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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

Sulfur Fertilization Changes the Community Structure of Rice Root-, and Soil- Associated Bacteria.

Under paddy field conditions, biological sulfur oxidation occurs in the oxidized surface soil layer and rhizosphere, in which oxygen leaks from the aerenchyma system of rice plants. In the present study, we examined community shifts in sulfur-oxidizing bacteria associated with the oxidized surface soil layer and rice roots under different sulfur fertilization conditions based on the 16S ribosomal RNA (rRNA) gene in order to explore the existence of oligotrophic sulfur-oxidizing bacteria in the paddy rice ecosystem. Rice plants were grown in pots with no fertilization (control) or CaCO3 or CaSO4 fertilization. A principal-coordinates analysis (PCoA) showed that CaSO4 fertilization markedly affected bacterial communities associated with rice roots and soil, whereas no significant differences were observed in plant growth among the fertilizer treatments examined. In rice roots, the relative abundance of Acidobacteria, Alphaproteobacteria, Gammaproteobacteria, and TM7 was significantly higher in CaSO4-fertilized pots than in control pots. Alphaproteobacteria, Bradyrhizobiaceae, and Methylocystaceae members were significantly more abundant in CaSO4-fertilized roots than in control roots. On the other hand, the abundance of Actinobacteria and Proteobacteria was lower in CaSO4-fertilized soil than in control soil. These results indicate that the bacteria associated with rice roots and soil responded to the sulfur amendment, suggesting that more diverse bacteria are involved in sulfur oxidation in the rice paddy ecosystem than previously considered.

Community Analysis of Root- and Tuber-Associated Bacteria in Field-Grown Potato Plants Harboring Different Resistance Levels against Common Scab.

Eight genotypes of potato plants with different resistance levels against common scab were grown in a field infested with Streptomyces turgidiscabies. DNA was extracted from the roots, tubers, and rhizosphere soils of each of the eight genotypes at the flowering stage, and the quantity of S. turgidiscabies genomic DNA was assessed by real-time PCR using a TaqMan probe. The results obtained showed that the different potato genotypes had significant impacts on the population levels of S. turgidiscabies between resistant and susceptible genotypes in the tubers, but not in the roots or rhizosphere soils. Clone analyses of 16S rRNA gene libraries from the eight potato genotypes identified three phyla (Proteobacteria, Firmicutes, and Actinobacteria) as dominant taxa in root and tuber clone libraries, while a clustering analysis identified 391 operational taxonomic units (OTUs) at the species level. Eleven OTUs closely related to Aquicella siphonis, Arthrobacter nicotinovorans, Streptomyces rishiriensis, Rhodococcus baikonurensis, Rhizobium radiobacter, Rhizobium etli, Phyllobacterium myrsinacearum, Paenibacillus pabuli, Paenibacillus alginolyticus, and Bacillus halmapalus were detected in the root or tuber libraries of all the potato genotypes examined. Furthermore, an abundance of OTUs related to Aquicella and Rhodococcus was observed in the rhizospheres of resistant and susceptible potato genotypes, respectively. Based on this ecological information, an efficient survey may be conducted for biological agents from the potato rhizosphere.

Characterization of leaf blade- and leaf sheath-associated bacterial communities and assessment of their responses to environmental changes in CO2, temperature, and nitrogen levels under field conditions.

Rice shoot-associated bacterial communities at the panicle initiation stage were characterized and their responses to elevated surface water-soil temperature (ET), low nitrogen (LN), and free-air CO2 enrichment (FACE) were assessed by clone library analyses of the 16S rRNA gene. Principal coordinate analyses combining all sequence data for leaf blade- and leaf sheath-associated bacteria revealed that each bacterial community had a distinct structure, as supported by PC1 (61.5%), that was mainly attributed to the high abundance of Planctomycetes in leaf sheaths. Our results also indicated that the community structures of leaf blade-associated bacteria were more sensitive than those of leaf sheath-associated bacteria to the environmental factors examined. Among these environmental factors, LN strongly affected the community structures of leaf blade-associated bacteria by increasing the relative abundance of Bacilli. The most significant effect of FACE was also observed on leaf blade-associated bacteria under the LN condition, which was explained by decreases and increases in Agrobacterium and Pantoea, respectively. The community structures of leaf blade-associated bacteria under the combination of FACE and ET were more similar to those of the control than to those under ET or FACE. Thus, the combined effects of environmental factors need to be considered in order to realistically assess the effects of environmental changes on microbial community structures.

Insecticide-degrading Burkholderia symbionts of the stinkbug naturally occupy various environments of sugarcane fields in a Southeast island of Japan.

The stinkbug Cavelerius saccharivorus, which harbors Burkholderia species capable of degrading the organophosphorus insecticide, fenitrothion, has been identified on a Japanese island in farmers' sugarcane fields that have been exposed to fenitrothion. A clearer understanding of the ecology of the symbiotic fenitrothion degraders of Burkholderia species in a free-living environment is vital for advancing our knowledge on the establishment of degrader-stinkbug symbiosis. In the present study, we analyzed the composition and abundance of degraders in sugarcane fields on the island. Degraders were recovered from field samples without an enrichment culture procedure. Degrader densities in the furrow soil in fields varied due to differences in insecticide treatment histories. Over 99% of the 659 isolated degraders belonged to the genus Burkholderia. The strains related to the stinkbug symbiotic group predominated among the degraders, indicating a selection for this group in response to fenitrothion. Degraders were also isolated from sugarcane stems, leaves, and rhizosphere in fields that were continuously exposed to fenitrothion. Their density was lower in the plant sections than in the rhizosphere. A phylogenetic analysis of 16S rRNA gene sequences demonstrated that most of the degraders from the plants and rhizosphere clustered with the stinkbug symbiotic group, and some were identical to the midgut symbionts of C. saccharivorus collected from the same field. Our results confirmed that plants and the rhizosphere constituted environmental reservoirs for stinkbug symbiotic degraders. To the best of our knowledge, this is the first study to investigate the composition and abundance of the symbiotic fenitrothion degraders of Burkholderia species in farmers' fields.

