Transcription factor PbbZIP4 is targeted for proteasome-mediated degradation by the ubiquitin ligase PbATL18 to influence pear's resistance to Colletotrichum fructicola by regulating the expression of PbNPR3.

Pear anthracnose caused by Colletotrichum fructicola is one of the main fungal diseases in all pear-producing areas. The degradation of ubiquitinated proteins by the 26S proteasome is a regulatory mechanism of eukaryotes. E3 ubiquitin ligase is substrate specific and is one of the most diversified and abundant enzymes in the regulation mechanism of plant ubiquitination. Although numerous studies in other plants have shown that the degradation of ubiquitinated proteins by the 26S proteasome is closely related to plant immunity, there are limited studies on them in pear trees. Here, we found that an E3 ubiquitin ligase, PbATL18, interacts with and ubiquitinates the transcription factor PbbZIP4, and this process is enhanced by C. fructicola infection. PbATL18 overexpression in pear callus enhanced resistance to C. fructicola infection, whereas PbbZIP4 overexpression increased sensitivity to C. fructicola infection. Silencing PbATL18 and PbbZIP4 in Pyrus betulaefolia seedlings resulted in opposite effects, with PbbZIP4 silencing enhancing resistance to C. fructicola infection and PbATL18 silencing increasing sensitivity to C. fructicola infection. Using yeast one-hybrid screens, an electrophoretic mobility shift assay, and dual-luciferase assays, we demonstrated that the transcription factor PbbZIP4 upregulated the expression of PbNPR3 by directly binding to its promoter. PbNPR3 is one of the key genes in the salicylic acid (SA) signal transduction pathway that can inhibit SA signal transduction. Here, we proposed a PbATL18-PbbZIP4-PbNPR3-SA model for plant response to C. fructicola infection. PbbZIP4 was ubiquitinated by PbATL18 and degraded by the 26S proteasome, which decreased the expression of PbNPR3 and promoted SA signal transduction, thereby enhancing plant C. fructicola resistance. Our study provides new insights into the molecular mechanism of pear response to C. fructicola infection, which can serve as a theoretical basis for breeding superior disease-resistant pear varieties.

Determination of anthracnose (Colletotrichum fructicola) resistance mechanism using transcriptome analysis of resistant and susceptible pear (Pyrus pyrifolia).

BACKGROUND: Anthracnose, mainly caused by Colletotrichum fructicola, leads to severe losses in pear production. However, there is limited information available regarding the molecular response to anthracnose in pears. RESULTS: In this study, the anthracnose-resistant variety 'Seli' and susceptible pear cultivar 'Cuiguan' were subjected to transcriptome analysis following C. fructicola inoculation at 6 and 24 h using RNA sequencing. A total of 3186 differentially expressed genes were detected in 'Seli' and 'Cuiguan' using Illumina sequencing technology. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the transcriptional response of pears to C. fructicola infection included responses to reactive oxygen species, phytohormone signaling, phenylpropanoid biosynthesis, and secondary metabolite biosynthetic processes. Moreover, the mitogen-activated protein kinase (MAPK) signaling pathway and phenylpropanoid biosynthesis were involved in the defense of 'Seli'. Furthermore, the gene coexpression network data showed that genes related to plant-pathogen interactions were associated with C. fructicola resistance in 'Seli' at the early stage. CONCLUSION: Our results showed that the activation of specific genes in MAPK, calcium signaling pathways and phenylpropanoid biosynthesis was highly related to C. fructicola resistance in 'Seli' and providing several potential candidate genes for breeding anthracnose-resistant pear varieties.

Biological characterization of emerging fungal pathogen Colletotrichum associated with mango (Mangifera indica L.) post-harvest anthracnose from Vietnam.

BACKGROUND: Post-harvest anthracnose (PHA) of mango is a devastating disease, which results in huge loss to mango producers and importers. Various species of PHA, diverse pathogenicity, and different resistance towards fungicides make it essential to evaluate the pathogen taxonomic status and biological characterization. METHODS AND RESULTS: Two strains DM-1 and DM-2 isolated from the fruit of DaQing mango from Vietnam were identified as Colletotrichum fructicola and C. asianum respectively, based on the morphological features, along with the phylogenetic tree of ITS and ApMat combined sequences. The growth status of different Colletotrichum strains under different conditions was analyzed to reveal the biological characteristics. The optimum growth temperature of DM-1 and DM-2 was 28 °C and mycelia grew rapidly in the dark. Both strains could grow in media with pH 4-11, while the optimum pH value was 6. Maltose and soluble starch were the most suitable carbon source for DM-1 and DM-2 respectively, and the peptone was the most suitable nitrogen source for both strains. The lethal temperatures were recorded as 55 °C 5 min for DM-1, and 50 °C 10 min for DM-2. CONCLUSIONS: To the best of our knowledge, it is the first study reporting the identification of the pathogens: C. fructicola and C. asianum responsible for postharvest fruit anthracnose of mango in Vietnam.

Functional Roles of Two ß-Tubulin Isotypes in Regulation of Sensitivity of Colletotrichum fructicola to Carbendazim.

Colletotrichum fructicola is the major pathogen of anthracnose in tea-oil trees in China. Control of anthracnose in tea-oil trees mainly depends on the application of chemical fungicides such as carbendazim. However, the current sensitivity of C. fructicola isolates in tea-oil trees to carbendazim has not been reported. Here, we tested the sensitivity of 121 C. fructicola isolates collected from Guangdong, Guangxi, Guizhou, Hainan, Hunan, Jiangsu, and Jiangxi provinces in China to carbendazim. One hundred and ten isolates were sensitive to carbendazim, and 11 isolates were highly resistant to carbendazim. The growth rates, morphology, and pathogenicity of three resistant isolates were identical to those of three sensitive isolates, which indicates that these resistant isolates could form a resistant population under carbendazim application. These results suggest that carbendazim should not be the sole fungicide in control of anthracnose in tea-oil trees; other fungicides with different mechanisms of action or mixtures of fungicides could be considered. In addition, bioinformatics analysis identified two ß-tubulin isotypes in C. fructicola: Cfß1tub and Cfß2tub. E198A mutation was discovered in the Cfß2tub of three carbendazim-resistant isolates. We also investigated the functional roles of two ß-tubulin isotypes. CfΔß1tub exhibited slightly increased sensitivity to carbendazim and normal phenotypes. Surprisingly, CfΔß2tub was highly resistant to carbendazim and showed a seriously decreased growth rate, conidial production, pathogenicity, and abnormal hyphae morphology. Promoter replacement mutant CfΔß2-2×ß1 showed partly restored phenotypes, but it was still highly resistant to carbendazim, which suggests that Cfß1tub and Cfß2tub are functionally interchangeable to a certain degree.

Fungicide resistance in Colletotrichum fructicola and Colletotrichum siamense causing peach anthracnose in China.

