3 gene revealed the presence of several potential cis-acting elements that could respond to the above-mentioned abiotic stresses. In addition to defence-related elements, we also found type I and II-/III-like elements, which are highly conserved motifs in the 5'-regulatory sequence of plant histone genes that are expressed specifically during the S-phase. Experiments using transgenic Italian ryegrass plants proved that the isolated 5'-flanking sequence of the replacement histone H3 gene, which was fused to a beta-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions.

Draft Genome Sequences of Streptomyces scabiei S58, Streptomyces turgidiscabies T45, and Streptomyces acidiscabies a10, the Pathogens of Potato Common Scab, Isolated in Japan.

The draft genome sequences of the three pathogens of potato common scab, Streptomyces scabiei S58, Streptomyces turgidiscabies T45, and Streptomyces acidiscabies a10, isolated in Japan, are presented here. The genome size of each strain is >10 Mb, and the three pathogenic strains share genes located in a pathogenicity island previously described in other pathogenic Streptomyces species.

Are Symbiotic Methanotrophs Key Microbes for N Acquisition in Paddy Rice Root?

The relationships between biogeochemical processes and microbial functions in rice (Oryza sativa) paddies have been the focus of a large number of studies. A mechanistic understanding of methane-nitrogen (CH4-N) cycle interactions is a key unresolved issue in research on rice paddies. This minireview is an opinion paper for highlighting the mechanisms underlying the interactions between biogeochemical processes and plant-associated microbes based on recent metagenomic, metaproteomic, and isotope analyses. A rice symbiotic gene, relevant to rhizobial nodulation and mycorrhization in plants, likely accommodates diazotrophic methanotrophs or the associated bacterial community in root tissues under low-N fertilizer management, which may permit rice plants to acquire N via N2 fixation. The amount of N fixed in rice roots was previously estimated to be approximately 12% of plant N based on measurements of (15)N natural abundance in a paddy field experiment. Community analyses also indicate that methanotroph populations in rice roots are susceptible to environmental conditions such as the microclimate of rice paddies. Therefore, CH4 oxidation by methanotrophs is a driving force in shaping bacterial communities in rice roots grown in CH4-rich environments. Based on these findings, we propose a hypothesis with unanswered questions to describe the interplay between rice plants, root microbiomes, and their biogeochemical functions (CH4 oxidation and N2 fixation).

Metagenomic Analysis Revealed Methylamine and Ureide Utilization of Soybean-Associated Methylobacterium.

Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere.