Gloeosporioides Species Complex Research Articles

Apple Bitter Rot and Glomerella Leaf Spot: A Comprehensive Review of Causal Species and Their Biology, Fungicide Sensitivities, and Management Strategies.

Bitter rot and Glomerella leaf spot (GLS) are two distinct diseases of apple fruit and foliage caused by members of the ascomycete fungal genus Colletotrichum. While GLS is restricted to subtropical and in some areas to temperate climates, bitter rot is responsible for significant yield loss worldwide, particularly during the post-harvest period. Initially thought to be caused by just two species of Colletotrichum, C. acutatum and C. gloeosporioides, advances in molecular biology and sequencing techniques enabled the identification of 25 different species capable of causing bitter rot and/or GLS of apple belongs to the C. gloeosporioides species complex (CGSC), C. acutatum species complex (CASC) and C. boninense species complex (CBSC). Three species (C. gloeosporioides, C. fructicola, and C. chrysophilum) of CGSC cause both bitter rot and GLS, 18 species (6 of CGSC and 12 of CASC) only cause bitter rot, and four species (C. aenigma and C. asianum of CGSC, C. limetticola of CASC and C. karstii of CBSC) only cause GLS. These species were found to differ in their geographical distribution, environmental and host tissue preference, pathogenicity, and fungicide sensitivities. In this review, we summarize the distribution, life cycle, and pathogenicity mechanisms of all currently known Colletotrichum species responsible for bitter rot and GLS of apple. Furthermore, we describe known apple defense mechanisms and management strategies for the control of these economically significant pathogens and identify gaps in our present understanding for future research.

First report of Colletotrichum queenslandicum associated with chilli anthracnose in Mauritius.

Globally, chilli (Capsicum annuum L.) is one of the most economically important and widely cultivated crop which elicits ethnomedicinal and nutritional potential as well as enhancing the taste and aroma of foods (Ayob et al., 2022; Kiran et al., 2020). Anthracnose disease is regarded as a prime constraint in chilli production, leading to enormous losses in tropical and subtropical countries. In September 2022, chilli fruit displaying sunken, shriveled and dark bown to black lesion with abundant acervuli on the surface was obtained from Flacq, Mauritius. From the symptomatic tissue, small pieces of the diseased tissue were excised, surface-disinfected using 1% sodium hypochlorite, twice rinsed using sterilized distilled water, air-dried and plated on PDA. After 7 days of incubation at room temperature, white to greyish white colony with dense white cottony aerial mycelium was recovered. Out of two isolates, CHF and CH10, the latter was considered for morphological and molecular characterization. The observed conidia (n=30) were unicellular, straight, cylindrical with rounded ends and slight constriction near the centre and had average length and width of 20.5 µm and 6 µm, respectively. For growth rate measurement of the isolate, two 5×5 mm of fungal agar plugs were taken from growing edge of colony, inoculated at centre of individual PDA plate and incubated at room temperature with a natural light/dark cycle. The diameter of the cultures were measured perpendicularly for a period of 7 days and the growth rate was calculated as 7-day average of daily growth (mm day-1). The growth rate of the fungal isolate (CH10) was 13.5 mm day-1 on PDA. Based on the morphological characters, the isolate was classified within the C. gloeosporioides species complex. For precise identification of the isolate, DNA was extracted from fungal mycelium using traditional DNA isolation methods (Ranghoo and Hyde, 2000), followed by PCR amplification and DNA sequencing using primer pairs ITS4/ITS5 (White et al., 1990), GDF/GDR and T1/Bt2b (Gan et al., 2016), respectively. ITS gene sequence (600 bp) confirmed that the isolate was Colletotrichum, with 99.83% similarity to KR704204 while GADPH (277 bp), TUB2 (733 bp) and ApMat (801 bp) gene sequences showed 99.64 to 100% similarity to C. queenslandicum with GenBank reference sequences, KT372374, KU221378 and MG674932 respectively. The gene sequences of isolate CH10 were deposited in GenBank database under the following accession numbers OR681557 (ITS), OR233734 (GADPH), OR475575 (TUB2) and PP622748 (ApMat). Koch's postulates were confirmed by spraying disease-free chilli plants with 10µL of conidial suspension (1 × 106 spores/ml) prepared from 7 days old colony of isolate CH10. Healthy chilli plants inoculated with sterile distilled water served as a negative control experiment. The plants were grown in pots in a moist chamber at 25˚C. After 5 days post-inoculation, anthracnose symptoms were developed on test plants while the control plant remained asymptomatic. The original isolate was successfully recovered from the test fruits, thus fulfilling Koch's postulates. The experiment was repeated thrice and revealed the same results. To the best of our knowledge, this is the first record of C. queenslandicum in Mauritius and is the first time to report anthracnose of chilli caused by this fungus. Colletotrichum queenslandicum has previously been reported in Europe, Mexico, US, Puerto Rico, Australia, Fiji, Brazil, Indonesia and China. Furthermore, the latter was associated with papaya, avocado, cashew, coffee, Persian lime, Licania tomentosa, white mangrove, lychee, mango, Nephelium lappaceum, olive, passionfruit, Dracaena cambodiana and Syzygium australe (Câmara and Vieira, 2022; Shidiq et al., 2024; Wang et al., 2022). This study will allow local farmers training and extension facilities to increase awareness among farmers about this disease-causing agent and allow them to take necessary measures for building up chilli crops resilience against this new and emerging pathogen in Mauritius.