Peach is one of the popular and economically important fruit crops in China. Peach cultivation is hampered due to attacks of anthracnose disease, causing significant economic losses. Colletotrichum fructicola and Colletotrichum siamense belong to the Colletotrichum gloeosporioides species complex and are considered major pathogens of peach anthracnose. Application of different groups of fungicides is a routine approach for controlling this disease. However, fungicide resistance is a significant drawback in managing peach anthracnose nowadays. In this study, 39 isolates of C. fructicola and 41 isolates of C. siamense were collected from different locations in various provinces in China. The sensitivity of C. fructicola and C. siamense to some commonly used fungicides, i.e., carbendazim, iprodione, fluopyram, and propiconazole, was determined. All the isolates of C. fructicola collected from Guangdong province showed high resistance to carbendazim, whereas isolates collected from Guizhou province were sensitive. In C. siamense, isolates collected from Hebei province showed moderate resistance, while those from Shandong province were sensitive to carbendazim. On the other hand, all the isolates of C. fructicola and C. siamense showed high resistance to the dicarboximide (DCF) fungicide iprodione and succinate dehydrogenase inhibitor (SDHI) fungicide fluopyram. However, they are all sensitive to the demethylation inhibitor (DMI) fungicide propiconazole. Positive cross-resistance was observed between carbendazim and benomyl as they are members of the same methyl benzimidazole carbamate (MBC) group. While no correlation of sensitivity was observed between different groups of fungicides. No significant differences were found in each fitness parameter between carbendazim-resistant and sensitive isolates in both species. Molecular characterization of the ß-tubulin 2 (TUB2) gene revealed that in C. fructicola, the E198A point mutation was the determinant for the high resistance to carbendazim, while the F200Y point mutation was linked with the moderate resistance to carbendazim in C. siamense. Based on the results of this study, DMI fungicides, e.g., propiconazole or prochloraz could be used to control peach anthracnose, especially at locations where the pathogens have already developed the resistance to carbendazim and other fungicides.

Fruit Anthracnose on Cavendish bananas Caused by Colletotrichum fructicola in Guangxi, China.

Cavendish banana (Musa spp. AAA group) is one of the main fruit crops worldwide. It is widely planted in Guangdong, Hainan, Guangxi, Fujian and Yunnan provinces in southern China. In November 2020, banana fruits with anthracnose symptoms were collected from Dayu Town (N 23.17°, E 109.80°), Guigang City, and Chengjun Town (N 22.60°, E 110.00°), Yulin City, Guangxi Province, China, where the disease was found on about 70% of the banana plants, and on individual fruit, up to 10% of the surface was covered with symptoms. The symptoms initially began with rust-colored spots on the surface of the immature fruit, which gradually became sunken and cracked as the disease progressed. Small tissues (5×5 mm) from the pericarp at the junction of disease and health were surface-disinfected in 75% ethanol for 10 s, 2% sodium hypochlorite (NaClO) for 1 min, and washed three times in sterile water. Tissue pieces were placed on potato dextrose ager (PDA) and incubated at 25°C. Fifty-nine morphologically similar colonies were obtained after 5 days of incubation, with 100% isolation frequency. Of 59 isolates, GG1-3 isolated from Guigang City and YL4-2 isolated from Yulin City were selected as representative strains for intensive study. Mycelia were off-white for both isolates and conidia obtained from PDA were cylindrical, unicellular, hyaline and obtuse ends, with sizes of 11.5 ± 1.8×3.9 ± 0.8 µm (n=60) and 11.5 ± 1.6×4.1 ± 0.6 µm (n=60) for GG1-3 and YL4-2, respectively (Prihastuti et al. 2009). Genomic DNA was extracted from 7-day-old aerial mycelia using a DNAsecure Plant Kit (Tiangen Biotech, China). The internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 (ApMAT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified and sequenced (White et al. 1990; Silva et al.2012; Templeton et al. 1992). Sequences were deposited in GenBank (ITS, OR596961 to OR596962; GAPDH, OR661771 to OR661772; APMAT, OR661773 to OR661774) and showed 100% identities with the corresponding type strains sequences of C. fructicola. Phylogenetic tree was constructed with software raxmlGUI v.2.0.0 based on sequences of multiple loci (ITS, GAPDH and ApMAT) and Maximum Likelihood method. Phylogenetic analysis revealed that the two isolates and C. fructicola were clustered in the same clade, with 94% bootstrap support. According to morphology and phylogenetic analysis, the two isolates GG1-3 and YL4-2 were identified as C. fructicola. For pathogenicity tests, healthy fruits were surface sterilized with 75% ethanol followed by a wash with sterilized water. Five adjacent needle punctures in a 5-mm-diameter circle were made with a sterilized needle on healthy fruits, followed by inoculation with 20 µL of conidial suspension (106 spores/ml), and sterilized water was used as controls. All banana fruit were incubated in a humid chamber at 28°C. After 4 days, all inoculated fruits showed visible symptoms and had rust-colored spots on the margins, while control banana fruits remained symptomless. The fungus was isolated from the inoculated fruit and the isolates were found to match the morphological and molecular characteristics of the original isolates, confirming Koch's hypothesis. To our knowledge, this is the first report of fruit anthracnose on Cavendish bananas caused by C. fructicola in China. This study will provide valuable information for prevention and management of anthracnose on banana fruit.

First Report of Colletotrichum fructicola Causing Anthracnose on Epimedium sagittatum (Sieb. et Zucc.) Maxim. in China.

