First Report of Colletotrichum siamense Causing Leaf Spot on Jasminum sambac in China.

Jasmine (Jasminum sambac (L.) Aiton) is cultivated as a commercial floricultural crop in many countries around the world (Gao et al., 2020). From June to August 2020, leaf spots on jasmine were observed on a jasmine plantation in Hengzhou of Guangxi province. Over 40% of the plants in 6 ha fields were infected. This disease was prevalent in jasmine production area of China (Chen et al., 2012; Du et al., 2020). Symptoms began as chlorotic regions (from 5 to 10 mm in diameter) with light brown necrotic centers, which gradually expanded to the entire leaf. Eventually, the disease leaded to defoliation and dieback. The edges of the affected parts from diseased leaves were cut into pieces (3 mm2). Pieces were treated with 75% ethanol for 10 s, soaked in 2% NaClO solution for 1 min, washed three times with sterile water, and then incubated on potato dextrose agar (PDA) plates at 28℃ for 5 days in the dark. Fungal cultures that showed similar morphological characteristics were isolated, and three representative isolates (HL6-1 to HL6-3) were purified following Mo et al. (2018). The cultures on PDA changed from white to dark grey after 7 days and produced conidiomata after 14 days. Conidia were hyaline, one-celled, guttulate, cylindrical, of 12.07 to 18.09 × 4.04 to 8.05 μm, 13.17 to 16.35 × 4.22 to 6.13 μm and 10.11 to 22.17 × 3.65 to 8.1 μm for HL6-1, HL6-2 and HL6-3, respectively. Gray-brown or dark brown appressoria formed from conidia were subglobose or elliptical. Conidial appressoria and mycelial appressoria were 5.53 to 13.96 × 3.58 to 13.95 μm and 4.24 to 14.01 × 2.4 to 10.86 μm. Genomic DNA was extracted from three isolates and the partial internal transcribed spacer (ITS) regions, intergenic region of apn2 and MAT1-2-1 (ApMAT), and fragments of actin (ACT), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and β-tubulin (TUB2) genes were amplified, sequenced and submitted to GenBank (ITS, ON115173 to ON115175; ApMat, ON156517 to ON156519; ACT, ON146469 to ON146471; GAPDH, ON156502 to ON156504; CHS-1, ON156507 to ON156509; TUB-2, ON156512 to ON156514). Phylogenetic tree was constructed with MrBayes v. 3.2.6 and MEGA v. 10.1.5 based on the concatenation of multiple sequences. Three isolates were grouped with strain C. siamense ICMP 18578. Results indicated three isolates were identified as Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde. To confirm the pathogenicity of the three isolates, four sets (five plants per set) of 160 healthy leaves of 2-year-old plants (J. sambac, eight leaves per plant) were slightly scratched with a sterilized toothpick at each of eight locations. Conidial suspension (1×106 conidia/mL) in 0.1% Tween 20 were inoculated onto each wounded spot of three sets as the treatment groups, while wounded leaves treated with sterile water as the control. All plants were covered with plastic bags and cultivated in phytotron (12 h/12 h light/dark, 28°C). After 7 days, irregular chlorotic regions with brown lesions were observed on inoculated leaves while no symptoms on controls. The same fungi were reisolated from inoculated leaves and confirmed by morphological and molecular identification, fulfilling Koch's postulates. Colletotrichum siamense has been associated with leaf anthracnose of J. sambac in Vietnam (Wikee et al., 2011) and J. mesnyi in China (Zhang et al., 2019). To our knowledge, this is the first report of C. siamense causing jasmine anthracnose in China, which provides a reference for the management of this disease.