Olive Anthracnose in Portugal Is Still Mostly Caused by Colletotrichum nymphaeae, but C. acutatum Is Spreading and C. alienum and C. cigarro Are Reported for the First Time

Olive anthracnose, caused by Colletotrichum fungi, is responsible for major fruit yield losses and poor olive oil quality worldwide. In the Mediterranean basin, some Colletotrichum spp. appear to be replacing others, possibly due to climate change and modification in cultural systems. To update the situation in Portugal, 525 olive groves were surveyed throughout the country over two years, revealing a decrease in disease incidence, associated with scarcer rainfall and new cropping systems using less susceptible cultivars. A collection of 212 isolates was obtained, and phylogenetic analyses using a multi-locus sequencing approach (five and six loci in the acutatum and gloeosporioides species complex, respectively) revealed the presence of seven Colletotrichum species within the collection. Compared to surveys conducted in the first decade of the 21st century, the species composition of olive anthracnose pathogens in Portugal remains mostly unchanged, with C. nymphaeae as the prevalent species, followed by C. godetiae, but with C. acutatum geographically expanding and with C. alienum and C. cigarro being reported for the first time as causal agents of olive anthracnose in Portugal. A close attention to pathogen population shifts, in the context of climate change and modification of cultivation systems, is fundamental for anticipating plant protection measures.

Characterization of Colletotrichum species obtained from citrus in northern Thailand and introducing a new host record for C. plurivorum

AbstractCitrus trees are affected by many plant‐pathogenic fungi. The present study aimed to characterize Colletotrichum species on citrus showing anthracnose symptoms, leaf and fruit spot and wither tip in northern Thailand using multilocus data and assay their pathogenicity. The surveys were carried out from December 2021 to January 2022 and 73 isolates were obtained from infected tissues. The fungal strains were identified based on morphological traits and were confirmed via amplification and sequencing of ITS, ACT, CHS‐1, GAPDH and TUB2. In all, 41 isolates were subjected to multilocus phylogeny. Forty of these isolates of Colletotrichum species belonged to the C. gloeosporioides species complex and one isolate belonged to the C. orchidearum species complex. Colletotrichum siamense, comprising 19 isolates, was the most dominant species associated with symptomatic citrus, followed by C. gloeosporioides (17 isolates), C. fructicola (three isolates), C. endophytica (one isolate) and C. plurivorum (one isolate). C. plurivorum is reported for the first time from citrus in the world. These isolates were obtained from mandarin (Citrus reticulata), pomelo (C. maxima), Persian lime (C. latifolia), Thai lime (C. hystrix) and acid lime (C. aurantifolia). The pathogenicity of the isolated fungi was tested through inoculation on branch, fruit and leaf using the detached method on mandarin, pomelo and Persian lime hosts, confirming Koch's postulate. In terms of disease severity, C. gloeosporioides and C. siamense were observed to be the most pathogenic species. This is the first pathogenic and molecular study of Colletotrichum species infecting citrus in northern Thailand.

