Use of PCR-DGGE-Based Molecular Methods to Analyze Nematode Community Diversity.

DGGE (denaturing gradient gel electrophoresis) is a nucleic acid separation technique applied to the evaluation of microbial biodiversity. This technique is quite rapid and cheap compared to other types of analysis. Here we describe the comparison of nematode communities inhabiting different ecosystems. After an ecologically representative sampling collection and the nematode extraction from soil, nematodes are centrifuged in Eppendorf tubes to facilitate DNA extraction. DNA from the whole community of each type of soil is extracted, amplified with primers for 18 S rDNA and used in DGGE analysis. The profiles of DGGE can be analyzed with appropriate software, and biodiversity indices can be estimated.

Genome wide association studies for japonica rice resistance to blast in field and controlled conditions.

BACKGROUND: Rice blast, caused by the fungus Pyricularia oryzae, represents the most damaging fungal disease of rice worldwide. Utilization of rice resistant cultivars represents a practical way to control the disease. Most of the rice varieties cultivated in Europe and several other temperate regions are severely depleted of blast resistance genes, making the identification of resistant sources in genetic background adapted to temperate environments a priority. Given these assumptions, a Genome Wide Association Study (GWAS) for rice blast resistance was undertaken using a panel of 311 temperate/tropical japonica and indica accessions adapted to temperate conditions and genotyped with 37,423 SNP markers. The panel was evaluated for blast resistance in field, under the pressure of the natural blast population, and in growth chamber, using a mixture of three different fungal strains. RESULTS: The parallel screening identified 11 accessions showing high levels of resistance in the two conditions, representing potential donors of resistance sources harbored in rice genotypes adapted to temperate conditions. A general higher resistance level was observed in tropical japonica and indica with respect to temperate japonica varieties. The GWAS identified 14 Marker-Traits Associations (MTAs), 8 of which discovered under field conditions and 6 under growth chamber screening. Three MTAs were identified in both conditions; five MTAs were specifically detected under field conditions while three for the growth chamber inoculation. Comparative analysis of physical/genetic positions of the MTAs showed that most of them were positionally-related with cloned or mapped blast resistance genes or with candidate genes whose functions were compatible for conferring pathogen resistance. However, for three MTAs, indicated as BRF10, BRF11-2 and BRGC11-3, no obvious candidate genes or positional relationships with blast resistance QTLs were identified, raising the possibility that they represent new sources of blast resistance. CONCLUSIONS: We identified 14 MTAs for blast resistance using both field and growth chamber screenings. A total of 11 accessions showing high levels of resistance in both conditions were discovered. Combinations of loci conferring blast resistance were identified in rice accessions adapted to temperate conditions, thus allowing the genetic dissection of affordable resistances present in the panel. The obtained information will provide useful bases for both resistance breeding and further characterization of the highlighted resistance loci.

Water management and phenology influence the root-associated rice field microbiota.

Microbial communities associated with plants are greatly influenced by water availability in soil. In flooded crops, such as rice, the impact of water management on microbial dynamics is not fully understood. Here, we present a comprehensive study of the rice microbiota investigated in an experimental field located in one of the most productive areas of northern Italy. The microbiota associated with paddy soil and root was investigated using 454 pyrosequencing of 16S, ITS and 18S rRNA gene amplicons under two different water managements, upland (non-flooded, aerobic) and lowland (traditional flooding, anaerobic), at three plant development stages. Results highlighted a major role of the soil water status in shaping microbial communities, while phenological stage had low impacts. Compositional shifts in prokaryotic and fungal communities upon water management consisted in significant abundance changes of Firmicutes, Methanobacteria, Chloroflexi, Sordariomycetes, Dothideomycetes and Glomeromycotina. A vicariance in plant beneficial microbes and between saprotrophs and pathotrophs was observed between lowland and upland. Moreover, through network analysis, we demonstrated different co-abundance dynamics between lowland and upland conditions with a major impact on microbial hubs (strongly interconnected microbes) that fully shifted to aerobic microbes in the absence of flooding.

Different Phenotypes, Similar Genomes: Three Newly Sequenced Fusarium fujikuroi Strains Induce Different Symptoms in Rice Depending on Temperature.

