Colletotrichum Gloeosporioides Species Complex Research Articles

First Report of Colletotrichum tropicale Causing Anthracnose on Pitahaya Fruit in Mexico.

Pitahaya (Hylocereus spp.), also called dragon fruit, is a cultivated cactus that is native to Mexico as well as Central and South America. In October 2021, anthracnose symptoms were observed on fruit of pitahaya (Hylocereus costaricensis) in a commercial orchard located in Culiacán, Sinaloa, Mexico. Lesions on fruit were circular, sunken, dark brown and with halo. To fungal isolation, small pieces from adjacent tissue to lesions of symptomatic fruits were surface disinfested by immersion in a 2% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently observed on PDA and five monoconidial isolates were obtained. An isolate was selected as a representative for morphological identification, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited as CCLF186 in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa). On PDA, initially white colonies turned grey with abundant orange conidia masses at 8 days after incubation at 25 ºC. Conidia were cylindrical, with ends rounded, aseptate, hyaline, and measuring 15.2 to 18.9 × 4.3 to 6.4 μm (n= 100). Appressoria were terminal, subglobose to clavate, of 7.4 to 11.6 × 5.9 to 8.2 µm (n= 30). Setae were not observed. These morphological characters were consistent with those reported for the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To determine the phylogenetic identity of the isolate CCLF186, genomic DNA was extracted following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region, the ApMat intergenic region, as well as partial sequences of actin (act) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were amplified and sequenced using the primers pairs ITS5/ITS4 (White et al. 1990), AM-F/AM-R (Silva et al. 2012), GDF/GDR, and ACT-512F/ACT-783R (Weir et al. 2012), respectively. The sequences were deposited in GenBank under accession nos. OP269659 (ITS), OP302778 (gapdh), OP302777 (act), and OP302779 (ApMat). BLASTn searches revealed high identity with sequences of C. tropicale (CBS 124949) for ITS (100%), ApMat (100%), act (100%), and gapdh (100%). A phylogenetic tree based on Bayesian inference and Maximum Likelihood methods, including published ITS, ApMat, act, and gapdh sequence datasets for isolates in the Colletotrichum gloeosporioides species complex was generated. The phylogenetic analysis based on the concatenated sequences clustered the isolate CCLF186 with the C. tropicale reference isolates. Pathogenicity of the isolate CCLF186 was confirmed on 10 healthy pitahaya fruits without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on two locations on each fruit. Ten control fruits were treated with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated pitahaya fruits exhibited sunken and necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation. Recently, C. tropicale causing anthracnose in dragon fruit (Selenicereus monacanthus) was reported from Philippines (Evallo et al. 2022). Now, this is the first report of C. tropicale causing fruit anthracnose in H. costaricensis in Mexico and worldwide. These findings provide a basis for research about the distribution and effective disease-management strategies.

Genetic Evidence for Colletotrichum gloeosporioides Transmission Between the Invasive Plant Ageratina adenophora and Co-occurring Neighbor Plants.

To understand the disease-mediated invasion of exotic plants and the potential risk of disease transmission in local ecosystems, it is necessary to characterize population genetic structure and spatio-temporal dynamics of fungal community associated with both invasive and co-occurring plants. In this study, multiple genes were used to characterize the genetic diversity of 165 strains of Colletotrichum gloeosporioides species complex (CGSC) isolated from healthy leaves and symptomatic leaves of invasive plant Ageratina adenophora, as well as symptomatic leaves of its neighbor plants from eleven geographic sites in China. The data showed that these CGSC strains had a high genetic diversity in each geographic site (all Hd > 0.67 and Pi > 0.01). Haplotype diversity and nucleotide diversity varied greatly in individual gene locus: gs had the highest haplotype diversity (Hd = 0.8972), gapdh had the highest nucleotide diversity (Pi = 0.0705), and ITS had the lowest nucleotide diversity (Pi = 0.0074). Haplotypes were not clustered by geographic site, invasive age, or isolation source. AMOVA revealed that the genetic variation was mainly from within-populations, regardless of geographic or isolation origin. Both AMOVA and neutrality tests indicated these CGSC strains occurred gene exchange among geographic populations but did not experience population expansion along with A. adenophora invasion progress. Our data indicated that A. adenophora primarily accumulated these CGSC fungi in the introduced range, suggesting a high frequency of CGSC transmission between A. adenophora and co-occurring neighbor plants. This study is valuable for understanding the disease-mediated plant invasion and the potential risk of disease transmission driven by exotic plants in local ecosystems.

First report of Colletotrichum siamense causing leaf spot on Machilus pauhoi in China.

