bioRxiv2025
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 (R{superscript 2} = 16.6%), followed by sampling campaign and production area (R{superscript 2} = 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 SummaryEnglish 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.