Bakanae, caused by the hemibiotrophic fungus Fusarium fujikuroi, is one of the most important diseases of rice and is attributed to up to 75% of losses, depending on the strain and environmental conditions. Some strains cause elongation and thin leaves, whereas others induce stunting and chlorotic seedlings. Differences in symptoms are attributed to genetic differences in the strains. F. fujikuroi strains Augusto2, CSV1, and I1.3 were sequenced with Illumina MiSeq, and pathogenicity trials were conducted on rice cultivar Galileo, which is susceptible to bakanae. By performing gene prediction, single nucleotide polymorphism (SNP) calling, and structural variant analysis with a reference genome, we show how an extremely limited number of polymorphisms in genes not commonly associated with bakanae disease can cause strong differences in phenotype. CSV1 and Augusto2 were particularly close, with only 21,887 SNPs between them, but they differed in virulence, reaction to temperature, induced symptoms, colony morphology and color, growth speed, fumonisin, and gibberellin production. Genes potentially involved in the shift in phenotype were identified. Furthermore, we show how temperature variation may result in different symptoms even in rice plants inoculated with the same F. fujikuroi strain. Moreover, all of the F. fujikuroi strains became more virulent at higher temperatures. Significant differences were likewise observed in gibberellic acid production and in the expression of both fungal and plant gibberellin biosynthetic genes.

Mixotrophic cultivation of Chlorella for local protein production using agro-food by-products.

A local strain of Chlorella vulgaris was cultivated by using cheese whey (CW), white wine lees (WL) and glycerol (Gly), coming from local agro-industrial activities, as C sources (2.2gCL-1) to support algae production under mixotrophic conditions in Lombardy. In continuous mode, Chlorella increased biomass production compared with autotrophic conditions by 1.5-2 times, with the best results obtained for the CW substrate, i.e. 0.52gL-1d-1 of algal biomass vs. 0.24gL-1d-1 of algal biomass for autotrophic conditions, and protein content for both conditions adopted close to 500gkg-1 DM. Mixotrophic conditions gave a much higher energy recovery efficiency (EF) than autotrophic conditions, i.e. organic carbon energy efficiency (EFoc) of 32% and total energy efficiency (Eft) of 8%, respectively, suggesting the potential for the culture of algae as a sustainable practice to recover efficiently waste-C and a means of local protein production.

Genome-Wide Association Study for Traits Related to Plant and Grain Morphology, and Root Architecture in Temperate Rice Accessions.

BACKGROUND: In this study we carried out a genome-wide association analysis for plant and grain morphology and root architecture in a unique panel of temperate rice accessions adapted to European pedo-climatic conditions. This is the first study to assess the association of selected phenotypic traits to specific genomic regions in the narrow genetic pool of temperate japonica. A set of 391 rice accessions were GBS-genotyped yielding-after data editing-57000 polymorphic and informative SNPS, among which 54% were in genic regions. RESULTS: In total, 42 significant genotype-phenotype associations were detected: 21 for plant morphology traits, 11 for grain quality traits, 10 for root architecture traits. The FDR of detected associations ranged from 3 · 10-7 to 0.92 (median: 0.25). In most cases, the significant detected associations co-localised with QTLs and candidate genes controlling the phenotypic variation of single or multiple traits. The most significant associations were those for flag leaf width on chromosome 4 (FDR = 3 · 10-7) and for plant height on chromosome 6 (FDR = 0.011). CONCLUSIONS: We demonstrate the effectiveness and resolution of the developed platform for high-throughput phenotyping, genotyping and GWAS in detecting major QTLs for relevant traits in rice. We identified strong associations that may be used for selection in temperate irrigated rice breeding: e.g. associations for flag leaf width, plant height, root volume and length, grain length, grain width and their ratio. Our findings pave the way to successfully exploit the narrow genetic pool of European temperate rice and to pinpoint the most relevant genetic components contributing to the adaptability and high yield of this germplasm. The generated data could be of direct use in genomic-assisted breeding strategies.

Rice bacterial endophytes: isolation of a collection, identification of beneficial strains and microbiome analysis.

Endophytes are harmless or beneficial microorganisms that live inside plants between cells. The relationship they develop with the plant as well as their potential role in plant health is at large unexplored and it is believed that the opportunity to find new and interesting endophytes among the large variety of plants is great. Here, we present the isolation and analysis of a large collection of endophytes from one cultivar of rice grown in Italy. A total 1318 putative endophytes were isolated from roots, leaves and stems from rice grown in submerged and dry conditions and a working collection of 229 isolates was created. Among these, several isolates were confirmed to be endophytes and a few displayed the trait of plant growth promotion. A cultivation independent analysis via 16S rDNA amplicons of the bacterial community of the endosphere was also performed providing information on bacterial diversity in the rice endopshere.

Genome sequencing and secondary metabolism of the postharvest pathogen Penicillium griseofulvum.