Machilus pauhoi Kaneh. is an excellent evergreen broad-leaved tree species widely grown in China for its ornamental and economic value (He et al. 2022). In September 2021, a leaf spot was observed on M. pauhoi plants on Guantian forest farm (27°06'15.6″N, 114°34'20.72″E) in ji' an city, Jiangxi province, China. The disease incidence was estimated to be above 20%. The symptoms began as brown irregular spots, then the spots gradually expand over time, with a gray-to-brown center and dark brown-to-black edges. Small infected tissues (3 to 5 mm2) were surface-sterilized in 70% ethanol for 30 s and 2% NaClO for 60 s, and rinsed three times with sterile water (Ju et al. 2021). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-two isolates of Colletotrichum ssp. were obtained (isolation frequency about 78%). Three representative single-spore isolates (PN-1, PN-4, and PN-9) were used for morphological studies and phylogenetic analyses. Colonies on the PDA of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 11.4-16.8 ×4.1-5.5 µm (13.2 ± 1.0 × 4.4 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.2-8.8 × 4.1-6.2 µm (6.7 ± 0.2 × 5.1 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. All sequences were deposited into GenBank (ITS, ON176154 - ON176156; ACT, ON185554 - ON185556; GAPDH, ON185563 - ON185565; TUB2, ON185566 - ON185568; CHS-1, ON185560 - ON185562; CAL, ON185557 - ON185559). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed PN-1, PN-4, and PN-9 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. The pathogenicity of three isolates was tested on nine M. pauhoi plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and inoculated with 10 µL of spore suspension (106 conidia/mL). The spore suspension of each isolate was inoculated onto six leaves. Another three plants inoculated with ddH2O served as the control (Wan et al. 2022). All the inoculated leaves were covered with plastic bags to keep them moist for 2 days (relative humidity > 80%). All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 7 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Up to now, Pestalotiopsis chamaeropis, Corynespora cassiicola and Arthrinium arundinis could infect M. pauhoi plants (Zhang et al. 2021), and cause leaf spots in China. To our knowledge, this is the first report of C. siamense causing leaf spots on M. pauhoi. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First Report of Colletotrichum aenigma Causing Anthracnose of tree peony in China.

Tree peonies (Paeonia suffruticosa Andr. and hybrids) are well-known ornamental and medicinal plants cultivated in temperate and subtropical regions around the world. From June to September 2021, severe leaf spot disease was observed on 3 tree peony cultivars ('Luoyanghong', 'Moyushenghui', 'Roufurong') in Xinxiang (35º29´N, 113º95´E) and Luoyang (34º64´N, 112º49´E), Henan Province, China. Leaf spot incidence was as high as 28% ('Luoyanghong'), 45% ('Moyushenghui') and 67% ('Roufurong'), respectively. Symptoms appeared initially as small purple spots less than 1 mm in diameter in the middle and upper parts of the leaves, and then evolved to coalescent lesions, causing brown scorch ultimately. From each cultivar, 5 diseased leaves were collected. Leaflet tissues (3-4 mm2) cut from spot margins were surface sterilized in 75% alcohol for 45 s, washed 5 times with sterile distilled water, and then cultivated on potato dextrose agar (PDA) medium at 28 °C in the dark. Eleven isolates were obtained, and colonies grown from single conidia on PDA were 80-85 mm in diameter after 10 d, with scattered small, dark-based spikes on the surface of the colonies. The aerial mycelium was cottony, dense, and dark gray near the center on the reverse side. Conidia were cylindrical to clavate, with rounded apex and rounded base, and the conidia contents were granular, 8.44-14.06×3.60-4.31 μm (mean=11.28×3.69 μm, n=40). Appressoria were mostly subglobose or with a few broad lobes, pale to medium brown, 3.36-6.72×3.35-5.60 μm (mean=5.02×4.55 μm, n=20). Based on the culture representation and conidial morphology, the isolates were characterized as Colletotrichum gloeosporioides species complex (Weir et al. 2012; Fu et al. 2019). To further identity the species, the actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the ribosomal internal transcribed spacers (ITS) loci of isolates PSW0002, PSW0008 and PSW0009 were amplified using ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF/GDR, and ITS1/ITS4, primers (Weir et al. 2012; Schena et al; 2014; Kim et al. 2021; Li et al. 2021). Fifteen sequences were deposited in GenBank (ACT for OP225605, OP225606, and OP225607, CAL for OP225608, OP225609 and OP225610, CHS for OP225611, OP225612 and OP225613, GAPDH for ON321897, OP225614, and OP225615, and ITS for ON323473, OP214349 and OP214350 ), which showed 100% sequence similarity to Colletotrichum aenigma (JX009443 and JX009519 for ACT, JX009683 and JX009684 for CAL, JX009774 and JX009903 for CHS-1, JX010244 and JX009913 for GAPDH, JX010243 and JX010148 for ITS). Three isolates clustered with C. aenigma (ex-holotype culture ICMP 18608) in the multi-locus phylogenetic tree with a bootstrap value of 100%. To achieve Koch's postulates, pathogenicity was tested on 5-year-old healthy potted plants ('Luoyanghong'). Thirty leaves were inoculated with 10 µL conidial suspension (isolate PSW0002, 1×106 conidia/mL) and the controls were inoculated with sterile water. Plants were placed in a greenhouse at 28°C under conditions with 12 h photoperiod and 90% relative humidity. After 5 to 10 days, distinct spots were observed on the inoculated leaves, while the control leaves showed no symptoms. C. aenigma was reisolated from all inoculated leaves, but not from the control. C. aenigma has been reported to cause anthracnose on Pyrus pyrifolia (Weir et al. 2012), Camellia sasanqua (Chen et al. 2019), Juglans regia (Wang et al. 2020), Paeonia ostii (Ren et al. 2020), and Capsicum annuum (Sharma et al. 2022). A previous study reported C. gloeosporioides as a pathogen of anthracnose in tree peonies in China (Xuan et al. 2017), the typical symptoms were big necrotic lesions (5-10 mm diam) on leaves，which were significantly different from those caused by C. aenigma. To our knowledge, this is the first report of C. aenigma causing anthracnose in tree peonies in China. This finding may help to take effective control of anthracnose in tree peonies.

Leaf Spots of Salix babylonica Caused by Colletotrichum gloeosporioides s.s. and C. siamense Newly Reported in China.

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.

First Report of Twig Anthracnose of Acacia melanoxylon Caused by Colletotrichum siamense in China.

