Molecular Identification and Pathogenicity of Gnomoniopsis castaneae Causing Nut Rot on Sweet Chestnut

Abstract

Nut rot symptoms characterized by internal kernel browning, necrosis, premature fruit drop, and post-harvest decay were observed in sweet chestnut (Castanea sativa) orchards, leading to significant yield and quality losses. Diseased nut samples collected from affected trees were used for pathogen isolation and characterization through morphological, pathogenicity, and molecular approaches. Fungal isolates consistently produced slow-growing colonies with whitish to grey mycelia, later developing dark pycnidia containing hyaline, ellipsoid conidia typical of Gnomoniopsis castaneae. Considerable variation in colony morphology, growth rate, and sporulation intensity was observed among isolates obtained from different chestnut-growing regions. Pathogenicity tests conducted on healthy chestnut nuts reproduced characteristic rot symptoms, including internal tissue necrosis and kernel collapse, whereas control nuts remained symptom-free. Re-isolation of the fungus from inoculated tissues fulfilled Koch’s postulates, confirming its pathogenic role. Molecular identification was performed using amplification and sequencing of the internal transcribed spacer (ITS) region along with additional conserved loci such as β-tubulin and translation elongation factor 1-α (TEF1-α). Sequence analysis revealed high similarity with authenticated G. castaneae isolates available in global sequence databases. Phylogenetic analysis grouped field isolates within the G. castaneae clade with strong bootstrap support, confirming species identity and revealing limited genetic variability among regional populations. Disease development was favored by warm, humid conditions and prolonged fruit surface wetness, which enhanced spore germination and infection efficiency. Severe infections resulted in significant nut quality deterioration and post-harvest losses, posing a serious threat to chestnut production systems.