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Plant compartments provide unique niches that lead to specific microbial associations. The microbiota colonizing the endophytic compartment (endorhizosphere) and the rhizosphere contribute to productivity, plant growth, phytoremediation and carbon sequestration. The main objective of this study was to investigate how fungal communities are enriched in different habitats outside and inside of grapevine roots. For this purpose, the spatial dynamics of the fungal communities associated with three soil-plant compartments (bulk soil, rhizosphere and endorhizosphere) were characterized by ITS high-throughput amplicon sequencing (HTAS). Fungal communities were largely affected in their diversity and composition by soil-plant compartments, whereas the spatial variation (i.e. across five vineyards) was low. The endorhizosphere compartment differed most from the other two, suggesting that the root tissues entail a barrier for fungal colonization. The results of functional prediction via FUNGuild suggested an increase in the relative abundances of potential plant pathogens, endophytes and arbuscular mycorrhiza, and a decrease in wood, dung and undefined saprotrophs from bulk soil towards the endorhizosphere. Roots of asymptomatic vines were a microbial niche that is inhabited by soilborne fungi associated with grapevine trunk diseases, which opens up new perspectives in the study of the endophytic role of these pathogens on grapevines. Results obtained in this study provide helpful information to better know how the host shapes its microbiome and the implications for vineyard productivity and management., David Gramaje was supported by the DOC-INIA program from theINIA, co-funded by the European Social Fund. María del Pilar Martínez-Diz was supported by the FPI-INIA program from the INIA.

Cadophora luteo-olivacea is the most prevalent Cadophora species associated with Petri disease and esca of grapevine. Accurate, early, and specific detection and quantification of C. luteo-olivacea are essential to alert growers and nurseries to the presence of the pathogens in soil and to prevent the spread of this pathogen through grapevine planting material. The aim of this study was to develop molecular tools to detect and quantify C. luteo-olivacea inoculum from environmental samples. Species specific primers based on the β-tubulin gene and a TaqMan probe for droplet digital PCR (ddPCR) and quantitative PCR (qPCR) were first developed to detect and quantify purified DNA of the target fungus. Specificity tests showed that the primers were able to amplify the C. luteo-olivacea DNA (20 isolates) while none of the 29 nontarget fungal species (58 isolates) tested were amplified. The ddPCR was shown to be more sensitive compared with qPCR in the detection and quantification of C. luteo-olivacea at very low concentrations and was further selected to accurately detect and quantify the fungus from environmental samples. Twenty-five of the 94 grafting plants (26.6%) analyzed by ddPCR tested positive to C. luteo-olivacea DNA (>3 copies/µl). C. luteo-olivacea was barely detected from vineyard soils. The procedure employed in this study revealed the presence of the pathogen in symptomless vines, which makes implementation of this technique suitable for certification schemes of C. luteo-olivacea-free grapevine planting material.

The grapevine trunk diseases (GTDs) Botryosphaeria dieback and esca threaten the sustainability of the grapevine industry worldwide. This study aimed to evaluate and compare the efficacy of various liquid (pyraclostrobin + boscalid and thiophanate methyl) and paste (paste + tebuconazole) formulation fungicide treatments, and biological control agents (Trichoderma atroviride SC1 and T. atroviride I-1237), for their potential to prevent infection of grapevine pruning wounds by Diplodia seriata and Phaeomoniella chlamydospora in two field trials over two growing seasons. Treatments were applied to freshly pruned wounds following their label dosages recommendations. After 24 hours, wounds were artificially inoculated with 400 spores of D. seriata or 800 spores of P. chlamydospora. Isolations were made from the treated pruning wounds after 12 months to evaluate the efficacy of the treatments. Fungicide formulations were superior to Trichoderma-based treatments for the control of both pathogens during both growing seasons, with mean percent disease control of 44 to 95% for D. seriata and 46 to 67% for P. chlamydospora. Pyraclostrobin + boscalid was the most effective treatment. Trichoderma atroviride-based treatments did not reduce infection by D. seriata or P. chlamydospora compared to the untreated inoculated control in both vineyards and seasons. This study represents the first vineyard assessment of several chemical and biological treatments to protect pruning wounds against GTDs fungi in Europe and provides growers with tangible preventative control practices to minimize yield losses due to GTDs.

