First Report of a Novel Female Cone Disease in <i>Torreya grandis</i> ‘Xifei’ Caused by <i>Cladosporium tenuissimum</i> in China

Torreya grandis is known for its medicinal seeds (Wu et al., 2018). Recently, moldy spots were observed on the micropyles of female cones, followed by necrosis of the entire nucellus, resulting in brown rot. In April 2022, the disease incidence reached 68% per cluster (n=132 clusters; 12-14 female cones per cluster) in a production greenhouse. To isolate the pathogen, diseased cones were surface-sterilized in 75% ethanol for 30 s, treated with 2% NaClO for 1 min, rinsed in sterile distilled water, and dried on sterile filter paper. Internal tissues were excised with sterile tweezers, plated on potato dextrose agar (PDA), and incubated at 25 °C in the dark for 7 days. Fungal colonies were purified by repeated subculturing on fresh PDA. Thirteen pure strains were isolated from infected nucellus tissue; 10 of them displayed identical morphology consistent with Cladosporium spp. observed under light microscopy. Colonies were gray-green, round, and velvety with white margins. Conidiophores were apically branched and formed chains of lemon-shaped conidia. Ramoconidia and conidia were cylindrical to globose or obovoid, measuring 4.50-31.40×1.86-5.43 μm and 2.15-9.25×1.06-4.21 μm (n=200), respectively. Based on cultural and morphological characteristics, the fungus was preliminarily identified as Cladosporium tenuissimum (Bensch et al., 2018). To confirm its identity, phylogenetic analysis was conducted using internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), and actin (ACT) gene regions (Yang et al., 2023). Three representative isolates (TGFC1, TGFC7, and TGFC9) were selected for multi-locus sequencing BLAST analysis showed 99% and 100% identity in ITS sequences with C. tenuissimum (OQ629133 and OR143776), and 100% identity in TEF and ACT sequences with reference strains (TEF: MT154177, OL504967; ACT: MT154174, HM148689). For phylogenetic reconstruction, 15 ex-type Cladosporium strains were included (Bensch et al., 2012). TGFC1, TGFC7, and TGFC9 formed a well-supported clade with C. tenuissimum (CBS 125995). Genetic homogeneity of TGFC1 (GenBank: PQ608378, PQ619083, PQ619082), TGFC7 (GenBank: PV300557, PV346768, PV346769), and TGFC9 (GenBank: PV544812, PV549291, PV549292) was confirmed. For the pathogenicity test, nine healthy female cone clusters of T. grandis "Xifei" were inoculated by spraying a conidial suspension (1×106 conidia/mL) of isolate TGFC1. Nine clusters, sprayed with sterilized water served as controls. The clusters were incubated at 25 °C and 90% relative humidity. After 14 days, the inoculated female cones developed yellow, moldy, and wrinkled symptoms, whereas the control cones remained healthy, showing no signs of fungal growth. C. tenuissimum was successfully reisolated from the diseased cones, thereby fulfilling Koch's postulates. This pathogen causes gray mold and blossom blight in strawberry (Santos et al. 2020). To the best of our knowledge, this is the first report of C. tenuissimum causing moldy spots on the female cones of T. grandis "Xifei". As the disease occurs during the flowering stage, it may impair pollination and seed development, leading to significant economic losses. Therefore, accurate identification of the causal agent is critical for developing effective disease management strategies. Reducing the incidence of female cone disease will improve both yield and quality, contributing to the sustainable development of the Torreya industry.