First report of Dactylonectria alcacerensis, Dactylonectria macrodidyma and Ilyonectria liriodendri associated with black-foot disease of grapevine in Argentina

Black-foot is a fungal disease that affects young vines and planting material in the countries where grapevines are cultivated. During the 2018 grape-growing season, symptoms of reduced vigor, short internodes, leaf chlorosis, root rot and necrosis in the base of plants were observed in vines between 2 to 10 years old from 400 ha of vineyards of Mendoza and Salta regions. A total of 30% of the vineyards of cultivars Malbec, Cabernet Sauvignon and Aspirant Bouchet, and rootstocks 101-14 Millardet et de Grasset and 1103 Paulsen, had vines with disease symptoms. Vines had 15% disease incidence and 30% disease severity. These symptoms have been described as characteristic of black-foot, caused by "Cylindrocarpon"-like asexual morph fungi. Isolations were made from roots and basal part of 30 symptomatic plants. Fragments of advanced necrotic tissue were washed with running water, surface sterilized with 70% alcohol for 20 seconds, 2% NaOCl solution for 4 minutes and rinsed in sterile distilled water twice. These fragments were placed onto Potato Dextrose Agar (PDA) and incubated at 25°C in the dark for 10 days to isolate fungal pathogens. A total of 50 colonies resembling black-foot disease pathogens were subcultured onto fresh PDA in order to obtain single spore cultures. The isolates developed buff to cinnamon and dark brown, and felty mycelium. Colonies subcultured in synthetic nutrient agar (SNA) produced hyaline, cylindrical, straight and/or slightly curved with one to three septate (35.6 × 5.3 µm) macroconidia and abundant, hyaline, ellipsoidal and zero to one septate (12.5 × 6.0 µm) microconidia. Cultures and conidia morphological characteristics were similar to those of Ilyonectria or Dactylonectria genera (Cabral et al. 2012a; Lombard et al. 2014). DNA sequence analysis of the partial histone H3 gene was obtained for isolates INTA SC1 (I. liriodendri), INTA LC2 (D. alcacerensis) and INTA LC1 (D. macrodidyma) and deposited in GenBank (accession nos. OK338901, OK338900 and OK338899). The BLAST search was conducted against type specimens. Sequences showed high similarity (99% to 100%) to the sequences of Ilyonectria liriodendri (Halleen, Rego & Crous) Chaverri & Salgado (GenBank accession no. JF735509), Dactylonectria alcacerensis (A. Cabral, Oliveira & Crous) L. Lombard & Crous (GenBank accession no. JF735630) and Dactylonectria macrodidyma (Halleen, Schroers & Crous) L. Lombard & Crous (GenBank accession no. JF735647). Pathogenicity tests were conducted under greenhouse conditions on 1-year-old rooted canes cv Malbec using the three isolates. Ten rooted cuttings with pruned roots were immersed in a suspension of 106 conidia ml-1 of each isolate for 60 min (Cabral et al. 2012b) while control cuttings were immersed in sterile distilled water. Immediately they were planted in pots and kept in a greenhouse at a temperature between 25 to 30°C. Symptoms developed on all plants 4 months after inoculation and consisted in necrotic lesions of roots and in the base of the canes, with a reduction in root biomass. Plants did not develop aerial symptoms. All fungi were re-isolated only from necrotic lesions of root and base of inoculated canes, fulfilling Koch's postulates. No symptoms were observed on the control plants. To our knowledge, this is the first report of the species I. liriodendri, D. alcacerensis and D. macrodidyma associated with black-foot disease of grapevine in Argentina. Black-foot disease has a negative effect on the viability of planting material and young vines, and this report will assist with monitoring distribution of the disease as well as developing management recommendations to nurseries and grape growers in Argentina.EEA MendozaFil: Longone, Maria Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Escoriaza, Maria Georgina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Paolinelli, Marcos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Gramaje, David. Universidad de la Rioja. Instituto de Ciencias de la Vid y del Vino; España. Consejo Superior de Investigaciones Científicas. Instituto de Ciencias de la Vid y del Vino; Españ

