First report of Colletotrichum plurivorum Causing Anthracnose Disease of Illicium verum in China

Aniseed (Illicium verum) is an evergreen tree of the genus Illicium in the family Schisandraceae. It is not only a kitchen seasoning, but also a traditional Chinese medicine. The peels, seeds, and leaves of aniseed contain aromatic oils (Ou et al. 2015). In August 2022 and June 2024, dark brown spots were observed on leaves and young branches of aniseed in Funing county of Yunnan province, China. The plantations of aniseed in Funing county had different degrees of disease. A survey of three plantations (each 2.9 ha and 1600 plants/ha) was conducted, with the incidence of anthracnose ranging from 45% to 63% of the total plants surveyed. 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 the lesion surface. As the disease progressed, branches rotted, broke, and leaves died and dropped, likely 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 s, 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 using the single-spore culture method, among which four isolates (FN-0021, FN-0047, FN-0084, and FN-0087) exhibited similar colony morphology. Based on the isolation frequency, isolate FN-0084 was selected for further research. After culturing for 7 d on PDA medium, the colony of isolate FN-0084 was cottony with white-to-gray aerial mycelium. The mycelial growth rate was 8.3 mm/d at 25℃. Conidia were single-celled, colorless, smooth-walled, and fusiform with acute ends, and measured 7.4 to 13.9 × 3.4 to 5.6 μm. Microscopic examinations showed that conidia of the FN-0084 were hyaline, aseptate, and sub-cylindrical. Mycelium was hyaline, branched, and septate. Based on these morphological features, the fungus was tentatively identified as belonging to Colletotrichum plurivorum species complex (Akinsanmi et al. 2017). For molecular identification, ITS, TUB2, ACT, and GAPDH were amplified and sequenced (Liu et al. 2022). The obtained sequences were deposited into the GenBank [accession numbers: PV603364, PV609720, PV609721, and PV609722]. A maximum likelihood phylogenetic tree was generated based on combining all sequenced loci in PAUP4.0b10. The isolate FN-0084 fell in the C. plurivorum clade with 99% bootstrap support. For all of these genes, isolate FN-0084 had a 99-100% similarity to multiple accessions of C. plurivorum. Live plant pathogenicity tests were performed with the representative isolates. Plugs were obtained from the margins of cultures growing on PDA. Ten healthy unwounded branches were inoculated with mycelial plugs (the mycelial side in contact with the plant), which were covered with moist sterile skimmed cotton wool (Kaiser et al. 2007). Plugs without mycelium were used as control. Plants were incubated at 25℃ with a 12 h photoperiod and 90% relative humidity. 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. plurivorum was re-isolated from the diseased branches and the morphology was consistent with original, fulfilling Koch's postulates. C. plurivorum was reported to cause severe anthracnose of many species of plants (Zhao et al. 2022). To our knowledge, this is the first report of C. plurivorum causing anthracnose on Illicium verum in China. The results will help to target the screening of control agents and provide guidance for disease prevention and control.