First report of Colletotrichum fructicola, C. perseae, and C. siamense causing anthracnose disease of avocado (Persea americana) in New Zealand.

Mexico is the largest avocado (Persea americana) producer and exporter in the world. In January of 2019, typical symptoms of fruit anthracnose were observed on approximately 90% of avocado trees in backyards localized in Leonardo Bravo municipality in Guerrero, Mexico. Lesions on avocado fruits were circular, necrotic, and sunken, whereas the mesocarp showed a soft rot with dark brown discoloration. To perform fungal isolation, small pieces from adjacent tissue to lesions of five symptomatic fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri dish containing potato dextrose agar (PDA). Plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and seven monoconidial isolates were obtained. An isolate was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession No. CSAEG-CJ19). After 8 days on PDA, the colonies were gray on the upper surface, and with orange conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with rounded ends, 14.4 to 18.5 × 4.5 to 6.2 μm. Based on morphological features, the isolate was tentatively identified in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), β-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced with primers ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), Bt2A/Bt2B (Glass and Donaldson 1995), and GDF/GDR (Templeton et al. 1992), respectively. BLAST analysis of the obtained sequences of the ITS, ACT, TUB2, and GAPDH genes revealed 100%, 99.63%, 99.77% and 100% identity with those of isolate LF687 of C. jiangxiense in GenBank (Accession numbers KJ955201, KJ954471, KJ955348, and KJ954902). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, and GAPDH data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CSAEG-CJ19 as C. jiangxiense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accession numbers ITS:MT011397; ACT:MN968784, TUB2:MN968786, and GAPDH:MN968785). To conduct Koch's postulates, 20 healthy avocado fruits (cv. Hass) were wounded with a sterile toothpick (2 mm in depth) and a drop of 15 µl of conidial suspension (1 × 105 spores/mL) was placed on each wound. Ten control fruit were wounded and treated with sterilized water. All the fruits were kept in a moist plastic chamber at 25°C for 8 days. All inoculated fruits developed circular and necrotic lesions (12 to 18 mm in diameter), 5 days after inoculation, whereas control fruits remained healthy. The fungus was consistently re-isolated from the inoculated fruits. Previously, C. jiangxiense has been reported as a pathogen on Camellia sinensis and Citrus sinensis in China (Farr and Rossman 2020). To our knowledge, this is the first report of C. jiangxiense causing anthracnose on avocado worldwide. This study shown another species in the C. gloeosporioides complex associated with avocado diseases in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

Pepper (Capsicum annuum L.) is an important solanaceous vegetable crop, with high nutritional and economic value. However, it is susceptible to Colletotrichum spp. infection during its growth and development, which seriously affects production yield and quality. Chili anthracnose, caused by Colletotrichum spp., is one of the most destructive diseases of pepper. In August 2020, chili anthracnose was observed with widespread distribution in the horticulture field of Northwest A&F University (34.16° N, 108.04° E) in Shaanxi Province, China. Approximately 60% of the pepper plants had disease symptoms typical of anthracnose. Lesions on pepper fruits were dark, circular, sunken, and necrotic, with the presence of orange to pink conidial masses (Figure S1A). To perform fungal isolation, the tissue at the lesion margin was cut from eight symptomatic fruits, surface disinfested with 75% ethanol for 30 s, and 2% NaClO for 1 min, then rinsed three times with sterile distilled water and dried on sterile filter paper. The tissues were placed on potato dextrose agar (PDA) and incubated at 28 ºC in the dark. After 3 days, hyphae growing from tissue of each lesion were recultured on PDA (Liu et al. 2016). A representative single-spore isolate (NWAFU2) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. NWAFU2 colonies had gray-white aerial mycelium, and the reverse side of the colonies was dark gray to light yellow after 10-days growth on PDA (Figure S1B-C). Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, and measured 10.1 to 16.9 (length) × 4.7 to 7.0 (width) μm (n=50) (Figure S1D). Based on morphological features, the isolate was consistent with the description of C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted using a CTAB method and the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial sequences of actin (ACT) genes were amplified and sequenced using primers ITS1F/ITS4, GDF1/GDR1 and ACT-512F/ACT-783R, respectively (Dowling et al. 2020). Using the BLAST, ITS, ACT, GAPDH gene sequences (GenBank accession nos. MW258690, MW258691 and MW258692, respectively) were 100%, 100% and 98.19% identical to ZJL-4 of C. gloeosporioides (GenBank accession nos. MN075757, MN058142 and MN075666, respectively). Phylogenetic analysis was conducted using MEGA-X (Version 10.0) based on the concatenated sequences of published ITS, ACT and GAPDH for Colletotrichum species using Neighbor-Joining algorithm. The identified isolate (NWAFU2) was closely related to C. gloeosporioides (Figure S2). To confirm the pathogenicity, ten healthy pepper fruits were surface-sterilized and 2 μL of conidial suspension (1×106 conidia/mL) was injected the surface of pepper. Five fruits were inoculated with 2μL sterile distilled water as controls. After inoculation, the fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. Anthracnose symptoms similar to those observed in the field, were observed 7 days after inoculation (Figure S1F) and control fruits remained healthy. A similarly inoculated detached leaf assay resulted in water-soaked lesions 3 days after inoculation. C. gloeosporioides was reisolated from the infected pepper fruits, fulfilling Koch's postulates. C. gloeosporioides has been reported to cause chili anthracnose in Sichuan Province, China (de Silva et al. 2019; Liu et al. 2016). However, Shaanxi is one of the main pepper producing areas in china and it is geographically distinct from Sichuan; its climate and environmental conditions are different from Sichuan. Knowledge that C. gloeosporioides causes chili anthracnose of pepper in Shaanxi province, China may aid in the selection of appropriate management tactics for this disease. Reference: de Silva, D. D., Groenewald, J. Z., Crous, P. W., Ades, P. K., Nasruddin, A., Mongkolporn, O., and Taylor, P. W. J. 2019. Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10:8. Dowling, M., Peres, N., Villani, S., and Schnabel, G. 2020. Managing Colletotrichum on Fruit Crops: A "Complex" Challenge. Plant Dis 104:2301-2316. Liu, F. L., Tang, G. T., Zheng, X. J., Li, Y., Sun, X. F., Qi, X. B., Zhou, Y., Xu, J., Chen, H. B., Chang, X. L., Zhang, S. R., and Gong, G. S. 2016. Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China. Sci Rep 6. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115-180.