Epimedium sagittatum (Sieb.et Zucc.) Maxim., belonging to the family Berberidaceae and genus Epimedium, is a perennial herb widely studied for its anti-osteoporosis, anti-cancer, and anti-sexual-dysfunction effects in Asian countries (Tan et al. 2016; Zhang et al. 2016). High levels of bioactive chemicals in Epimedium spp. has endowed it with important clinical and commercial values (Liu et al. 2013). In September 2021, a leaf disease was found in Zhumadian City, China (32°58'12" N, 114°37'48" E). Survey statistics indicated that disease prevalence in a 266-ha planting area was approximately 29.6%. The lesions appeared at the leaf tips, gradually enlarged, and were brown with a yellow halo. Further, the lesions were dry with distributed black spots. Thirty infected leaves collected from five sites within the planting base . The collected leaves were cut into 5×5 mm pieces , surface-sterilized in 75% alcohol for 15 s, triple washed with sterile ddH2O, disinfested with 0.1% HgCl2 solution for 30 s (Liu et al. 2021), triple washed again with sterilized ddH2O, and then placed onto PDA and incubated in the dark for 3 d at 28°C. Subsequently, five fungal strains were purified; among them, only the isolate HY3-2 infected the host plant and was selected for further morphological characterization. The colonies of HY3-2 initially appeared white, their mycelia became gray at the center after 4 d, and orange-red conidial clumps appeared in them after 7 d. Conidia (10.0-19.5 µm × 4.5-5.6 µm, n=50) were single celled, nearly spherical or stick-shaped and colorless. Morphological characteristics of the isolate were consistent with those of Colletotrichum species. Additionally, glycerol-3-phosphate dehydrogenase (gapdh), actin (act), calmodulin (cal), ß-tubulin 2 (tub2), and chitin synthase-1 (chs-1), (Weir et al. 2012) were amplified and sequenced using the primers GDF/GDR, ACT-512F/783R, CL1C/CL2C, T1/Bt2b, and CHS-79F/354R, respectively for molecular identification. The resulting sequences were deposited in GenBank: gapdh (ON351609), act (ON351608), tub2 (ON351610), chs-1 (ON532788), and cal (ON532787). Phylogenetic analyses were performed by concatenating all the sequenced loci using the Bayesian method (Zhang et al. 2020). The phylogenetic tree showed that the isolate belongs to C. fructicola clade with a credibility value of 85%.To satisfy Koch's postulates, a conidial suspension (106 conidia/mL) of the isolate HY3-2 were prepared with sterile ddH2O to infect the leaves. Ninety healthy leaves from 30 plants in pots were punctured using a sterilized needle (Huang et al. 2022), and inoculated by spraying the conidial suspension on the leaves in a greenhouse at 25°C and 80% relative humidity. In the control plants, the suspension was replaced with water. After 7 d, the inoculated plants showed symptoms similar to those of the original infected plant, whereas the control showed no symptoms. C. fructicola was isolated and identified again as previously described. A pathogenicity test was also conducted in the field using the same method as that used in the greenhouse in July 2022, the results of which were consistent with those of the greenhouse. In China, C. fructicola has been reported on Walnut (Wang et al. 2022), Punica granatum (Hu et al. 2023) and others. To our knowledge, this is the first report of C. fructicola causing anthracnose in E. sagittatum in China. This report provides an important basis for further disease control research.

First report of anthracnose of Lithocarpus polystachyus caused by Colletotrichum fructicola in China.

Lithocarpus polystachyus (Wall. ex A. DC.), an economically valuable plant species belonging to the Fagaceae family, has been used as herbal tea to prevent diabetes because of the high content of flavonoids and dihydrochalcones in the leaves (Shang et al. 2022). In July 2022, the severe leaf lesion on L. polystachyus was first observed in Yongshun County, Xiangxi autonomous prefecture (28°45'34''N, 109°40'11''E), Hunan province, China. Yongshun County is characterized by hills and mountains, situated in a subtropical region with a mild and humid climate. A second outbreak in July 2023 was observed in the same area. The observed incident rates in the past two years were 87.3% and 90.6%, respectively. Once infected, almost all plant leaves will be infected, leading to a substantial reduction in the yield of L. polystachyus. The disease presented symptoms characterized by round or irregularly shaped lesions that initially manifested as brown spots. These lesions frequently merged into larger, dark-brown areas along the leaf margins before eventually wilting. To ascertain the pathogenic species responsible for this disease, fungal isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected with 75% ethanol and 0.1% HgCl then small pieces (1×1 cm), were placed onto potato dextrose agar (PDA) medium (Sigma-Aldrich, 70139) and incubated at 28°C for 6-9 days. Colonies were villiform and initially white, becoming gray after 6 days. Sterilized dissecting needles were used to pick single hyphal tips from the edge of the colonies and placed onto PDA for strain purification. After 15 days, the purified colonies grew fluffy white hyphae with abundant conidia. The conidia were cylindrical, had round ends, and ranged from 5.75 to 14.83 µm long and 1.75 to 2.38 µm wide (n=50). According to morphological and cultural characteristics, these isolates were preliminarily identified as Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Damm et al. 2012). To further affirm the identity of the pathogen, DNA was extracted from mycelia using a DNA extraction kit (Sigma-Aldrich, G2N70). The internal transcribed spacer (ITS) region, the transcription elongation factor (TEF), and the actin (ACT) gene were then amplified from genomic DNA extracted from three isolates (Cof1, Cof2, and Cof3) using specific primers. The primers utilized were ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and ACT-512F/ACT-783R (Carbone and Kohn 1999) for ITS region, transcription elongation factor gene and actin gene amplification, respectively. Sequence identity indicated that these isolates were highly homologous to C. fructicola. The ITS (Genbank No. PP002156, OR880553 and OR880554), TEF (No. PP061421, PP061422 and PP061423), and ACT (No. PP061418, PP061419 and PP061420) sequences of the isolates Cof1, Cof2, and Cof3 shared 99 to 100% identity with their counterparts (No. OR083309, MF627961, and OQ427895) in C. fructicola, respectively. A neighbor-joining phylogenetic tree constructed using MEGA11 (Tamura et al. 2021) also indicated that these isolates were C. fructicola. Both morphological and molecular characteristics confirmed the identification of this pathogen as C. fructicola. Colletotrichum species are known to cause anthracnose disease in a variety of economically important crops (Sharma and Kulshrestha 2015). To further validate the ability of the isolated C. fructicola to induce the same symptoms as observed in the field, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspensions (1×105 conidia per mL) from three isolates were individually inoculated onto artificially wounded leaves of 3-year-old L. polystachyus. Negative controls were established by inoculating leaf wounds with sterile distilled water. The plants were incubated in a greenhouse at 28°C and 90% humidity with a 12-h photoperiod. The experiment was replicated three times. Necrotic lesions were observed on all pathogen-inoculated wounds within 6 days after inoculation, whereas controls showed no observable symptoms. Morphological and molecular characterization of re-isolated pathogens from infected leaves indicated that the pathogens were identical. To our knowledge, this is the first report of anthracnose of L. polystachyus caused by C. fructicola in China. Farmers in the local mountainous areas are economically reliant on L. polystachyus production, while anthracnose has caused over half of the trees to lose their commercial value, resulting in significant economic losses. Our findings hold great promise for advancing strategies in the prevention and treatment of anthracnose in L. polystachyus.

First Report of Colletotrichum fructicola Causing Anthracnose on Canna indica L. in Guangxi, China.

