First report of Anthracnose Disease on Bletilla striata (Thunb.) Reichb. f. (Baiji) caused by Colletotrichum orchidophilum in Yunnan, China

Bletilla striata (Thunb.) Reichb. f. has high ornamental and medicinal value. B. striata has been used in Chinese medicine for thousands of years to treat various diseases, such as peptic ulcers, gastrointestinal diseases, lung diseases, and traumatic bleeding (Jiang et al. 2021). In September 2020, about 30% of B. striata plants in a plantation in Lancang County (Puer City, Yunnan Province, 22°48'17 N, 99°46'58 E) were found with symptoms resembling Bletilla anthracnose disease. In May 2021, symptomatic leaves were observed again with a disease incidence rate reached 54.05%, and were collected. The initial symptoms were the appearance of one or more round light brown spots on the leaves. Subsequently, the lesions became larger, forming oval brown-black spots. On severely affected leaves, several spots coalesced, causing withering and death of the leaves. To isolate the pathogen, 10 symptomatic tissues (5mm2) of 10 infected leaves were disinfected in 75% ethanol for 30s then in 2% sodium hypochlorite for 3 minutes, rinsed with sterile water for 3 times, incubated on potato dextrose agar (PDA) plates at 25℃ for 5 to 7 days with a 12 h light/dark photoperiodic cycle. Finally, ten pathogen strains with similar morphology were isolated and pure cultures were obtained by single spore isolation. The morphology of fungal colonies was round, carpet-like, flat and neat, and the color was gray black. The conidiophores were colorless to brown and oval or stick-shaped. The conidia were cylindrical or oblong, consisted of a single cell with blunt round ends, and were on average 12.92 (8.13-21.21) μm×3.96 (2.55-5.90) μm (n=200) in size. Based on the morphology and conidial characteristics, the pathogen was identified as Colletotrichum sp. (Damm et al. 2012). The total genomic DNA of representative isolates (LCTJ-02, LCTJ-03, LCTJ-04, LCTJ-05 and LCTJ-06) was extracted and the ITS region, GAPDH, ACT and HIS3 gene regions were amplified and sequenced using the primer pairs ITS1/ITS4, GDF1/GDR1, 512F/783R, and CylH3F/CylH3R, respectively (Cai et al. 2009). The sequences were deposited in GenBank (MZ433189 to MZ433193 for ITS, MZ436430 to MZ436434 for GAPDH, MZ436435 to MZ436439 for HIS3 and MZ448474 to MZ448478 for ACT). BLAST search revealed that all sequences showed 98% to 100% homology with the corresponding sequences of C. orchidophilum ex-type (CBS 632.80). Phylogenetic trees were constructed using neighbor-joining and maximum likelihood methods for the combined data set of the ITS, GAPDH, ACT, and HIS3 genes by MEGA-X (Kumar et al.2018). In general, LCTJ-02, LCTJ-03, LCTJ-04, LCTJ-05, LCTJ-06, and C. orchidophilum type strains clustered on the same clade. To confirm pathogenicity, the five isolates of C. orchidophilum were inoculated in healthy leaves of potted B. striata, as described by Cai et al. (2009), with slight modifications. Each leaf was inoculated with three drops(10 µl drop-1) of conidia suspension (106 spores mL-1). The control group was mock inoculated with sterile water the same way. All samples were covered with plastic bags and maintained at 70% to 80% relative humidity for seven days. After this period, all inoculated leaves showed similar lesions, and the symptoms were identical to those observed in the field. The control plants remained healthy. The pathogens were re-isolated from two leaves of each treatment and re-identified as C. orchidophilum. To our knowledge, this is the first report of C. orchidophilum causing anthracnose on B. striata in China. In the future, the occurrence and transmission of this pathogen should be further studied in order to develop reasonable control measures.