BACKGROUND: Penicillium griseofulvum is associated in stored apples with blue mould, the most important postharvest disease of pome fruit. This pathogen can simultaneously produce both detrimental and beneficial secondary metabolites (SM). In order to gain insight into SM synthesis in P. griseofulvum in vitro and during disease development on apple, we sequenced the genome of P. griseofulvum strain PG3 and analysed important SM clusters. RESULTS: PG3 genome sequence (29.3 Mb) shows that P. griseofulvum branched off after the divergence of P. oxalicum but before the divergence of P. chrysogenum. Genome-wide analysis of P. griseofulvum revealed putative gene clusters for patulin, griseofulvin and roquefortine C biosynthesis. Furthermore, we quantified the SM production in vitro and on apples during the course of infection. The expression kinetics of key genes of SM produced in infected apple were examined. We found additional SM clusters, including those potentially responsible for the synthesis of penicillin, yanuthone D, cyclopiazonic acid and we predicted a cluster putatively responsible for the synthesis of chanoclavine I. CONCLUSIONS: These findings provide relevant information to understand the molecular basis of SM biosynthesis in P. griseofulvum, to allow further research directed to the overexpression or blocking the synthesis of specific SM.

Genetic analysis of durable resistance to Magnaporthe oryzae in the rice accession Gigante Vercelli identified two blast resistance loci.

Rice cultivars exhibiting durable resistance to blast, the most important rice fungal disease provoking up to 30 % of rice losses, are very rare and searching for sources of such a resistance represents a priority for rice-breeding programs. To this aim we analyzed Gigante Vercelli (GV) and Vialone Nano (VN), two temperate japonica rice cultivars in Italy displaying contrasting response to blast, with GV showing a durable and broad-spectrum resistance, whereas VN being highly susceptible. An SSR-based genetic map developed using a GV × VN population segregating for blast resistance identified two blast resistance loci, localized to the long arm of chromosomes 1 and 4 explaining more than 78 % of the observed phenotypic variation for blast resistance. The pyramiding of two blast resistance QTLs was therefore involved in the observed durable resistance in GV. Mapping data were integrated with information obtained from RNA-seq expression profiling of all classes of resistance protein genes (resistance gene analogs, RGAs) and with the map position of known cloned or mapped blast resistance genes to search candidates for the GV resistant response. A co-localization of RGAs with the LOD peak or the marker interval of the chromosome 1 QTL was highlighted and a valuable tool for selecting the resistance gene during breeding programs was developed. Comparative analysis with known blast resistance genes revealed co-positional relationships between the chromosome 1 QTL with the Pi35/Pish blast resistance alleles and showed that the chromosome 4 QTL represents a newly identified blast resistance gene. The present genetic analysis has therefore allowed the identification of two blast resistance loci in the durable blast-resistant rice cultivar GV and tools for molecular selection of these resistance genes.

Draft Genome Sequence of Rice Endophyte-Associated Isolate Kosakonia oryzae KO348.

Kosakonia oryzae KO348 is an endophytic and plant growth-promoting strain isolated from the roots of rice in Italy. Here, we report the draft genome sequence of Kosakonia oryzae KO348.

CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis.

In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.

Sapogenin content variation in Medicago inter-specific hybrid derivatives highlights some aspects of saponin synthesis and control.

In the Medicago genus, saponins are a complex mixture of triterpene glycosides showing a broad spectrum of biological properties. Here we analyzed the variation in the sapogenin content and composition of inter-specific hybrid Medicago sativa × Medicago arborea derivatives to highlight the pattern of this variation in plant organs (leaves/roots) and the possible mechanisms underlying it. In Sativa Arborea Cross (SAC) leaves and roots, saponins and sapogenins were evaluated using chromatographic methods. Phenotypic correlations between sapogenin content and bio-agronomic traits were examined. Expression studies on ß-amyrin synthase and four cytochromes P450 (CYPs) involved in sapogenin biosynthesis and sequence analysis of the key gene of the hemolytic sapogenin pathway (CYP716A12) were performed. Chromatographic analyses revealed a different pattern of among-family variation for hemolytic and nonhemolytic sapogenins and saponins and for the two organs/tissues. Different correlation patterns of gene expression in roots and leaves were found. Diachronic analysis revealed a relationship between sapogenin content and gene transcriptional levels in the early stages of the productive cycle. The results suggest that there are different control mechanisms acting on sapogenin biosynthesis for leaves and roots, which are discussed. A key role for medicagenic acid in the control of sapogenin content in both the tissues is proposed and discussed.