Colletotrichum xishanense as a new etiological anthracnose agent on Fagopyrum gracilipes in Yunnan Province, China

Buckwheat (Fagopyrum spp.) is an important agricultural crop in some remote regions of China. In this study, two strains of Colletotrichum were isolated from brown and grey-white spots on the leaves of Fagopyrum gracilipes in Yunnan Province, China. Phylogenetic analyses inferred from combined ITS, act, chs-1, cal, gapdh, apmat, gs, and tub2 sequences revealed that these isolates are a new species, belonging to the C. gloeosporioides species complex and therefore we introduce as Colletotrichum xishanense. Pathogenicity tests indicated that C. xishanense infects not only herbaceous plant but also woody plant. Given the broader host range and stronger pathogenicity, it is time to focus on disease management and resistance breeding.

Genome comparisons reveal accessory genes crucial for the evolution of apple Glomerella leaf spot pathogenicity in Colletotrichum fungi.

Apple Glomerella leaf spot (GLS) is an emerging fungal disease caused by Colletotrichum fructicola and other Colletotrichum species. These species are polyphyletic and it is currently unknown how these pathogens convergently evolved to infect apple. We generated chromosome-level genome assemblies of a GLS-adapted isolate and a non-adapted isolate in C. fructicola using long-read sequencing. Additionally, we resequenced 17 C. fructicola and C. aenigma isolates varying in GLS pathogenicity using short-read sequencing. Genome comparisons revealed a conserved bipartite genome architecture involving minichromosomes (accessory chromosomes) shared by C.fructicola and other closely related species within the C. gloeosporioides species complex. Moreover, two repeat-rich genomic regions (1.61 Mb in total) were specifically conserved among GLS-pathogenic isolates in C. fructicola and C. aenigma. Single-gene deletion of 10 accessory genes within the GLS-specific regions of C. fructicola identified three that were essential for GLS pathogenicity. These genes encoded a putative non-ribosomal peptide synthetase, a flavin-binding monooxygenase and a small protein with unknown function. These results highlight the crucial role accessory genes play in the evolution of Colletotrichum pathogenicity and imply the significance of an unidentified secondary metabolite in GLS pathogenesis.

The pathogenic diversity and host range of Colletotrichum spp. causing pepper spot and anthracnose of lychee (Litchi chinensis) in Australia

AbstractLychee pepper spot, a field disease affecting lychee fruit skin, pedicels and petioles, is caused by Colletotrichum siamense, a fungal pathogen within the gloeosporioides species complex. Members of Colletotrichum from the gloeosporioides species complex and occasionally those from the acutatum species complex also cause postharvest anthracnose of lychee. Pepper spot was first described in Australia many years after anthracnose on lychee was first described, giving rise to the hypothesis that a novel species or strain within the gloeosporioides species complex causes pepper spot. In the present study, 19 isolates of Colletotrichum spp., collected from pepper spot and anthracnose symptoms on lychee fruit, representing 13 different genotypes across five species, were inoculated onto lychee fruit in the field or on detached fruit in the laboratory, to understand more about their pathogenic diversity. We found that symptoms were specific to genotype of the pathogen, as three genetically similar isolates of C. siamense consistently caused pepper spot and anthracnose, whilst other isolates caused anthracnose only. Cross‐inoculation studies on detached fruit of lychee, banana, avocado and mango also provided some evidence of host specialization in isolates of C. siamense infecting lychee in Australia. Our experiments provided further evidence that detached fruit assays cannot be used as a reliable proxy for field inoculation studies. This research confirms that C. siamense is a causal agent of both lychee pepper spot and lychee anthracnose in Australia, and Colletotrichum alienum and Colletotrichum queenslandicum are reported as causal agents of anthracnose of lychee for the first time.

First Report of Anthracnose Caused by Colletotrichum siamense on Machilus thunbergii Siebold & Zucc. in South Korea.