Acacia melanoxylon R. Br. native to Australia, is a high-quality timber tree with wide genetic and phenotypic diversity. In recent years, A. melanoxylon has been extensively cultivated in some provinces in southern China. In December 2019, anthracnose-like symptoms were observed on twigs of A. melanoxylon in China. In certain valleys in south China, the disease incidence on plants and shoots was 60-75% and 80-90%, respectively. The wither rate of disease branches was 30-40% in dry seasons from September to November. The appearance of symptoms occurred in a humid and warm valley. Symptoms were initially observed on the young branches as brown spindle shaped sunken spots. At later stages, the disease spots girdled the whole branch, which became wilted and its leaves turned reddish-brown. For pathogen isolation, diseased branches were sampled and 55 pieces (5× 5 mm) of these branches section were surface-sterilized in 75% ethanol for 30 seconds, followed by 0.5% NaClO for 5 min and then were rinsed three times in distilled water. After drying with sterilized filter paper, the surface-sterilized sections were transferred to potato dextrose agar medium (PDA) and incubated at 25 °C for 7 days in the dark. Three isolates were obtained as representatives for morphological characterization and were labeled as 1A912, 1B912, and 1C912. These specimens were deposited in the Guangdong Province Key Laboratory of Microbial Signals and Disease Control at the South China Agricultural University (China). Purified isolates were initially white, cottony and with dense aerial mycelium on PDA at 25 ℃, ten days later their colonies turned grayish white with orange conidial masses. Conidia were one-celled, hyaline, straight, cylindrical, with round obtuse ends, and measured 11.0 to16.3× 4.0 to 6.0 μm (n= 100), appressoria were 5.86 to 9.07 × 3.55 to 6.96 μm (n= 100). Morphological characteristics of selected isolates matched the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For further identification, the internal transcribed spacer (ITS) region, and partial sequences of the actin (ACT), beta-tubulin (TUB2), and glycerol dehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced, using primer pairs ITS1/ ITS4 (White et al. 1990), Bt2a/ Bt2b (Donaldson and Glass 1995), ACT512F/ ACT783R, GDF1/ GDR1(Weir et al. 2012). The sequences were deposited in GenBank (ITS: MW228101-MW228103; TUB2: MW250346, MW320707, MW320708; ACT: MW250347, MW320703, MW320704; GAPDH: MW250348, MW320705, MW320706). The multilocus phylogenetic analysis distinguished the isolates 1A912, 1B912, and 1C912 as C. siamense. Pathogenicity of those three isolates of C. siamense was tested on healthy twigs of the one clone of A. melanoxylon. 27 young twigs of nine 1-year-old plants were inoculated with the mycelium of the 7 days-old isolates 1A912, 1B912, and 1C912(Each isolate infected three plants and each infected three young twigs) through an artificial wound. The same nine plants were inoculated with PDA medium alone (each infected three young twigs) as a negative control. Five days after inoculation, brown spindle spots similar to the field disease symptoms were observed on the twigs. No symptoms were observed on the control plants. The experiment was repeated twice. The fungus was successfully reisolated from the symptomatic plants, and had identical morphological and molecular characteristics to the initial isolates, fulfilling Koch´s postulates. To our knowledge, this is the first report of anthracnose caused by C. siamense on A. melanoxylon in China. Twig anthracnose can reduce the growth of A. melanoxylon. Further research on management options for this disease is required.

Natural Flora Is Indiscriminately Hosting High Loads of Generalist Fungal Pathogen Colletotrichum gloeosporioides Complex over Forest Niches, Vegetation Strata and Elevation Gradient.

Crop pathogenic fungi may originate from reservoir pools including wild vegetation surrounding fields, and it is thus important to characterize any potential source of pathogens. We therefore investigated natural vegetation's potential for hosting a widespread pathogenic group, Colletotrichum gloeosporioides species complex. We stratified sampling in different forest environments and natural vegetation strata to determine whether the fungi were found preferentially in specific niches and areas. We found that the fungi complex was fairly broadly distributed in the wild flora, with high prevalence in every study environment and stratum. Some significant variation in prevalence nevertheless occurred and was possibly associated with fungal growth conditions (more humid areas had greater prevalence levels while drier places had slightly lower presence). Results also highlighted potential differences in disease effects of strains between strata components of study flora, suggesting that while natural vegetation is a highly probable source of inoculums for local crops nearby, differences in aggressiveness between vegetation strata might also lead to differential impact on cultivated crops.

First report of anthracnose on Acer fabric caused by Colletotrichum siamense in China.

Acer fabri Hance, an evergreen tree, is widely cultivated in China for its ornamental value (Lin. 2020). In July 2020, a leaf spot disease, with an incidence of Approximately 48% (12 out of 25), was observed on A. fabri plants (almost 9-year-old) at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E). On average, 30% of the leaves per individual tree were affected. Small spots initially formed along the edge or tip of the leaves and gradually expanded into dark brown spots, and eventually the diseased leaves withered. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Wan et al. 2020). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, LFY-1, LFY-5, and LFY-8 were used for morphological studies and phylogenetic analyses. Colonies on PDA of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 12.8-17.4 ×4.3-5.7 µm (14.3 ± 1.1 × 4.6 ± 0.4 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.3 × 4.7-6.6 µm (7.4 ± 0.3 × 5.5 ± 0.4 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. All sequences were deposited into GenBank (ITS, OL818322- OL818324; ACT, OL830175 - OL830177; GAPDH, OL830166 - OL830168; TUB2, OL830163 - OL830165; CHS-1, OL830169 - OL830171; CAL, OL830172 - OL830174). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed LFY-1, LFY-5, and LFY-8 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. The pathogenicity of three isolates was tested on six A. fabri plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and inoculated with 10 µL of spore suspension (106 conidia/mL). The spore suspension of each isolate was inoculated onto five leaves. Another three plants inoculated with ddH2O served as the control (Si et al. 2019). All the inoculated leaves were covered with plastic bags to keep a high-humidity for 2 days. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. The pathogen was previously reported to cause anthracnose on Kadsura coccinea (Jiang et al. 2022), Carica papaya (Zhang et al. 2021), Michelia alba (Qin et al. 2021). This study is the first to report C. siamense causing anthracnose on A. fabric. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First Report of Colletotrichum aenigma Causing Anthracnose on Mulberry Leaves in China.