Triterpenoid glycosides from Medicago sativa as antifungal agents against Pyricularia oryzae.

The antifungal properties of saponin mixtures from alfalfa (Medicago sativa L.) tops and roots, the corresponding mixtures of prosapogenins from tops, and purified saponins and sapogenins against the causal agent of rice blast Pyricularia oryzae isolates are presented. In vitro experiments highlighted a range of activities, depending upon the assayed metabolite. The antifungal effects of the most promising prosapogenin mixture from alfalfa tops were confirmed by means of in planta tests using three different Italian cultivars of rice (Oryza sativa L. ssp. japonica), known to possess high, medium, and low blast resistance. The evidenced antifungal properties of the tested metabolites allowed some considerations on their structure-activity relationship. Results indicate that prosapogenins are active compounds to prevent the fungal attack of P. oryzae on different rice cultivars. Therefore, if properly formulated, these substances could represent a promising and environmentally friendly treatment to control rice blast.

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots.

Draft Genome Sequence of the Plant Pathogen Dickeya zeae DZ2Q, Isolated from Rice in Italy.

Dickeya zeae is an emerging rice (Oryza sativa) pathogen causing bacterial foot rot. Related pathogens affect maize (Zea mays) and potato (Solanum tuberosum) and a variety of important ornamental and floral plants. Here, we present the draft genome sequence of D. zeae DZ2Q, an isolate obtained from rice grown in Italy.

Analysis of transcript and metabolite levels in Italian rice (Oryza sativa L.) cultivars subjected to osmotic stress or benzothiadiazole treatment.

One of the major objectives of rice (Oryza sativa L.) breeding programs is the development of new varieties with higher tolerance/resistance to both abiotic and biotic stresses. In this study, Italian rice cultivars were subjected to osmotic stress or benzothiadiazole (BTH) treatments. An analysis of the expression of selected genes known to be involved in the stress response and (1)H nuclear magnetic resonance ((1)H NMR) metabolic profiling were combined with multivariate statistical analyses to elucidate potential correlations between gene expression or metabolite content and observed tolerant/resistant phenotypes. We observed that the expression of three chosen genes (two WRKY genes and one peroxidase encoding gene) differed between susceptible and resistant cultivars in response to BTH treatments. Moreover, the analysis of metabolite content, in particular in the osmotic stress experiment, enabled discrimination between selected cultivars based on differences in the accumulation of some primary metabolites, primarily sugars. This research highlights the potential usefulness of this approach to characterise rice varieties based on transcriptional or metabolic changes due to adverse environmental conditions.

Comparative transcriptome profiling of the early response to Magnaporthe oryzae in durable resistant vs susceptible rice (Oryza sativa L.) genotypes.

Durable resistance to blast, the most significant fungal disease of rice, represents an agronomically relevant character. Gigante Vercelli (GV) and Vialone Nano (VN) are two old temperate japonica Italian rice cultivars with contrasting response to blast infection: GV displays durable and broad resistance while VN is highly susceptible. RNA-seq was used to dissect the early molecular processes deployed during the resistance response of GV at 24 h after blast inoculation. Differential gene expression analysis identified 1,070 and 1,484 modulated genes, of which 726 and 699 were up regulated in response to infection in GV and VN, respectively. Gene ontology (GO) enrichment analyses revealed a set of GO terms enriched in both varieties but, despite this commonality, the gene sets contributing to common GO enriched terms were dissimilar. The expression patterns of genes grouped in GV-specific enriched GO terms were examined in detail including at the transcript isoform level. GV exhibited a dramatic up-regulation of genes encoding diterpene phytoalexin biosynthetic enzymes, flavin-containing monooxygenase, class I chitinase and glycosyl hydrolase 17. The sensitivity and high dynamic range of RNA-seq allowed the identification of genes critically involved in conferring GV resistance during the early steps of defence perception-signalling. These included chitin oligosaccharides sensing factors, wall associated kinases, MAPK cascades and WRKY transcription factors. Candidate genes with expression patterns consistent with a potential role as GV-specific functional resistance (R) gene(s) were also identified. This first application of RNA-seq to dissect durable blast resistance supports a crucial role of the prompt induction of a battery of responses including defence-related genes as well as members of gene families involved in signalling and pathogen-related gene expression regulation.

OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast.