Machilus thunbergii Siebold & Zucc., known as Japanese bay tree, is an evergreen tree distributed widely in East Asia, including South Korea, where the species is of ecological importance. Machilus thunbergii provides habitat for wildlife species and is a common urban tree. In September 2022, anthracnose symptoms on leaves were observed in Jeju (33°26'02.4"N, 126°19'48.8"E) and Tongyeong (34°49'27.1"N, 128°24'01.8"E) in South Korea. Disease incidence on leaves of each affected tree, naturally growing in an urban forest area covering approximately 0.5 ha was approximately ~ 70 % in each study area. Anthracnose symptoms that were observed on 70 to 80% leaves per tree in each study area included orbicular or irregular, whitish-grey spots on leaves that were 1.5 to 3.0 cm in diam. In some cases where leaves were severely affected, larger blotches were formed, leading to bleaching symptoms and eventually defoliation. For pathogen isolation, two or three leaves showing anthracnose symptoms from each of the 15 trees were randomly selected and brought to the laboratory. Fungal isolations were then directly made by transferring spores from acervuli that developed on diseased leaves onto potato dextrose agar (PDA) media. Cushion shaped acervuli filled with salmon to orange-colored conidial masses were produced on media approximately two weeks after the incubation at 25 ± 1°C with a photoperiod of 12 h. Conidia were single celled, hyaline, cylindrical with rounded ends, smooth walls, 13.7 to 18.1 μm long and 3.1 to 4.5 μm wide (n=30). Among 15 cultures that were successfully isolated, 10 isolates were retained based on culture characteristics, and two randomly selected monoconidial cultures were deposited in the culture collection (CDH) of the Chungnam National University, Republic of Korea (Accession No. CDH057-58). Two isolates selected, CDH057 and CDH058, were subjected to identification, and this was achieved based on multiplesequence comparisons using on internal transcribed spacer regions of rDNA (ITS1 and ITS2), partial sequences of actin (ACT) and β-tubulin (TUB2) gene regions amplified using ITS1F / ITS4, ACT-512F / ACT-783R and T1 / Bt2b, respectively (Weir et al. 2012). The representative sequence data were deposited in GenBank under the accession numbers OR473277 and OR473278 for the ITS, OR480772 and OR480773 for ACT, and OR480774 and OR480775 for TUB2. The resulting sequences were further used for a phylogenetic analysis based on the maximum likelihood method using a concatenated dataset of the ITS, ACT and TUB2 gene sequences for Colletotrichum species in the C. gloeosporioides clade. The results showed that the pathogen isolated in this study clustered with Colletotrichum siamense (Vouchered specimens: MFLU 090230, COUFPI291, and COUFPI294) (Prihastuti et al. 2009). Sequence comparisons revealed that the isolates obtained in this study differed from the type species of C. siamense (MFLU 090230; FJ972613 for ITS, FJ 907423 for ACT, FJ907438 for TUB2) at 2 of 258 bp (∼0.8%) and 6 of 387 bp (∼1.6%) in the ACT and TUB2 sequences, respectively, while the ITS was identical to the type species. For pathogenicity tests, a total of ten three-year-old seedlings of M. thunbergii were used. The leaves of each tree were sprayed with 5 ml of conidial suspension (105 conidia/ml, isolate CDH057). Three control plants were sprayed with sterile water. After being sprayed, treated areas were sealed with a plastic bag for 24 hours to preserve humidity. Anthracnose symptoms, identical to those observed in the field, appeared five to seven days after the inoculations, while no symptoms were observed on control plants. The isolates used in the pathogenicity test were reisolated from 90% of lesions, and their identity was confirmed based on sequence comparisons, thus fulfilling Koch's postulates. Species of the C. gloeosporioides species complex include important plant pathogens, particularly C. siamense, which cause significant losses of economic and ecological relevance on a wide range of hosts (~ 100 hosts) (Talhinhas and Baroncelli 2021). Although C. fioriniae in the C. acutatum species complex, was found on M. thunbergii in South Korea (Thao et al. 2023), anthracnose associated with C. siamense on M. thunbergii has not been reported in the country. In this regard, this is the first report of anthracnose caused by C. siamense on M. thunbergii in South Korea. To effectively control the disease, more attention should be paid on the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

