Botryosphaeriaceae are a family of fungi associated with the decay of a large number of woody plants with economic importance and causing particularly great losses in viticulture due to grapevine trunk diseases. In recent years, major advances in the knowledge of the pathogenicity factors of these pathogens have been made possible by the development of next-generation sequencing. This review highlights the knowledge gained on genes encoding small secreted proteins such as effectors, carbohydrate-associated enzymes, transporters and genes associated with secondary metabolism, their representativeness within the Botryosphaeriaceae family and their expression during grapevine infection. These pathogenicity factors are particularly expressed during host–pathogen interactions, facilitating fungal development and nutrition, wood colonization, as well as manipulating defense pathways and inducing impacts at the cellular level and phytotoxicity. This work highlights the need for further research to continue the effort to elucidate the pathogenicity mechanisms of this family of fungi infecting grapevine in order to improve the development of control methods and varietal resistance and to reduce the development and the effects of the disease on grapevine harvest quality and yield.

Walnut dieback can be caused by several fungal pathogenic species, which are associated with symptoms ranging from branch dieback to fruit necrosis and blight, challenging the one pathogen–one disease concept. Therefore, an accurate and extensive description of the walnut fungal pathobiome is crucial. To this end, DNA metabarcoding represents a powerful approach provided that bioinformatic pipelines are evaluated to avoid misinterpretation. In this context, this study aimed to determine (i) the performance of five primer pairs targeting the ITS region in amplifying genera of interest and estimating their relative abundance based on mock communities and (ii) the degree of taxonomic resolution using phylogenetic trees. Furthermore, our pipelines were also applied to DNA sequences from symptomatic walnut husks and twigs. Overall, our results showed that the ITS2 region was a better barcode than ITS1 and ITS, resulting in significantly higher sensitivity and/or similarity of composition values. The ITS3/ITS4_KYO1 primer set allowed to cover a wider range of fungal diversity, compared to the other primer sets also targeting the ITS2 region, namely, GTAA and GTAAm. Adding an extraction step to the ITS2 sequence influenced both positively and negatively the taxonomic resolution at the genus and species level, depending on the primer pair considered. Taken together, these results suggested that Kyo set without ITS2 extraction was the best pipeline to assess the broadest fungal diversity, with a more accurate taxonomic assignment, in walnut organs with dieback symptoms.

English walnut (Juglans regia L.) is an economically important fruit crop worldwide and ranks second in France in cultivated area after apples. This study presents the first comprehensive investigation into the etiology and fungal community analysis associated with dieback symptoms in walnut trees in France, where this disease has been increasingly reported since 2015. Both symptomatic and asymptomatic twigs and husks were collected from 12 commercial walnut orchards in the two primary production regions from 2020 to 2022. Several fungal species—among Botryosphaeriaceae, Colletotrichum, Diaporthe, and Fusarium taxa—were consistently isolated from symptomatic husks and twigs. Among these, Botryosphaeria dothidea and Colletotrichum godetiae were significantly associated with symptomatic husks, and Diaporthe eres and Fusarium juglandicola with symptomatic twigs, as confirmed by ITS2 metabarcoding sequencing. Additionally, Neofusicoccum and Diaporthe were significantly associated with warm and dry years, unlike Colletotrichum. Following pathogenicity assays on twigs and fruits, N. parvum emerged as the most aggressive species. These results suggest a complex interplay of factors influencing disease dynamics, further compounded by potential interactions among the main pathogenic species. Notably, in twigs, we found that the prevalence of D. eres was negatively correlated to that of N. parvum while the association between D. eres and F. juglandicola was the most frequent one, suggesting both competitive and facilitative interactions. This research underscores the importance of understanding the fungal pathobiome and its interactions in the development of walnut tree diseases.