With the aim of identifying novel regulators of host and nonhost resistance to fungi in rice, we carried out a systematic mutant screen of mutagenized lines. Two mutant wrky22 knockout lines revealed clear-cut enhanced susceptibility to both virulent and avirulent Magnaporthe oryzae strains and altered cellular responses to nonhost Magnaporthe grisea and Blumeria graminis fungi. In addition, the analysis of the pathogen responses of 24 overexpressor OsWRKY22 lines revealed enhanced resistance phenotypes on infection with virulent M. oryzae strain, confirming that OsWRKY22 is involved in rice resistance to blast. Bioinformatic analyses determined that the OsWRKY22 gene belongs to a well-defined cluster of monocot-specific WRKYs. The co-regulatory analysis revealed no significant co-regulation of OsWRKY22 with a representative panel of OsWRKYs, supporting its unique role in a series of transcriptional responses. In contrast, inquiring a subset of biotic stress-related Affymetrix data, a large number of resistance and defence-related genes were found to be putatively co-expressed with OsWRKY22. Taken together, all gathered experimental evidence places the monocot-specific OsWRKY22 gene at the convergence point of signal transduction circuits in response to both host and nonhost fungi encountering rice plants.

Characterization of WRKY co-regulatory networks in rice and Arabidopsis.

BACKGROUND: The WRKY transcription factor gene family has a very ancient origin and has undergone extensive duplications in the plant kingdom. Several studies have pointed out their involvement in a range of biological processes, revealing that a large number of WRKY genes are transcriptionally regulated under conditions of biotic and/or abiotic stress. To investigate the existence of WRKY co-regulatory networks in plants, a whole gene family WRKYs expression study was carried out in rice (Oryza sativa). This analysis was extended to Arabidopsis thaliana taking advantage of an extensive repository of gene expression data. RESULTS: The presented results suggested that 24 members of the rice WRKY gene family (22% of the total) were differentially-regulated in response to at least one of the stress conditions tested. We defined the existence of nine OsWRKY gene clusters comprising both phylogenetically related and unrelated genes that were significantly co-expressed, suggesting that specific sets of WRKY genes might act in co-regulatory networks. This hypothesis was tested by Pearson Correlation Coefficient analysis of the Arabidopsis WRKY gene family in a large set of Affymetrix microarray experiments. AtWRKYs were found to belong to two main co-regulatory networks (COR-A, COR-B) and two smaller ones (COR-C and COR-D), all including genes belonging to distinct phylogenetic groups. The COR-A network contained several AtWRKY genes known to be involved mostly in response to pathogens, whose physical and/or genetic interaction was experimentally proven. We also showed that specific co-regulatory networks were conserved between the two model species by identifying Arabidopsis orthologs of the co-expressed OsWRKY genes. CONCLUSION: In this work we identified sets of co-expressed WRKY genes in both rice and Arabidopsis that are functionally likely to cooperate in the same signal transduction pathways. We propose that, making use of data from co-regulatory networks, it is possible to highlight novel clusters of plant genes contributing to the same biological processes or signal transduction pathways. Our approach will contribute to unveil gene cooperation pathways not yet identified by classical genetic analyses. This information will open new routes contributing to the dissection of WRKY signal transduction pathways in plants.

Art-loving bugs: the resurrection of Spinello Aretino from Pisa's cemetery.

During 1391-1392, Spinello Aretino painted a cycle of frescoes in Pisa's cemetery on the theme "Storie dei Santi Martiri Efisio e Potito", highly prized by contemporaries and by Vasari. Twenty years ago, one of these frescoes, "Conversione di S. Efisio e Battaglia" (Conversion of S. Efisio and Battle), because of discolouring and bad damage caused by humidity and atmospheric pollution, was removed from the walls using the "tear-off" technique, consisting in covering the surface with a strong cloth bound to the surface with generous layers of formaldehyde-treated glue. As luck would have it, this large fresco (3.50x7.80 m) was abandoned in a storehouse for more than 20 years. When the curators attempted to remove the cloth, much to their dismay they found that the glue resisted any attempt at digestion, even when treated with concoctions of the most aggressive proteolytic enzymes available on the market. It is likely that during the long storage the glue became slowly cross-linked by the formaldehyde to the point of forming an intricate mass of untreatable proteinaceous material. Thus, although poor Spinello died presumably as a bona fide Christian, his painting was condemned to wear the burka, Muslim-fashion, for the rest of its life. When we recently treated the fresco with a suspension of viable Pseudomonas stutzeri cells, these bacteria, although agnostic, were able to fully digest the hardened glue and restore to life Spinello's glorious painting. We show here how proteomics helped us solve the riddle of how these bacteria acted on the burka-obscured fresco.