Identification and pathogenicity of Colletotrichum species associated with twig dieback of citrus in Western Australia

AbstractUp to 32 Colletotrichum species have been reported to be associated with pre‐ or postharvest diseases of citrus globally, while in Australia, six species have been reported to cause citrus leaf and fruit disease. Twig or shoot dieback has recently been observed as an emerging disease in citrus orchards in Western Australia. Colletotrichum species were isolated from diseased twigs showing dieback (withertip) or lesions, with or without gummosis, collected from 12 varieties of orange, mandarin and lemon. Colletotrichum gloeosporioides sensu stricto, Colletotrichum karstii and Colletotrichum novae‐zelandiae were identified using a polyphasic approach that included multigene phylogenetic analysis using sequences of internal transcribed spacer and intervening 5.8S nrDNA (ITS), glyceraldehyde‐3‐phosphate dehydrogenase (gapdh), β‐tubulin (tub2), actin (act) and histone (his3) for isolates in the boninense species complex, and Apn2–Mat1–2 intergenic spacer and partial mating type (Mat1–2) (ApMat) and glutamine synthetase (gs) for isolates in the gloeosporioides species complex, as well as morphological characteristics. C. gloeosporioides was the most prevalent species associated with twig dieback in Western Australia, while C. novae‐zelandiae was reported for the first time in Australia. Pathogenicity tests on shoot twigs from lemon and orange trees confirmed C. gloeosporioides, C. karstii and C. novae‐zelandiae as the cause of twig dieback, with C. gloeosporioides being the most aggressive species. Knowledge of the species causing twig dieback and their lifestyle will assist the development of integrated control methods.

First report of Colletotrichum queenslandicum and Colletotrichum endophyticum causing citrus anthracnose in Indonesia

Citrus (Citrus spp.), including orange as an important one, has a high economic value and significant role both in global market as well as in Indonesia. However, citrus anthracnose caused by Colletotrichum spp. constantly becomes a limiting factor in citrus production. Nevertheless, there is no report on molecular basis about the species associates to the citrus anthracnose in Indonesia up to date. Therefore, this research aims to identify the isolates of Colletotrichum spp. based on polyphasic approach which combining morphological character, pathogenicity and multigene analysis using actin, β-tubulin, and GAPDH primers. Twenty-two isolates were obtained from leaves and citrus fruits with anthracnose symptoms which showed various morphological characteristics, both macroscopic and microscopic. Pathogenicity test results showed that all 22 isolates were virulent to Keprok Batu 55 orange and California lemon. The anthracnose symptoms produced are identical to the symptoms in the field, however the symptoms on the California lemon were milder than on the Keprok Batu 55 mandarin. The results of multigene analysis showed that there were three species causing citrus anthracnose in Indonesia, which were C. endophytichum, C. queenslandicum, and C. gloeosporioides. Those three species are C. gloeosporioides species complex, and this is the first report of C. endophytichum, C. queenslandicum causing citrus anthracnose in Indonesia which needs further concern for biosecurity. All isolate sequences based on actin, β-tubulin, and GAPDH gene markers were submitted into the GenBank and got accession numbers of OR188855–OR188875.

First report of Colletotrichum siamense causing fruit anthracnose on Acer fabric in China.

