First Report of Colletotrichum truncatum Causing Anthracnose in Tobacco in China.

Abstract

In July 2022, large spots were observed on the leaves of tobacco in Guangxi province, China, whose shape was round and elliptical or irregular. The margins of spots were brown or dark brown with a pale yellow centre and several small black fruiting bodies. The pathogen was isolated by tissue isolation. Diseased leaves collected were cut into small pieces, sterilized with 75% ethanol for 30s and 2% sodium hypochlorite (NaCIO) for 60s, and rinsed with sterile deionized water for three times. Each air-dried tissue segment was cultured on potato dextrose agar (PDA) and incubated at 28℃ for 5 to 7 days in the dark (Wang et al. 2022). A total of six isolates were isolated, with differences in colony shape, edge type and colony colour, and aerial mycelium morphology, with the colony shape round or subrounded, and the edge rounded crenate, dentate or sinuate. The color of the colony was initially light yellow, then gradually changed to yellow and dark yellow. After 3-4 days, white aerial mycelia gradually grew up, which was peony-like or covered the whole colony, thus the color of the colony appeared white, and then gradually changed to orange, gray or nearly black, and all six isolates rarely produced conidia, which was consistent with the description of previous reports(Mayonjo and Kapooria 2003, Feng et al. 2021, Xiao et al. 2018). Conidia were hyaline, aseptate, and falcate, with the size of 7.8 to 12.9 × 2.2 to 3.5 μm. For molecular identification, the colony PCR method was used to amplify the internal transcribed spacer(ITS), actin(ACT), chitin synthase(CHS), and beta-tubulin(TUB2) loci of the six isolates using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and T1/Bt2b, respectively(Cheng et al. 2014). Partial sequences were amplified, sequenced, and uploaded to GenBank (GenBank accession Nos. OP484886，OP518265，OP518266，OP756065，OP756066, and OP756067 for ITS, OP620430 to OP620435 for ACT, OP620436 to OP620441 for CHS, and OP603924 to OP603929 for TUB2). These sequences had 99 to 100% similarity with C. truncatum isolates C-118(ITS), TM19(ACT), OCC69(CHS), and CBS 120709(TUB2) in GenBank. Homology matching was performed using BLAST and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA (7.0) software based on ITS, ACT, CHS, and TUB2 sequences, which showed that all six isolates clustered in the same score as the C. truncatum. A pathogenicity test was performed with healthy tobacco infected with mycelial plugs (about 5 mm in diameter) of six isolates of C. truncatum from a 5-day-old culture, while negative controls on the other leaves were inoculated with sterile PDA plugs. All plants were placed in a greenhouse at 25℃ to 30℃ with 90% relative humidity. The experiment was conducted three times. Five days later, all inoculated leaves had diseased spots, whereas no symptoms appeared on negative controls. The same pathogen, C. truncatum, was identified from the inoculated leaves on the basis of morphological and molecular charchseristics as described above, fulfilling Koch's postulates. In this study, it is the first time to report that the anthracnose on tobacco was caused by C. truncatum. Thus, this work provides a foundation for controlling tobacco anthracnose in the future.