Identification of the Pathogen Causing Black Spot and Soft Rot of Torreya grandis During Post-ripening Stage

Torreya grandis ‘Merrillii’ is a rare nut species endemic to southern China, with kernel being highly nutritious. however, black spot soft and rot disease often occurs during the post-ripening stage, manifesting as black spots on the seed shell and seed coat, accompanied by off-flavor, and in severe cases, this disease leads to kernel rot, causing substantial economic losses. Currently, the pathogen species of this disease still remain unclear. In this study, pathogens were isolated and purified from naturally diseased black-spotted seeds after harvest using the tissue isolation method. The pathogenic fungi were identified through morphological observation, and multi-gene (ITS, tef1-α, and tub2) phylogenetic analysis, and the extracellular enzyme activities of representative strains were detected. A total of 9 pathogenic strains were isolated and identified as four species: Lasiodiplodia theobromae (strains TGBR9 and TGBR10), L. parva (TGBR8 and TGBR11), L. pseudotheobromae (TG-BR2, TGBR3, and TGBR4, and L. iraniensis (TGBR1 and TGBR5). Pathogenicity test showed that all strains could infect through cracks in the aril, causing blackening of the seed shell and internal kernel rot. The representative strains (TGBR1, TGBR4, TGBR8, and TGBR9) could all colonize the seed shell and invade the internal kernel, among which L. theobromae TGBR9 exhibited the strongest virulence. Extracellular enzyme activity assays showed that all four representative strains secreted cellulase, pectin lyase, pectinase, amylase and protease. Among them, TGBR1 had the highest activity on pectinase, TGBR8 had the highest activity on amylase, and TGBR9 had the highest pectin lyase and protease activities. In conclusion, L. theobromae, L. parva, L. pseudotheobromae, and L. iraniensis are the main pathogens causing black spot and soft rot disease during the post-ripening period. The results can provide a theoretical basis for pathogen diagnosis and prevention and control of this disease in T. grandis ‘Merrillii’.