Acer fabric Hance is an evergreen tree widely cultivated in China for its ornamental value (Liu et al. 2021). In August 2021, serious fruit anthracnose, with brown to black irregular sunken lesions, occurred on A. fabric plants at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E) in Nanchang, Jiangxi province, China. On average, 25% of the fruit per individual tree was affected. Small spots initially formed along the edge of the fruit and gradually expanded into dark brown spots, and eventually the diseased fruit withered. Small pieces (4 × 4 mm) from the affected fruits were surface sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Liao et al. 2023). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, AFG-3, AFG-7, and AFG-12, 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 under incubation at 25°C in the dark for 7 days. Conidia were single-celled, hyaline, cylindrical, clavate, and measured 12.6-17.3 × 4.2-5.6 µm (14.2 ± 0.9 × 4.5 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.5-9.2 × 4.5-6.7 µm (7.2 ± 0.2 × 5.4 ± 0.5 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 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, OQ184035 - OQ184037; ACT, OQ196109 - OQ196111; GAPDH, OQ196115 - OQ196117; TUB2, OQ196121 - OQ196123; CHS-1, OQ196112 - OQ196114; CAL, OQ196118 - OQ196120). A maximum likelihood and Bayesian posterior probability analysis using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed AFG-3, AFG-7, and AFG-12 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. Pathogenicity tests were conducted on six 9-year-old A. fabric plants. Healthy fruits were wounded with a sterile needle and inoculated by placing a drop on the surface of wounded tissue (106 conidia/mL; 20 µL) prepared from the three isolates. The conidial suspension of each isolate was inoculated onto three fruit per plant. Another nine fruit on three plants inoculated with sterile water served as the control (Wan et al. 2022). All the inoculated fruit were covered with plastic bags to keep the humidity for two days. All the inoculated fruit showed dark brown spots similar to those symptoms observed on naturally infected fruit on campus, whereas control fruit were asymptomatic. C. siamense was reisolated from the inoculated fruit. The pathogen was previously reported to cause fruit anthracnose on Carya illinoinensis (Zhuo et al. 2022), Chili Pepper (May et al. 2021), and Salix babylonica (Zhang et al. 2023). This study confirmed that C. siamense also causes anthracnose on fruit of A. fabric. This work contributes to a better understanding of the etiology of fruit anthracnose on A. fabric in south China and helps develop effective control strategies.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Peanut in Chongqing, China.

In July 2022, dieback and discoloration were detected on infected stems of peanut in Qijiang District of Chongqing (106.56°E,29.41°N), China, with an incidence up to 5%. These peanut stems had disease symptoms typical of anthracnose with irregular gray-brown spots with dark brown edges, sunken, and necrotic. High temperature and high humidity were favorable for the growth of the pathogen. To isolate the pathogen, we collected 10 typical infected peanuts and cut one piece from each of symptomatic stems, surface sterilized with 0.5% NaClO for 1 min, and 75% ethanol for 30 s, then rinsed three times with sterile distilled water and dried on sterilized filter paper. These pieces were incubated on potato dextrose agar (PDA) at 25°C in the dark. Pure cultures were obtained from hyphal tips of each colony. It was found that isolates with the same colony morphology were isolated from each infected stem. A representative isolate (L7) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. The colonies appeared white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, 13.4 to 18.8 × 4.2 to 5.8 μm (n=50). Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Cannon et al., 2012). For molecular identification, genomic DNA was extracted using a CTAB method and partial sequences of β-tubulin (TUB2), actin (ACT) genes, chitin synthase (CHS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced using primers T1/T2, ACT-512F/ACT-783R, CHS-79F/CHS-345R, and GDF1/GDR1, respectively (Damm et al., 2012; Dowling et al., 2020). Using the BLAST, TUB2, ACT, CHS and GAPDH gene sequences (GenBank accession No. OR714793, OP168707, OP168708 and OR714794, respectively) were100% (429 bp out of 429 bp), 99.22% (256 bp out of 258 bp), 99.64% (276 bp out of 277 bp) and 100% (253 bp out of 253 bp) identical to C. gloeosporioides CBS:112999 (JQ005587, JQ005500, JQ005326, and JQ005239), respectively. Using Neighbor-Joining algorithm, phylogenetic analysis was conducted based on the concatenated sequences of published TUB2, ACT, CHS and GAPDH genes. The identified isolate (L7) was closely related to C. gloeosporioides. To evaluate pathogenicity, the stems of ten peanut (Zhonghua12) seedlings (2 weeks) were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) or 20 μl of conidial suspension (105/ml) were inoculated. Non-colonized agar plugs or 20 μl of sterile distilled water were treated as control. After inoculation, the peanuts were kept in a moist chamber at 28°C with 80% humidity in the dark for 24 h, and subsequently transferred to the moist chamber with 12 h light and darkness cycle for 6 days, similar symptoms were observed on all inoculated peanuts. Controls remained asymptomatic. C. gloeosporioides was reisolated from the diseased stems and confirmed using morphological features and sequence analysis of TUB2, ACT, CHS and GAPDH. Anthracnose caused by C. truncatum and C. fructicola has been reported on peanut leaves in China (Gong et al., 2023; Yu et al., 2019). To our knowledge, this is the first report of anthracnose on peanut stem caused by C. gloeosporioides in Chongqing. Our report will provide crucial information for studying on epidemiology and management of this disease.