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.

First report of black pepper (Piper nigrum L.) anthracnose caused by Colletotrichum siamense in north-east India.

Black pepper (Piper nigrum L.) has been commonly cultivated as a spice crop in northeast India. In August 2021, anthracnose leaf spot was observed on black pepper vines with 50 to 60% of disease incidence in Assam Agricultural University, Jorhat (26.7509° N, 94.2037° E), Assam, India. On average, 80% of the leaves per individual vine were affected by this disease. Foliar symptoms initially appeared as chlorotic circular spots, which then coalesced into larger irregular lesions. The centers of the spots were brown, papery in texture, and surrounded by a yellow halo. Numerous acervuli at the center of the spots were observed. Ten vines from the orchard were sampled to identify the causal agent. Symptomatic leaves along with some healthy portion were cut (3 to 4.5 mm2), surface-sterilized in 70% ethanol for 30 s, rinsed in sterile distilled water twice, dried on sterilized filter paper, aseptically plated on potato dextrose agar (PDA) amended with Streptomycin sulphate (30 mg/L), and then incubated at 25°C for four days. Two Colletotrichum isolates were recovered from infected tissues and purified by the hyphal tip method. Fungal colonies on PDA were cottony, dense, white to gray in color, and with salmon pink conidial masses. Conidia (n = 50) were 13.6 to 19.8 × 4.2 to 6.4 μm, cylindrical, hyaline, single-celled, smooth-walled, and with rounded ends. Conidiophores were aseptate, hyaline, short and branched. Based on morphological features, the isolates were identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For accurate identification of two isolates, the DNA was extracted from pure culture. The internal transcribed spacer (ITS) region, actin (ACT), β-tubulin 2 (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by polymerase chain reaction (Weir et al. 2012) and sequenced. The sequences were deposited in the GenBank database (ITS: OP297054 and OP296876; ACT: OP327082 and OP327081; TUB2; OP327086 and OP327085; GAPDH: OP327084 and OP327083). A BLAST analysis of ITS, ACT, TUB2 and GAPDH sequences revealed 99.5-100%, 99.9-100%, 99.9-100% and 99.8-100% similarity respectively to C. siamense for both isolates in NCBI database. The pathogenicity tests were carried out on potted four months old vine cuttings of P. nigrum L., which were kept in a greenhouse. Ten healthy plants were sprayed with 50 µl of conidial suspension of each isolate (107 conidia ml-1, 10 ml/plant). Five control plants were sprayed with sterile distilled water. The plants were covered with sterilized plastic bags after inoculation to maintain humidity and kept in a greenhouse at day/night temperatures of 25 ± 2°C and 17 ± 2°C (Zhang et al., 2021). Within eight days, all the inoculated plants showed symptoms similar to those observed in the field, whereas control plants were asymptomatic. The pathogenicity test was repeated twice. C. siamense was consistently reisolated from the lesions and was confirmed by morphological characterization and molecular assays as described above in this note, whereas no fungus was isolated from control leaves. To our knowledge this is the first report of C. siamense causing black pepper anthracnose in northeast India. The pathogen has significant potential for causing high losses in black pepper production. These data will help researchers to develop effective management strategies for this disease.

Sensitivity of <i>Colletotrichum gloeosporioides</i> species complex (CGSC) isolated from strawberry in Taiwan to benzimidazole and strobilurin

Colletotrichum gloeosporioides species complex (CGSC) is the major pathogen causing strawberry anthracnose in Taiwan. Benzimidazoles and strobilurins are common fungicides used to control strawberry anthracnose. A total of 108 CGSC isolates were collected from five major strawberry-producing areas in Taiwan. The half-maximal effective concentration (EC50) values of most CGSC isolates for benomyl (59 isolates), carbendazim (70 isolates), and thiabendazole (63 isolates) were higher than 500 µg a.i./mL. Strobilurin tests showed that the EC50 values of most CGSC isolates for azoxystrobin (66 isolates), kresoxim-methyl (42 isolates), and trifloxystrobin (56 isolates) were higher than 500 µg a.i./mL. However, most CGSC isolates were sensitive to pyraclostrobin at 100 µg a.i./mL. Fungicide tests indicated that CGSC isolates show multi-resistance to benzimidazoles and strobilurins. Benzimidazole-resistant isolates were associated with a point mutation in codon 198 of the β-tubulin gene, and strobilurin-resistant isolates did not correspond with mutation in the cyt b gene or alternative oxidase activity.

A real-time PCR for detection of pathogens of anthracnose on Chinese fir using TaqMan probe targeting ApMat gene.