Canna indica L., a well-known wetland plant (Lei et al. 2023), was found with leaf spots in a planting area (∼667 m2) in Tiandong, Guangxi, China, in June 2022. 5500 plants were affected by this disease. Symptoms began as yellow lesions, and then developed brown sub-ellipsoid spots with yellow borders, then gradually expanded and encompassed the entire leaves until leaves wilted. 18 diseased leaves were collected and cut into small pieces (3 ×3 mm) from the brown margins. The pieces were moistened with 75% ethanol for 10 seconds, disinfected with 2% NaClO for two minutes and rinsed with sterile water three times. Pieces were placed on potato dextrose agar (PDA) and incubated at 28°C for four days. 15 isolates with similar morphological characterizations were isolated and purified (about 68% isolation frequency) from 18 diseased leaves. Three isolates (CI1-1, CI1-2 and CI1-3) were selected for further morphological and molecular identification. Fungi mycelia on PDA were grayish white initially, and became dark gray after seven days. Conidia were hyaline, guttulate, unicellular, cylindrical, and averaged 15.09 × 5.72 µm. To confirm the identification, genomic DNA was extracted from mycelium of the three isolates, and the partial internal transcribed spacer (ITS) regions, intergenic region of apn2 and MAT1-2-1 (ApMAT), fragments of actin (ACT), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and ß-tubulin (TUB2) genes were amplified, sequenced and submitted to GenBank (ITS: OR501461 to OR501463; ApMat: OR684455-OR684457; ACT: OR765956-OR765958; GAPDH: OR779527-OR779529; CHS-1: OR797622-OR797624; TUB2: OR820537-OR820539). The sequences of the three isolates were 99%-100% identical (ApMat, 882/882 bp; ACT, 228/230 bp; GAPDH, 278/280 bp; CHS-1, 298/299 bp and TUB2, 298/299 bp) with those of Colletotrichum fructicola isolate ICMP18581 (JQ807838, FJ907426, JX010033, JX009866 and JX010405) (Liu et al. 2015). Compared with C. fructicola isolate ICMP18581 (JX010165), the ITS sequence identities were 94% (556/594 bp). A Maximum Likelihood phylogenetic tree was constructed by using MEGA v. 10.1.5 based on the concatenation of multiple sequences. Based on these results, the three isolates were identified as C. fructicola. Pathogenicity tests of three isolates were conducted on nine one-year-old seedlings. Three leaves per plant (six sites per leaf) were inoculated with the adjusted conidial suspension of each isolate. Ten µl suspension (106 conidia/ml) was dripped on each inoculation site without wounding. Three additional plants were inoculated with sterile water as negative controls. All plants were covered with plastic bags sprayed with sterile water, and cultured in a light incubator at 28°C, with 14:10 h light/dark cycle. After five days, dark-brown spots (0.1-1.4cm×0.2-1.6cm) appeared on the leaves of experimental groups, while no lesions were found in the controls. The pathogen was reisolated from the symptomatic leaves and confirmed as C. fructicola based on molecular and morphological methods, fulfilling Koch's hypothesis. C. fructicola has been reported in various ornamental plants (Silva-Cabral et al. 2019, Guarnaccia et al. 2021, Sun et al. 2020). This is the first report of C. fructicola causing anthracnose on C. indica in China, according to literature analysis. The findings will help growers to prevent and control this pathogen, and improve the landscape effect.

First Report of Colletotrichum fructicola Causing Leaf Spot on Smilax glabra Roxb in China.

Smilax glabra Roxb is a medicinal plant distributed in 17 countries and used in the production of food and tea (Wu et al. 2022). In May 2021, a leaf spot disease was observed on ~60% of S. glabra plants in a field (∼0.4 ha) in Qinzhou City, Guangxi Province. Initially, small, circular, brown spots appeared on the leaf surfaces, which then gradually expanded into large, sunken, dark brown necrotic areas. As disease progressed, lesions merged into large spots, eventually leading to defoliation. To determine the causal agent, six symptomatic plants were collected from the field. Small pieces (∼5 mm2) were cut from the infected leaves (n = 12), sterilized for two min in 1% NaOCl, and rinsed three times in sterile water. Then, the leaf tissues were placed on potato dextrose agar (PDA) with chloramphenicol (0.1 g/liter) and incubated for 3 days at 28°C (12-h photoperiod). Pure cultures were obtained by transferring hyphal tips from recently germinated spores or colony edges onto PDA. Among the 17 isolates, 15 exhibited similar morphologies. Two single-spore isolates (TFL45.1 and TFL46.2) were subjected to further morphological and molecular characterization. Colonies on PDA were grayish green with a white outer ring and cottony surface, and pale blackish green on the reverse side. Conidia were hyaline, aseptate, straight, and cylindrical, with rounded ends, and 11.4 to 16.5 µm × 4.1 to 6.1 µm (average 13.9 × 4.8 µm, n = 100). Appressoria were brown to dark brown, with a smooth edge and different shapes such as ovoid, elliptical or irregular, and 6.8 to 8.9 µm × 5.9 to 7.8 µm (average 7.7 × 6.6 µm, n = 25). For molecular identification, eight target gene sequences, internal transcribed spacer (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPHD), calmodulin (CAL), partial actin (ACT), chitin synthase (CHS-1), glutamine synthetase (GS), manganese superoxide dismutase (SOD2), and ß-tubulin (TUB) were selected for PCR amplification (Weir et al. 2012). The resulting sequences were deposited in GenBank (OR399160-61 and OR432537-50). BLASTn analysis of the obtained sequences showed 99-100% identity with those of the ex-type strain C. fructicola ICMP:18581 (JX010165, JX010033, FJ917508, FJ907426, JX009866, JX010095, JX010327, JX010405) (Weir et al. 2012). In addition, a phylogenetic analysis confirmed the isolates as C. fructicola. Therefore, based on morphological and molecular characteristics (Park et al. 2018; Weir et al. 2012), the isolates were identified as C. fructicola. To verify pathogenicity, three healthy leaves on each of six two-year-old S. glabra plants were inoculated with ∼5 mm2 mycelial discs or aliquots of 10 µl suspension (106 conidia/ml) of the strain TFL46.2, and six control plants were inoculated with sterile PDA discs or sterile water. All plants were enclosed in plastic bags and incubated in a greenhouse at 25°C (12-h photoperiod). Six days post-inoculation, leaf spot symptoms appeared on the inoculated leaves. No symptoms were detected in the controls. Experiments were replicated three times with similar results. To fulfill Koch's postulates, C. fructicola was consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing of the eight genes, whereas no fungus was isolated from the control plants. To our knowledge, this is the first report of C. fructicola causing leaf spot disease on S. glabra. Further studies will be needed to develop strategies against this disease based on the identification of this pathogen.

A Novel Heptasegmented Positive-Sense Single-Stranded RNA Virus from the Phytopathogenic Fungus Colletotrichum fructicola.

