First Report of Leaf Blight in Cathaya argyrophylla Caused by Botryosphaeria dothidea in China

Cathaya argyrophylla Chun et Kuang is a plant belonging to the family Pinaceae, known for its ancient origin and multiple ecological and economic values. It is classified as a Class I protected species in China. In 2022, leaf blight in C. argyrophylla was first detected in the Dasha River Nature Reserve (107. 6° E, 28. 89° N), located in Daozhen County, Guizhou Province. The incidence of this disease reached 25% (n = 100). The symptoms initially appeared as yellowing at the needle tips, which progressively developed into brown, desiccated, and deformed areas. As the disease advanced, the lesions gradually expanded to complete leaf necrosis, followed by defoliation. To identify the causal pathogen, 10 diseased leaves were collected and processed. Tissue samples (2 × 5 mm) from the margins of leaf lesions were sterilized in 75% alcohol for 15 seconds and 1. 5% NaClO solution for 1 minute, rinsed with sterile water, and cultured on potato dextrose agar (PDA) at 28°C for 7 days. Eight isolates were obtained, of which six (KS1-1 to KS1-6) showed consistent morphological characteristics on PDA plates (isolation frequency 75%), with colonies exhibiting a ring-shaped pattern characterized by a black center and white periphery. After 14 days, the mycelium turned black. Microscopic examination revealed septate hyphae and unicellular, fusiform conidia measuring 23. 6 to 26. 8 × 4. 8 to 5. 2 μm (n = 50), leading to the tentative identification of the strains as Botryosphaeria dothidea based on morphological characteristics. To confirm the identification, genomic DNA was extracted from the isolates, and the internal transcribed spacer (ITS) regions, beta-tubulin gene fragment (TUB2) of rDNA were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990) and primers Bt2a/Bt2b (Varga et al. 2011). BLAST analysis demonstrated that the ITS and TUB2 sequences of the six isolates were identical and had 100% homology with B. dothidea, and we selected one representative isolate named KS1-2 for further study. The sequence of the KS1-2 isolate has been deposited in GenBank (ITS: PP838632; TUB2: PP853612), showing 100% homology to B. dothidea strain XL-M-7 (ITS, 100% homology to OQ555078; TUB2, 100% homology to OR025341). A phylogenetic tree was constructed based on ITS and TUB2 genes using Bayesian analysis with PhyloSuiteᵥ1. 2. 3, confirming that the strains under study clustered within the same branch as B. dothidea. The pathogenicity of 10 five-year-old C. argyrophylla seedlings was verified by pricking part of the leaf tips with a 0. 7-mm syringe needle, then spraying conidial suspension (106 conidia ml-1) of strain KS1-2 to inoculate wounded and unwounded leaves to the point of dripping at the tip of the leaf, and then moisturising the leaves with cotton balls soaked in 60μL of spore suspension. Control seedlings were inoculated with sterile water. Seedlings were incubated in a greenhouse. After 7 days, all wounded leaves and about 20% of the unwounded leaves showed symptoms similar to those observed in the field, while the control plants remained symptom-free. The pathogen was reisolated from diseased plants with the same morphology and molecular sequence (ITS and TUB2) as the original isolate, thus fulfilling Koch's postulates. B. dothidea is a globally significant pathogen affecting agroforestry (Tang et al. 2012; Marsberg et al. 2017), yet this study marks the first report of it causing leaf blight in C. argyrophylla in China.