Expanded Fungal Diversity associated with branch and twig dieback of English Walnut in Central Chile

Branch and twig dieback is recognized as an important fungal disease of walnut worldwide and is caused by multiple fungi, mainly species from the Botryosphaeriaceae and Diaporthaceae families. The current study was conducted to identify and characterize associated fungi causing dieback disease in English walnut (Juglans regia) orchards in the Maule Region of Central Chile. During 2022 and 2023, field surveys were conducted in seven 'Chandler' walnut orchards (7 to 30 years old). Disease incidence ranged from 20 to 76%. In this study, a total of 65 fungal isolates were recovered from 210 affected samples. Fungal isolates were identified based on morphological characteristics and multigene phylogenetic analyses of the ITS region and partial sequences of the beta-tubulin and translation elongation factor 1-alpha genes. These analyses identified four species from the Botryosphaeriaceae family: Diplodia mutila, Dothiorella sarmentorum, Neofusicoccum nonquaesitum, and N. parvum. Three species from the Diaporthaceae family were also identified: Diaporthe australafricana, D. foeniculina, and D. patagonica. Pathogenicity tests conducted on detached and attached host tissues, including fruits, leaves, green shoots, and twigs, confirmed that isolates from both families were pathogenic. However, Botryosphaeriaceae species showed significantly greater aggressiveness, inducing larger necrotic lesions across all tested tissues compared to Diaporthaceae species. This research reports for the first time D. foeniculina, and D. patagonica causing branch and twig dieback in Chile.