In this study, a novel positive-sense single-stranded RNA (+ssRNA) mycovirus, tentatively named Colletotrichum fructicola RNA virus 1 (CfRV1), was identified in the phytopathogenic fungus Colletotrichum fructicola. CfRV1 has seven genomic components, encoding seven proteins from open reading frames (ORFs) flanked by highly conserved untranslated regions (UTRs). Proteins encoded by ORFs 1, 2, 3, 5, and 6 are more similar to the putative RNA-dependent RNA polymerase (RdRp), hypothetical protein (P2), methyltransferase, and two hypothetical proteins of Hadaka virus 1 (HadV1), a capsidless 10- or 11-segmented +ssRNA virus, while proteins encoded by ORFs 4 and 7 showed no detectable similarity to any known proteins. Notably, proteins encoded by ORFs 1 to 3 also share considerably high similarity with the corresponding proteins of polymycoviruses. Phylogenetic analysis conducted based on the amino acid sequence of CfRV1 RdRp and related viruses placed CfRV1 and HadV1 together in the same clade, close to polymycoviruses and astroviruses. CfRV1-infected C. fructicola strains demonstrate a moderately attenuated growth rate and virulence compared to uninfected isolates. CfRV1 is capsidless and potentially encapsulated in vesicles inside fungal cells, as revealed by transmission electron microscopy. CfRV1 and HadV1 are +ssRNA mycoviruses closely related to polymycoviruses and astroviruses, represent a new linkage between +ssRNA viruses and the intermediate double-stranded RNA (dsRNA) polymycoviruses, and expand our understanding of virus diversity, taxonomy, evolution, and biological traits. IMPORTANCE A scenario proposing that dsRNA viruses evolved from +ssRNA viruses is still considered controversial due to intergroup knowledge gaps in virus diversity. Recently, polymycoviruses and hadakaviruses were found as intermediate dsRNA and +ssRNA stages, respectively, between +ssRNA and dsRNA viruses. Here, we identified a novel +ssRNA mycovirus, Colletotrichum fructicola RNA virus 1 (CfRV1), isolated from Colletotrichum fructicola in China. CfRV1 is phylogenetically related to the 10- or 11-segmented Hadaka virus 1 (HadV1) but consists of only seven genomic segments encoding two novel proteins. CfRV1 is naked and may be encapsulated in vesicles inside fungal cells, representing a potential novel lifestyle for multisegmented RNA viruses. CfRV1 and HadV1 are intermediate +ssRNA mycoviruses in the linkage between +ssRNA viruses and the intermediate dsRNA polymycoviruses and expand our understanding of virus diversity, taxonomy, and evolution.

First Report of Colletotrichum fructicola Causing Anthracnose on Glycine max in China.

Soybean (Glycine max L.) is one of the important oilseed and vegetable crop worldwide and provides the main source of vegetable oil and proteins for human and livestock (Hartman et al. 2011). In October 2021, approximately 35% of soybean pods suffered from anthracnose in the farmer's field in Chongzhou, Sichuan Province, China (103°40'12"E, 30°37'48"N), and the occurrence area accounted for about 3.3 hm2. Symptoms of soybean were characterized by yellow spots at the initial stage, gradually expanded into dark brown spots, and eventually amounts of small black particles were densely arranged in the wheel shape on dead spots. Diseased spots of soybean pods were cut into pieces and sequentially sterilized in 75% alcohol for 30 s, 4% sodium hypochlorite for 30 s, sterile water for 3 times. After that, these pieces were placed on potato dextrose agar (PDA), and incubated at 25±2°C in the dark for 5-7 days. Single spore was separately picked and transferred to a fresh PDA plate to obtain pure culture isolates. Total six pure isolates were collected, and among them the hyphae of representative isolate 8-B were initially white, turned grey gradually on PDA medium, and the colonial reverse were radiating, whorled or a mixture of both. Conidia of 8-B were septate, hyaline, unicellular, cylindrical, obtusely rounded at both ends with 1 or 2 oil balls inside, and 10.5-17.6 µm in length and 7.0 µm-3.6 µm in width (n=100). The conidial appressoria were brown subspherical, 6.9 µm-13.3 µm in length and 5.6 µm-10.1 µm (n=50) in width. Based on morphological and cultural characteristics, the isolate 8-B was tentatively identified as Colletotrichum gloeosporioides species complex（Weir et al. 2012). To test pathogenicity, the mycelial plugs were inoculated on 20 detached soybean pods at full seed (R6) stage, and three areas of each pod were lightly scratched using a needle prior to inoculation. As controls, the PDA plugs were attached to the pinned-treated pods. Three independent replicates were conducted for control and inoculated pods, respectively. All pods were incubated in a greenhouse at 25 ± 2°C with a relative humidity of approximately 90%. After 4-5 days post-inoculation, typical anthracnose lesions were observed on the inoculated pods while the control pods remained healthy only with small wound spots. The pathogen re-isolated from all the inoculated pods were morphologically identical to the inoculation isolate (8-B). For further molecular verification, the six gene fragments including the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), ß-tubulin 2 (TUB2) and calmodulin (CAL) were amplified and sequenced (Weir et al. 2012, Damm et al. 2012), and the obtained sequences were deposited in GenBank (Accession numbers ON960278, ON685214, ON964475, ON974476, ON685215 and ON964477, respectively). All six gene sequences of 8-B had a high identity to C. fructicola (the stand isolate ICMP 18581) with the accession numbers ON960278 (100%), ON974476 (96%), ON685214 (99%), ON964475 (99%), ON685215 (100%), and ON964477 100%), respectively. Anthracnose disease caused by C. fructicola has previously been reported to affect a range of plant hosts worldwide (Guarnaccia et al. 2017). However, it is still unknown on C. fructicola causing anthracnose in soybean in China. This study firstly reports C. fructicola as the causal agent of anthracnose on soybean in the country, and provides a theoretical basis for the diagnosis and control of this disease.

First report of Colletotrichum fructicola causing anthracnose on peanut (Arachis hypogaea L.) in China.