Metagenomic analysis of the bacterial community associated with the taproot of sugar beet.

We analyzed a metagenome of the bacterial community associated with the taproot of sugar beet (Beta vulgaris L.) in order to investigate the genes involved in plant growth-promoting traits (PGPTs), namely 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, indole acetic acid (IAA), N2 fixation, phosphate solubilization, pyrroloquinoline quinone, siderophores, and plant disease suppression as well as methanol, sucrose, and betaine utilization. The most frequently detected gene among the PGPT categories encoded ß-1,3-glucanase (18 per 10(5) reads), which plays a role in the suppression of plant diseases. Genes involved in phosphate solubilization (e.g., for quinoprotein glucose dehydrogenase), methanol utilization (e.g., for methanol dehydrogenase), siderophore production (e.g. isochorismate pyruvate lyase), and ACC deaminase were also abundant. These results suggested that such PGPTs are crucially involved in supporting the growth of sugar beet. In contrast, genes for IAA production (iaaM and ipdC) were less abundant (~1 per 10(5) reads). N2 fixation genes (nifHDK) were not detected; bacterial N2 -fixing activity was not observed in the (15)N2 -feeding experiment. An analysis of nitrogen metabolism suggested that the sugar beet microbiome mainly utilized ammonium and nitroalkane as nitrogen sources. Thus, N2 fixation and IAA production did not appear to contribute to sugar beet growth. Taxonomic assignment of this metagenome revealed the high abundance of Mesorhizobium, Bradyrhizobium, and Streptomyces, suggesting that these genera have ecologically important roles in the taproot of sugar beet. Bradyrhizobium-assigned reads in particular were found in almost all categories of dominant PGPTs with high abundance. The present study revealed the characteristic functional genes in the taproot-associated microbiome of sugar beet, and suggest the opportunity to select sugar beet growth-promoting bacteria.

Elevated atmospheric CO2 levels affect community structure of rice root-associated bacteria.

A number of studies have shown that elevated atmospheric CO2 ([CO2]) affects rice yields and grain quality. However, the responses of root-associated bacteria to [CO2] elevation have not been characterized in a large-scale field study. We conducted a free-air CO2 enrichment (FACE) experiment (ambient + 200 µmol.mol(-1)) using three rice cultivars (Akita 63, Takanari, and Koshihikari) and two experimental lines of Koshihikari [chromosome segment substitution and near-isogenic lines (NILs)] to determine the effects of [CO2] elevation on the community structure of rice root-associated bacteria. Microbial DNA was extracted from rice roots at the panicle formation stage and analyzed by pyrosequencing the bacterial 16S rRNA gene to characterize the members of the bacterial community. Principal coordinate analysis of a weighted UniFrac distance matrix revealed that the community structure was clearly affected by elevated [CO2]. The predominant community members at class level were Alpha-, Beta-, and Gamma-proteobacteria in the control (ambient) and FACE plots. The relative abundance of Methylocystaceae, the major methane-oxidizing bacteria in rice roots, tended to decrease with increasing [CO2] levels. Quantitative PCR revealed a decreased copy number of the methane monooxygenase (pmoA) gene and increased methyl coenzyme M reductase (mcrA) in elevated [CO2]. These results suggest elevated [CO2] suppresses methane oxidation and promotes methanogenesis in rice roots; this process affects the carbon cycle in rice paddy fields.

Phylogeny and functions of bacterial communities associated with field-grown rice shoots.

Metagenomic analysis was applied to bacterial communities associated with the shoots of two field-grown rice cultivars, Nipponbare and Kasalath. In both cultivars, shoot microbiomes were dominated by Alphaproteobacteria (51-52%), Actinobacteria (11-15%), Gammaproteobacteria (9-10%), and Betaproteobacteria (4-10%). Compared with other rice microbiomes (root, rhizosphere, and phyllosphere) in public databases, the shoot microbiomes harbored abundant genes for C1 compound metabolism and 1-aminocyclopropane-1-carboxylate catabolism, but fewer genes for indole-3-acetic acid production and nitrogen fixation. Salicylate hydroxylase was detected in all microbiomes, except the rhizosphere. These genomic features facilitate understanding of plant-microbe interactions and biogeochemical metabolism in rice shoots.

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.

An assessment of urea-formaldehyde fertilizer on the diversity of bacterial communities in onion and sugar beet.

The impact of a urea-formaldehyde (UF) fertilizer on bacterial diversity in onion bulbs and main roots of sugar beet were examined using a 16S rRNA gene clone library. The UF fertilizer markedly increased bacterial diversity in both plants. The results of principal coordinates analysis (PCoA) revealed that nearly 30% of the variance observed in bacterial diversity in both the onion and sugar beet was attributed to the fertilization conditions and also that the community structures in both plants shifted unidirectionally in response to the UF fertilizer.