Anthracnose of Dendrobium brymerianum Caused by Colletotrichum fructicola and C. jiangxiense in Yunnan, China.

Dendrobium brymerianum Rchb. f. is a species of Dendrobium in the Orchidaceae family, with important medicinal and ornamental values. In November 2019, anthracnose symptoms were observed on D. brymerianum in Xishuangbanna Prefecture, Yunnan, China (N22°5', E100°34'), with approximately 60% disease incidence in a 2.5-ha planting area (n=100). Each plant was investigated approximately 30% leaves. The disease spot first extends from the leaf tip to the root and stem, initially reddish brown, later enlarging into necrotic lesions, resulting in many dry black spots or forming large brown lesions. In severe cases, it can lead to the death of the entire plant. Three diseased samples in total were collected from plants in three different locations. Leaf pieces (3×3 mm) were surface-sterilized with 75% ethanol (10 s) and 2% sodium hypochlorite (1 min), rinsed three times in sterile water, then plated on potato dextrose agar (PDA). Forty-four isolates were obtained and thirty-eight of them were morphologically similar to Colletotrichum (average isolation frequency 86%). Based on morphology, one representative Colletotrichum-like isolate from each sample (CSSH1, CSSH2, and CSSH3) was purified using the single-spore separation method (Chen et al. 2019). Colonies were villiform, initially white, later producing olive green to black pigment in 7 days on PDA at 25°C. Conidia were hyaline, celliptical to cylindrical, and averaged 14.28 μm × 5.03 μm, 14.85 μm × 5.33 μm and 14.93 μm × 5.16 μm (n=90) for CSSH1, CSSH2 and CSSH3, respectively. Based on morphological features, the isolates were identified as in the C. gloeosporioides species complex (Weir et al. 2012). Genomic DNA was extracted using the CTAB method (Yan et al. 2018). The isolates were further identified by sequencing the ITS, GAPDH, ACT, TUB2, CHS-1 and CAL with the primers ITS1/ITS4 (White et al. 1990), GDF/GDR, ACT-512F/ACT-783R, T1/Bt2b, CHS-79F/CHS-354R and CL1C/CL2C (Weir et al. 2012), respectively. All sequences were deposited in GenBank (ITS: PQ495644-PQ495646; GAPDH: PQ498501-PQ498503; ACT: PQ498495-PQ498497; TUB2: PQ510845-PQ510847; CHS-1: PQ498498-PQ498500 and CAL: PQ510842-PQ510844). A phylogenetic analysis was made via maximum Likelihood (ML) based on sequences of multiple loci (ITS, GAPDH, ACT, TUB2, CHS-1 and CAL). Based on morphology and phylogenetic analysis, CSSH1 was identified as C. jiangxiense, and CSSH2 and CSSH3 were identified as C. fructicola. Pathogenicity tests were confirmed on asymptomatic leaves without wounds of 12 two-year-old D. brymerianum plants in a greenhouse kept at 25°C. Unwounded leaves were sprayed with conidial suspension (106 spores/ml）until runoff with an atomizer (Lorenz et al. 1995) and control plants were sprayed with sterile distilled water (6 leaves/plant and 3 plants/treatment). All plants were covered with plastic bags to maintain high humidity. After 7 days, typical reddish brown spots or brown lesions were observed on all inoculated leaves, while the control remained asymptomatic. The pathogen was reisolated from the symptomatic leaves, which was confirmed both morphologically and molecularly as described earlier. In contrast, no fungi were isolated or recovered from the plants inoculated with water. To our knowledge, this is the first report of C. fructicola and C. jiangxiense causing D. brymerianum leaf anthracnose in China. The results will provide valuable information for prevention and management of anthracnose in D. brymerianum.