Peanut (Arachis hypogaea L.) is an important cash crop and oil crop around the world. In August 2021, symptoms of leaf spot were found on nearly 50% of peanut plants in the peanut planting base of Xuzhou Academy of Agriculture Sciences, Jiangsu, China. Symptoms began as small, round or oval, dark brown spots on the leaf. As the spot expanded, the center of the spot became gray to light brown and the spot was covered with small black dots. Fifteen leaves with typical symptoms were randomly collected from fifteen plants in three fields about a kilometer apart from each other. Leaf pieces (5 × 5 mm) were cut from the junction part of diseased and healthy leaf tissue, sterilized with 75% ethanol for 30 s and 5% NaClO for 30 s, washed 3 times with sterile water, placed on full strength potato dextrose agar (PDA) and incubated at 28°C in darkness. Five days after incubation, 12 isolates were obtained. Fungal colonies were white to gray on the upper surface and orange to gray on the reverse side. Conidia were single-celled, cylindrical and colorless after maturation, and were 12 - 16.5 × 4.5 - 5.5 µm (n = 50) in size. Ascospores were one-celled, hyaline, with tapering ends and one or two large guttulates at the center, and measured 9.4 - 21.5 × 4.3 - 6.4 µm (n = 50). Based on morphological characteristics, the fungi were preliminarily identified as Colletotrichum fructicola (Prihastuti et al. 2009; Rojas et al. 2010). Single spore isolates were cultured on PDA medium and two representative strains (Y18-3 and Y23-4) were selected for DNA extraction. The internal transcribed spacer (ITS) rDNA region, partial actin gene (ACT), partial calmodulin gene (CAL), partial chitin synthase gene (CHS), partial glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), and partial beta-tubulin 2 gene (TUB2) were amplified. The nucleotide sequences were submitted to Genbank (accession numbers of strain Y18-3: ITS: ON619598; ACT: ON638735; CAL: ON773430; CHS: ON773432; GAPDH: ON773436; TUB2: ON773434; accession numbers of strain Y23-4: ITS: ON620093; ACT: ON773438; CAL: ON773431; CHS: ON773433; GAPDH: ON773437; TUB2: ON773435). The phylogenetic tree was constructed using MEGA 7 based on the tandem of six genes (ITS-ACT-CAL-CHS-GAPDH-TUB2). The result showed that isolates Y18-3 and Y23-4 reside in the clade of C. fructicola species. To determine pathogenicity, conidial suspensions (107/mL) of isolate Y18-3 and Y23-4 were sprayed on ten 30-day-old healthy peanut seedlings per isolate. Five control plants were sprayed with sterile water. All plants were kept moist at 28°C in the dark (> 85% RH) for 48 h and then transferred to a moist chamber at 25°C with a 14-h photoperiod. After two weeks, typical anthracnose symptoms similar to those observed in the field appeared on leaves of inoculated plants, whereas controls remained asymptomatic. C. fructicola was re-isolated from symptomatic leaves but not from controls. Koch's postulates verified that C. fructicola was the pathogen of peanut anthracnose. C. fructicola is a well-known fungus causing anthracnose on many plant species worldwide. In recent years, new plant species infected by C. fructicola have been reported, like cherry, water hyacinth and Phoebe sheareri (Tang et al. 2021; Huang et al. 2021; Huang et al. 2022). To our knowledge, this is the first report of C. fructicola causing peanut anthracnose in China. Thus, it is recommended to pay close attention and take necessary prevention and control measures against potential spread of peanut anthracnose in China. .

First report of Colletotrichum fructicola causing anthracnose on Carya cathayensis Sarg. in China.

Carya cathayensis Sarg. (Chinese hickory) is one of the important economic forest plants, mainly distributed in Zhejiang and Anhui provinces in China. In September 2020, leaf spot disease occurred on 90% C. cathayensis in a 2.6 km2 plantation with 500 hickorys in Shangshu Village (30°26'N, 119°32'E), Huzhou, Zhejiang, China. Symptoms initially appeared as small brown spots. Later, the spots became dark brown, and joined into irregular shapes. Twenty diseased leaves with typical symptoms were collected and used to isolate the pathogen. The leaf tissues (5 × 5 mm) at junction of diseased and healthy portion were cut and surface-sterilized with 75% ethanol for 15 s, 0.1% NaClO for 2 min, and rinsed 3 times in sterile water, then placed on potato dextrose agar (PDA) plates and incubated at 25°C in the darkness for 3 days. Eight isolates with similar morphological characterizations were obtained after pure cultures by transferring hyphal tips. The colony growing on PDA for 7 days was circular, dense, white cotton-like hyphae, and light gray-black hyphae can be seen inside. The conidia were cylindrical, aseptate, hyaline, with rounded ends, and 12.5 to 20.0 × 5.0 to 7.5 µm (n = 50). The appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and were in the range of 6.4 to 10.2 × 5.0 to 6.7 µm (n = 50). The morphologies of the isolates were consistent with the genus description of Colletotrichum (Fuentes-Aragón et al. 2018; Liu et al. 2015). The internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), beta-tubulin 2 (TUB2) and actin (ACT) genes were amplified from genomic DNA for the isolates using the primers described by Weir et al. (2012). The sequences of eight isolates were consistent and the representative isolate CFZJ-64 were deposited in GenBank under the following accession numbers: ITS, OK145563; ACT, OK216738; CAL, OK216739; CHS-1, OK216740; GAPDH, OK216741; and TUB2, OK216742. A phylogenetic tree was generated by combining ITS, ACT, CAL, CHS-1, TUB2, and GAPDH sequences in MEGA11. Three representative isolates CFZJ-42, CFZJ-53 and CFZJ-64 clustered in the C. fructicola clade with 90% bootstrap support. Based on morphological characteristics and phylogenetic analysis, the isolates were identified as C. fructicola. To confirm pathogenicity, 9 detached healthy leaves and 9 healthy leaves on 3-year-old C. cathayensis seedlings were inoculated with conidial suspension of each isolate (20 µL, 1 × 106 conidia/mL). The control leaves were treated with distilled water (20 µL). Each tested leaf was covered with a clean ziplock bag and incubated for 48h at about 27°C, and 14h photoperiod. After five days, 7 of 8 isolates caused on all detached leaves or part of the leaves on the seedlings developed lesions similar to those observed in the field, whereas controls were asymptomatic. The same fungus was re-isolated from all the diseased leaves and identified by sequencing, confirming Koch's postulates. As far as we know, this is the first report of C. fructicola causing anthracnose on C. cathayensis. This study not only expands the knowledge on this important pathogen of C. cathayensis in China, but also provides the foundation to further investigate the biology, epidemiology, and control of the disease.

First Report of Anthracnose on Tetrapanax papyriferus Caused by Colletotrichum fructicola in China.

Tetrapanax papyriferus is an evergreen shrub native to China and traditionally used as a herbal medicine (Li et al., 2021). In September 2021, a serious leaf spot disease with symptoms similar to anthracnose was extensively observed on T. papyriferus in Shibing county (E 127°12'0", N 25°11'60"), Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou province, China. Field surveys were conducted in about 1000 T. papyriferus plants in Shibing in September 2021. The incidence of the leaf spot on leaves was 45% to 60%, significantly reducing the quality of medicinal materials. The symptoms began as small yellow spots, developing a brown center and dark brown to black margin, and eventually the diseased leaves were wiltered and rotted. Symptomatic leaves were collected from 20 trees. Symptomatic tissue from diseased leaves was surface desinfected (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days (Fang. 2007). Three single-spore isolates were obtained (GUTC37, GUTC310 and GUTC311) and deposited in the collection of the Plant Pathology Deparment, College of Agriculture, Guizhou University, China (GUCC) (with the accession numbers, GUCC220241, GUCC220242, GUCC220243 respectively). These isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS], glyceraldehy-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], actin [ACT], and calmodulin [CAL] genes (White et al. 1990; Carbone and Kohn 1999; Templeton et al. 1992). Therefore, the representative isolate GUTC37 was used for further analysis. The pathogenicity of GUTC37 was tested through a pot assay. Plants were inoculated by spraying a spore suspension (106 spores·ml-1) of isolated strains onto leaves until runoff, and the control leaves sprayed with sterile water. The inoculated plants were incubated in a growth chamber at 28 ℃ and 95% relative humidity for 10 days. Pathogenicity tests were repeated three times (Fang. 2007). The symptoms developed on the inoculated leaves, while control remained asymptomatic. The lesions were first visible 72 h after inoculation, and typical lesions like those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the non-inoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white or greyish, aerial mycelium pale grey, dense, surface partly covered with orange conidial masses. The conidia were abundant, oval-ellipsoid, aseptate, and 13.89 (11.62 to 15.21) × 5.21 (4.39 to 5.65) µm (n=50). Appressorium were greyish green, nearly ovoid to cylindrical, 9.64 (6.62 to 14.61) × 6.33 (5.45-7.72) µm (n=50). The morphological features were consistent with the descriptions of Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Prihastuti et al. 2009). The pathogen was identified to be C. fructicola by amplification and sequencing of the five genes. The sequences of the PCR products were deposited in GenBank with accession numbers OP143657 (ITS), OP177868 (GAPDH), OP177865 (CHS-1), OP278677 (ACT) and OP177862 (CAL). BLAST searches of the obtained sequences revealed 100% (509/509 nucleotides), 99.63% (269/270 nucleotides), 99.31% (287/289 nucleotides), 99.29% (280/282 nucleotides), and 99.86% (728/729 nucleotides) homology with those of C. fructicola in GenBank (JX010165, JX010033, JX009866, FJ907426, and JX009676, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUTC37 in a well-supported cluster with C. fructicola. To our knowledge, this is the first report of anthracnose on T. papyriferus caused by C. fructicola in Guizhou, China. This study provides valuable information for the identification and control of the anthracnose on T. papyriferus.

