First report of Alternaria alternata causing winged leaf dieback disease on Rhus chinensis in Yunnan Province, China
Authors: Wen Lianhao , Li Yanjie , Gu Ju , Zhang Yu , Chen Jingjing , Wu Ziqiang , Wu Yandi , Wang Chao 
Published: January 2025

Rhus chinensis, a deciduous tree of the genus Rhus (family Anacardiaceae), is widely cultivated in China for its medicinal, edible, and ornamental value (Zhang et al., 2022). In April 2022, symptoms of winged leaf dieback disease were observed at Southwest Forestry University in Kunming, Yunnan Province, China (E102°45′42.122″, N25°4′1.36″, H1951m). A plantation survey revealed that 82% of R. chinensis individuals had symptoms consistent with winged leaf dieback disease, representing the disease severity across the district. The early symptoms of the disease included small, irregular dark brown or black spots on the leaves, which progressively expanded into necrotic patches, leading to the withering and shedding of adjacent leaves. A total of 25 samples were collected from different R. chinensis individuals and winged leaves in the plantation at Southwest Forestry University, Kunming, with sampling conducted in April 2022. Fifteen fungal isolates with similar morphological characteristics were purified using the tissue isolation method described by Li et al (Li et al., 2019). Initially, the fungal colonies appeared white, later turned grey, and eventually became greyish-black. The conidia were light brown to brown, ovate to oblong-ellipsoid, and slightly constricted near some septa. They measured 12.4 to 41.8 µm in length and 6.1 to 14.2 µm in width, containing 1 to 7 transverse septa and 0 to 3 longitudinal septa. The conidiophores were straight or slightly curved, light brown to brown, and measured 13 to 30 by 1.5 to 6 µm. These morphological characteristics were consistent with descriptions of A. alternata (Zhang et al., 2023). For molecular identification, four representative fungal strains (RCM1, RCM2, RCM3, and RCM4) were selected. Multilocus analysis was conducted on five genes: internal transcribed spacer (ITS), 18S ribosomal RNA (SSU), 28S ribosomal RNA (LSU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (EF1-α). The gene sequences were subjected to a BLAST search in NCBI, and the corresponding sequences were downloaded from the GenBank database. A phylogenetic tree was then constructed using MEGA11, employing the Maximum Likelihood method for multi-gene association analysis. The resulting sequences were deposited in GenBank with accession numbers: ITS (OR649186, OR649187, OR649188, OR649189), GAPDH (OR727018, OR727019, OR727020, OR723970), SSU (OR649193, OR649194, OR649195, OR649196), LSU (OR649198, OR649199, OR649200, OR649201), and EF1-α (OR683440, OR683441, OR727016, OR727017). Phylogenetic analysis grouped these isolates within the A. alternata clade, supported by a 99% bootstrap value. To verify Koch’s postulates, a spore suspension of the pathogen (1.2×105 spores/ml) and distilled water were separately inoculated onto 20 three-year-old potted R. chinensis winged leaves, using the RCM1 isolate. The inoculated winged leaves were covered with cotton to maintain moisture. After 10 days, lesions consistent with those observed in the field developed at all inoculated sites, whereas the control group, inoculated with sterile water, remained symptom-free. The pathogen was successfully reisolated from the diseased winged leaves, fulfilling Koch’s postulates. This study confirmed A. alternata as the causative agent of winged leaf dieback disease in R. chinensis. To the best of our knowledge, this is the first report in China identifying A. alternata as responsible for this disease. R. chinensis is an economically important forest species in China, and this disease significantly reduces its yield, leading to substantial economic losses for plantation companies and farmers. These findings provide a theoretical foundation for developing effective strategies to prevent and control winged leaf dieback disease in R. chinensis.