Characterization of Cadophora luteo-olivacea and C. melinii isolates obtained from grapevines and environmental samples from grapevine nurseries in Spain

[EN] Fifty-eight Cadophora luteo-olivacea and three C. melinii isolates were recovered from grapevines showing black vascular streaking and decline symptoms characteristic of Petri disease, and from different stages of the grapevine nursery process in Spain. The isolates were studied by means of phenotypical characterization, DNA analysis and pathogenicity tests. The morphological characters studied included conidiophore, phialide and conidial morphology. Colony characters and pigment production on MEA, PDA and OA were also examined. Phenotypical data were subjected to cluster analysis, which clearly separated C. luteo-olivacea isolates into four groups. Mating tests were performed on all possible combinations for each Cadophora species but no sexual fruiting bodies were produced. Partial sequences of the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (BT) and the elongation factor 1¿ (EF) were analysed, but no genetic variation occurred within the C. luteo-olivacea isolates or within the C. melinii isolates in any of the regions studied. Pathogenicity tests were conducted on 1-year-old grapevine cuttings of four different rootstocks using four C. luteo-olivacea isolates and one isolate of C. melinii. All Cadophora isolates except the C. melinii isolate caused significantly longer lesions in the xylem of grapevine rootstocks than in the controls.This research was financially supported by the Projects AGL2006-11884-C04-01 (Ministerio de Educación y Ciencia, Spain), RTA2007-00023-C04-03 and RTA2010-00009-C03-03 (Programa Nacional de Recursos y Tecnologías Agrarias, Ministerio de Educación y Ciencia, Spain), and the European Regional Development Fund (ERDF). We thank V. Garrigues for technical assistance.Gramaje, D.; Mostert, L.; Armengol Fortí, J. (2011). Characterization of Cadophora luteo-olivacea and C. melinii isolates obtained from grapevines and environmental samples from grapevine nurseries in Spain. Phytopathologia Mediterranea. 50(Supplement):112-126. https://doi.org/10.14601/Phytopathol_Mediterr-8723S11212650Supplemen

Identifying practices likely to have impacts on grapevine trunk disease infections: a European nursery survey

A questionnaire covering all aspects of grapevine propagation including cultural and sanitation practices in mother blocks and harvest and transport of cuttings from mother blocks to nurseries, nursery operations and field nursery management, was mailed to all Management Committee members of the European COST Action FA1303 “Sustainable Control of Grapevine Trunk Diseases” for distribution to the identifiable nurseries in each European country. The main objective was to develop understanding of the current propagation practices and to identify those likely to have the greatest impacts on the quality of planting material, especially with regard to the control measures used against fungal trunk pathogen infections. The questionnaire was sent to 666 vine nurseries, and 146 replies were received (21.9% response rate) The study identified several risks factors which could increase infection by fungal trunk pathogens during the propagation processes, as well as a clear need for further research into the effects of treatments on grapevine viability, including hot water treatment, and the potential of biological agents and other strategies such as ozonation to control grapevine trunk diseases in nurseries

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

Diversity of Phytophthora Species Associated with Quercus ilex L. in Three Spanish Regions Evaluated by NGS

[EN] The diversity of Phytophthora species in declining Fagaceae forests in Europe is increasing in the last years. The genus Quercus is one of the most extended Fagaceae genera in Europe, and Q. ilex is the dominant tree in Spain. The introduction of soil-borne pathogens, such as Phytophthora in Fagaceae forests modifies the microbial community present in the rhizosphere, and has relevant environmental and economic consequences. A better understanding of the diversity of Phytophthora spp. associated with Q. ilex is proposed in this study by using Next Generation Sequencing (NGS) in six Q. ilex stands located in three regions in Spain. Thirty-seven Phytophthora phylotypes belonging to clades 1 to 12, except for clades 4, 5 and 11, are detected in this study, which represents a high diversity of Phytophthora species in holm oak Spanish forests. Phytophthora chlamydospora, P. citrophthora, P. gonapodyides, P. lacustris, P. meadii, P. plurivora, P. pseudocryptogea, P. psychrophila and P. quercina were present in the three regions. Seven phylotypes could not be associated with known Phytophthora species, so they were putatively named as Phytophthora sp. Most of the detected phylotypes corresponded to terrestrial Phytophthora species but aquatic species from clades 6 and 9 were also present in all regions.We would like to thank M. Leon from the Instituto Agroforestal Mediterraneo-UPV (Spain) for its technical assistance. This research was supported by funding from the project AGL2011-30438-C02-01 (Ministerio de Economia y Competitividad, Spain) and Euphresco [Instituto Nacional de Investigacion y Tecnologia Agraria y Agroalimentaria (EUPHESCO-CEP: "Current and Emerging Phytophthoras: Research Supporting Risk Assesssment and Risk Management")].Mora-Sala, B.; Gramaje Pérez, D.; Abad Campos, P.; Berbegal Martinez, M. (2019). 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Evaluation of fungicides to protect pruning wounds from Botryosphaeriaceae species infections on almond trees