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

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 ß-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.

Colletotrichum fructicola Causal agent of Shot-Hole Symptoms on Leaves of Prunus sibirica in China.

Prunus sibirica L. (Siberian apricot) is a member of the Rosaceae family and an ecologically important tree species in China (Buer et al., 2022). Shot hole symptoms on the leaves were observed in five Siberian apricot groves in Chengdu (103.81 E, 30.97 N), Sichuan province in July 2020. The symptoms first appeared as small purplish-brown spots with yellow rings around them. As the disease progressed, the damaged area (diameter 1.5-3.0 cm) became necrotic and fell off. The disease incidence was about 60% and the disease index was 28.6 of leaves in the grove. in most severe cases. Fifteen symptomatic leaves were collected from 5 different trees in an orchard. Pathogen isolation was performed from symptomatic leaf tissue (5 × 5 mm) though surface disinfection (in 70% ethanol and 2% NaClO) and incubation on Potato Dextrose Agar (PDA) at 28℃ for 3 days. Overall 10 isolates with similar colony morphology were obtained from the 15 infected tissue pieces, and three representative isolates (XCK 2-4) were selected for further study. Colonies of the isolates on PDA were initially cottony, pale white to grayish-green with abundant aerial hyphae and produced conidial masses after 7 days. Conidiogenous cells were clavate and aggregated in acervuli. Conidia were smooth-walled, single-celled, straight, and slightly obtusely rounded at both ends, 12.8 to 18.7 × 4.3 to 5.7 µm in size (Fig. 1). The morphological characteristics of the three isolates were consistent with the description of species in the Colletotrichum gloeosporioides complex. DNA was amplified using the following primers pairs for the internal transcribed spacer (ITS) region of rDNA and partial sequences of beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and translation elongation factor (TEF-1), respectively: ITS1/ITS4, T1/Bt2b, GDF/GDR, CHS-F/CHS-R, and EF-F/EF-R (Vieira et al., 2014). Accession numbers (MW228049, MW284974, MW284976, MW284975 and MW284977, respectively) were obtained afterepositing all the resulting sequences in GenBank. Nucleotide blast showed 99 to 100% identities with Colletotrichum fructicola (GenBank accessions nos. MZ961683, MW284974, MN525881, MN525860, MF627961). Phylogenetic analysis of combined ITS-TUB-GAPDH genes using the Mrbayes inference method showed that the three isolates clustered with three reference isolates of C. fructicola as a distinct clade (Fig. 2). To verify Koch's postulates, ten 3-year-old healthy potted plants of P. sibirica were inoculated by spraying a conidial suspension (6 × 105 conidia/mL) of isolate XCK2 on both sides of leaves, and the control leaves were sprayed with sterile water. Then, all treatments were placed in a moist environment (25±2°C, 80% relative humidity, natural light). The inoculated plants showed typical symptoms of plants with natural infections, while the controls remained asymptomatic after 14 days. The pathogen C. fructicola was re-isolated from all inoculated plants, and the culture and fungus characteristics were the same as those of the original isolate. Colletotrichum fructicola was not isolated from the control plants. The results indicated that C. fructicola is the causal agent of the disease. Colletotrichum fructicola was reported as a leaf pathogen on Camellia chrysantha in China (Zhao et al., 2021). This is the first report of C. fructicola causing P. sibirica leaf shot-hole in the world. The identification of C. fructicola could provide relevant information for applying management strategies and research on the Siberian apricot disease.

Detection and Quantification of Anthracnose Pathogen Colletotrichum fructicola in Cultivated Tea-Oil Camellia Species from Southern China Using a DNA-Based qPCR Assay.

Tea-oil Camellia species as edible-oil producing trees are widely cultivated in southern China. Camellia anthracnose that is mainly caused by Colletotrichum fructicola is a major disease of tea-oil trees. However, rapid detection and precise quantification of C. fructicola in different Camellia species that are crucial for the fundamental study of this pathosystem and effective disease management remain largely unexplored. Here, we developed a sensitive, rapid, and accurate method for quantifying C. fructicola growth in different Camellia species using a quantitative PCR assay. Amplified C. fructicola DNA using ITS-specific primers is relatively compared with the amplification of Camellia oleifera using the TUB gene. We determined that the fungal growth is tightly associated with the disease development in Ca. oleifera following C. fructicola infection in a time-course manner. This assay is highly sensitive, as fungal growth was detected in six different inoculated tea-oil Camellia species without visible disease lesion symptoms. Additionally, this method was validated by quantifying the Camellia anthracnose in orchards that did not show any disease symptoms. This assay enables the rapid, highly sensitive, and precise detection and quantification of C. fructicola growth in different tea-oil Camellia species, which will have a practical application for early diagnosis of anthracnose disease under asymptomatic conditions in Camellia breeding and field and will facilitate the development of tea-oil trees and C. fructicola interaction as a mold system to study woody plant and fungal pathogens interaction.

First Report of Colletotrichum fructicola Causing Anthracnose on Paeonia lactiflora in China.