Re-identification of Colletotrichum gloeosporioides Species Complex Isolates in Korea and Their Host Plants.

The Colletotrichum gloeosporioides species complex includes many phytopathogenic species, causing anthracnose disease on a wide range of host plants and appearing to be globally distributed. Seventy-one Colletotrichum isolates in the complex from different plants and geographic regions in Korea were preserved in the Korean Agricultural Culture Collection (KACC). Most of them had been identified based on hosts and morphological features, this could lead to inaccurate species names. Therefore, the KACC isolates were re-identified using DNA sequence analyses of six loci, comprising internal transcribed spacer, gapdh, chs-1, his3, act, and tub2 in this study. Based on the combined phylogenetic analysis, KACC strains were assigned to 12 known species and three new species candidates. The detected species are C. siamense (n = 20), C. fructicola (n = 19), C. gloeosporioides (n = 9), C. aenigma (n = 5), C. camelliae (n = 3), C. temperatum (n = 3), C. musae (n = 2), C. theobromicola (n = 2), C. viniferum (n = 2), C. alatae (n = 1), C. jiangxiense (n = 1), and C. yulongense (n = 1). Of these, C. jiangxiense, C. temperatum, C. theobromicola and C. yulongense are unrecorded species in Korea. Host plant comparisons showed that 27 fungus-host associations are newly reported in the country. However, plant-fungus interactions need to be investigated by pathogenicity tests.

First report of Colletotrichum siamense causing leaf spot on Macropanax rosthornii in China.

Macropanax rosthornii (Harms) C. Y. Wu ex Hoo is an evergreen broadleaf species cultivated in subtropical China as an ornamental (Liang et al. 2015). In August 2020, leaf spot symptoms were observed on the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E), Jiangxi province, China. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became grayish brown with reddish egdes, eventually developing large irregular lesions. The disease incidence was estimated at 45%. Leaf pieces (5 × 5 mm) from the lesion borders were surface disinfested in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Li et al. 2023). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Three representative single-spore isolates (DS-2, DS-3, and DS-5) were used for morphological studies and phylogenetic analyses. Colonies on PDA of the three isolates were white-to-gray with cottony mycelia. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.3-18.1 ×4.3-6.9 µm (15.8 ± 1.1 × 5.3 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.4 × 4.5-6.9 µm (7.7 ± 0.3 × 5.5 ± 0.2 µm, n=100). Morphological features were similar to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, calmodulin (CAL), actin (ACT), β-tubulin 2 (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (CHS-1) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, CL1/CL2, ACT-512F/ACT-783R, T1/Bt2b, GDF/GDR and CHS-79F/CHS-354R (Weir et al. 2012), respectively. Sequences were deposited in GenBank under nos. OL895315 - OL895316 (ITS), OL830190 - OL830192 (ACT), OL830181 - OL830183 (GAPDH), OL830178 - OL830180 (TUB2), OL830184 - OL830186 (CHS-1), and OL830187 - OL830189 (CAL). 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 DS-2, DS-3, and DS-5 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. Pathogenicity of the three isolates was verified on six 5-year-old Macropanax rosthornii plants, which were grown in the field. Three healthy leaves per plant were wounded using a sterile needle (Φ=0.5 mm) and inoculated with a 20-μL conidial suspension per leaf (106 conidia/mL). Another six control plants were treated with sterile water. Eighteen leaves were used for the pathogenicity test of three isolates. All leaves were covered with plastic bags to maintain humidity for 2 days. The inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic after 8 days. The fungi were consistently reisolated only from the inoculated and symptomatic leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, including Liriodendron chinense × tulipifera (Zhu et al. 2019), Salix matsudana (Zhang et al. 2021), Carya illinoinensis (Zhuo et al. 2023). However, this is the first report of C. siamense infecting Macropanax rosthornii in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy).

