Esca is a disease complex belonging to the grapevine trunk diseases cluster. It comprises five syndromes, three main fungal pathogenic agents and several symptoms, both internal (i.e., affecting woody tissue) and external (e.g., affecting leaves and bunches). The etiology and epidemiology of this disease complex remain, in part, unclear. Some of the points that are still under discussion concern the sudden rise in disease incidence, the simultaneous presence of multiple wood pathogens in affected grapevines, the causal agents and the discontinuity in time of leaf symptoms manifestation. The standard approach to the study of esca has been mostly through culture-dependent studies, yet, leaving many questions unanswered. In this study, we used Illumina^® next-generation amplicon sequencing to investigate the mycobiome of grapevines wood in a vineyard with history of esca. We characterized the wood mycobiome composition, investigated the spatial dynamics of the fungal communities in different areas of the stem and in canes, and assessed the putative link between mycobiome and leaf symptoms. An unprecedented diversity of fungi is presented (289 taxa), including five genera reported for the first time in association with grapevines wood (Debaryomyces, Trematosphaeria, Biatriospora, Lopadostoma, and Malassezia) and numerous hitherto unreported species. Esca-associated fungi Phaeomoniella chlamydospora and Fomitiporia sp. dominate the fungal community, and numerous other fungi associated with wood syndromes are also encountered (e.g., Eutypa spp., Inonotus hispidus). The spatial analysis revealed differences in diversity, evenness and taxa abundances, the unique presence of certain fungi in specific areas of the plants, and tissue specificity. Lastly, the mycobiome composition of the woody tissue in proximity to leaves manifesting ‘tiger stripes’ symptoms of esca, as well as in leaf-symptomatic canes, was highly similar to that of plants not exhibiting any leaf symptomatology. This observation supports the current understanding that leaf symptoms are not directly linked with the fungal communities in the wood. This work builds to the understanding of the microbial ecology of the grapevines wood, offering insights and a critical view on the current knowledge of the etiology of esca.

Epicoccum is a genus of ascomycetes often associated with the mycobiome of grapevines (Vitis vinifera). Epicoccum spp. are found in the soil, phyllosphere, as well as in the wood, where they interact both with the plant and with other endophytes and pathogens. Wood pathogens involved in the esca disease complex, a grapevine trunk disease, are particularly concerning in viticulture, as current control strategies have proven unsatisfactory. This study investigated the interaction among Epicoccum spp. and three esca-associated fungi, with the aim of establishing whether they are suitable candidates for biological control.A screening conducted in vitro, by means of dual culture, revealed that all tested Epicoccum spp. inhibited the growth of pathogens Phaeomoniella chlamydospora and Fomitiporia mediterranea, while only some of them inhibited Phaeoacremonium minimum. Epicoccum layuense E24, identified as the most efficient antagonist, was tested in rooted grapevine cuttings of cultivars Cabernet Sauvignon and Touriga Nacional, under greenhouse conditions, against P. chlamydospora and P. minimum. This study revealed that the inoculation of E. layuense E24 produced a successful colonization of the wood of grapevines; in addition it did not impair the growth of the plants or induce the appearance of symptoms in leaves or in wood. Moreover, grapevines colonized by E. layuense E24 showed a considerable decrease in the wood symptomatology caused by the inoculated pathogens (by 31–82%, depending on the pathogen/grapevine cultivar), as well as a reduction in their frequency of re-isolation (60–74%).Our findings suggest that E. layuense E24 is a promising candidate for its application in biological control, due to its antagonistic interaction with some esca-associated fungal pathogens.

Esca is a major grapevine trunk disease that heavily affects vineyards in the Northern hemisphere. The etiology and epidemiology of this disease have been subject of dispute ever since the earliest disease reports. The reason behind such debate is the presence of multiple internal and external symptoms, as well as several putative and confirmed wood pathogens. While the role of pathogenic fungi, as causal agents of wood symptoms, has been thoroughly assessed, their role in the expression of leaf symptoms remains to be fully elucidated. In this review, we analyzed etiological and epidemiological data, with a special focus on the microbiological aspect of esca and the involvement of Hymenochaetales (Basidiomycota). Vineyard studies have associated leaf symptoms with the presence of white rot, most frequently caused by Fomitiporia mediterranea (Hymenochaetales), while tracheomycotic fungi are commonly found, with similar abundance, in symptomatic and asymptomatic vines. Pathogenicity trials have excluded a direct effect of Hymenochaetales species in triggering leaf symptoms, while the data concerning the role of tracheomycotic fungi remains controversial. Recent microbiome studies confirmed that F. mediterranea is more abundant in leaf-symptomatic vines, and treatments that effectively control leaf symptoms, such as sodium arsenite spray and trunk surgery, act directly on the abundance of F. mediterranea or on the presence of white rot. This suggest that the simultaneous presence of Hymenochaetales and tracheomycotic fungi is a pre-requisite for leaf symptoms; however, the relation among fungal pathogens, grapevine and other biotic and abiotic factors needs further investigation.