In vitro efficacy of ten fungicides was evaluated against four Botryosphaeriaceae spp. (Diplodia seriata, Neofusicoccum luteum, N. mediterraneum and N. parvum) associated with branch cankers on almond trees. Cyproconazole, pyraclostrobin, tebuconazole, and thiophanate-methyl were effective for the inhibition of mycelial growth of most of these fungi. An experiment on 3-year-old almond trees evaluated boscalid, mancozeb, thiophanate-methyl, pyraclostrobin and tebuconazole for preventative ability against infections caused by the four pathogens. Five months after pruning and fungicide application, lesion length measurements and isolation percentages showed no significant differences among the four pathogens after they were inoculated onto the trees, and also between the two inoculation times tested (1 or 7 d after fungicide application). Thiophanate-methyl was the most effective fungicide, resulting in the shortest lesion lengths and the lowest isolation percentages from artificially inoculated pruning wounds. This chemical is therefore a candidate for inclusion in integrated disease management, to protect pruning wounds from infections caused by species of Botryosphaeriaceae. This study represents the first approach to development of chemical control strategies for the management of canker diseases caused by Botryosphaeriaceae fungi on almond trees.

Temporal dispersal patterns of Phaeomoniella chlamydospora, causal agent of Petri disease and esca, in vineyards

[EN] Although the fungus Phaeomoniella chlamydospora is the most commonly detected causal agent of Petri disease and esca, two important fungal grapevine trunk diseases, little is known about the dispersal patterns of P. chlamydospora inoculum. In this work, we studied the dispersal of P. chlamydospora airborne inoculum from 2016 to 2018 in two viticultural areas of eastern (Ontinyent) and northern (Logroño) Spain. The vineyards were monitored weekly from November to April using microscope slide traps, and P. chlamydospora was detected and quantified by a specific real-time quantitative (qPCR) method set up in this work. The method was found to be sensitive, and a good correlation was observed between numbers of P. chlamydospora conidia (counted by microscope) and DNA copy numbers (quantified by qPCR). We consistently detected DNA of P. chlamydospora at both locations and in all seasons but in different quantities. In most cases, DNA was first detected in the last half of November, and most of the DNA was detected from December to early April. When rain was used as a predictor of P. chlamydospora DNA detection in traps, false-negative detections were observed, but these involved only 4% of the total. The dispersal pattern of P. chlamydospora DNA over time was best described (R2 = 0.765 and concordance correlation coefficient = 0.870) by a Gompertz equation, with time expressed as hydrothermal time (a physiological time accounting for the effects of temperature and rain). This equation could be used to predict periods with a high risk of dispersal of P. chlamydosporaFinancial support for carrying out this research was provided by transnational funding bodies, being partners of the H2020 ERA-net project, CORE Organic Cofund, and the Cofund from the European Commission (PCI2018-093015/Project BIOVINE). Part of the research was funded by CAR (Government of La Rioja, Spain), project number R-03-16. C. Berlanas was supported by the FPI-INIA program from the INIA. D. Gramaje was supported by the Ramon y Cajal program, Spanish Government (RYC-2017-23098). Financial support for C. Berlanas during her 1-month stay at Universitat Polit`ecnica de Val`encia was provided by the Viticulture Research Network RedVitis (AGL2015-70931-REDT)González-Domínguez, E.; Berlanas, C.; Gramaje, D.; Armengol Fortí, J.; Rossi, V.; Berbegal Martinez, M. (2020). Temporal dispersal patterns of Phaeomoniella chlamydospora, causal agent of Petri disease and esca, in vineyards. Plant Disease. 110(6):1216-1225. https://doi.org/10.1094/PHYTO-10-19-0400-RS12161225110

Cultivar Contributes to the Beneficial Effects of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to Protect Grapevine Against Neofusicoccum parvum

