First Report of <i>Alternaria chlamydosporigena</i> Causing <i>Poa poophagorum</i> Root Rot in China

In July 2023, a survey conducted in the natural grasslands of Wulan County (36°38'N, 98°45'E), Qinghai Province, China, identified Poa poophagorum plants exhibiting symptoms of brown root lesions, with a disease incidence of approximately 60%. Symptomatic roots from five diseased plants were surface-sterilized, cultured on potato dextrose agar (PDA) medium, and incubation at 25°C. A fungus was consistently isolated, and eight representative isolates (LYZ1295-LYZ1302) were subsequently purified. Colonies on PDA displayed olive-colored on both the surface and reverse, with undulate margins. Aerial mycelium was sparse and extremely short, while substrate mycelium exhibits abundant growth. No conidia or distinguishing morphological features were observed on any of the six media types tested: PDA, Potato-Carrot Agar, Potato Sucrose Agar, Wheat Straw Decoction Agar, Oatmeal Agar, or Corn Meal Agar. Genomic DNA was extracted using the Omega Fungal DNA Kit. Four loci were amplified and sequenced: the internal transcribed spacer (ITS) region with primers ITS1/ITS4 (White et al. 1990), large subunit (LSU) of nuclear ribosomal DNA with LROR/LR5 (Vilgalys and Hester 1990; Rehner and Samuels 1994), translation elongation factor 1-alpha (TEF-1α) with EF1-728F/EF1-986R (Carbone and Kohn 1999), and RNA polymerase II second largest subunit (RPB2) with RPB2-5F2/fRPB2-7cR (Liu et al. 1999). BLASTn analysis of ITS (PV926699-PV926706), LSU (PV926707-PV926714), TEF-1α (PV972343-PV972350), and RPB2 (PV972351-PV972358) sequences revealed greater than 99.8% identity with ex-type strains of Alternaria chlamydosporigena. A maximum-likelihood phylogenetic tree constructed from concatenated ITS-LSU-TEF-1α-RPB2 sequences in MEGA placed all isolates in a clade with A. chlamydosporigena strain CBS 341.71 (Woudenberg et al. 2013) with 98% bootstrap support, confirming their identity. The pathogenicity of isolate LYZ1295 was evaluated by placing the roots of germinated seedlings (approximately 1 cm in length) at the periphery of actively growing colonies on PDA for 48 h. Control seedlings were placed onto sterile PDA medium before transplanting. The seedlings were transplanted into sterile loess soil. Each pot contained five seedlings, with four replicate pots for each treatment group (inoculated and control). The plants were maintained at a temperature of 28 °C and a relative humidity of 80%. Observations revealed yellowing and wilting of leaves in inoculated plants 12 days post-transplantation (dpt). By 30 dpt, 80% of the inoculated plants exhibited wilting and mortality, whereas control plants remained asymptomatic. The fungus was successfully re-isolated from symptomatic roots of inoculated plants and confirmed as the inoculated strain based on morphological and molecular analysis, fulfilling Koch's postulates. A. chlamydosporigena has been reported causing leaf spot disease on Solanum lycopersicum (Sadeghi and Mirzaei 2018) and dry rot on Lens culinaris (Kouadri et al. 2023). To our knowledge, this represents the first report of A. chlamydosporigena causing root rot on P. poophagorum in China. This study not only provides a theoretical basis for disease diagnosis and effective control measures but also emphasizes the necessity of further investigating its ecological role in natural grasslands.