First Report of Colletotrichum karstii Causing Anthracnose of Begonia lanternaria Irmsch. in China.

Abstract

Begonia lanternaria Irmsch., an ornamental plant endemic in China, which is commonly used in landscape and interior decoration. In March 2021, an estimated 30% B. lanternaria plants were observed with anthracnose-like symptoms at a botanical garden conservation greenhouse in Mengla County of Yunnan Province (21.91° N, 101.21°E). Initially, small black spots developed on the disease leaves, which gradually expanded into irregular necrotic lesions surrounded by a yellowish halo, eventually turned wilting and defoliating. Twenty diseased leaves were collected and surface-disinfested with 75% ethanol for 30 s. Small fragments (5 × 5 mm) from the margin of lesions were disinfected with 1% NaClO for 120 s, washed with sterile water three times, and cultured on potato dextrose agar (PDA) at 28 ± 1℃. After 3 days single spores from four fungal colonies with identical morphology were isolated. Colonies on PDA were 70-75 mm diam in 7 d (7.5-10.6 mm/d), with dense white to gray-white mycelia attached with brown to black-brown acervulus. The underside of the culture was yellow to yellowish-brown concentric circle. Conidia were single-celled, hyaline, straight to slightly curved, cylindrical, 12.88 to 16.66 × 6.25 to 7.97 μm (av=14.65 μm × 7.22 μm, n=50) in size. For molecular identification, genomic DNA was extracted from a representative isolate, and the internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, calmodulin gene, β-tublin, actin, and chitin synthase 1 genes were amplified with ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Li et al, 2018), Bt2a/Bt2b (Prihastuti et al, 2009), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999) primers, respectively. The obtained DNA sequences showed over 99% homology with Colletotrichum karsti (GenBank Accession No. ITS: NR144790; GAPDH: KX578772; CAL: KY039988; TUB2: KX578804; ACT: LC412408; CHS1: KU251855), and the results of sequences were deposited into GenBank with accession No. MZ496954 (522/522 bp), MZ504978 (238/238 bp), MZ504979 (737/737 bp), MZ504982 (472/472 bp), MZ504981 (273/273 bp), MZ504980 (282/284 bp). The phylogenetic tree combined with ITS-ACT-GAPDH-CHS 1-CAL-TUB2 concatenated sequences using the maximum likelihood methods showed that the isolate was C. karsti. To confirm pathogenicity, Koch's postulates were conducted on intact plants, 10 μl spore suspension (1.0 × 106 conidia/ml) of each of four isolates (7-day-old culture on PDA) was inoculated on 15 wounded with a sterilized needle or non-wounded healthy living leaves, and 15 wounded leaves were inoculated with sterile water as controls. All leaves were incubated at 28 ± 1°C and 90% relative humidity (12 h/12 h light/dark). After 5 days, all wounded leaves inoculated with C. karsti showed symptoms similar to those previously observed, while the control and non-wounded leaves remained healthy. Colletotrichum karsti was re-isolated from inoculated leaves. C. karsti was previously reported to cause disease on Nicotiana tabacum L. (Zhao et al, 2020), Stylosanthes guianensis (Jia et al, 2017) and Fatsia japonica (Xu et al, 2020) in China. To our knowledge, this is the first report of C. karsti causing anthracnose of B. lanternaria Irmsch. in China. This disease reduces the ornamental and economic value of B. lanternaria Irmsch., and this work will provide a basis for the prevention and treatment of the disease in the future.