Evaluating treatments for the protection of grapevine pruning wounds from natural infection by trunk disease fungi.

Infection of grapevines by fungal pathogens causing grapevine trunk diseases (GTDs) primarily arises from annual pruning wounds made during the dormant season. While various studies have showcased the efficacy of products in shielding pruning wounds against GTDs infections, most of these investigations hinge on artificial pathogen inoculations, which may not faithfully mirror real field conditions. This study aimed to evaluate and compare the efficacy of various liquid formulation fungicides (pyraclostrobin + boscalid) and paste treatments, as well as biological control agents (BCA: Trichoderma atroviride SC1, T. atroviride I-1237, and T. asperellum ICC012 + T. gamsii ICC080), for their potential to prevent natural infection of grapevine pruning wounds by trunk disease fungi in two field trials located in Samaniego (Northern Spain) and Madiran (Southern France) over three growing seasons. Wound treatments were applied immediately after pruning in February. One year after pruning, canes were harvested from vines and brought to the laboratory for assessment of Trichoderma spp. and fungal trunk pathogens. More than 1,200 fungal isolates associated with five GTDs (esca, Botryophaeria, Diaporthe and Eutypa diebacks, and Cytospora canker) were collected from the two vineyards each growing season. Our findings reveal that none of the products under investigation exhibited complete effectiveness against all the GTDs. The efficacy of these products was particularly influenced by the specific year of study. A notable exception was observed with the biocontrol agent T. atroviride I-1237, which consistently demonstrated effectiveness against Botryosphaeria dieback infections throughout each year of the study, irrespective of the location. The remaining products exhibited efficacy in specific years or locations against particular diseases, with the physical barrier (paste) showing the least overall effectiveness. The recovery rates of Trichoderma spp. in treated plants were highly variable, ranging from 17% to 100%, with both strains of T. atroviride yielding the highest isolation rates. This study underscores the importance of customizing treatments for specific diseases, taking into account the influence of environmental factors for BCA applications.

Droplet Digital PCR Technology for Detection of Ilyonectria liriodendri from Grapevine Environmental Samples.

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.

Quantification of Cadophora luteo-olivacea From Grapevine Nursery Stock and Vineyard Soil Using Droplet Digital PCR.

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 ß-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.

Occurrence and Diversity of Black-Foot Disease Fungi in Symptomless Grapevine Nursery Stock in Spain.

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 ß-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.

Exploring the Temporal Dynamics of the Fungal Microbiome in Rootstocks, the Lesser-Known Half of the Grapevine Crop.

Rootstocks are the link between the soil and scion in grapevines, can provide tolerance to abiotic and biotic stresses, and regulate yield and grape quality. The vascular system of grapevine rootstocks in nurseries is still an underexplored niche for research, despite its potential for hosting beneficial and pathogenic microorganisms. The purpose of this study was to investigate the changes in the composition of fungal communities in 110 Richter and 41 Berlandieri rootstocks at four stages of the grapevine propagation process. Taxonomic analysis revealed that the fungal community predominantly consisted of phylum Ascomycota in all stages of the propagation process. The alpha-diversity of fungal communities differed among sampling times for both rootstocks, with richness and fungal diversity in the vascular system decreasing through the propagation process. The core microbiome was composed of the genera Cadophora, Cladosporium, Penicillium and Alternaria in both rootstocks, while the pathogenic genus Neofusicoccum was identified as a persistent taxon throughout the propagation process. FUNguild analysis showed that the relative abundance of plant pathogens associated with trunk diseases increased towards the last stage in nurseries. Fungal communities in the vascular system of grapevine rootstocks differed between the different stages of the propagation process in nurseries. Numerous genera associated with potential biocontrol activity and grapevine trunk diseases were identified. Understanding the large diversity of fungi in the rootstock vascular tissue and the interactions between fungal microbiota and grapevine will help to develop sustainable strategies for grapevine protection.

Drought Influences Fungal Community Dynamics in the Grapevine Rhizosphere and Root Microbiome.

Plant roots support complex microbial communities that can influence nutrition, plant growth, and health. In grapevine, little is known about the impact of abiotic stresses on the belowground microbiome. In this study, we examined the drought-induced shifts in fungal composition in the root endosphere, the rhizosphere and bulk soil by internal transcribed spacer (ITS) high-throughput amplicon sequencing (HTAS). We imposed three irrigation regimes (100%, 50%, and 25% of the field capacity) to one-year old grapevine rootstock plants cv. SO4 when plants had developed 2-3 roots. Root endosphere, rhizosphere, and bulk soil samples were collected 6- and 12-months post-plantation. Drought significantly modified the overall fungal composition of all three compartments, with the root endosphere compartment showing the greatest divergence from well-watered control (100%). The overall response of the fungal microbiota associated with black-foot disease (Dactylonectria and "Cylindrocarpon" genera) and the potential biocontrol agent Trichoderma to drought stress was consistent across compartments, namely that their relative abundances were significantly higher at 50-100% than at 25% irrigation regime. We identified a significant enrichment in several fungal genera such as the arbuscular mycorrhizal fungus Funneliformis during drought at 25% watering regime within the roots. Our results reveal that drought stress, in addition to its well-characterized effects on plant physiology, also results in the restructuring of grapevine root microbial communities, and suggest the possibility that members of the altered grapevine microbiota might contribute to plant survival under extreme environmental conditions.

Field evaluation of biocontrol agents against black-foot and Petri diseases of grapevine.

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.