First report of Colletotrichum fioriniae Causing Anthracnose Disease of Rhus chinensis in China.

Abstract

Rhus chinensis, a tree of major economic importance in China, belongs to the Anacardiaceae. It is the summer host of the aphidMelaphis chinensis which produces a leaf gall utilized for medicinal purposes (Li et al. 2022). In August 2021 and June 2022, dark brown spots were observed on young branches of R. chinensis in Wufeng, Hubei province, China. The plantations of R. chinensis in Wufeng County had different degrees of disease. We focused our survey on three plantations, each with an area of 1.5 hectares and 1600 R. chinensis plants per hectare, and the incidence of the disease was found to be around 70%. Symptoms began as small brown spots that expanded with time and eventually led to large, irregular, dark brown and sunken lesions. Under high temperature and humidity, orange conidiomata appeared on top of the lesions. As the disease progressed, branches rotted, broke, and leaves died and dropped, eventually causing the death of trees. The fungus was isolated from infected branches. Branch pieces were cut and surface disinfested in 75% (v/v) alcohol for 30 sec, then sterilized in 4% sodium hypochlorite for 1 min, and washed three times with sterile distilled water before incubated on potato dextrose agar (PDA) at 25℃.Ten isolates were obtained by a single-spore culturing method, considering HTK-3 isolate showed more pathogenic and grew faster than other isolates, it was selected for further research. After culturing for 7 days on PDA medium, the colony of isolate HTK-3 was cottony with white-to-gray aerial mycelium. The mycelial growth rate was 8.7 mm/day at 25℃. Conidia were single-celled, colorless, smooth-walled, fusiform with acute ends, and measured 7.7 to 14.3 × 3.2 to 5.3μm (mean 11.8 ± 1.3 to 4.2 ± 0.5μm, n = 50). Appressoria were single, medium brown, ovate to ellipsoid, 5.8 to 8.5× 3.7 to 6.1μm (mean 7.2 ± 0.7 × 4.9 ± 0.4 μm, n=50). Microscopic examinations showed conidia of the HTK-3 were hyaline, aseptate, and sub-cylindrical, with obtuse apices and tapering bases. Mycelium of which was hyaline, branched and septate. Based on these morphological features, the fungus was tentatively identified as belonging to Colletotrichum acutatum species complex (Damm et al. 2012). For molecular identification, the ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), beta-tubulin 2 (TUB2), and actin (ACT) were amplified and sequenced (Liu et al., 2022). The obtained sequences were deposited into the GenBank [accession numbers: OP630818 (ITS); OP649736 (GAPDH); OP649735 (TUB2); OP649738 (CHS-1); OP649737 (ACT)]. For all of these genes, isolates HTK-3 had a 99-100% similarity to multiple C. fioriniae accessions. A maximum likelihood tree was generated from a multiple sequence alignment of reported isolates (Liu et al. 2022) and HTK-3 was identified as C. fioriniae. To fulfill Koch's postulates, ten healthy branches were inoculated with 5-mm-diameter mycelial plugs of each of ten fungal isolates (Wang et al., 2022). PDAs plug without mycelium was used as control. Six days post-inoculation, all branches developed anthracnose symptoms similar to those observed in the field, while the control remained healthy. The pathogenicity tests were repeated twice with the same results. C. fioriniae was re-isolated from the disease branches and the morphology was consistent with original, fulfilling Koch's postulates. The species of C. fioriniae has been reported to cause severe anthracnose of many species of plants (Eaton et al. 2021). To our knowledge, this is the first report of C. fioriniae as a pathogen of R. chinensis in China. The results will help to target the screening of control agents and provide guidance for disease prevention and control.