Crataegus is a genus classified in family Rosaceae and includes several tree species commonly called tejocote that are widely cultivated for their pome fruits in Mexico. During fall of 2014, 2015, and 2016, severe symptoms of anthracnose were observed on approximately 60% of tejocote (Crataegus gracilior) fruits in an orchard located in Tulancingo, Oaxaca, Mexico. Affected fruits showed sunken, prominent, dark brown to black necrotic lesions and were exuding salmon spore masses. To isolate the fungus, small pieces from tissue adjacent to the lesions of 10 symptomatic fruits were excised and surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed three times in sterile distilled water, placed in Petri plates containing potato dextrose agar (PDA), and incubated at 25°C for 5 to 7 days in darkness. Mycelial plugs were excised from the edge of the actively growing fungal colony and aseptically transferred to fresh PDA medium and incubated at 25°C for 6 days. Five monoconidial cultures were obtained by transferring germinated spores to Petri plates with fresh PDA. One isolate was selected as representative for morphological and molecular identification. Colonies of pure cultures exhibited greyish-white aerial mycelium and abundant salmon-pink conidial masses. Conidia (n = 100) were subcylindrical, hyaline, straight, one-celled, with rounded ends, measuring 13.6 to 17.7 × 4.4 to 5.9 μm. Conidial appressoria were ovoid and brown to dark brown. Based on morphological characteristics, the fungus was identified within the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The isolate was designated UACH-177 and deposited in the Culture Collection of Phytopathogenic Fungi at the Chapingo Autonomous University. For molecular identification, the internal transcribed spacer (ITS) region (White et al. 1990) and fragments of Apn2 (Rojas et al. 2010), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin 2 (TUB2) genes (Weir et al. 2012) were amplified by polymerase chain reaction and sequenced. The sequences were deposited in GenBank (accession nos.: ITS, MG821312; Apn2, MG821310; GAPDH, MG821311; and TUB2, MG821313). A phylogenetic analysis using Bayesian inference and including published ITS, Apn2, GAPDH, and TUB2 data for C. gloeosporioides and other Colletotrichum species was performed. The phylogenetic analysis showed the sequences were grouped into the clade of C. gloeosporioides. To confirm the pathogenicity of the fungus, 20 tejocote fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 1 min, washed three times with sterile distilled water, and dried on sterilized filter paper. Inoculations were performed by deposition of 10 μl of a conidial suspension (10⁶ spores/ml) on the fruit surface. Ten fruit were mock inoculated with distilled water as a control. All fruits were kept in a moist chamber at 25°C for 10 days. The pathogenicity test was repeated twice. Disease symptoms were observed on all inoculated fruit after 7 days, whereas control fruit did not develop symptoms. Fungal colonies were reisolated from all symptomatic fruits and were found to be morphologically identical to the original isolate inoculated on tejocote fruits, thus fulfilling Koch’s postulates. In Mexico, Garcia-Alvarez (1976) reported Colletotrichum sp. on fruits of Crataegus mexicana; however, that report was not supported by morphological characterization nor pathogenicity tests. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of C. gracilior in Mexico and worldwide.

Tomatoes (Solanum lycopersicum) are among the most economically important vegetables in Mexico. During 2016, more than 52,000 ha of tomatoes were planted, both in open fields and in greenhouses, yielding more than 2,875,164 t nationwide and placing Mexico as one of the main producers and an important exporter of this product worldwide (SAGARPA 2016; SIAP 2016). Anthracnose is a disease caused by several species of the genus Colletotrichum in several economically important hosts, affecting both pre- and postharvest fruits (Damm et al. 2009). During October 2017, 240 tomato fruits with symptoms were collected from an open field in the state of Morelos, Mexico. During this time, anthracnose was also observed in the harvest fruits, with circular and sunken lesions covered with black acervuli containing spores and dark setae in a group. Epidermal tissue sections of 1.0 cm were cut from 30 fruits and disinfested with 1.5% sodium hypochlorite for 3 min. These tissues were washed twice with sterile distilled water and allowed to dry on absorbent paper towels. They were planted in potato dextrose agar (PDA) culture medium and incubated at 25 ± 2°C under white light. Pure cultures were obtained from monosporic cultures on agar-agar and transferred to PDA after 8 days. The initial growth was white, turning pale gray, with numerous black structures (microsclerotia) and setae. Conidia were produced in masses and were hyaline, unicellular, fusiform, and apex-acute and measured 20 to 22.35 μm long and 2.4 to 2.6 μm wide. The setae were 75 to 85 μm long and 4 to 5 μm wide, with elliptic to claviform or slightly lobed appressoria. The cultural and morphological characteristics agreed with descriptions of Colletotrichum truncatum (Sutton 1980). To confirm the molecular identity, DNA was extracted using a DNeasy minikit (Qiagen) and amplified using ITS, actin, GAPDH, and EF1-728 primers. Consensus sequences were deposited in GenBank, and the identity match with C. truncatum was 97% for ITS (MH448905), actin (MH493053), and GAPDH (MH4930952) and 100% for EF1-728 (MH493054). The sequences were aligned with the same species from the NCBI GenBank database. To confirm pathogenicity, 48 tomato fruits were superficially disinfested in a 1.5% sodium hypochlorite solution, washed twice with sterile distilled water, and placed on absorbent presterilized paper towels. A conidial suspension (10⁶ conidia/ml) from the fungus was placed on the tomato’s surface. Nine fruits were used as controls, with only sterile distilled water, and stored in a humidified chamber at temperature 26°C for 8 days. Pathogenicity tests were repeated twice. Symptoms developed 8 days after inoculation, and anthracnose was observed in all inoculated fruits, whereas no symptoms were present in the control fruit. Koch’s postulates were fulfilled when the pathogen was isolated from the tomato fruits but not from the control fruits. C. truncatum is reported to cause anthracnose in different hosts, including Solanaceae species (He et al. 2016; Ranathunge and Sandani 2016). Based on a literature review, this is the first report of C. truncatum affecting S. lycopersicum fruits in Mexico.