English walnut ( Juglans regia L.) is an economically-important fruit crop worldwide, particularly in France, where it is the second most important fruit crop after apples in terms of cultivated area. Walnut orchards are targeted by numerous diseases, but new symptoms have been widely observed since 2015 in France, consisting in typical branch dieback and fruit blight and necrosis. Herein, both symptomatic and asymptomatic twigs and husks were collected from 12 commercial French walnut orchards from the two main production areas located in the South of France from 2020 to 2022. Overall, Botryosphaeriaceae, Colletotrichum, Diaporthe , and Fusarium species were consistently isolated from symptomatic husks and twigs. Among these taxa, B. dothidea and C. godetiae were significantly more associated with symptomatic husks, contrary to D. eres and F. juglandicola, more frequently isolated from symptomatic twigs, as also confirmed by ITS2 metabarcoding sequencing. Tissue type was the primary factor significantly impacting the composition of the fungal communities (R² = 16.6%), followed by sampling campaign and production area (R² = 11.6% and 4.6%, respectively). We also found that Neofusicoccum and Diaporthe were significantly more associated with warm and dry years, unlike Colletotrichum . Pathogenicity tests revealed that only Botryosphaeriaceae and Diaporthe species were pathogenic on walnut fruits and twigs, with B. dothidea and N. parvum being the most aggressive species. Several significant positive and negative correlations were identified based on SparCC co-occurrence networks and correlograms based on prevalence data. In particular, the positive correlation between Colletotrichum and Fusarium species on symptomatic twigs was highlighted by both methods. This work is the first comprehensive etiological and epidemiological study of branch dieback and fruit blight and necrosis of English walnut in France. Author Summary: English walnut is an economically important crop, mainly produced in China, in the United States and in Turkey while in Europe, France is one of the biggest producers. Although widespread in nut orchards abroad, new symptoms, including fruit necroses and branch dieback, were observed for the first time in 2015 in France. We then conducted sampling campaigns between 2020 and 2022 to collect walnut husks and twigs in the two main French production areas. Exploration of the associated microbiota revealed the presence of six predominant fungal species, even in samples without symptoms. We then investigated the capacity of these species to cause lesions on twigs and fruits. Among all species, Neofusicoccum parvum was the most aggressive on both organs while other species were mainly aggressive on only twigs or husks. In addition, we evidenced that the presence of some species was favored by warm and dry weather or inversely cold and wet weather. We also found negative correlations between certain species (in particular between N. parvum and Diaporthe eres ), suggesting that some species probably prevented the other from further colonization. Overall, we identified the species specifically associated with the disease in France and the factors influencing the disease risk. This work will enable us to better identify the best strategies to combat this disease.

Depuis 2015, les nuciculteurs français font face une forte occurrence de symptômes jusqu’alors peu constatés, tels que des brûlures, nécroses et/ou décoloration sur rameaux, jeunes pousses et/ou fruits. Décrits dans la littérature depuis le début des années 2000 en Californie, ces symptômes sont caractéristiques de la maladie du dépérissement du noyer et principalement associés à des espèces de Botryosphaeriaceae, retrouvées en co-occurrence avec des espèces de Diaporthe. Afin de mieux comprendre l’étiologie et l’épidémiologie de cette maladie, cette thèse visait à étudier le pathobiome fongique français, via une double approche culturale et par séquençage métabarcoding. Après avoir mis au point une méthode d’analyse des échantillons de noyers par séquençage métabarcoding, l’étude approfondie de brous et de rameaux de noyers a permis de mettre en évidence un cortège de six principales espèces pathogènes : B. dothidea, N. parvum, D. eres, F. juglandicola, C. godetiae et C. fioriniae. Puis, les conditions météorologiques favorisant leur présence ainsi que les interactions entre ces espèces et avec d’autres espèces du phytomicrobiome ont été décrites, montrant à la fois des interactions compétitives, mettant en jeu N. parvum, et facilitatrices entre F. juglandicola et D. eres. Dans un second temps, l’étude de la diversité et de la structure des populations des deux espèces pathogènes de Botryosphaeriaceae à l’aide de marqueurs microsatellites a mis en évidence leur mode de reproduction majoritairement clonal et une différenciation géographique des populations de B. dothidea. L’exploration de l’origine de l’introduction de ces espèces sur noyers a révélé une forte proximité génétique avec des isolats provenant de vignes françaises, à la différence des populations californiennes ou espagnoles.