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

Growth response of Saccharomyces cerevisiae strains to stressors associated to the vine cycle

Saccharomyces cerevisiae isolates from grapes, soil, vine bark and buds collected at seven phenological stages of an annual growth cycle, were molecular typed by Microsatellite Multiplex PCR. Subsequently 30 S. cerevisiae genotypes were selected and the effect of vineyard environmental stressors, in both sublethal upper and lower levels, on their growth parameters was evaluated. The effect of low and high temperature (7–40 ◦C), pH (2.5–8.0), glucose concentration (3.0–300.0 g/L), nitrogen concentration (0.008–8.0 g/L), and copper presence (24 mg/L) were modelled individually using the reparametrized Gompertz equation. Multivariate ANOVA and Generalized Procrustes Analysis were used to determine the environmental stressor’s influence over the lag phase (λ) and the maximum specific growth rate (μmax). Both parameters were significantly affected by the S. cerevisiae genotype, the treatments, and the interaction between them. Despite a generalized reduction in μmax and a variable answer in λ, the 30 S. cerevisiae genotypes were able to overcome all the treatments. Extreme glucose limitation, copper presence and low temperature had the highest impact over the growth parameters. Interestingly, ten genotypes mostly distributed in the vineyard were the least affected, suggesting a greater acclimatization fitness and the possibility to persist in the changing conditions of the vine annual cycle.EEA MendozaFil: Gonzalez, Magali Lucia Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez, Magali Lucia Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Valero, Eva. Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería Bioquímica; EspañaFil: Chimeno, Selva Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Garrido Fernandez, Antonio. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos, Instituto de la Grasa (IG); España. Consejo Superior de Investigaciones Científicas (CSIC); EspañaFil: Rodriguez Gomez, Francisco. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos, Instituto de la Grasa (IG); España. Consejo Superior de Investigaciones Científicas (CSIC); EspañaFil: Rojo, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Rojo, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paolinelli, Marcos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Arroyo Lopez, Francisco Noe. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos, Instituto de la Grasa (IG); España. Consejo Superior de Investigaciones Científicas (CSIC); EspañaFil: Combina, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Mercado, Laura Analia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; Argentin

Occurrence of Eutypella microtheca in grapevine cankers in Mexico

Trunk diseases cause slow decline and loss in productivity of grapevine. In surveys carried out from 2010 to 2012 in vineyards in Baja California (Mexico), fungal isolates were obtained from Cabernet Sauvignon grapevines with cankers. Morphological studies and phylogenetic analyses, using the internal transcribed spacer (ITS) region of the rDNA and a partial sequence of the β-tubulin gene, identified two isolates as Eutypella microtheca. Pathogenicity tests, using grapevine green tissue, fulfilled Koch´s postulates. To our knowledge, this is the first report of E. microtheca occurring on grapevine in Mexico and highlights the need of considering this fungus as another potential threat to vineyards in this region

Lasiodiplodia gilanensis used as model for understanding the pathogenicity of Botryosphaeriaceae