HomePlant DiseaseVol. 101, No. 9First Report of Anthracnose on Peach Fruit Caused by Colletotrichum acutatum in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Anthracnose on Peach Fruit Caused by Colletotrichum acutatum in ChinaY. X. Du, H. C. Ruan, N. N. Shi, L. Gan, X. J. Yang, Y. L. Dai, and F. R. ChenY. X. DuSearch for more papers by this author, H. C. RuanSearch for more papers by this author, N. N. ShiSearch for more papers by this author, L. GanSearch for more papers by this author, X. J. YangSearch for more papers by this author, Y. L. DaiSearch for more papers by this author, and F. R. ChenSearch for more papers by this authorAffiliationsAuthors and Affiliations Y. X. Du H. C. Ruan N. N. Shi L. Gan X. J. Yang Y. L. Dai F. R. Chen , Institute of Plant Protection, Fujian Academy of Agricultural Science, Fuzhou, Fujian, 350013, China; and Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Fuzhou, Fujian, 350013, China. Published Online:27 Jun 2017https://doi.org/10.1094/PDIS-11-16-1568-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Peach (Prunus persica L.) is an economically important fruit crop in most parts of China. Anthracnose of peach fruit causes significant production and economic losses worldwide every year. In South Carolina, anthracnose is caused mainly by species of the genus Colletotrichum, e.g., C. gloeosporioides, C. acutatum, and C. truncatum (Bernstein et al. 1995; Hu et al. 2015). In August 2012, 2013, and 2014, typical symptoms of anthracnose were observed on peach fruits (Baifeng) near maturity in commercial orchards in Ningde, Fujian Province, China. Initially, symptoms appeared as small, light brown, water-soaked, slightly sunken lesions, circular to semicircular spots. As infection continued, the lesions expanded and became dark brown to black and produced wrinkled concentric rings and orange conidial masses in the center of the lesions under high humidity. Ten peach fruits with a single lesion were collected, surface sterilized for 1 min in 0.5% sodium hypochlorite, and rinsed with sterile distilled water. Then 5 × 5 mm necrotic tissue was placed on acidified potato dextrose agar (APDA) and incubated at 25°C for 3 days. The pure cultures were obtained by single conidia isolation. Ten isolates produced pale gray, dense aerial hyphae, in reverse brown with concentric rings at 25°C on PDA after 6 days. Conidia were fusiform-elliptical, sometimes long obclavate to oblong-elliptical, pointed at one or both ends, one celled, aseptate, hyaline, guttulate, and 11.6 to 16.1 × 3.1 to 5.2 μm in size (n = 50). The internal transcribed spacer (ITS) regions of rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, β-tubulin (TUB2) gene, and partial sequence of the actin (ACT) gene of representative isolate C1 were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), GDF1/GDR1, Bt2F/Bt2R, and ACT-512F/ACT-783R, respectively. The ITS consensus sequence (GenBank accession no. KX611163) showed 99% homology with the ITS sequence of C. acutatum (KF928293). The other three consensus sequences (KY049983, KY049984, and KY049982, respectively) revealed 100% identity to the corresponding sequences of C. acutatum in GenBank (JQ948685, JQ950015, and JQ949720, respectively). Based on the above, the isolates were identified as C. acutatum (Sutton 1980; Zhang et al. 2008). To confirm pathogenicity, six mature, healthy peach fruits purchased commercially were surface disinfested with 70% ethanol, and then wounded in four locations on each fruit with a sterile needle. Sterile filter paper disks, dipped in conidial suspension (1 × 105 conidia/ml) or sterile water, were placed on each wound. Three fruits were inoculated per treatment and incubated at 25°C for 2 days at 100% humidity and then another 7 days at 75% humidity. After removal of the filter paper disks 3 days later, symptoms (small, dark lesions) appeared. Six days after inoculation, abundant orange conidial masses were observed. Ten days later, the inoculated areas had decayed. Control fruits remained healthy. C. acutatum was reisolated from symptomatic fruits, and the recovered isolates were identified as C. acutatum through morphological and molecular characterizations. To our knowledge, this is the first report of anthracnose of peach fruit caused by C. acutatum in Fujian Province, China. This disease can seriously affect peach quality and yield, so effective measures should be implemented to control it.

