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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. 1H 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. [ABSTRACT FROM AUTHOR]

The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [RI) 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 Rhizobials 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 OsCGaMK 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. [ABSTRACT FROM AUTHOR]

The diversities leaf-associated bacteria on nonnodulated (Nod-), wild-type nodulated (Nod+), and hyper-nodulated (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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

The diversity of stem-associated bacteria of non-nodulated (Nod−), wild-type nodulated (Nod+) and hypernodulated (Nod++) soybeans were evaluated by clone library analyses of the 16S ribosomal RNA gene. Soybeans were dressed with standard nitrogen (SN) fertilization (15 kg N ha–1) and heavy nitrogen (HN) fertilization (615 kg N ha–1). The relative abundance of Alphaproteobacteria in Nod+ soybeans (66%) was smaller than that in Nod− and Nod++ soybeans (75–76%) under SN fertilization, whereas that of Gammaproteobacteria showed the opposite pattern (23% in Nod+ and 12–16% in Nod− and Nod++ soybeans). Principal coordinate analysis showed that the bacterial communities of Nod− and Nod++ soybeans were more similar to each other than to that of Nod+ soybeans under SN fertilization. HN fertilization increased the relative abundance of Gammaproteobacteria in all nodulation phenotypes (33–57%) and caused drastic shifts of the bacterial community. The clustering analyses identified a subset of operational taxonomic units (OTUs) at the species level in Alpha- and Gammaproteobacteria responding to both the nodulation phenotypes and nitrogen fertilization levels. Meanwhile, the abundance of Betaproteobacteria was relatively constant in all libraries constructed under these environmental conditions. The relative abundances of two OTUs in Alphaproteobacteria (Aurantimonas sp. and Methylobacterium sp.) were especially sensitive to nodulation phenotype and were drastically decreased under HN fertilization. These results suggested that a subpopulation of proteobacteria in soybeans is controlled in a similar manner through both the regulation systems of plant–rhizobia symbiosis and the nitrogen signaling pathway in plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The molecular diversity of bacterial chitinases in the bulk soils of arable land was investigated using culture-independent methods. The results demonstrate that bacterial chitinases in arable soils are highly diverse and comprise unique groups when their sequences were compared to those in public databases. The diversity of bacterial chitinases in arable soil was further evaluated using conventional phylogenetic analysis, the UniFrac analysis of the phylogenetic data, and the multidimensional scaling (MDS) analysis of T-RFLP profiles to elucidate the relationship between the diversity of bacterial chitinases and soil characteristics. These analyses indicate that environmental factors such as soil type and pH are responsible for shaping the composition of bacterial chitinases. [Copyright &y& Elsevier]

Microorganisms associated with the stems and roots of nonnodulated (Nod-), wild-type nodulated (Nod+), and hypernodulated (Nod++) soybeans [Glycine max (L.) Merrill 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. [ABSTRACT FROM AUTHOR]

A simple detection procedure was developed for terminal restriction fragment length polymorphism and ribosomal intergenic spacer analysis for microbial community analysis. A fluorescence scanner enables generation of fingerprinting data in these methods within 3 h of obtaining PCR products of microbial communities. [Copyright &y& Elsevier]

Presents the results of a study investigating the association between self-compatibility and the absence of transcripts encoded by an aquaporin-related gene in mod mutants of Brassica. The suggestion that a water channel is required for the self-incompatibility response; The concept that regulation of water transfer from the stigma to pollen is a checkpoint in the early events of pollination in the crucifer family.

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 soxY1 gene at the sox locus I, homologous to the sulfur-oxidizing (Sox) system in Aiphaproteobacteria, left B. japonicum unable to oxidize thiosulfate and grow under chemolithotrophic conditions, whereas the deletion mutation of the soxY2 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 O2 respiration was observed only in USDA110 and the soxY2 mutant and not in the soxY1 mutant. In the cells, 1 mol of thiosulfate was stoichiometrically converted to approximately 2 mol of sulfate and consumed approximately 2 mol of O2. B.japonicum USDA110 showed 14CO2 fixation under chemolithotrophic growth conditions. The CO2 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 soxY1 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 soxY1 gene of B. japonicum USDA110. [ABSTRACT FROM AUTHOR]

Summary: 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 β-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions. [Copyright &y& Elsevier]