Anthracnose is one of the most widespread and destructive diseases on Chinese fir. Colletotrichum cangyuanense, Colletotrichum fructicola, Colletotrichum gloeosporioides, and Colletotrichum siamense are the causal agents of anthracnose on Chinese fir. A rapid and accurate diagnosis of different pathogens is critical for the disease management. Phylogenetic tree and sequence similarity analysis showed that the single-locus ApMat provides superior phylogenetic information and is an appropriate target to distinguish C. cangyuanense, C. fructicola, C. gloeosporioides, and C. siamense. The real-time PCR assays with the primer sets of MQ-F/R, 1#C-F/R, YK-F/R, and WZ-F/R, and corresponding TaqMan probes of MQ-P, 1#C-P, YK-P, and WZ-P were specific for C. cangyuanense, C. fructicola, C. gloeosporioides, and C. siamense, respectively. The sensitivity tests showed that the lowest amount of gDNA that the PCRs can detect was 1ng of genomic DNA. The validity of the assays was confirmed by directly detecting the pathogens from both the fungal culture and infected Chinese fir. These results demonstrated the potential of the TaqMan real-time PCR targeting the ApMat gene to provide rapid, specific, and reliable molecular detection of C. fructicola, C. gloeosporioides, C. siamense, and C. cangyuanense, respectively. The data also provided a reference solution for the identification of species within Colletotrichum gloeosporioides species complex (CGSC), which share similar morphological characteristics. © 2022 Society of Chemical Industry.

First Report of Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde causing anthracnose disease in Santalum album Linn. in India.

Indian sandalwood (Santalum album), valued for its medicinal properties, is an indigenous species of India. Circular or irregular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the centre of the lesion were observed on leaves of sandalwood seedlings in a nursery located in Karnataka, with a disease incidence of 75% (n = 100 investigated plants) during July 2020. The disease prevailed in monsoon followed by winter season (July 2020 - January 2021); summer was less supportive for the disease incidence. As the disease progressed, lesions expanded and merged, causing necrosis of the whole leaf. Isolation of the pathogen involved excision of small sections of diseased tissues from the lesions followed by surface sterilization with 70% ethanol for 30 s and in 1% NaClO for 1 min. Sections were rinsed in sterile distilled water, placed on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Ten isolates of Colletotrichum ssp. were obtained with an isolation frequency = 10/12×100 = 83%. One representative single-spore isolate (CSSA-1) was used for further study. Initially, pure cultures exhibited a white mycelium which later turned gray with time, and had orange conidial masses in a concentric ring pattern with the aggregation of black acervuli at the center of the culture Conidia were single celled, hyaline, and cylindrical having smooth rounded ends and the size ranged from 12.6 to 18.5 µm in length, and 3.5 to 5.6 µm in width (n = 100). The morphological characteristics were in agreement with the species description of fungi in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm the species designation of the isolate CSSA-1, a multilocus phylogenetic analysis was performed using six genomic loci (Weir et al. 2012; Marin-Felix et al. 2017). The internal transcribed spacer (ITS) region of rDNA and a partial sequence of the beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and glutamine synthetase (GS) genes were amplified using ITS1/ITS4, BT2F/BT4R, GAPDHF/R, 79F/354R, 512F/783R and GSF/GSR primers, respectively. The ITS (OK254122), TUB2 (OL462863), GAPDH (OL462859), CHS-1 (OL462860), actin (OL462861), and glutamine synthetase (OL961822) sequences of representative isolate CSSA-1 showed 99 to 100% identity with sequences MZ148628, MK967339, MN525882, MW192791, MT263504, MF111030, MH370542 and KX578767, respectively to the holotype isolate of Colletotrichum siamense (Prihastuti et al. 2009). The sequences were analysed with representative sequences of Colletotrichum and a multilocus Bayesian inference phylogenetic tree with ITS-GAPDH-ACT-CHS1-GS-TUB2 concatenated data sets (concatenated with Sequence Matrix v.1.8 (Vaidya et al. 2011)) was constructed using Beast version 1.8.4 to confirm the isolate identification (Drummond et al. 2012; Hyde et al. 2014; Weir et al. 2012). Isolate CSSA-1 clustered with C. siamense isolates. To complete Koch's postulates, for the characterized isolate CSSA-1, a pathogenicity test was conducted on 3-month-old sandalwood seedlings by spore spray inoculation. Ten plants were inoculated with a conidial suspension (106 conidia/ml) and control plants inoculated with sterilized water then kept in a glass house at 25°C and >85% relative humidity with a 12-h photoperiod. Humidity was maintained by spraying the plants with water in the morning and evening to enhance the infection. Typical symptoms of anthracnose disease similar to naturally infected leaves were observed, which included circular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the center of the lesion 7 days after inoculation, while the control plants remained unaffected. The pathogen was reisolated from infected leaves and its identity was confirmed as C. siamense based on morphological characteristics. Previously, C. siamense was identified causing disease on chili in India (Gunjan and Shenoy, 2014), but to our knowledge this is the first report of leaf anthracnose caused by C. siamense on Indian sandalwood in India or globally. This study documents crucial information, paving way for epidemiologic studies and design of control strategies to combat this newly emerging disease.

First Report of Colletotrichum chrysophilum Causing Papaya Anthracnose in Mexico.