ilamentous fungi of the genus Colletotrichum and its teleomorph Glomerella are considered major plant pathogens worldwide. They cause significant economic damage to crops in tropical, subtropical, and temperate regions. Cereals, legumes, ornamentals, vegetables, and fruit trees may be seriously affected by the pathogen (3). Although many cultivated fruit crops are infected by Colletotrichum species, the most significant economic losses are incurred when the fruiting stage is attacked. Colletotrichum species cause typical disease symptoms known as anthracnose, characterized by sunken necrotic tissue where orange conidial masses are produced. Anthracnose diseases appear in both developing and mature plant tissues (4). Two distinct types of diseases occur: those affecting developing fruit in the field (preharvest) and those damaging mature fruit during storage (postharvest). The ability to cause latent or quiescent infections has grouped Colletotrichum among the most important postharvest pathogens. Species of the pathogen appear predominantly on aboveground plant tissues; however, belowground organs, such as roots and tubers, may also be affected. In this article, we deal in particular with methods used to identify and characterize Colletotrichum species and genotypes from almond, avocado, and strawberry, as examples, using traditional and molecular tools. The three pathosystems chosen represent different disease patterns of fruitassociated Colletotrichum. Multiple Species on a Single Host Numerous cases have been reported in which several Colletotrichum species or biotypes are associated with a single host. For example, avocado and mango anthracnose, caused by both C. acutatum and C. gloeosporioides, affect fruit predominantly as postharvest diseases (25,40,41). Strawberry may be infected by three Colletotrichum species, C. fragariae, C. acutatum, and C. gloeosporioides, causing anthracnose of fruit and other plant parts (31). Almond and other deciduous fruits may be infected by C. acutatum or C. gloeosporioides (Table 1) (1,5,46,50). Citrus can be affected by four different Colletotrichum diseases (61): postbloom fruit drop and key lime anthracnose, both caused by C. acutatum, and shoot dieback and leaf spot, and postharvest fruit decay, both caused by C. gloeosporioides. Additional examples of hosts affected by multiple Colletotrichum species include coffee, cucurbits, pepper, and tomato. Single Species on Multiple Hosts It is common to find that a single botanical species of Colletotrichum infects multiple hosts. For example, C. gloeosporioides (Penz.) Penz. & Sacc. in Penz. (teleomorph: Glomerella cingulata (Stoneman) Spauld. & H. Schrenk), which is considered a cumulative species and forms the sexual stage in some instances, is found on a wide variety of fruits, including almond, avocado, apple, and strawberry (Table 2) (6,15,31,46). Likewise, C. acutatum J.H. Simmonds has been reported to infect a large number of fruit crops, including avocado, strawberry, almond, apple, and peach (1,5,16,25,27). Examples of other species with multiple host ranges include C. coccodes, C. capsici, and C. dematium (14,56).

Bitter rot of apple (Malus × domestica Borkh.) is a cosmopolitan disease affecting fruit and causes considerable losses worldwide. In September 2020, symptoms of bitter rot were observed on 'Pink Lady' apples in two orchards (~2.5 ha each) in Gualta, Catalonia, Spain (42.03803 N, 3.09831 E, and 42.03942 N, 3.10931 E). Early symptoms consisted of light brown and sunken circular lesions (1-4 mm) that enlarged over time, later becoming dark brown and water soaked, and extending cone-shaped toward the core. Sporulation was mostly noticed in larger lesions. Estimated incidence was 2% and 20% of 150 trees surveyed in each orchard, respectively. Twenty-one fungal isolates were obtained from diseased fruit by culturing small pieces of necrotic tissue on potato dextrose agar (PDA) amended with rifampicin at 50 mg/liter. Colonies on PDA looked identical. They were cottony, initially light-gray colored on top and darkening with age; colony reverse initially cream colored and darkening with age. Conidia were produced in orange acervular masses on Spezieller Nährstoffarmer Agar, and were aseptate, hyaline, cylindrical with obtuse ends, and measuring 10.1 to 14.7 × 4.5 to 7.1 μm (average 13.1 ± 1.04 × 5.3 ± 0.67 μm [mean ± SD], n = 50), with a mean length/width ratio 2.6 ± 0.39 (n = 16 isolates). Perithecia were not observed. Based on the conidial morphology, the isolates were tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). Total genomic DNA was extracted from all isolates and six nuclear regions were amplified and partially sequenced: the internal transcribed spacer region of rDNA (ITS), the mating type protein 1-2-1 gene and the Mat1-2-1-Apn2 intergenic spacer region (ApMAT), actin (ACT), calmodulin (CAL), glyceraldehyde 3-P dehydrogenase (GAPDH), and tubulin (TUB2). The sequences for each region were 100% identical across all isolates. BLAST searches in GenBank showed 99-100% identity with sequences of various C. chrysophilum W.A.S. Vieira, W.G. Lima, M.P.S. Câmara & V.P. Doyle strains including the ex-type CMM4268 (Vieira et al. 2017). Sequences of the representative isolate CJL1080 were deposited in GenBank (ACT, MZ488944; ApMAT, MZ442299; CAL, MZ488945; GAPDH, MZ488946; ITS, MZ443972; TUB2, MZ442300). A multilocus phylogenetic analysis through Bayesian inference conducted with the obtained sequences and reference ones (Khodadadi et al. 2020) revealed that our isolates clustered well within C. chrysophilum, as suggested by BLAST results. To confirm Koch's postulates, isolates CJL1080 and CJL1095 were inoculated on 'Pink Lady' apples. Six surface-sterilized fruits per isolate were wound-inoculated four times each with either 20 μl of a conidial suspension (105 conidia/ml) or sterile distilled water (control). After 7 days of incubation in a moist chamber at 22°C, symptoms compatible with Colletotrichum infection were observed around the wounds, whereas control inoculations remained symptomless. The fungus was reisolated from all the lesions and identified through its morphological traits and DNA sequencing (ApMat, CAL, and GAPDH). No fungus was isolated from the controls. Taxa of the C. gloeosporioides species complex causing bitter rot have been recently reported in Europe (Grammen et al. 2019; Nodet et al. 2019). This is the first report of C. chrysophilum causing apple bitter rot in Spain, which expands the knowledge on the geographic distribution of this important pathogen of apple in Europe.