Phaeomoniella chlamydospora is a tracheomycotic fungus that colonizes the xylem of grapevines (Vitis vinifera L.), causing wood discoloration, brown wood streaking, gummosis, and wood necrosis, which negatively affect the overall health, productivity, and life span of vines. Current control strategies to prevent or cope with P. chlamydospora infections are frequently ineffective. Moreover, it is unclear how fungicides commonly applied in vineyards against downy and powdery mildew agents affect the wood mycobiome, including wood pathogens such as P. chlamydospora. In this study, we used next-generation sequencing to assess the effects of foliar spray of grapevines with inorganic (copper oxychloride and sulfur), synthetic (penconazole and fosetyl-aluminum), and natural (Blad) fungicides currently used against the downy and powdery mildews. The subjects of our investigation were (i) the resident wood mycobiome, (ii) the early colonization by a consortium of fungal wood endophytes (ACEA1), (iii) the wood colonization success of P. chlamydospora, and (iv) the in planta interaction between P. chlamydospora and ACEA1, under greenhouse conditions, in rooted grapevine cuttings of cv. Cabernet Sauvignon. The data obtained suggest that the resident mycobiome is affected by different fungicide treatments. In addition, the early colonization success of the endophytes composing ACEA1 varied in response to fungicides, with relative abundances of some taxa being overrepresented or underrepresented when compared with the control. The wood colonization by P. chlamydospora comported significant changes in the mycobiome composition, and in addition, it was greatly affected by the foliar spray with Blad, which decreased the relative abundance of this pathogen 12-fold (4.9%) when compared with the control (60.7%) and other treatments. The presence of the pathogen also decreased considerably when co-inoculated into the plant with ACEA1, reaching relative abundances between 13.9% and 2.0%, depending on the fungicide treatment applied. This study shows that fungicides sprayed to prevent infections of powdery and downy mildews have an effect on non-target fungi that colonize the endosphere of grapevines. We suggest two potential control strategies to fight P. chlamydospora, namely, the foliar spray with Blad and the use of ACEA1. Further studies to confirm these results are required.

Fungal pathogens involved in grapevine trunk diseases (GTDs) may infect grapevines throughout their lifetime, from nursery to vineyard, via open wounds in stems, canes or roots. In vineyards, pruning wound protection products (PWPPs) offer the best means to reduce the chance of infection by GTD fungi. However, PWPPs may affect non-target microorganisms that comprise the natural endophytic mycobiome residing in treated canes, disrupting microbial homeostasis and indirectly influencing grapevine health. Using DNA metabarcoding, we characterized the endophytic mycobiome of one-year-old canes of cultivars Cabernet Sauvignon and Syrah in two vineyards in Portugal and Italy and assessed the impact of established and novel PWPPs on the fungal communities of treated canes. Our results reveal a large fungal diversity (176 taxa), and we report multiple genera never detected before in grapevine wood (e.g., Symmetrospora and Akenomyces). We found differences in mycobiome beta diversity when comparing vineyards (p = 0.01) but not cultivars (p > 0.05). When examining PWPP-treated canes, we detected cultivar- and vineyard-dependent alterations in both alpha and beta diversity. In addition, numerous fungal taxa were over- or under-represented when compared to control canes. Among them, Epicoccum sp., a beneficial genus with biological control potential, was negatively affected by selected PWPPs. This study demonstrates that PWPPs induce alterations in the fungal communities of grapevines, requiring an urgent evaluation of their direct and indirect effects on plants health with consideration of factors such as climatic conditions and yearly variations, in order to better advise viticulturists and policy makers.

A peculiar symptom that may develop in grapevines affected by wood pathogens involved in the esca complex of diseases is the leaf stripe symptom, which also gives the name to the Grapevine Leaf Stripe Disease. Multiple studies have revealed strong links between fungal presence, wood symptomatology and expression of the leaf stripe symptom. However, numerous other factors have been shown to play roles in symptom onset, incidence, severity and yearly fluctuation of this disease. While the factors triggering the leaf stripe symptom are still under investigation, three control strategies have been proven effective for substantially reducing its expression, namely trunk surgery, and applications of sodium arsenite or a fertilizer mixture. These control strategies are examined here, including their (putative or confirmed) modes of action, and how they may influence the leaf stripe symptom development. In this article, we also propose the ‘edge’ hypothesis to tentatively explain symptoms onset, keeping in consideration past knowledge and recent advances in the understanding of the esca leaf stripe symptom. Ultimately, it is our intention to offer food-for-thought and stimulate debate within the phytopathological community.