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

Rhus chinensis, a tree of major economic importance in China, belongs to the Anacardiaceae. It is the summer host of the aphid Melaphis 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.

Molecular characterization of a novel penoulivirus from the phytopathogenic fungus Colletotrichum camelliae.

Colletotrichum camelliae is a widespread filamentous phytopathogenic fungus. In this study, a novel mycovirus designated as "Colletotrichum camelliae botourmiavirus 1" (CcBV1) was isolated from strain ZJQT11 of C. camelliae, and its complete genome sequence was determined. CcBV1 has a genome of 2,506 nucleotides and contains a large open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp) with 672 amino acids and a predicted molecular mass of 75.23 kDa. A BLASTp search showed that RdRp encoded by CcBV1 is closely related to that of Pyricularia oryzae ourmia-like virus 1 with 73.22% identity. Phylogenetic analysis indicated that CcBV1 clustered in the penoulivirus clade within the family Botourmiaviridae. To the best of our knowledge, this is the first report of a penoulivirus in C. camelliae.

First Report of Colletotrichum nymphaeae Causing Walnut Anthracnose in China.

Walnut (Juglans regia L.) is a high quality woody nut and edible oil tree with a planting area of about 5,000,000 hectare in China. Walnut anthracnose is a serious disease, infecting approximately 50% of the fruits and causing a great yield losses (Wang et al. 2016). In 2019 to 2020, walnut fruits with anthracnose symptoms were collected from walnut orchards in province of Hubei, Sichuan procinve and Chongqing municipality, China. Symptoms on fruits were circular or subcircular or irregular shaped, with brown to black water soaked and sunken lesions. The black lesions enlarged and amalgamated into large necrotic areas. The older spots in the center became blackish with acervuli causing the complete mummification of the fruit, and orange conidial masses appeared under wet conditions. Necrotic tissues of the fruits were sterilized in 75% ethanol solution for 30 s, then sterilized in 4% sodium hypochlorite for 1min, and washed 3 times with sterile distilled water. The tissues were put on potato dextrose agar (PDA) and incubated at 25℃. Pure cultures were obtained by single-spore culturing method and the isolate HBBK4-4 was deposited into the China's Forestry Culture Collection Center (CFCC 57388). On PDA, the colonies were cottony, white to pale gray with aerial mycelium on the upper side and pink with black spots on the reverse. The mycelial growth rate was 9.6 mm/day at 25°C. Conidia were 1-celled, colorless, smooth-walled, straight, cylindrical to cylindrical-clavate with acute ends, 12.5 to 18.2 × 3.9 to 5.4 µm (mean 15.3 ± 3.7 × 4.9 ± 0.6 µm, n = 40). Most conidia germinated and developed one pleurogenous, 1-celled appressorium. Appressoria were single, medium brown, smooth-walled, ovate to ellipsoid, 5.4 to 7.8 × 5.4 to 7.8 µm (mean 6.7 ± 0.6 × 6.3 ± 0.5 µm, n = 30). These morphological characteristics were in concordance with published descriptions of Collectotrichum acutatum species complex. To further confirm the identity, internal transcribed spacer (ITS), beta-tubulin (TUB2), chitin synthase 1 (CHS-I) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced (Damm et al. 2012). The ITS (OM189549) and TUB2 (OM273642) sequences of isolate HBBK4-4 showed 100% similarity, and GAPDH (OM249791) and CHS-1 (OM273641) sequences showed 98.7% and 99.6% similarity with C. nymphaeae CBS100064 respectively. A maximum likelihood phylogenetic tree was generated based on combining all sequenced loci in MEGA5. 18 isolates including HBBK4-4 fell in the C. nymphaeae clade with 96% bootstrap support. To verify Koch's postulates, six isolates were used for pathogenicity test, and 20 healthy fruits and 15 fully expanded leaves for each isolate were inoculated with 5-mm-diameter mycelial plugs. Controls consisted of detached premature fruits inoculated with a PDA plug without the fungus. Six days after inoculation, all fruits and leaves developed anthracnose symptoms similar to those observed in the field, while the controls remained healthy. The pathogenicity tests were repeated twice with the same results. The morphology of the reisolated fungi was consistent with the inoculated one, fulfilling Koch's postulates. The isolate HBBK4-4 was identified as C. nymphaeae, based on the description by Damm et al. (2012). The species C. nymphaeae has been previously reported to cause severe anthracnose on walnut in France (Da Lio et al., 2018), Brazil (Savian et al., 2019) and Italy (Luongo et al., 2022). To our knowledge, this is the first report of C. nymphaeae as a pathogen of walnut anthracnose in China. The result provided crucial information for epidemiologic studies and management of this disease.