In July 2013, two diseased peach fruit (Prunus persica (L.) Stokes) of the cv. Sweet Dream were collected from a commercial orchard in Ridge Springs, South Carolina. Affected peaches were at or near maturity and symptoms resembled anthracnose disease caused by Colletotrichum spp. with circular sunken tan to brown lesions that were firm in touch, and had wrinkled concentric rings. The center of the lesion was covered with black acervuli containing setae. To isolate the causal agent, the two symptomatic fruit were surface-sterilized in 10% bleach for 2 min and rinsed with sterile distilled water. Lesions were cut in half, and necrotic tissue from the inside of the fruit was placed on acidified potato dextrose agar (APDA). Flat colonies covered with olive-gray to iron-gray acervuli developed on APDA incubated at 22°C with a 12-h cycle of fluorescent light and darkness. Morphology of acervuli, setae (avg. 90 to 160 μm), conidiophores (up to 90 um long), and conidia (avg. 22 × 3.8 μm) of single spore isolates were consistent with descriptions of Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore (3), a causal agent of anthracnose disease. Genomic DNA was extracted from isolate Ct_RR13_1 using the MasterPure Yeast DNA Purification Kit (Epicentre, Madison, WI). The ribosomal ITS1-5.8S-ITS2 region and a partial sequence of the actin gene were amplified with primer pair ITS1 and ITS4 (4), and primer pair ACT-512F and ACT-783A (2), respectively. A multilocus sequence identification in Q-bank Fungi revealed a 100% similarity with C. truncatum (1). The C. truncatum sequences from the peach isolate were submitted to GenBank (accessions KF906258 and KF906259). Pathogenicity of isolate Ct_RR13_1 was confirmed by inoculating five mature but still firm peach fruits with a conidial suspension of C. truncatum. Peaches were washed with soap and water, surface-disinfected for 2 min with 10% bleach, rinsed with sterile distilled water, and air dried. Dried fruit were stabbed at three equidistant points, each about 2 cm apart, to a depth of 9.5 mm using a sterile 26G3/8 beveled needle (Becton Dickinson & Co., Rutherford, NJ). For inoculation, a 30-μl droplet of conidia suspension prepared in distilled, sterile water (1 to 2 × 104 spores/ml) was placed on each wound; control fruit received sterile water without conidia. Fruit were incubated at 22°C for 2 days at 100% humidity and another 12 days at 70% humidity. Inoculated fruit developed anthracnose symptoms with sporulating areas as described above and the fungus was re-isolated. All control fruit remained healthy. C. truncatum has a wide host range, including legumes and solanaceous plants of the tropics, and is especially common in the Fabaceae family. Its occurrence in a commercial peach orchard is worrisome because control measures may need to be developed that are different from those developed for endemic species, i.e. C. acutatum and C. gloeoporioides, due to differences in disease cycle or fungicide sensitivity. To our knowledge, this is the first report of C. truncatum causing anthracnose on a member of the genus Prunus.

