First Report of Leaf Anthracnose on Dendrobium nobile Caused by Colletotrichum citricola in China.

Abstract

Dendrobium nobile, a rare orchid in Orchidaceae, is a traditional Chinese medicinal herb known for its diverse biological activities, including anti-tumor, anti-aging, and hypoglycemic effects (Fan et al. 2023). In February 2025, symptoms of leaf anthracnose were observed on D. nobile plants growing in the medicinal plant plantation of Zhejiang Chinese Medical University (30.08°N, 119.88°E), China. The affected area covered approximately 25 m2, with a disease incidence of around 55%. The disease symptoms began at the leaf edges, where light yellow spots first appeared. As the lesions expanded, they turned into irregular brown or black patches. The edges of the infected leaves dried out, forming a large necrotic area with dark brown spots, ultimately leading to defoliation. Six symptomatic leaf samples (5×5 mm) were surface sterilized with 75% ethanol for 30 s, followed by 2.5% NaClO for 1 minute. Afterair-drying, the tissues were placed on potato dextrose agar (PDA) medium and incubated at 28°C for five days. Two fungal isolates, DNLP07 and DNLP08, were obtained using the hyphal-tip isolation method. On PDA, the colonies appeared white and cottony on the surface, with a white reverse side, yellow-brown discoloration, and concentric rings visible on both sides. The conidia were single-celled, aseptate, cylindrical to oval, measuring 21.6-31.1 × 9.5-14.9 μm (average 25.9 × 11.7 μm; n=50). These morphological characteristics were consistent with those of Colletotrichum citricola (Fu et al. 2019). Six gene regions were amplified: internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (TUB2). The following primer sets were used: ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF/GDR, and T1/Bt2b, respectively (Weir et al. 2012). The resulting sequences were submitted to GenBank with the following accession numbers: ITS: PV271806, PV271807; ACT: PV287640, PV287641; CAL: PV287638, PV287639; CHS: PV287642, PV287643; GAPDH: PV287644, PV287645; TUB2: PV287646, PV287647. BLASTn analysis showed that the isolate DNLP08 shared 99% to 100% sequence identity with the C. citricola type strain CBS 134228, including: ITS (KC293576; 468/468 bp), ACT (KC293616; 268/270 bp), CAL (KC293696; 424/425 bp), CHS (KC293792; 278/278 bp), GAPDH (KC293736; 239/239 bp), and TUB2 (KC293656; 487/488 bp). A maximum likelihood phylogenetic tree was constructed using the concatenated sequence dataset in MEGA 11, which showed that both isolates clustered with C. citricola. To confirm pathogenicity, a conidial suspension of isolate DNLP08 (1×106 spores/mL) was sprayed onto three healthy D. nobile plants. Three additional plants were treated with sterile distilled water as controls. All plants were maintained at 28°C with 85% relative humidity and a 12-hour photoperiod. After 12 days, the inoculated plants developed symptoms identical to those observed in the field, while the control plants remained asymptomatic. The pathogen was successfully re-isolated from symptomatic leaves and identified based on morphological features and sequence data from the six genes, fulfilling Koch's postulates. No pathogens were isolated from the control plants. This study first reports C. citricola causing leaf anthracnose on D. nobile in China. Accurate pathogen identification lays a crucial foundation for future effective prevention and control of the disease.