Etiology of Branch Dieback and Shoot Blight of English Walnut Caused by Botryosphaeriaceae and Diaporthe Species in Southern Spain

Abstract

English walnut (Juglans regia L.) is considered an economically important fruit crop worldwide. In Spain, little attention has been given to walnut diseases owing to the minor economic importance of the walnut crop in the country until recently. In 2017, typical symptoms of branch dieback and shoot blight of English walnut were observed in southern Spain. From 2017 to 2018, 10 commercial walnut orchards showing disease symptoms were surveyed. Botryosphaeriaceae and Diaporthe fungi were consistently isolated from affected shoots. Cytospora isolates were also recovered with minor relevance. Representative isolates of each fungal group were characterized based on colony and conidial morphology, optimum growth temperature, and comparison of DNA sequence data from the internal transcribed spacer, elongation factor 1-α, and β-tubulin genomic areas. Pathogenicity tests were performed on detached and attached shoots and on detached fruit by inoculating them with mycelial plugs. Botryosphaeriaceae and Diaporthe isolates had higher optimum growth temperatures (≈25 to 27°C) than Cytospora sp. (19.5°C). The following species were identified: Botryosphaeriaceae: Botryosphaeria dothidea, Diplodia seriata, Dothiorella sarmentorum, Dothiorella sp., Neofusicoccum mediterraneum, and N. parvum; Diaporthe: Diaporthe neotheicola, Dia. rhusicola, Diaporthe sp., and Phomopsis amygdali; and Cytospora sp. Botryosphaeriaceae isolates were the most aggressive fungi to walnut in all tissues evaluated, followed by Diaporthe isolates and Cytospora sp. N. parvum was the most virulent among the remaining species tested in any of the tissues evaluated, followed by B. dothidea or N. mediterraneum. This work is the first report to identify the fungal species causing this complex disease of English walnut in Spain and Europe.