Anthracnose, caused by Colletotrichum spp., is the most important fungal disease of papaya (Carica papaya L.) worldwide. In March 2020, mature papaya fruit (cv. Maradol) showing typical symptoms of anthracnose were observed in an orchard located in Pinotepa Nacional, Oaxaca, Mexico. Disease incidence of 100 papaya plants surveyed in the orchard was estimated at about 45%. Initially, small and water-soaked lesions appeared on the fruit surface, which later enlarged to circular sunken lesions with translucent light brown margins. On advanced infections, salmon-pink masses of spores were observed on the lesions. Twenty Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium at 25°C in the dark for 6 days and 10 monoconidial isolates were obtained. An isolate was selected as representative for further characterization. The isolate was deposited as CPM-H4 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. On PDA, the colonies were initially light grey then later became dark grey with orange conidial masses after incubation for 7 days. Conidia (n= 50) were hyaline, aseptate, cylindrical with rounded ends, and measured 10.2 to 13.6 × 4.1 to 5.3 μm. Appressoria (n= 20) were mostly simple, solitary and smooth-walled, dark brown, and clavate, measuring 6.8 to 14.8 × 5.5 to 7.7 μm. Based on morphology, the isolate was tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes were amplified (Weir et al. 2012), and sequenced. The sequences were deposited in GenBank (accessions nos. OM965612 (ITS), OM959540 (ACT), ON065005 (TUB2), ON065003 (CHS-1), ON065004 (GAPDH). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, GAPDH, and CHS-1 sequence dataset for Colletotrichum spp. was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CPM-H4 as Colletotrichum chrysophilum. Pathogenicity of the fungus was verified on 10 healthy papaya fruits (cv. Maradol) without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on three locations on each fruit. Ten control fruit were treated in the same way but with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated papaya fruits developed sunken necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Colletotrichum chrysophilum has been previously reported to cause anthracnose on mango (Fuentes-Aragón et al. 2020a), avocado (Fuentes-Aragón et al. 2020b), and banana (Fuentes-Aragón et al. 2021) in Mexico; however, to our knowledge, this is the first report of C. chrysophilum causing papaya anthracnose in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species associated with papaya in Mexico through subsequent phylogenetic studies as well as to monitor the possible movement and distribution of this pathogen into other Mexican regions.

First report of Colletotrichum siamense associated with anthracnose on Theobroma cacao fruits in Venezuela

Cacao (Theobroma cacao) plantations are spread throughout Venezuela, but the cultivar “Sweet cacao” or “Criollo (= Creole)” is concentrated in the states of Mérida, Táchira, and Zulia (Palma, 1953). In April 2016, brown and black sunken lesions typical of anthracnose symptoms were observed on cacao fruits in plantations from the municipalities Alberto Adriani, Andrés Bello, Antonio Pinto Salinas, Obispo Ramos de Lora, and Sucre in Mérida State, Venezuela (Figure 1). For isolation of the pathogen, fruits were cut into c. 5 mm2 sections at the healthy and diseased margin. Tissues were then surface disinfected for 30 seconds in 70% ethanol, 60 seconds in 0.5% sodium hypochlorite, washed three times with sterilised water for 60 seconds, and dried with sterilised filter paper. Disinfected samples were plated onto 2% malt extract agar and incubated at 25°C for seven days or until mycelia were observed growing from the samples. The fungal mycelium was sub-cultured onto potato dextrose agar (PDA), and hyphal tipping methods were performed to obtain pure cultures that were used for morphological analysis. Nine strains were isolated and evaluated. Colonies on PDA grew to 60–80 mm (mean = 70), in diameter in seven days, and >90 mm diameter in 10 days at 25°C. After 15 days, colonies were flat with an entire edge, with aerial mycelium that were dense, cottony, pale olivaceous grey on the surface and buff to pale luteous on the underside of the culture (Figure 2). Conidiomata were acervular with conidiophores formed on a cushion of roundish and medium olive-brown cells; setae were not observed. Conidiogenous cells were hyaline, cylindrical to ampulliform, simple, short, straight, measuring 9–14 × 1.1–1.2 µm. Conidial mass orange in colour and visible through the mycelium in the reverse of the culture. Conidia were hyaline, smooth-walled, cylindrical, aseptate, with ends rounded and often tapering slightly from the middle towards the base or only sometimes narrowed at the centre (sub-cylindrical), 8–13 × 2.4–3.8 µm, mean ± SD = 10.1 ± 1.4 × 3 ± 0.3 µm, L/W ratio = 3.4 (Figure 3). Appressoria measuring 5–9 µm diameter, terminal, subglobose or with one or two borders lobed, brown, solitary were observed (Figure 4). No chlamydospores were observed. The morphological characteristics of these isolates placed this fungus within the Colletotrichum gloeosporioides species complex (Weir et al., 2012). The glutamine synthetase (GS) and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) were amplified using the primer pairs GSLF2/GSLR1 (Liu et al., 2016) and AMF1/AMR1 (Silva et al., 2012), respectively. The ApMat sequences were deposited in GenBank (Accession Nos. MF436054-MF436062) and GS sequences (MF436063-MF436071). Maximum likelihood (ML) was evaluated with a bootstrapping method and Bayesian inference (BI) analysis was performed with MrBayes v. 3.2.6 phylogenies, both calculated in Geneious 2021.0.3.0. The posterior probabilities from ML/BI analyses (87/0.99) in the combined dataset supported the inclusion of T. cacao isolates within the Colletotrichum siamense clade (Figure 5). Therefore, using phylogenetic analyses based on sequence data from the two loci (GS and ApMat) and morphological characteristics of the fungus, we identified the isolates as C. siamense. ApMAT and GS loci provided robust support to separate all Colletotrichum spp. inside the C. gloeosporioides complex species, confirming that these loci provide higher phylogenetic resolution compared to other standard markers (Silva et al., 2012; Liu et al., 2015). Colletotrichum gloeosporioides has been reported to cause anthracnose on leaves, flowers and unripe cacao fruits in Venezuela (Reyes & Capriles de Reyes, 2000), and was also present at high incidence on fruits in the municipality of Sucre, Merida state (Parra et al., 2009). Morphological similarities between C. gloeosporioides and C. siamense, such as the orange colour of conidial masses that developed in cultures, could have led to misidentification of the causal agent of the disease previously in Venezuela. To our knowledge, this is the first report of C. siamense associated with anthracnose on cacao fruits in Venezuela. We are grateful to the U. S. Department of State Fulbright Scholar Program for supporting a one-year exchange visit and providing financial support to Prof. Mohali Castillo, ID assigned: PS00236102, as researcher in the Department Agricultural Biology, Colorado State University. Further, we thank Kristen Otto for excellent laboratory assistance.

