Occurrence of Ophiopogon bodinieri anthracnose caused by Colletotrichum siamense in China.

Abstract

Ophiopogon bodinieri H.Lév. (Asparagaceae) is a perennial flowering herbaceous plant native to China, and widely cultivated as ornamental plant in South China. During May 2024, an anthracnose was observed on approximately 70% of O. bodinieri in a farm at Mazhang, Zhanjiang, Guangdong Province (22°6'14.7''N,110°27'26.8''E). The symptoms appeared first as small spots with irregular yellow or brown halos, subsequently, the lesions darkened and expanded, covering the leaves. Twenty four plants infected with the disease were sampled, and the pathogen was isolated from sixteen of the plants. Disease tissues (5×5mm) were surface disinfected with 75% ethanol and 3% hydrogen peroxide, rinsed with sterilized distilled water three times, and placed onto potato dextrose agar (PDA) medium containing 50 mg/L of penicillin. The plates were incubated in the dark at 28°C for 3 days. Isolates obtained by transferring the hyphal tips onto fresh PDA were further cultured by single spore dilution method (Choi et al. 1999). Colonies with dense, cottony aerial mycelia were initially white and became grey after 8 days. Conidia were hyaline, one-celled, guttulate, oblong with rounded ends, 13.2 to 17.9 µm × 3.8 to 5.7 µm (n=50). Appressoria formed from mycelia were dark brown, mostly irregular lobes and becoming complex with age, 12.9 to 15.6µm × 7.8 to 11.9 µm (n=50). Setae were absent. Morphological and cultural characteristics were identical to Colletotrichum siamense. To further identify, internal transcribed spacer (ITS) region, B-tubulin (tub), glyceraldehydes-3-phosphate dehydrogenase (gapdh), chitin synthase (chs), actin (act) and calmodulin (cal) genes of isolate YJ8-2 and YJ1-3 were amplified and sequenced with the primer pairs of ITS1/ITS4 (White et al. 1990), TUB-2Fd/4Rd, GAPDH-F/R, CHS-345R/79F, ACT-Rd/512F, CAL-CL1C/CL2C respectively (Weir et al. 2012). The sequences were submitted to GenBank (ITS: PQ327950 and PQ060497; TUB: PQ340881 and PQ061099; ACT: PQ340884 and PQ061095; GAPDH: PQ340882 and PQ061098; CHS: PQ340883 and PQ061097; CAL: PQ340885 and PQ061096). BLAST research showed the sequences of YJ8-2 and YJ1-3 had above 98% identity with C. siamense ex-type ICMP: 18578 (ITS: JX010171 (548/548 100%) and (545/546 99%); GAPDH:JX009924 (271/277 98%) and (260/266 98%); CAL:JX009714 (720/728 99%) and (726/731 99%); TUB: JX010404 (479/485 99%) and (466/467 99%); CHS:JX009865 (294/299 98%) and (254/256 99%); ACT:JX009518 (268/271 99%) and (270/271 99%)). A phylogenetic tree based on concatenated sequences of the six genes using maximum-likelihood method revealed that isolate YJ1-3 and YJ8-2 clustered in the same clade with C. siamense. To confirm pathogenicity, five healthy leaves of one-year-old O. bodinieri in the field were wiped with 75% ethanol and sterile water, wounded with a sterile needle. Three leaves were inoculated with 10 µl of spore suspension (1×105 conidia/ml), and two leaves were inoculated with sterile water as controls. The experiment was repeated three times. After 10 days, symptoms of anthracnose were observed on leaves similar to the disease described above, whereas no symptoms were observed on the control leaves. The same fungus was reisolated from the inoculated leaves and confirmed by morphology and molecular analysis (ITS TUB CHS GAPDH ACT CAL). To our knowledge, this is the first report of C. siamense causing O. bodinieri anthracnose in China. This report is crucial to implement disease prevention and control measures.