First Report of Colletotrichum fructicola Causing Anthracnose on Rosa chinensis in China.

Abstract

China rose (Rosa chinensis Jacq.) is a popular ornamental plant grown widely in China. In September 2021, a serious leaf spot disease was observed on R. chinensis in Rose plantation of Nanyang Academy of Agricultural Sciences in Nanyang (112°25'41″N, 32°54'28″E), Henan Province, causing severe defoliation of infected plants with a foliar disease incidence of 50 to 70% (n = 100). The early symptoms were irregular brown specks on the leaves, mostly at the tip and margin of the leaves. Then the specks gradually expanded into round amorphous and became dark brown, eventually leading to large irregular or circular lesions. Twenty symptomatic samples were collected from several individual plants, and the junction areas between infected and healthy tissues were cut into 3×3 mm pieces. These tissues were sterilized in 75% ethanol for 30 seconds and 1% HgCl solution for 3 min, rinsed thrice in sterile water, and placed on potato dextrose agar (PDA) plates, incubated at 25°C for 3 days. The edges of the colony were cut and transferred to new PDA plates for purification. These isolates were isolated from the original diseased leaves and showed similar phenotypes in morphological characters. Three representative purified strains (YJY20, YJY21, and YJY30) were used for further study. Colonies were villiform, initially white, later turning gray and greyish-green. Conidia were unitunicate, clavate, and averaged 17.36 (11.61 to 22.12) - 5.29 (3.92 to 7.04) µm in diameter (n=100). The characteristics were close to those of Colletotrichum spp. (Weir et al. 2012). The genomic DNA was extracted, and the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), calmodulin genes (CAL), actin genes (ACT), chitin synthase 1 genes (CHS-1), manganesesuperoxide dismutase (SOD2), and β-tubulin 2 genes (TUB2) were amplified from genomic DNA by primers ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-345R, SODglo2-F/SODglo2-R, and Bt2a/Bt2b, respectively (Weir et al. 2012). Sequences were submitted to GenBank with accession numbers OP535983, OP535993, OP535994(ITS), OP554748, OP546349, OP546350(GAPDH), OP546351-OP546353(CAL), OP546354-OP546356(ACT), OP554742-OP554744(CHS-1), OP554745-OP554747(SOD2), and OP554749-OP554751(TUB2). BLASTn analyses of ITS, GAPDH, CAL, ACT, CHS-1, SDO2 and TUB2 sequences exhibited 99.62%, 98.40%, 99.72%-99.86%, 96.85%-96.86%, 99.26%-100%, 100% and 99.33% similarity to the sequences of Colletotrichum fructicola strain ICMP 18581, respectively in GenBank. These morphological features and molecular identification indicated that the pathogen possessed identical characteristics as C. fructicola (Weir et al. 2012). Pathogenicity was tested through in vivo experiments. Six 1-year-old intact plants were used per isolate. The test leaves of plants were gently scratched with a sterilized needle. Conidial suspension of the pathogen strains were inoculated on the wounded leaves at a concentration of 107 conidial/mL. The control leaves were inoculated with distilled water. The inoculated plants were placed in greenhouse at 28℃ and 90% humidity. After 3-6 days，anthracnose-like symptoms were observed on inoculated leaves of five plants while the control plants remained healthy. The strains of C. fructicola were reisolated from the symptomatic inoculated leaves, confirming Koch's postulates. To our knowledge, this is the first report of C. fructicola causing anthracnose on Rosa chinensis in China. C. fructicola has been reported to affect numerous plants worldwide, including grape, citrus, apple, cassava, mango （Qili Li et al. 2019）, and tea-oil tree (X. G. Chen et al. 2022), among others (Oliveira et al. 2018). This identification research will facilitate subsequent assistance with disease control and field management of plants.