Magnolia wufengensis belongs to the Magnoliaceae family. Its variation-rich flowers (tepal number from 9 to 46, tepal color from pink to bright red) and excellent wood characteristics (strong, straight, texture) have important ornamental and economic value (Duan et al. 2019; Luyi et al. 2006). M. wufengensis is popularly cultivated in parks, courtyards, mountains, and along roadsides. In May 2020, leaf spot symptoms were observed on over 85% of M. wufengensis in Yuyangguan Township, Wufeng County, Hubei Province (110.60°E, 30.21°N). The damaged area was over 18.7 hectares. Early symptoms began as small brown spots with a light-yellow halo. Gradual lesions expanded, and the center was withered, gray, and necrotic with a dark brown border. Eventually, several spots combined with larger irregular lesions, turning the leaves yellow and causing them to fall off. The border of lesions and healthy tissues were cut into small pieces (5×5 mm), and surface sterilized with 1% sodium hypochlorite solution for three minutes, rinsed three times with sterile water, and plated on potato dextrose agar (PDA) medium at 25±2 °C with a 12h photoperiod under fluorescent lighting. Pure isolates (MCS1228.1, MCS1228.4, MCS1228.9) were gray to pale grayish, and their average growth rate was 10.5±1.23 mm/day. Conidiophores were hyaline, aseptate, branched. Conidia were hyaline, aseptate, cylindrical, and 14.00 to 25.17 × 4.74 to 6.56 μm in size (average 17.48 × 5.58 μm) (n=50). Appressoria were brown and showed multivariate shape. The morphological characteristics of the isolates corresponded to the description given for Colletotrichum fructicola (Liu et al. 2015). Molecular identification was accomplished through amplification of the internal transcribed spacer (IST), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) genes (Fu et al. 2018). The ITS (OL800580.1, OL800581.1, OL800582.1), ACT (GenBank accession No. OL873155- OL873157), CAL (GenBank accession No. OL873158- OL873160), CHS-1 (GenBank accession No. OL873161- OL873163), GAPDH (GenBank accession No. OL873164- OL873166) and TUB2 (GenBank accession No. OL873167- OL873169) sequences were deposited in GenBank. A Bayesian inference phylogenetic tree based on multilocus sequences was constructed, and the sequences of the 3 isolations showed the same homology with C. fructicola (Fu et al. 2018). To fulfill Koch's postulates, 30 potted seedlings were inoculated with 1×10^6 conidia/ml suspension of each isolate by spraying the leaves, and 30 potted seedlings were sprayed with sterile distilled water as control. Inoculated and control plants were kept in a greenhouse with 25/15°C (day/night) temperature and 80% relative humidity. In addition, 30 healthy detached leaves free of pests and diseases were washed three times with sterile distilled water, air-dried, and artificially inoculated using a 6 mm (diameter) PDA medium (5 days incubation) with mycelium. 30 leaves were inoculated with sterile PDA medium as control. All leaves were sprayed with sterile water every 24 hours, covered with plastic wrap, and incubated at 25±2 °C, 100% humidity. The experiment was repeated three times. Similar symptoms to those found initially were both observed on all the inoculated potted seedlings and detached leaves after 14 days and 5 days post inoculation (dpi), respectively. Whereas the controls remained symptomless. The reisolated pathogens from symptomatic tissues were identical to the original isolates. In this study, isolated fungi associated with M. wufengensis leaf spot were identified as C. fructicola based on morphological and multiloci phylogenetic analyses, and Koch's postulates. Colletotrichum species are important plant pathogens and cause diseases in a wide variety of woody and herbaceous plants (Cannon et al. 2012). C. fructicola has been identified as a responsible pathogen for apple (Casanova et al. 2016), Fatsia japonica (Shi et al. 2017), and Rubus corchorifolius (Wu et al. 2021) leaf spot. To our knowledge, this is the first report of C. fructicola causing leaf spot in M. wufengensis in China. This research may contribute to the development of management strategies for this disease.

Chili (Capsicum annuum L.) is an economically important crop worldwide, valued for its culinary uses. In South Korea, anthracnose caused by Colletotrichum spp. including C. truncatum, C. gloeosporioides, C. coccodes, C. acutatum, and C. scovillei incurs on substantial economic loss (Kim et al. 2008; Oo and Oh 2020). In August 2022, somewhat different types of symptoms that was not typical on chilli fruits were observed in a field in Yereonggwang (GPS: 35.2579° N, 126.4742° E), South Korea. The disease symptoms appeared as sunken, necrotic lesions with dense black spore masses forming in concentric rings. The estimated disease incidence the 0.2 ha field showing up to 1% of fruits affected. To isolate the pathogen, six symptomatic chilli fruits were collected. Small pieces (5 mm²) were cut from the margins of the lesions, surface-sterilized in 70% ethanol for 30 sec, followed by 1% sodium hypochlorite for 1 minute, and then rinsed three times in sterile distilled water. The tissue pieces were placed on potato dextrose agar (PDA) plates and incubated at 25°C in the dark. After 3 to 5 days, emerging fungal colonies were sub-cultured to obtain pure isolates. A total of five isolates were obtained and initially identified as Colletotrichum spp. based on morphological characteristics. Seven-day old colonies were initially white, turning light orange with age on PDA. Setae (observed on lesion) were dark brown, verruculose and septate. Conidia were cylindrical, hyaline, and measured 14.8 to 19.9 × 4.2 to 6.5 µm (mean 16.7 × 5.6 μm, n = 70) in size; appressoria were brown to dark brown and irregularly shaped. These morphological characteristics of the isolates agree with those reported for the morphology of C. sojae by Damm et al. (2019). To confirm the identity of the isolates, DNA was extracted and specific gene regions were amplified and sequenced using the following primer sets: ITS (ITS1 and ITS4), GAPDH (GDF1 and GDR1), ACT (ACT-512F and ACT-783R), TUB (T1 and Bt2b), HIS3 (CYLH3F and CYLH3R), and CHS-1 (CHS-79F and CHS-345R). The resulting sequences were deposited in the NCBI GenBank with accession numbers (LC830742 to LC830766). Maximum likelihood phylogenetic analysis using combine sequences of ITS, GAPDH, ACT, TUB, HIS3 and CHS-1 in MEGA X confirmed the isolates as C. sojae, marking the first report of this pathogen on chilli in South Korea, previously known to infect soybean. Pathogenicity tests were conducted on wound and nonwounded healthy and mature-green chili fruits (cv. Bicksita) to confirm the pathogenicity of the isolated C. sojae. The fruits were surface-sterilized using 70% ethanol and then rinsed with sterile distilled water. The fruits were wounded using a sterile needle to facilitate infection. A conidial suspension (1x106 conidia/mL) was prepared from 7-day-old PDA cultures. Each fruit was inoculated by placing a 10 µL drop of the conidial suspension onto the wounded and nonwounded sites (4 to 5) of the wound and unwound fruits, respectively. Control fruits were inoculated with sterile water. A total of 40 fruits per treatment were used and the experiment repeated twice. The fruits were placed in plastic box lined with moist paper towels to maintain high humidity and incubated at 25°C. Anthracnose symptoms developed on the inoculated fruits within 7 days, while control and unwounded fruits remained symptom-free. Colletotrichum sojae was successfully reisolated from the symptomatic fruits, fulfilling Koch's postulates and confirming its role as the causal agent of the disease. Colletotrichum sojae is known to infect Fabaceae species worldwide such as Glycine max, Medicago sativa, Phaseolus vulgaris, Atractylodes ovata and Vigna unguiculata (Damm et al. 2019; Talhinhas and Baroncelli 2021), Atractylodes ovata in South Korea (Hassan et al. 2021) and chili pepper in China (Zhanget al. 2023). The first report of C. sojae causing chili anthracnose in South Korea represents a new challenge for chili growers. Integrated disease management strategies need to be developed and implemented to mitigate its impact.