Black-foot disease is one of the most important soilborne diseases affecting planting material in grapevine nurseries and young vineyards. Accurate, early, and specific detection and quantification of black-foot disease causing fungi are essential to alert growers and nurseries to the presence of the pathogens in soil, and to prevent the spread of these pathogens through grapevines using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, efficiency, and specificity of droplet digital PCR (ddPCR) and real-time PCR (qPCR) techniques for the detection and quantification of

[EN] Black-foot disease is one of the most important soilborne diseases affecting planting material in grapevine nurseries and young vineyards. Accurate, early, and specific detection and quantification of black-foot disease causing fungi are essential to alert growers and nurseries to the presence of the pathogens in soil, and to prevent the spread of these pathogens through grapevines using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, efficiency, and specificity of droplet digital PCR (ddPCR) and real-time PCR (qPCR) techniques for the detection and quantification of Ilyonectria liriodendri in bulk and rhizosphere soils, as well as grapevine endorhizosphere. Fungal abundance was not affected by soil-plant fractions. Both techniques showed a high degree of correlation across the samples assessed (R2 = 0.95) with ddPCR being more sensitive to lower target concentrations. Roots of asymptomatic vines were found to be a microbial niche that is inhabited by black-foot disease fungi., David Gramaje was supported by the DOC-INIA program from the INIA, co-funded by the European Social Fund. María del Pilar Martínez-Diz was supported by the FPI-INIA program from the INIA.

[Background] Black‐foot and Petri diseases are the main fungal diseases associated with young grapevine decline. Two field experiments were established to evaluate the preventive effect of two potential biocontrol agents (BCAs), that is Streptomyces sp. E1 + R4 and Pythium oligandrum Po37, and three BCA‐commercial products containing Trichoderma atroviride SC1, Trichoderma koningii TK7 and Pseudomonas fluorescens + Bacillus atrophaeus on fungal infection in grafted plants and plant growth parameters. [Results] The effectiveness of some BCA in reducing the incidence and severity of both diseases was dependent on the plant part analyzed and the plant age. No single BCA application was able to control both diseases. Streptomyces sp. E1 + R4 were able to reduce significantly the infection of the most prevalent black‐foot disease fungi while P. oligandrum Po37 and Trichoderma spp. were able to reduce significantly Phaeomoniella chlamydospora and Phaeoacremonium minimum (Petri disease) infection. BCA treatments had no effect on the shoot weight, and root weight was significantly lower in all BCA treatments with respect to the control. [Conclusions] The combination of the disease‐suppressive activity of two or more beneficial microbes in a biocontrol preparation is required to prevent infection by black‐foot and Petri disease fungi in vineyards.

In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and β-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar ‘Tempranillo’. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines., CAR (Government of La Rioja, Spain), under the project ‘Characterization, epidemiology and control of fungal trunk pathogens of grapevine in La Rioja’ Grant/Award Number: R-03-16

Esca and leaf stripe diseases are two of the most destructive grapevine trunk diseases which are caused by a complex of fungal species, being the Ascomycete Phaeomoniella chlamydospora the most frequently isolated species from affected vines. To date, no curative measures are known for control these diseases; therefore, planting disease-resistant cultivars is a time-tested and sustainable approach for disease management. The aim of this study was to identify sources of resistance to P. chlamydospora among minority and commercial grapevine germplasm collections in Spain. For this purpose, 15 and 38 cultivars from the ITACyL and the EVEGA collections, respectively, were selected based on the visual assessment of symptom expression in standing vines over the last 3 years, and inoculated with P. chlamydospora in a detached cutting assay under greenhouse conditions over two consecutive years. Seven months after inoculation, cuttings were collected and inspected for lesion length. The severity of internal wood symptoms caused by P. chlamydospora varied considerably amongst the cultivars. No foliar symptoms were observed during the experiment. All cultivars were susceptible to fungal necrotic infection, indicating that there is no evidence of qualitative resistance to this Ascomycete fungus., The research was financially supported by The National Institute for Agricultural and Food Research and Technology (INIA) under the project RTA2015-00015-C02-01. David Gramaje and David Ruano-Rosa were supported by the DOC-INIA program from the INIA, co-funded by the European Social Fund. María del Pilar Martínez-Diz was supported by the FPI-INIA program from the INIA. Marcos Andrés-Sodupe was supported by a grant from the Government of La Rioja

