Draft Genome Sequence of the Ascomycete Phaeoacremonium aleophilum Strain UCR-PA7, a Causal Agent of the Esca Disease Complex in Grapevines.

Grapevine infections by Phaeoacremonium aleophilum in association with other pathogenic fungi cause complex and economically important vascular diseases. Here we present the first draft of the P. aleophilum genome sequence, which comprises 624 scaffolds with a total length of 47.5 Mb (L50, 45; N50, 336 kb) and 8,926 predicted protein-coding genes

Draft Genome Sequence of the Grapevine Dieback Fungus Eutypa lata UCR-EL1.

The vascular pathogen Eutypa lata, which causes Eutypa dieback in grapevines, is a major threat to grape production worldwide. Here, we present the first draft genome sequence of E. lata (UCR-EL1). The computational prediction and annotation of the protein-coding genes of UCR-EL1 provide an initial inventory of its potential virulence factors

The role of melanin in the grapevine trunk disease pathogen Lasiodiplodia gilanensis

Lasiodiplodia (Botryosphaeriaceae) includes fungi that are considered among the most aggressive to grapevine, capable of causing cankers and necrotic lesions which eventually lead to death of host plants. A common characteristic of this genus is the presence of melanin in conidia and mycelium. Melanin is produced by the oxidation of phenolic and/or indolic compounds. For some fungi, this pigment is an essential factor for pathogenicity. This study characterized the types and the roles of melanin produced by Lasiodiplodia gilanensis. Using specific melanin inhibitors, L. gilanensis was shown to synthesize DOPA-melanin, DHN-melanin, and pyomelanin. DOPA-melanin was shown to be involved in production of aerial mycelium and protection against enzymatic lysis and oxidative stress; DHN-melanin to be involved in ramification of mycelium when exposed to nutrient deficiency; and pyomelanin to be related with hyphae development. The fungus used tyrosine as a precursor of DOPAmelanin and as carbon and nitrogen sources, and produced melanin inside the piths of infected plants. Genes involved in melanin synthesis were conserved among the Botryosphaeriaceae, highlighting the importance of melanin in this family.EEA MendozaFil: Rangel Montoya, Edelweiss Airam. Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE). Departamento de Microbiología; MéxicoFil: Paolinelli, Marcos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Paolinelli, Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina; ArgentinaFil: Rolshausen, Philippe. University of California Riverside. Department of Botany and Plant Sciences; Estados UnidosFil: Hernandez Martinez, Rufina. Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE). Departamento de Microbiología; Méxic

Characterization of Lasiodiplodia species associated with grapevines in Mexico

Botryosphaeria dieback is one of the most prevalent grapevine trunk diseases (GTDs), and is caused by fungi in the Botryosphaeriaceae. Fungi invade grapevine vascular systems mainly through pruning wounds, and cause cankers and necrotic lesions, which lead to grapevine decline and death. Lasiodiplodia theobromae has been reported as a highly virulent pathogen of grapevine, and was previously reported in Mexican vineyards. The taxonomy of Lasiodiplodia was recently revised, adding new species, and some were reduced to synonymy. This study aimed to characterize Lasiodiplodia producing grapevine dieback symptoms in Sonora and Baja California, Mexico. Using the phylogenetic markers tef1-α and ITS regions, Lasiodiplodia brasiliensis, L. crassispora, L. exigua, and L. gilanensis were identified. Lasidiplodia exigua was the most prevalent species. Lasiodiplodia brasiliensis and L. gilanensis were very virulent to ‘Cabernet Sauvignon’ plants, while L. exigua and L. gilanensis were less virulent, and L. crassispora did not produce lesions at 2 months post-inoculation. The optimum temperature of the Lasiodiplodia spp. was 28°C, but all four species grew up to 37°C, and the isolates of L. exigua grew slowly at 40°C. This is the first report of the four of Lasiodiplodia species in vineyards of Mexico.EEA MendozaFil: Rangel Montoya, Edelweiss A. Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE). Departamento de Microbiología; MéxicoFil: Paolinelli, Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paolinelli, Marcos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Rolshausen, Philippe E. University of California, Riverside. Department of Botany and Plant Sciences; Estados UnidosFil: Valenzuela Solano, Cesar. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Campo Experimental Costa de Ensenada; MéxicoFil.: Hernandez Martinez, Rufina. Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE). Departamento de Microbiología; Méxic

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens

BackgroundTrunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development.ResultsThe integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution.ConclusionsThis study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management

Draft Genome Sequence of Neofusicoccum parvum Isolate UCR-NP2, a Fungal Vascular Pathogen Associated with Grapevine Cankers.

Neofusicoccum parvum, a member of the Botryosphaeriaceae family, is a vascular pathogen that causes severe decline and dieback symptoms in grapevines worldwide. The draft genome of the grapevine isolate N. parvum UCR-NP2 provides a first glimpse into the complex set of putative virulence factors that this pathogen may use to rapidly colonize plants

Xylem Vessel Diameter Affects the Compartmentalization of the Vascular Pathogen Phaeomoniella chlamydospora in Grapevine

Fungal wilt diseases are a threat to global food safety. Previous studies in perennial crops showed that xylem vessel diameter affects disease susceptibility. We tested the hypothesis that xylem vessel diameter impacts occlusion processes and pathogen compartmentalization in Vitis vinifera L. We studied the interaction between four grape commercial cultivars with the vascular wilt pathogen Phaeomoniella chlamydospora. We used qPCR and wood necrotic lesion length to measure fungal colonization coupled with histological studies to assess differences in xylem morphology, pathogen compartmentalization, and fungal colonization strategy. We provided evidence that grape cultivar with wide xylem vessel diameter showed increased susceptibility to P. chlamydospora. The host response to pathogen included vessel occlusion with tyloses and gels, deposition of non-structural phenolic compounds and suberin in vessel walls and depletion of starch in parenchyma cells. Pathogen compartmentalization was less efficient in wide xylem vessels than in narrow diameter vessels. Large vessels displayed higher number of tyloses and gel pockets, which provided substrate for P. chlamydospora growth and routes to escape occluded vessels. We discuss in which capacity xylem vessel diameter is a key determinant of the compartmentalization process and in turn grape cultivar resistance to disease caused by P. chlamydospora