Mulberry (Morus alba L.) has been grown worldwide as a crop for silkworm rearing for over five thousand years (Jiao et al. 2020). In July 2021, a leaf spot disease was observed on mulberry leaves in Wuhan city (114°33'E, 30°48'N), Hubei province, China, with approximately 40% of leaves (about 300 trees) affected. Early symptoms were light brown, with small lesions subsequently expanding to larger sometimes irregular dark brown or black spots surrounded by yellow-brown margins, with easily perforated necrotic lesions. Leaf tissues (5 mm×5 mm) were excised from the border between diseased and healthy tissues, surface sterilized with 75% ethanol solution for 30 s and 2.5% sodium hypochlorite for 2 min, washed thrice in sterile distilled water, and then placed on potato dextrose agar (PDA), and incubated at 25°C in darkness. Four isolates (C1, C9, CHS2, and CHS6) were subcultured using the single-spore method. On PDA, colonies were cottony, pale white from above, and white to grayish-green on the reverse side. Conidia were aseptate, hyaline, subcylindrical with broadly rounded ends, 8.4 to 18.3×4.1 to 7.7 μm (mean = 13.9×5.5 μm, n = 30). Appressoria were typically elliptic or irregular with a few lobes, dark brown, 5.9 to 9.6×4.2 to 8.1 μm (mean = 7.9 ×5.7 μm, n = 30). The morphological characteristics of the isolates matched the descriptions of Colletotrichum gloeosporioides species complex (Weir et al. 2012). The isolates were further identified by analysis of the ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), glutamine synthetase (GS), and β-tubulin 2 (TUB2) genes, amplified respectively with ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GSF1/GSR, and Bt2a/Bt2b (Glass and Donaldson 1995; Weir et al. 2012; White et al. 1990). The sequences were deposited in GenBank (ON492187-ON492214). Concatenated sequences of the seven genes in addition to Colletotrichum species sequences from GenBank were used to conduct a phylogenetic analysis using Maximum-Likelihood (ML) method in MEGA7. The four isolates were grouped into a clade with Colletotrichum aenigma supported by a high bootstrap value (89%), and hence, they were identified as C. aenigma based on the morphological and molecular analyses. To confirm Koch's postulates, wounded leaves of six healthy 2-month-old seedlings made by a sterile needle were inoculated with each isolate by spraying 10 ml of conidial suspensions (105 conidia/ml) on each plant, and the control plants were treated with sterile distilled water. All the treated plants were kept in a plastic box containing sterile water and incubated at 28°C in a 12 h/12 h light/dark cycle. The test was performed three times. After 7 days, typical anthracnose lesions appeared on all inoculated leaves, whereas control plants remained asymptotic. Furthermore, C. aenigma was only reisolated from the symptomatic leaves. Previous studies reported five Colletotrichum species (C. morifolium, C. fioriniae, C. brevisporum, C. karstii, and C. kahawae subsp. ciggaro) to cause this disease on mulberry in China (Tian, 1981; Xue et al. 2019). To our knowledge, this is the first report of C. aenigma causing anthracnose on mulberry in China. The finding will facilitate epidemiological studies and the development of effective control strategies for the disease.

Lifestyles of Colletotrichum acutatum.

Foliar anthracnose is one of the main diseases of onion (Allium cepa L.) under tropical and subtropical conditions. Thus far, only Colletotrichum gloeosporioides has been reported as the causal agent of this disease in Brazil. However, there are no extensive studies characterizing Colletotrichum isolates associated with onion anthracnose in the country. Here, 38 Colletotrichum isolates obtained from onion plants displaying foliar anthracnose across major Brazilian onion-producing regions were characterized using morphometric and molecular information. The Bayesian and Maximum Liklihood methods were used for an initial analysis of the β-tubulin gene (tub2) sequences of all isolates, resulting in the discrimination of nine haplotypes. Three haplotypes grouped with the reference species of the C. acutatum complex and six with the C. gloeosporioides complex. Sequences of either the glyceraldehyde-3-phosphate dehydrogenase (gapdh), actin (act), and calmodulin genes or the intergenic spacer (IGS) region between DNA lyase (apn2) gene and the mating-type mat1–2-1 locus were used to characterize a subset of isolates representing these nine distinct tub2 gene haplotypes. These analyses revealed five anthracnose-inducing Colletotrichum species, including three members of the C. acutatum species complex (C. nymphaeae, C. scovillei, and C. tamarilloi) and two of the C. gloeosporioides species complex (C. fructicola and C. theobromicola). Bioassays confirmed that all these Colletotrichum species are pathogenic to onion, inducing typical anthracnose symptoms on bulbs and leaves. Twenty-six out of 38 isolates were identified as C. theobromicola, indicating this fungus as the prevalent anthracnose pathogen under Brazilian conditions. This Colletotrichum species diversity will affect anthracnose management strategies, including chemical and cultural control as well as the identification and deployment of onion cultivars with species-specific and/or wide-spectrum tolerance/resistance.

