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.

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.

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.

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.

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.

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.

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.

An assessment of the diversity of culturable bacteria from main root of sugar beet.

The partial sequences of the 16S rRNA genes of 531 bacteria isolated from the main root of the sugar beet (Beta vulgaris L.) were determined and subsequently grouped into 155 operational taxonomic units by clustering analysis (≥99% identity). The most abundant phylum was Proteobacteria (72.5-77.2%), followed by Actinobacteria (9.8-16.6%) and Bacteroidetes (4.3-15.4%). Alphaproteobacteria (46.7-64.8%) was the most dominant class within Proteobacteria. Four strains belonging to Verrucomicrobia were also isolated. Phylogenetic analysis revealed that the Verrucomicrobia bacterial strains were closely related to Haloferula or Verrucomicrobium.

Molecular characterization of the bacterial community in a potato phytosphere.

The bacterial community of a potato phytosphere at the flowering stage was examined using both culture-dependent and -independent methods. Tissues (leaves, stems, roots and tubers) were sampled from field-grown potato plants (cultivar Matilda), and the clone libraries of 16S rRNA genes and the isolate collections using R2A medium were constructed. By analyzing the combined data set of 16S rRNA gene sequences from both clone libraries and isolate collections, 82 genera from 8 phyla were found and 237 OTUs (≥97% identity) at species level were identified across the potato phytosphere. The statistical analyses of clone libraries suggested that stems harbor the lowest diversity among the tissues examined. The phylogenetic analyses revealed that the most dominant phylum was shown to be Proteobacteria for all tissues (62.0%-89.7% and 57.7%-72.9%, respectively), followed by Actinobacteria (5.0%-10.7% and 14.6%-39.4%, respectively). The results of principal coordinates analyses of both clone libraries and isolate collections indicated that distinct differences were observed between above- and below-ground tissues for bacterial community structures. The results also revealed that leaves harbored highly similar community structures to stems, while the tuber community was shown to be distinctly different from the stem and root communities.

Genetic diversity and ecological evaluation of fluorescent pseudomonads isolated from the leaves and roots of potato plants.

A total of 828 isolates of fluorescent pseudomonads (FPs) were obtained from the leaves (305 isolates) and roots (523 isolates) of potato plants grown in different geographical locations in Japan, and 16S rRNA gene sequences of 776 isolates were successfully determined by direct PCR sequencing. Clustering analysis (≥99% identity) identified 13 and 26 operational taxonomic units (OTUs) for leaf- and root-associated FPs, respectively, and 29 OTUs were identified in the phytosphere of potato plants. Among them, 7 and 9 OTUs showed a significantly biased distribution to the leaves and roots, respectively. Phylogenetic analysis revealed that 3 dominant OTUs for leaf-associated FPs were grouped in a cluster of leaf-associated pathogens, such as Pseudomonas cichorii and Pseudomonas viridiflava. In contrast, 4 OTUs were located in a cluster of saprophytic pseudomonads. Among them, 3 OTUs showed high similarity to Pseudomonas koreensis and Pseudomonas vancouverensis, both of which have been reported to be beneficial for biological control or plant growth promotion. These data provide key information for efficient surveying and utilization of beneficial FPs in agricultural practices.