Paeonia lactiflora Pall is a traditional famous flower with long cultivated history in China, and has important medical and ornamental functions (Duan et al. 2022). In the middle of June 2022, anthracnose disease was observed nearly 25% (n=90) on P. lactiflora in Poyang County, Shangrao City, Jiangxi Province (29.00° N, 116.67° E) (Figure 1 E). The symptoms of the disease were small, round, light brown spots then grew bigger to round or irregular dark brown lesions (5 to 7 mm diameter) progressively on the leaves with disease spread (Figure 1 A). Subsequently, necrotic tissue was formed in the center and caused fade and wilt on the leaves ultimately, which reduced the medicinal and aesthetic value severely. Small pieces of diseased tissue (5 × 5 mm) were cut from the diseased junction, disinfected with 75% ethanol for 30 to 45 seconds, then 1% NaClO for 1 to 2 minutes, rinsed three times with sterile water. To identify the pathogen, tissues were placed on PDA and incubated for 3 days at 28°C. Single spore isolates were cultured on PDA, the colonies of one representative strain (SY4) were originally white with a lot of aerial mycelium after 5 to 7 days at 28°C in the incubator. The center of the colony turned greyish-white, released tiny orange-yellow particles (conidia) (Figure 1 F and 1 G), which were single, colorless, elongated ovals with rounded ends and measured 11.29 to 23.24 × 3.94 to 5.60 µm (av=15.89 µm × 4.74 µm, n=50) (Figure 1 H and 1 I). The isolate SY4 was identified to Colletotrichum fructicola based on morphological characteristics (Yang et al. 2021; Li et al. 2022b). For further molecular identification, the rDNA-ITS, actin gene (ACT), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), chitin synthase (CHS) and calmodulin gene (CAL) genes were amplified and sequenced with primers of ITS1/ITS4 (Gardes et al. 1993), ACT-512F/ACT-783R, GDF/GDR (Templeton et al. 1992), CHS-79F/CHS-345R (Carbone et al. 1999) and CL1C/ CL2C (Weir et al. 2012) respectively. The accession numbers in GenBank were OP523977 (ITS-rDNA), OP547618 (ACT), OP605733 (GAPDH), OP605732 (CHS), and OP605731 (CAL). The BLAST analysis revealed that these sequences were identical more than 99% with those of C. fructicola (GenBank accession Nos. MZ437948.1, MN525803.1, MN525860.1, MZ13360.1 and ON188684.1) (Figure 2). To confirm pathogenicity, the leaves were cleaned with 75% ethanol, rinsed with sterile water. After the leaf surface was dried naturally, 20 leaves were pricked at two symmetrical places on either side of the main veins of the leaf with a sterilized inoculum needle (2.0 mm in diameter), half of the wounded leaves were inoculated with 20 µL spore suspension (1.0 × 106 spores/mL) (Figure 1 C and 1 D), while the other half were inoculated with sterile water as controls (Figure 1 B). Inoculated leaves were grown for 5 days in an incubator at 28 °C and above 90% relative humidity, repeated three times. The results demonstrated that the wounded leaves with C. fructicola showed the same signs of wilting with the original disease leaves, while control leaves remained healthy. The same fungus was reisolated from the diseased leaves which confirmed with Koch's postulates. The same fungus was re-isolated from the diseased leaves while it was not isolated from control leaves, confirmed with Koch's postulates. In China, it had been reported that C. fructicola caused anthracnose on Persea americana (Li et al. 2022a) and Myrica rubra (Li et al. 2022b). To the best of our knowledge, this is the first report of anthracnose on P. lactiflora caused by C. fructicola in China. The results will help to develop effective control strategies for anthracnose on P. lactiflora.

First report of anthracnose on spotted laurel caused by Colletotrichum fructicola in South Korea.

Spotted laurel (Aucuba japonica) is a popular ornamental bush (it has two-colored leaves and red berries) and is used outdoors and indoors for decoration in South Korea. Anthracnose reduces the aesthetic value of spotted laurel leaves. In August 2022, anthracnose symptoms were observed on leaves in a park at Jeju Island, South Korea. Approximately 55% of bushes were infected by this disease. Symptoms consisted of round or irregular lesions that initially appeared as black spots and coalesced into larger, black lesions covering whole leaves and twigs. Entire leaves wither and finally die. To identify the putative causal agent, 12 affected leaves were collected, placed in a plastic box containing moist tissue, and incubated at 25 ºC in the dark to obtain conidial mass. Conidial masses were produced on leaf lesions after 2 days, and then 12 morphologically similar fungal isolates were recovered following single the spore isolation technique on solid potato dextrose agar (PDA) (Cai et al. 2009). Ten-day-old colonies were olivaceous gray with immersed perithecia on the upper side and black at the center on the reverse side. Conidia were aseptate, cylindrical with round ends and measured 14.9 - 22.7 × 5.5 - 9.4 µm (n = 80). Appressoria were brown, irregular in shape, and 7.0 - 16.1 × 5.00 - 9.9 µm (n = 50). Asci were eight-spored, banana-shaped, and measuring 60.8 - 123.1 × 13.00 - 18.9 µm (n = 30). Hyaline ascospores were single-celled, curved or straight with round ends, and ranged in size was 15.5 - 23.3 × 5.1 - 11.8 µm (n = 50). The morphological characteristics of the isolates overlapped with those of Colletotrichum species within the C. gloeosporioides complex, including Colletotrichum fructicola (Weir et al. 2012). Five genomic DNA loci of the isolates, including the partial ITS rDNA region, ACT, GAPDH, TUB, and ApMat genes, were amplified and sequenced using ITSF1/ITS4, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting consensus sequences were deposited in the GenBank and the accession numbers (ITS = LC739331- LC739334, TUB = LC739335- LC739338, GAPDH = LC739339- LC739342, ACT = LC739343 -LC739346, ApMat = LC742925 - LC742928) were obtained. A maximum phylogenetic tree was constructed based on the combined data sets of ITS, ACT, GAPDH, TUB, ApMat sequences. The isolates were clustered with reference isolates of C. fructicola (isolates ICMP18581). The pathogenicity test was performed on uninfected, healthy spotted laurel cuttings in the pot. Five leaves per seedling were selected, surface sterilized with 70% ethanol, and rinsed with sterile distilled water (SDW). A sterile pin was used to make 3 to 4 wounds on each side of the leaf from the midrib. 10 µl of spore suspension per wound spot (1 × 106 spores/ml) was applied on the wounds of one site from midrib, and SDW was placed on the wounds of other site as a control. The treated seedlings were covered with sterile plastic bag and kept in a 12-h fluorescent light/dark cycle under greenhouse conditions at 25 ± 2°C and 80% relative humidity. Two seedlings were inoculated with a single isolate, and this experiment was repeated twice. Circular or irregular lesions appeared after 5 days of inoculation, while the control remained asymptotic. Koch's postulates were fulfilled by reisolating and reidentifying the causal agent from the lesions of inoculated leaves. Colletotrichum fructicola has been reported as the causal agent of anthracnose on mango (Joa et al. 2016), apple (Kim et al. 2018), grapes (Lim et al. 2019), peaches (Lee et al. 2020), and hybrid pear (Choi et al. 2021) in South Korea. To the best of our knowledge, it is the first report of C. fructicola causing anthracnose on spotted laurel. This study will be helpful to develop effective management strategies to minimize leaf lesions.