Salix babylonica L. shows a great potential for restoration of contaminated water or soils and has a high ornamental value (Li et al. 2015). In mid-October 2021, a leaf spot disease, with an incidence of approximately 61%, occurred on leaves of 25-year-old S. babylonica on the campus of Nanjing Forestry University. On average, 65% of the leaves per tree were infected. Symptoms began as dark brown, irregular spots, and the centers were grayish white. The spots gradually enlarged with time. Fresh specimens were collected from 3 trees (10 leaves/tree). Small tissue pieces cut from lesion margins were surface-sterilized (Mao et al. 2021), plated on potato dextrose agar (PDA), and incubated at 25°C. Three representative isolates (NL1-7, NL1-10, and NL1-13) were obtained and deposited in The China Forestry Culture Collection Center. The colonies of 3 isolates were white, grayish white at the center. The conidia of 3 isolates were one-celled, straight, subcylindrical, hyaline, smooth, 14.6-18.6 × 4.3-6.7 µm, 13.8-16.7 × 4.7-6.0 µm and 12.1-16.9 × 5.4-7.5 µm (n = 50) for NL1-7, NL1-10, and NL1-13, respectively. The conidiophores of NL1-7 were hyaline to pale brown, septate, and branched, 18.9-48.0 µm (n = 50). Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled. The conidiophores and appressoria of the other two isolates were almost identical to NL1-7. Based on morphological characteristics, the 3 isolates matched the Colletotrichum gloeosporioides species complex (Weir et al. 2012). DNA of the 3 isolates was extracted. The internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin 2 (TUB2) loci were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-354R, GDF1/GDR1, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. ON870951 and ON858477 to ON858481 for NL1-7; ON908707 and ON858482 to ON858486 for NL1-10; ON870949 and ON858487 to ON858491 for NL1-13]. BLAST result showed that ITS, ACT, CAL, CHS-1, GAPDH, and TUB2 sequences of NL1-7 were identical to C. gloeosporioides at a high level (>99%). The sequences of NL1-10 and NL1-13 were consistent with C. siamense at a high level (>99%). A maximum likelihood and Bayesian Inference analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, and TUB2) placed NL1-7 in the clade of C. gloeosporioides sensu stricto and NL1-10 and NL1-13 in the clade of C. siamense. To confirm their pathogenicity, 9 healthy 3-yr-old seedlings, and 10 leaves/seedling were wounded with a sterile needle and inoculated with 10 µL of conidial suspension (106 conidia/mL) of the 3 isolates, respectively. Three control plants were treated with sterile water. Seedlings were covered with plastic bags after inoculation and kept in a greenhouse at 25 ± 2°C and RH 80%. Within 7 days, all inoculated leaves showed lesions similar to those in the field, and controls were asymptomatic. C. gloeosporioides s.s. and C. siamense were reisolated from the infected tissues. It was reported that Colletotrichum species can cause many plant diseases, for example, C. acutatum causes twig canker (Swain et al. 2012), and C. salicis causes willow anthracnose (Okorski et al. 2018), etc. However, some Colletotrichum species are endophytic (Martin et al. 2021) and may only become pathogenic under the right conditions. This is the first report of C. gloeosporioides s.s. and C. siamense causing leaf spots on S. babylonica in the world. These data will help select appropriate strategies for managing this disease and further studies on the pathogen and the host.

Guava (Psidium guajava L.) is a small tree belonging to the Myrtaceae family and it is distributed worldwide in the tropical and subtropical areas. During the summer of 2019, symptoms of fruit anthracnose were observed on approx. 90% of 250 guava trees located in backyards in Juan Jose Rios, Sinaloa, Mexico. Lesions on guava fruit were irregular, necrotic, and sunken. On advanced infections, acervuli containing salmon-pink masses of spores were observed on the lesions. Twenty fruits were collected from 10 trees (2 fruits per tree). Colletotrichum-like colonies were consistently isolated on PDA medium and 20 monoconidial isolates were obtained. Four isolates were selected as representatives for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of El Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF205-FAVF208). Colonies on PDA medium were flat with an entire margin, with abundant felty and white aerial mycelium, with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with ends rounded, and measuring 14.8 to 18.1 × 4.4 to 5.3 μm. Based on morphological features, the isolates were tentatively allocated in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), as well as partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2), chitin synthase (CHS-1) and glutamine synthetase (GS) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference and including published ITS, GAPDH, TUB2, ACT, CHS-1, and GS data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the four isolates FAVF205-FAVF208 as C. siamense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accessions nos. ITS: MW598512-MW598515; GAPDH: MW595216-MW595219; TUB2: MW618012-MW618015; ACT: MW595208-MW595211; CHS-1: MW595212-MW595215; and GS: MW618008-MW618011). Pathogenicity of the four isolates was verified on 40 healthy guava fruits. Twenty fruits were wounded with a sterile toothpick (2 mm in depth) and a mycelial plug (6 mm of diameter) was placed on each wound. Ten fruits inoculated with a PDA plug without mycelial growth served as controls. The fruit was kept in a moist plastic chamber at 25°C for 7 days. Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. The experiments were repeated twice with similar results. All inoculated fruits developed sunken necrotic lesions 4 days after inoculation, whereas no symptoms were observed on the control fruits. The fungi were consistently re-isolated only from the diseased fruits, fulfilling Koch´s postulates. Colletotrichum siamense has been previously reported on guava fruit in India (Sharma et al. 2015). However, to our best knowledge, this is the first report of C. siamense causing fruit anthracnose on guava in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species on guava in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.