Pathogenicity and Phylogenetic Placement of Gnomonia leptostyla Causing Walnut Anthracnose in Temperate Orchards

Abstract

Walnut anthracnose, attributed to Gnomonia leptostyla (Fr.) Ces. & De Not. (syn. Ophiognomonia leptostyla), is a recurrent and economically significant fungal disease affecting common walnut (Juglans regia L.) orchards throughout temperate growing regions, manifesting as necrotic lesions on leaves, petioles, rachises, and immature nuts that collectively precipitate premature defoliation, reduced photosynthetic capacity, and substantial kernel quality deterioration. Despite the recognized agronomic importance of walnut anthracnose, detailed investigations into pathogen virulence variability, host infection mechanisms, and the precise phylogenetic positioning of G. leptostyla within the order Diaporthales remain insufficiently resolved, limiting the formulation of durable disease management frameworks. This study evaluated the pathogenicity and phylogenetic placement of G. leptostyla isolates recovered from symptomatic walnut tissues exhibiting anthracnose lesions across geographically diverse temperate orchard sites. Isolates were characterized morphologically through examination of ascomatal architecture, ascospore dimensions, conidiomatal structure, and cultural growth parameters under standardized laboratory conditions. Pathogenicity was rigorously established through inoculation of detached walnut leaflets, rachises, and immature husks with conidial suspensions of representative isolates, fulfilling Koch's postulates under controlled greenhouse conditions. Disease severity was quantitatively assessed using standardized rating scales at defined post-inoculation intervals. Multilocus phylogenetic analyses incorporating the internal transcribed spacer region, the large subunit ribosomal RNA gene, RPB2, and TEF1-α sequence datasets were performed using maximum likelihood and Bayesian inference frameworks to resolve the taxonomic placement of recovered isolates within Gnomoniaceae. Phylogenetic reconstructions robustly confirmed the identity of isolates as G. leptostyla and clarified intergeneric relationships within the family. These findings establish a comprehensive pathological and molecular reference for walnut anthracnose surveillance, cultivar resistance evaluation, and the implementation of fungicide-based management programs in temperate walnut production systems.