Lasiodiplodia spp. cause degenerative diseases, dieback and plant death in a wide range of woody plants. To understand the fungal behavior of the genus we are using Lasiodiplodia gilanensis as a model. A transcriptional study revealed that the fungus is capable of using the phenylpropanoid precursors and salicylic acid to avoid the host defense response of the plant. On another hand, several genes encoding enzymes involved in different melanin synthesis pathways; 3,4-dihydroxyphenylalanine (DOPA)-melanin, 1,8-dihydroxynaphthalene (DHN)-melanin and pyomelanin; were identified and their production evauated, concluding that the fungus use diferent types of melanin to overcome environmental stress. An in silico analysis shows the presence of those genes in all the available genomes of Botryosphaeriaceae in the GeneBank, evidencing the importance of the melanin in this family. L. gilanensis produces siderophores of catechol and hydroxamate-type as well as naturally esterified fatty acids that might have a role in plant growth regulation. A plethora of hydrolytic enzymes is also produced, including xylanases, ligninases, cellulases, pectinases, cutinases, and hemicelluloses. Another secondary metabolite produced is oxalic acid, reported as a pathogenicity factor in other fungi, which role is under evaluation. Microscopical observations showed that the fungus uses the starch deposited in the ray cells as carbon source, induces the production of suberin and phenolic compounds and colonizes the vascular cambium, ray parenchyma, and the vascular bundles. These studies extend our understanding of the pathogenicity of a widely distributed pathogenic fungus with our final goal to control the diseases it causes.Fil: Rangel Montoya Edelweiss. Consejo Nacional de Ciencia y Tecnología de México. Centro de Investigación Científica y de Educación Superior de Ensenada Baja California; MéxicoFil: Paolinelli, Marcos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria Mendoza; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hernández Martínez Rufina. Consejo Nacional de Ciencia y Tecnología de México. Centro de Investigación Científica y de Educación Superior de Ensenada Baja California; México11th International Workshop on grapevine trunk diseasesPentictonCanadáOregon Wine Research Institut

Characterization of grapevine wood microbiome through a metatranscriptomic approach

Grapevine trunk diseases threaten wine and table grape production worldwide, primarily by reducing yields and, in its advanced stages, causing plant death. Among those diseases, the complex etiology disease known as hoja de malvón (HDM) significantly concerns Argentinian and Uruguayan viticulture. At least four different fungi are associated with this disease, but their role and interactions with other wood microorganisms are understudied. In this sense, analyzing grapevine wood microbiome composition could help understand microbial interactions occurring in HDM onset. Hence, a metatranscriptomic study was performed for the microbiome characterization of mature field-grown Vitis vinifera cv. Malbec, leaf-symptomatic or leaf-asymptomatic. The microbiome was mainly represented by Dothideomycetes and Actinobacteria. In the plant with more marked symptoms, higher levels of the Basidiomycota Arambarria destruens and Phellinus laevigatus were detected. Despite this particular difference, discriminating symptomatic from asymptomatic plants based on the presence or abundance of HDM pathogens was not possible. Alpha diversity and rank-abundance curve analyses indicated that plants with foliar symptoms have lower microbial evenness than asymptomatic plants. The co-occurrence network modeled microbial interkingdom interactions. Molecular data generated in this study will help develop future targeted molecular quantification for specific taxa.EEA MendozaFil: Paolinelli, Marcos. Instituto Nacional de Investigación Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Escoriaza, Maria Georgina. Instituto Nacional de Investigación Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Cesari, Cecilia Alicia. Instituto Nacional de Investigación Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Garcia Lampasona, Sandra Claudia. Instituto Nacional de Investigación Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Garcia Lampasona, Sandra Claudia. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias, Instituto de Biología Agrícola de Mendoza (IBAM), Mendoza, ArgentinaFil: Garcia Lampasona, Sandra Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mendoza; ArgentinaFil: Hernandez Martinez, Rufina. Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE); Méxic

The Role of Diversity in Mediating Microbiota Structural and Functional Differences in Two Sympatric Species of Abalone Under Stressed Withering Syndrome Conditions

