Morphological and molecular identification of Alternaria dumosa causing post-harvest black spot disease in persimmon (Diospyros Kaki l.) in Pakistan

A survey was conducted from October to January 2021 in local fruit markets in Lahore and surrounding areas to collect persimmon fruit (Diospyros kaki) displaying small black spots, ranging from 3-5 mm in size. To isolate the pathogen, tissue segments (3-5 mm) from the symptomatic fruit were placed on Malt Extract Agar and incubated at 28°C. After 7 days, light brown mycelia appeared. Based on morphological characteristics, the preliminary pathogen was tentatively identified as Alternaria dumosa. To confirm the identification, genomic DNA was extracted and amplified using primer pairs for ITS, GAPDH, Calmodulin, Actin, and TEF. The resulting sequences were compared to those in GenBank using BLAST searches in NCBI, confirming a close match to A. dumosa. The sequences were deposited in GenBank under accession numbers OK447926, OL754642, OL804135, OL830275, and OL962418. For pathogenicity testing, surface-sterilized, uninfected persimmon fruits were inoculated with a conidial suspension (106 spores/ml) of the isolate from this study, while sterile distilled water was used for the control treatment. The fruits were then incubated at 28°C for 7 days. The experiment was repeated twice for validation. Pathogenicity tests showed that 85% of the inoculated persimmon fruits developed characteristic black spots within 7 days, indicating the high virulence of A. dumosa under controlled conditions. The inoculated fruits displayed symptoms similar to those observed in naturally infected fruits, while the control fruits remained symptom-free. Based on these results, we concluded that A. dumosa is the causative agent of leaf spot disease in persimmon fruit.