The grapevine trunk diseases (GTD) Botryosphaeria dieback and esca threaten the sustainability of the grapevine industry worldwide. This study aimed to evaluate and compare the efficacy of various liquid (pyraclostrobin + boscalid and thiophanate methyl) and paste (paste + tebuconazole) formulation fungicide treatments as well as biological control agents (Trichoderma atroviride SC1 and T. atroviride I-1237), for their potential to prevent infection of grapevine pruning wounds by Diplodia seriata and Phaeomoniella chlamydospora in two field trials over two growing seasons. Treatments were applied to freshly pruned wounds following label dosages’ recommendations. After 24 h, wounds were artificially inoculated with 400 spores of D. seriata or 800 spores of P. chlamydospora. Isolations were made from the treated pruning wounds after 12 months to evaluate the efficacy of the treatments. Fungicide formulations were superior to Trichoderma-based treatments for the control of both pathogens during both growing seasons, with mean percent disease control of 44–95% for D. seriata and 46–67% for P. chlamydospora. Pyraclostrobin + boscalid was the most effective treatment. Trichoderma atroviride-based treatments did not reduce infection by D. seriata or P. chlamydospora compared to the untreated inoculated control in both vineyards and seasons. This study represents the first scientific comparative vineyard assessment of both commercial and biological treatments to protect pruning wounds against GTD fungi in Europe and provides growers with tangible preventative control practices to minimize yield losses due to GTD., This work has been funded by European funds in collaboration with the ‘Ministerio de Agricultura, Pesca y Alimentación (MAPA)’ (Spanish Government) and the ‘Xunta de Galicia’ (Regional Government) through the project ‘EVID: Innovative practices to combat grapevine diseases’ (FEADER 2017/003B). The authors acknowledge Bodegas Godeval for the provision of field sites and technical assistance for this study. We also thank Dr. J. Luque for the provision of Diplodia seriata isolate JL-398. M.P. Martínez-Diz was supported by the FPI-INIA program from the INIA (Spain, CPD2015-0188). D. Gramaje was supported by the Ramón y Cajal program, Spanish Government (RYC-2017-23098).

Timing of pruning may affect wound susceptibility to wood-invading fungi, such as those associated with grapevine trunk diseases (GTDs). This study aimed to determine the effect of pruning time on natural fungal infection in six vineyards in Galicia, Spain, during two periods of three months each, from November to February and from February to May by ITS2 high-throughput amplicon sequencing. Fungal microbiome composition did not differ significantly with year of sampling. Pruned canes harboured a core community of fungal species, which appeared to be independent of the infection period. Accumulated rainfall over 8 and 11 weeks after pruning positively correlated with the total fungal microbiome. A seasonal effect on GTD fungal infection was detected for most genera, with a higher percent detected after pruning in February (winter) as compared with that of pruning in November (mid-autumn). Early pruning is recommended to reduce the infections caused by GTD fungi in Galicia., Funding was provided by Ministerstvo Školství, Mládeže a Tělovýchovy (CZ.02.1.01./0.0/0.0/16_017/0002334).

In June 2017, symptoms of black foot disease were observed on 2-year-old grapevines (Vitis vinifera L.) cultivar Malvasía grafted on 110 Richter rootstock and on 2-year-old grapevines cultivar Garnacha Tintorera grafted on 110 Richter rootstock in Mendavia (Navarra Province, northern Spain) and Requena (Valencia Province, eastern Spain), respectively. Affected plants showed delayed budding with low vigor and leaf chlorosis. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto malt extract agar supplemented with 0.4 g/liter of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days, and all colonies were transferred to potato dextrose agar (PDA). A Cylindrocarpon-like asexual morph fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and incubated at 25°C for 10 days in darkness. The isolates developed buff to cinnamon and felty abundant mycelium. All isolates produced slightly curved one-septate (16.0 to) 21.6 (to 26.5) × (4.5 to) 6.1 (to 7.8) μm, two-septate (19.5 to) 25.9 (to 35.0) × (5.0 to) 6.6 (to 7.9) μm, and three-septate (22.5 to) 31.3 (to 47.5) × (5.5 to) 7.1 (to 8.9) μm macroconidia, and aseptate (4.0 to) 7.7 (to 12.5) × (2.5 to) 3.3 (to 5.4) μm and one-septate (8.5 to) 12.5 (to 17.5) × (3.5 to) 5.1 (to 6.2) μm microconidia. DNA sequence analysis of the partial histone H3 gene was obtained for isolates BV-816 and GIHF-156 and deposited in GenBank (accession nos. MH229866 and MH229867). These sequences showed high similarity (99%) to the sequence of Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous (GenBank accession no. JF735517), in agreement with morphological features (Cabral et al. 2012a). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using both isolates. Fifteen seedlings per isolate were inoculated when two to three leaves had emerged by immersing the roots in a 106/ml conidial suspension for 30 s (Cabral et al. 2012b). Fifteen control plants were inoculated with sterile distilled water. Seedlings were maintained in a growth chamber at 25°C. Symptoms developed on all plants 15 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as I. robusta, completing Koch’s postulates. No symptoms were observed on the control plants. To our knowledge, this is the first report of I. robusta causing black foot disease of grapevine in Spain. I. robusta is one of the Cylindrocarpon-like asexual morphs included in the complex of fungal species belonging to the genera Campylocarpon, Cylindrocladiella, Dactylonectria, Ilyonectria, Neonectria, and Thelonectria, which are associated with black-foot disease, causing untenable economic losses to the grapevine industry in Spain and worldwide