Close-Range Thermography and Reflectance Spectroscopy Support InVitro and InVivo Characterization of Colletotrichum spp. Isolates from Mango Fruits.

Colletotrichum causing anthracnose in mango is known for its variable virulence that may have an effect on disease development and efficacy of management strategies. In this study, we characterized Colletotrichum spp. isolated from mango fruits under invitro and invivo conditions using close-range thermography and reflectance spectroscopy. Twenty-six isolates were phylogenetically characterized to ascertain species using the internal transcribed spacer sequence. Virulence, spectral (invivo and invitro), and thermographic responses (invivo) of these isolates were analyzed. Isolates were grouped into the Colletotrichum gloeosporioides species complex and classified into eight morphotypes. Mycelial growth, conidia production, sporulation abundance, and area under disease progress curve (AUDPC) varied largely among isolates. Disease symptoms were observed 4 days after inoculation (dai), and, for most morphotypes, changes in tissue temperature were registered at 11 dai, with the greatest decrease at 14 dai with pathogen sporulation. In vitro and invivo morphotypes shared changes in the spectrum range, and main variations were found in the number of informative spectral bands. In vivo average gross reflectance was higher in disease-inoculated tissue than in healthy uninoculated tissue. Morphotype responses varied depending on AUDPC values and postinoculation time. Discriminant analysis of the spectral response using principal component analysis and partial least squares regression explained 94 to 96.3 and 98 to 99.9% of the variance from invitro and invivo tests, respectively. Spectral markers were obtained for four distinct morphotype groups. We found three (550 to 650, 650.1 to 790, and 1,300 to 1,400 nm) and two (520 to 830 and 1,100 to 1,450 nm) regions with highly (P < 0.05) discriminant spectral bands for diseased fruits and morphotype characterization.

Reclassification of the Main Causal Agent of Glomerella Leaf Spot on Apple into Colletotrichum chrysophilum in Southern Brazil and Uruguay.

Glomerella leaf spot (GLS) is one of the most important diseases of apple, affecting a wide range of economically important cultivars, particularly Golden Delicious and its descendants. Caused mainly by species of the Colletotrichum gloeosporioides species complex (CGSC), C. fructicola has been described as the most prevalent and aggressive species associated with GLS and apple bitter rot (ABR) in Brazil and Uruguay. Recently, new CGSC species, closely related to C. fructicola, have been identified causing ABR. To verify the accuracy of species identification within the CGSC, we aimed to reevaluate the identity of representative GLS-causing isolates from Brazilian and Uruguayan populations, previously identified as C. fructicola. Multilocus phylogenetic analysis based on APN2, ApMAT, CAL, GAPDH, GS, ITS, and TUB2 allocated these isolates in a monophyletic clade with C. chrysophilum. This species was first described as the causal agent of anthracnose in banana fruits in Brazil, and recent reports indicate its association with ABR in the United States. This is the first report of C. chrysophilum causing GLS disease on apple worldwide.

First Report of Anthracnose Caused by Colletotrichum fructicola on Loropetalum chinense in China.

Loropetalum chinense Oliv. (Hamamelidaceae), an evergreen shrub or small tree, is widely used as a bonsai and landscape plant because of its aesthetic value. In addition, it has medicinal value, its leaves, flowers and roots can be used to treat diarrhea, coughs, hemorrhaging and burn (Zhang et al. 2013). In October 2020, leaf spot symptoms were observed on L. chinense distributed in Meiling Scenic Spot of Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). We surveyed about 500 m2 of the mountain area which holds about 60 trees of L. chinense, with an incidence up to 20%. Initially, small gray-brown spots with dark brown edges appeared on the leaf surface, becoming large circular or irregular brown necrotic lesions over time. To isolate the pathogen, ten leaves of infected tissues were cut into 4 mm2 pieces, and surface disinfected with 75% ethanol for 30s and 1% hypochlorite for 1 min, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 to 7 days. Five isolates with similar morphological characteristics were obtained. Conidia were hyaline, aseptate, smooth-walled, cylindrical with obtuse to slightly rounded ends, and their dimensions varied from 8.57 to 15.80 × 1.56 to 4.65 μm (n = 20). The morphological characteristics of the isolates matched the descriptions of Colletotrichum gloeosporioides species complex (Cai et al. 2009; Weir et al. 2012; Liu et al. 2015). For molecular identification, two representative isolates (JAUCC L003 and JAUCC L004) were selected for genomic DNA extraction, and the internal transcribed spacer (ITS), partial calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) were amplified, using primer pairs ITS1/ITS4, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (White et al. 1990; Weir et al. 2012; Yang et al.2020). The sequenced loci (GenBank accession nos. ITS: MZ054401, MZ054400; CAL: MZ056750, MZ056754; CHS-1: MZ056751, MZ056755; GAPDH: MZ056752, MZ056756; ACT: MZ056753, MZ056757) exhibited up to 99% homology with corresponding sequences of C. fructicola Prihast., L. Cai & K.D. Hyde strains (GenBank accession nos. LC494271, MN525833, MN525854, MN525875 and MN525814). Concatenated sequences of the five genes were used to conduct a phylogenetic analysis using Maximum-Likelihood (ML) method in MEGA7. The isolates were identified as C. fructicola based on morphology and a multigene phylogenetic analyses. Pathogenicity of one representative isolate (JAUCC L003) was tested indoor by inoculating the top wounded leaves of six healthy L. chinense plants, rather than inoculating unwounded leaves due to the low incidence. Three leaves from each of three plants were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and three leaves from each of other three plants were wounded and inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each. Treated plants were incubated in an artificial climate box at 25 °C, 90% relative humidity, with a 12-h photoperiod. All leaves inoculated with conidial suspension and mycelial plugs produced similar symptoms as described above 10 days postinoculation, whereas the mock inoculated plants remained asymptomatic. The fungus isolated from inoculated leaves was identical to the original pathogen on account of morphological and molecular data, confirming Koch's postulates, but not from the mock inoculated plants. Anthracnose disease caused by C. fructicola has been reported affecting numerous plants worldwide, including cotton, coffea, grape, citrus, mango, apple, pear, and cassava, among others (Guarnaccia et al. 2017, Oliveira et al. 2018). To our knowledge, this is the first report of C. fructicola causing anthracnose on L. chinense in China. This disease is significant concern in horticulture due to its impact on the aesthetics of ornamentals used in landscape plantings.