Withering syndrome (WS) is a gastro-intestinal (GI) infectious disease likely affecting all abalone species worldwide. Structural and functional changes in abalone GI microbiotas under WS-stressed conditions remain poorly investigated. It is unclear if interspecific microbiota differences, such as the presence of certain microbes, their abundance, and functional capabilities, may be involved in the occurrence of this disease. Bacterial microbiotas of healthy Haliotis fulgens and Haliotis corrugata are mainly composed by Tenericutes, Proteobacteria, Fusobacteria, and Spirochaetes. We previously reported species-specific structural and functional profiles of those communities and suggested that they are of consequence to the different susceptibility of each species to WS. Here, we address this question by comparing the structure and function of healthy and dysbiotic microbiota through 454 pyrosequencing and PICRUSt 2, respectively. Our findings suggest that the extent to which WS-stressed conditions may explain structural and functional differences in GI microbiota is contingent on the microbiota diversity itself. Indeed, microbiota differences between stressed and healthy abalone were marginal in the more complex bacterial communities of H. corrugata, in which no significant structural or functional changes were detected. Conversely, significant structural changes were observed in the less complex bacterial microbiota of H. fulgens. Moreover, structural alterations led to a significant downregulation of some metabolic activities conducted by GI bacteria. Accordingly, results suggest that gastro-intestinal bacterial diversity appears to be related with both the health of abalone and the etiology of WS.EEA MendozaFil: Cicala, Francesco. CICESE. Department of Biological Oceanography; MéxicoFil: Cicala, Francesco. CICESE. Department of Biomedical Innovation; MéxicoFil: Cisterna-Céliz, José Alejandro. CICESE. Department of Biological Oceanography; MéxicoFil: Paolinelli, Marcos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Paolinelli, Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moore, James D. University of California at Davis. Bodega Marine Laboratory; Estados UnidosFil: Sevigny, Joseph. University of New Hampshire. Hubbard Center for Genome Studies; Estados UnidosFil: Rocha-Olivares, Axayácatl. CICESE. Department of Biological Oceanography; Méxic

Prioritization of vigor QTL-associated genes for future genome-directed Vitis breeding: Priorización de genes relacionados a vigor en Vitis

Vigor control in grapevine may become especially important under climate change. A better understanding of gene-phenotype relationships is required in order to exploit plant genomics for breeding purposes. This research aims to use quantitative trait loci (QTLs) for vigor identified in the progeny from a cross of Ramsey (Vitis champinii) × Riparia Gloire (V. riparia). Genes located 700 kb up and downstream from each QTL position were interrogated for functional enrichment through ShinyGO online tool, based on the gene ontology annotation of Vitis vinifera PN40024. Key biological processes like phloem and xylem development, cell cycle, response to hormones, amino acid transport, tissue development, sugar metabolism, nitrogen transport, and stress/immune responses, showed functional enrichment. Integral response to light and auxin might be required for fine molecular tuning of vegetative growth in Vitis. Fifty out of 1318 candidate genes were prioritized, reducing their amount to a manageable number of candidates for further directed breeding strategies. Highlights Plant vigor control may become especially important under climate change. Genes from various vigor-related QTLs were interrogated for functional enrichment. The analysis reduced candidate gene number based on marker proximity and functional enrichment, constituting a suitable shortcut for target-directed genome-guided breeding strategies. Three TFs are strong candidates for targeted breeding: TIF - HY5, TIF - SUS1, TIF - VOZ1 potentially enhance growth by relating light response to hormone activation, and then to photosynthesis and morphogenesis.  Vigor control in grapevine may become especially important under climate change. A better understanding of gene-phenotype relationships is required in order to exploit plant genomics for breeding purposes. This research aims to use quantitative trait loci (QTLs) for vigor identified in the progeny from a cross of Ramsey (Vitis champinii) × Riparia Gloire (V. riparia). Genes located 700 kb up and downstream from each QTL position were interrogated for functional enrichment through ShinyGO online tool, based on the gene ontology annotation of Vitis vinifera PN40024. Key biological processes like phloem and xylem development, cell cycle, response to hormones, amino acid transport, tissue development, sugar metabolism, nitrogen transport, and stress/immune responses, showed functional enrichment. Integral response to light and auxin might be required for fine molecular tuning of vegetative growth in Vitis. Fifty out of 1318 candidate genes were prioritized, reducing their amount to a manageable number of candidates for further directed breeding strategies. Highlights Plant vigor control may become especially important under climate change. Genes from various vigor-related QTLs were interrogated for functional enrichment. The analysis reduced candidate gene number based on marker proximity and functional enrichment, constituting a suitable shortcut for target-directed genome-guided breeding strategies. Three TFs are strong candidates for targeted breeding: TIF - HY5, TIF - SUS1, TIF - VOZ1 potentially enhance growth by relating light response to hormone activation, and then to photosynthesis and morphogenesis