First Report of Brown Leaf Spot Disease on <i>Dendrobium denneanum</i> Caused by <i>Phyllosticta capitalensis</i> in China

Dendrobium denneanum, a rare orchid in the family Orchidaceae, is a traditional Chinese medicinal herb known for its various biological activities, including immune enhancement, antioxidant effects, and anti-tumor properties (Fan et al. 2022). In February 2025, symptoms of brown leaf spot disease were observed on D. denneanum plants in the medicinal plant plantation of Zhejiang Chinese Medical University (30.08°N, 119.88°E), Zhejiang Province. The affected area spanned approximately 20 m2, with a disease incidence of around 30%. Symptoms included yellow to brown leaf spots, which progressively led to leaf drying, curling, and eventual defoliation. Six symptomatic leaves tissues (5×5 mm) were surface-sterilized with 75% ethanol for 30 s, followed by 2.5% NaClO for 1 min. After rinsing three times in sterile water and drying, the tissues were placed on potato dextrose agar (PDA) medium and incubated at 28°C in darkness for five days. Two fungal isolates (DDLP05 and DDLP06) were obtained using hyphal-tip method. On PDA, the colonies initially appeared grayish-white and uneven, later darkening to black with a granular texture and irregular white edges. The conidia were single-celled, colorless, round to oval, measuring 7.3-11.0 × 4.7-7.7 μm (average 8.9 × 6.2 μm; n=30), with some bearing a layer of transparent mucus and a transparent accessory filament at the distal end. These morphological characteristics closely resembled Phyllosticta spp. (Wikee et al. 2013a). The internal transcribed spacer (ITS), translation elongation factor (TEF1), actin (ACT), and glyceradehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using the primers ITS1/ITS4, EF1-728F/EF1-986R, ACT-512F/ACT-783R, and Gpd1-LM/Gpd2-LM, respectively (Wikee et al. 2013b). The sequences were deposited in GenBank under the following accession Nos. ITS: PV361569 and PV361570; TEF1: PV287629 and PV287630; ACT: PV278758 and PV278759; GAPDH: PV287635 and PV287636. BLASTn analysis showed that the DDLP05 isolate shares 99% to 100% sequence similarity with P. capitalensis CPC18848, including ITS (JF261465; 557/559 bp), TEF1 (JF261507; 209/210 bp), ACT (JX010033; 232/232 bp), and GADPH (JX010405; 646/650 bp). A Bayesian inference phylogenetic tree was constructed using a combined dataset of gene sequences with MrBayes 3.2.7. The two isolates clustered with P. capitalensis (Wikee et al. 2013a). To confirm pathogenicity, a conidial suspension of isolate DDLP05 (1×106 spores/mL) was sprayed onto three healthy D. denneanum plants. Three additional plants were treated with sterilized distilled water as controls. All plants were incubated at 28°C with 85% relative humidity and a 12-hour light/dark cycle. After 10 days, symptoms identical to those observed in the field appeared on inoculated plants, while control plants remained asymptomatic. The pathogen was successfully re-isolated from symptomatic leaves and identified based on morphology and ITS, TEF1, ACT, GAPDH sequences, fulfilling Koch’s postulates. No pathogens were isolated from the three control plants. P. capitalensis has been reported to cause disease in various fruits (Chaiwong et al. 2024), ornamental plants (Wu et al. 2024), and medicinal plants (Xiong et al. 2023). This is the first report of P. capitalensis causing brown leaf spot disease on D. denneanum in China. Accurate identification of the pathogen provides a crucial foundation for the effective prevention and control of this disease.