First Report of Colletotrichum chrysophilum Causing Apple Bitter Rot in Spain.

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.

First report of Colletotrichum henanense causing anthracnose on Chinese chestnut in the United States.

Culinary chestnut production in the United States (US) is a rapidly growing industry supporting fresh market and value-added industries. An estimated 200-400 new acres of chestnuts are planted every year in the US, with most growers east of the Rocky Mountains planting Chinese chestnut (Castanea mollissima) or Chinese chestnut hybrids. In 2018, Ohio producers of Chinese chestnut reported losses of up to 80% to blossom end rot. Symptoms were like those reported by Fowler and Berry (1958), including black spots on the chestnut shell, often at the stylar end, and blackening of the kernels. Spots covered 1-100% of the kernel, however, no signs of any pathogen were present on the shell or kernel. In 2020, cankers that were brown/black in color, sunken, and ~1 cm in length were observed on 1-yr twigs of chestnut seedlings from a nursery operation on the same farm from which the symptomatic kernels were observed. In some seedlings, distal portions of the twigs died, while in other seedlings only shoots and leaves within the cankered areas died. Black acervuli were observed erupting from the cankers. Colletotrichum spp. were isolated and cultured on potato dextrose agar from surface-sterilized tissue from kernel lesions (MLI246-21 to MLI249-21) and twig cankers (MLI250-21 and MLI251-21). All isolates produced grey aerial mycelia, pink sporodochia, and cylindrical conidia with rounded ends ranging in size from 12-20 um long by 5-8 um wide. Isolates were preliminarily identified as belonging to the Colletotrichum gloeosporioides species complex (CGSC). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, β-tubulin (TUB2) gene, and the intergenic spacer ApMat were amplified from genomic DNA and sequenced (Dowling et al. 2020). These genes are suitable for identifying species within the CGSC (Eaton et al. 2021). Sequences were submitted to the GenBank database (GAPDH OL687148 to OL687153, TUB2 OL741624 to OL741629 and ApMAT OL695914 to OL695919). BLASTn queries of NCBI GenBank showed that the GAPDH, TUB2, and ApMat sequences from all isolates had 98%, 98% and 99% identity with C. henanense isolates MT513015.1, MT513080.1, and MT512917.1 from apple (Martin et al. 2021). Representative isolates were used to demonstrate Koch's postulates and confirm pathogenicity on kernels (MLI246-21 and MLI249-21) and twigs (MLI250-21). Developing chestnuts in burs (n=4 per isolate) were gathered from the field and surface-sterilized with 70% ethanol. Nuts (n=12 per isolate) inside burs were inoculated by injecting 50uL of inoculum (~1.0 x 106 conidia/mL) directly into each kernel with a hypodermic needle and sterile syringe. Burs were incubated at room temperature in a moist chamber for 14 days. One-year-old seedlings (n=6) grown in containers were used for twig inoculations. Twig nodes (one/seedling) were surface sterilized with 70% ethanol and inoculated by wounding the stem with a sterile probe and dropping 100uL of inoculum into each wound. Seedlings were incubated in a glass house for 60 days and monitored daily for symptom development. Nuts (n=9) and twigs (n=3) were inoculated with sterile water using the same inoculation and incubation conditions to serve as negative controls. Inoculated kernels developed characteristic brown/black lesions at the wound site and inoculated twigs developed brown/black, slightly sunken cankers with acervuli. No symptoms developed on the control kernels or twigs. Fungi with the same colony, conidial, and molecular characteristics as C. henanense were re-isolated from inoculated kernels and twigs. Blossom end rot caused by Glomerella cingulata has been reported on chestnut kernels in Georgia (Fowler and Berry 1958), but this is the first report of C. henanense causing rot on kernels and twigs in the US. Since 2018, C. henanense has been isolated from infected nuts received from commercial orchards in Pennsylvania, Missouri, and Alabama. We propose to retire the name "blossom end rot" for the symptoms found in kernels and replace it with the name "chestnut anthracnose" for this disease that affects both twigs and kernels of Chinese chestnut.