A mutant, leafy head 1 (lhd 1), was discovered in Japan from the progeny of Italian ryegrass (Lolium multiflorum Lam.) 'Nioudachi' and local line 'Aichikei #3'. Compared with normal plants, the mutant plant is a dwarf with a larger number of internodes per stem, shorter internodes, and smaller leaves. The plants also head later in the season. Aerial roots are usually produced from the stem nodes during rainy seasons. In characterizing lhd 1, it was found to have many branches with small leaves and many small panicles on the upper part of the plant. Panicle development was severely disturbed in lhd 1 mutants, and the number of leaves produced in the vegetative phase was nearly twice that produced in the wild-type counterpart. The lhd 1 mutant appears to be a heterochronic mutation that is able to extend the vegetative period during development. The frequency of mutants in segregating populations indicated that lhd 1 is a recessive allele. To determine the linkage relationship between the lhd 1 gene and AFLP markers, 768 primer combinations were screened for polymorphisms using bulked segregant analyses in two populations with 316 and 30 plants, respectively. Five AFLP markers were linked to the lhd 1 locus. E3/M41-1 and E16/M14-2 cosegregated with lhd 1. E16/M14-1 and E30/M10-1 flanked the gene at a distance of 0.3 cM and E30/M14-2 was linked to lhd 1 at a distance of 0.6 cM.Key words: Lolium multiflorum, leafy head mutant, mapping.Un mutant, nommé « leafy head 1 » (lhd 1), a été trouvé au Japon parmi la progéniture du cultivar 'Nioudachi' et d'une lignée locale, 'Aichikei #3, de l'ivraie multiflore (Lolium multiflorum Lam.). Par rapport à des plantes normales, le mutant est nain, présente un plus grand nombre d'entre-noeuds par tige, les entre-noeuds sont plus courts et des feuilles plus petites. Les plants du mutant fleurissent aussi plus tardivement. Des racines aériennes sont habituellement produites à partir des nœuds de la tige au cours des saisons de pluie. Au cours de la caractérisation de lhd 1, il a été noté qu'il présentait plusieurs branches avec de petites feuilles et plusieurs petites panicules produites dans la partie supérieure de la plante. Le développement de la panicule était très anormal chez lhd 1 et le nombre de feuilles produites au cours de la phase végétative était près du double du nombre produit chez le type sauvage. lhd 1 semble être un mutant hétérochronique, chez lequel la phase végétative du développement est prolongée. La fréquence de mutants au sein de populations en ségrégation indique que l'allèle lhd 1 est récessif. Afin de déterminer la liaison entre lhd 1 et des marqueurs AFLP, 768 combinaisons d'amorces ont été examinées pour du polymorphisme par analyse de ségrégants en mélange (" bulked segregant analysis ") au sein de deux populations comptant respectivement 316 et 30 plantes. Cinq marqueurs AFLP liés au locus lhd 1 ont été identifiés. Les marqueurs E3/M41-1 et E16/M14-2 ont montré une parfaite co-ségrégation avec lhd 1. Les marqueurs E16/M14-1 et E30/M10-1 bordaient le gène à 0,3 cM tandis que le marqueur E30/M14-2 était situé à 0,6 cM de lhd 1.Mots clés: Lolium multiflorum, mutant « leafy head », cartographie.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Comparative genomic hybridization (CGH) was performed with nine strains of Bradyrhizobium japonicum (a symbiotic nitrogen-fixing bacterium associated with soybean) and eight other members of the Bradyrhizobiaceae by DNA macroarray of B. japonicum USDA110. CGH clearly discriminated genomic variations in B. japonicum strains, but similar CGH patterns were observed in other members of the Bradyrhizobiaceae. The most variable regions were 14 genomic islands (4–97 kb) and low G+C regions on the USDA110 genome, some of which were missing in several strains of B. japonicum and other members of the Bradyrhizobiaceae. The CGH profiles of B. japonicum were classified into three genome types: 110, 122 and 6. Analysis of DNA sequences around the boundary regions showed that at least seven genomic islands were missing in genome type 122 as compared with type 110. Phylogenetic analysis for internal transcribed sequences revealed that strains belonging to genome types 110 and 122 formed separate clades. Thus genomic islands were horizontally inserted into the ancestor genome of type 110 after divergence of the type 110 and 122 strains. To search for functional relationships of variable genomic islands, we conducted linear models of the correlation between the existence of genomic regions and the parameters associated with symbiotic nitrogen fixation in soybean. Variable genomic regions including genomic islands were associated with the enhancement of symbiotic nitrogen fixation in B. japonicum USDA110.The ISME Journal (2009) 3, 326–339; doi:10.1038/ismej.2008.88; published online 30 October 2008 [ABSTRACT FROM AUTHOR]