[EN] Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective chemicals, biocontrol strategies are developed as alternatives to better cope with environmental concerns. A combination of biological control agents (BCAs) could even improve sustainable disease management through complementary ways of protection. In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their combination were then characterized in planta, as well as their direct benefits in vitro. Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation test. We also report for the first time the beneficial potential of a combination of BCA against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1) the high basal expression of SA-dependent defenses in Tempranillo explains its highest susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial Bs PTA-271 and Ta SC1 remain to be further investigated.Funding This work was supported by a French Grant from the Region GRAND-EST France and the City of GRAND-REIMS France through the BIOVIGNE Ph.D. program, whose functioning is supported by BELCHIM Crop Protection France. DG was supported by the Ramon y Cajal program, Spanish Government (RyC-2017-23098).Leal, C.; Richet, N.; Guise, J.; Gramaje, D.; Armengol Fortí, J.; Fontaine, F.; Trotel-Aziz, P. (2021). Cultivar Contributes to the Beneficial Effects of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to Protect Grapevine Against Neofusicoccum parvum. Frontiers in Microbiology. 12:1-17. https://doi.org/10.3389/fmicb.2021.726132S1171

The road to molecular identification and detection of fungal grapevine trunk diseases

Grapevine is regarded as a highly profitable culture, being well spread worldwide and mostly directed to the wine-producing industry. Practices to maintain the vineyard in healthy conditions are tenuous and are exacerbated due to abiotic and biotic stresses, where fungal grapevine trunk diseases (GTDs) play a major role. The abolishment of chemical treatments and the intensification of several management practices led to an uprise in GTD outbreaks. Symptomatology of GTDs is very similar among diseases, leading to underdevelopment of the vines and death in extreme scenarios. Disease progression is widely affected by biotic and abiotic factors, and the prevalence of the pathogens varies with country and region. In this review, the state-of-the-art regarding identification and detection of GTDs is vastly analyzed. Methods and protocols used for the identification of GTDs, which are currently rather limited, are highlighted. The main conclusion is the utter need for the development of new technologies to easily and precisely detect the presence of the pathogens related to GTDs, allowing to readily take phytosanitary measures and/or proceed to plant removal in order to establish better vineyard management practices. Moreover, new practices and methods of detection, identification, and quantification of infectious material would allow imposing greater control on nurseries and plant exportation, limiting the movement of infected vines and thus avoiding the propagation of fungal inoculum throughout wine regionsinfo:eu-repo/semantics/publishedVersio

High-throughput amplicon sequencing-based analysis of active fungal communities inhabiting grapevine after hot-water treatments reveals unexpectedly high fungal diversity

[EN] The ecology of total fungal communities in grapevine is so far largely derived from studies on culture-dependent methods or cultivation-independent rDNA approaches. Sequencing the ribosomal RNA transcripts (rRNA) would rather reveal the functionally and metabolically active important taxa of the fungal community and provide insights into its activity in the wood. The present study investigated changes in the potentially active fungal communities of internal grapevine wood after Hot-Water Treatment (HWT) in planting material from Czech Republic and Spain at two different times during the propagation process and from two plant zones. We examined fungal communities using both traditional isolation into culture and high-throughput amplicon sequencing (HTAS) of the internal transcribed spacer 2 (ITS2) region in extracted total RNA. HTAS from metatranscriptomic RNA increased the resolution of the fungal community analysis and revealed a highly diverse mycobiota of grapevine wood compared to the traditional method. Fungal diversity differed between grapevine genotypes and showed a temporal variation over the vegetative period. Grapevine planting materials exhibited high fungal diversity after HWT, which demonstrates that the HWT process does not sterilize the internal wood of grapevine. HWT reduced the infection caused by fungal trunk disease pathogens but was not completely effective in eliminating their growth. This study provides important and practically useful insights into the dynamics of active fungal communities in hot-water treated plants, and represents the first study of active fungal communities on grapevine grafted plants by comparing traditional and next-generation sequencing methods. (C) 2018 Elsevier Ltd and British Mycological Society. All rights reserved.This work was supported by Projects No. LD14051 from the Ministry of Education, Youth and Sports, Czech Republic covered by COST Action FA1303, and No. CZ.02.1.01/0.0/0.0/16_025/0007314 "Multidisciplinary research to increase application potential of nanomaterials in agricultural practice" from EFRR (Grant provider: Ministerstvo Skolstvi, mladeze a telovychovy Ceske republiky (MSMT). Access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum provided under the programme "Projects of Large Research, Development, and Innovations Infrastructures" (CESNET LM2015042), is greatly appreciated. David Gramaje was supported by the DOC-INIA program from the National Institute for Agronomic Research (INIA), co-funded by the European Social Fund. The authors are grateful for the technical assistance of Jesus Alfonso Crespo and Aitor Gavara Vidal.Eichmeier, A.; Pecenka, J.; Penazova, E.; Baranek, M.; Català, S.; León Santana, M.; Armengol Fortí, J.... (2018). High-throughput amplicon sequencing-based analysis of active fungal communities inhabiting grapevine after hot-water treatments reveals unexpectedly high fungal diversity. Fungal Ecology. 36:26-38. https://doi.org/10.1016/j.funeco.2018.07.011S26383