Apple bitter rot is a globally widespread disease that is observed on fruits both pre-harvest and post-harvest, contributing to considerable economic losses. While the Colletotrichum acutatum species complex are predominant in Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021), in recent years, the Colletotrichum gloeosporioides species complex are emerging, raising many concerns (Amaral Carneiro et al. 2023). Circular, slightly sunken, brown lesions with acervuli produced in concentric spots were observed on 'Story® Inored' cultivar harvested in September 2022 from an organic orchard in Masi (Padova province, Italy), with a disease incidence close to 30%. From ten diseased apples, tissue samples were excised under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to potato dextrose agar medium and incubated in the dark at 25 °C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25 °C for 7 days appeared light gray-white on the upper side with floccose aerial mycelium, while the reverse side was dark gray with a distinct margin. Conidia were hyaline, cylindrical in shape with both ends rounded or one end acute and measured 16.6 ± 1.4 × 6.1 ± 0.5 μm [mean ± SD] (n=50) as described by Diao et al. 2017. To identify the species, genomic DNA of a representative isolate (C38) was extracted, beta-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), Apn2-Mat1-2 intergenic spacer (ApnMat) genes and the internal transcribed spacer (ITS) region, were amplified by PCR and Sanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtained DNA sequences of, TUB2, CAL, GAPDH, GS, ApnMat and ITS were submitted to GenBank under the accession numbers OR025589, OR025586, OR025587, OR025588, OR025585 and OR004800, respectively. A MegaBLAST analysis resulted 100 % identity to the epitype CAUG7 of Colletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159), for TUB2 (KP890171), 99.85% for CAL (KP890147) and 99,5 % for ITS (KP890165). The phylogenetic tree constructed by concatenation with the obtained sequences, as well as references, revealed that the C38 isolate clustered within C. grossum, confirming the BLAST approach. Pathogenicity tests were performed on 40 'Story® Inored' apples cleaned and wounded with a sterilized needle and exposed to two different conditions: 20 apples (10 inoculated with 20 μl of spore suspension (105 ml-1) and 10 with sterile water as control) were incubated at 20°C with a 12-hour photoperiod for 14 days, while the remaining 20 apples, prepared with same approach, were placed at 1°C for 3 months, then at room temperature for 14 days. Symptoms appeared after 6 days on apples incubated at 20°C, whereas those stored at 1°C displayed symptoms at 11 days after being placed at room temperature. In both conditions, lesions were similar to those observed on the original fruits; while the controls remained asymptomatic. Identity of reisolated fungal colonies was confirmed by CAL, GAPDH and GS region sequence analysis. C. grossum has been reported rarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 on Mangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoides in Italy (Diao et al. 2017; Manzano León et al. 2018; Guarnaccia et al. 2021). To the best of our knowledge, this is the first report of apple bitter rot caused by Colletotrichum grossum worldwide.

First Report of Colletotrichum chrysophilum Causing Anthracnose on Nance (Byrsonima crassifolia) in Mexico.

Nance fruit [Byrsonima crassifolia (L.) HBK] is a native crop widely distributed in Mexico and South America (Medina-Torres et al. 2018). It has been reported that nance is a good source of active compounds with anti-inflammatory, neuropharmacological and antioxidant effects. In 2022, the annual production of nance fruit in Mexico was of 7,713.13 tons and average yield of 5.64 t/ha with economic value of 51,952.66 million pesos (SIAP, 2022). This production generated significant economic income for the communities at a local, regional, and national level. In January 2023, irregular necrotic spots were observed on leaves and fruit of nance in an orchard of 50 nance trees located in San Sebastián Nopalera (16°54'52.73"N; 97°47'50.35"W), Oaxaca, Mexico. The incidence of the disease ranged from 50 to 60% of the trees. Infected fruit first showed dark-brown lesions with defined borders that coalesced to form large necrotic area. Isolates were purified by single spore isolation method (Choi et al. 1999).Colletotrichum strains were grown in PDA medium and five monoconidial isolates were obtained. A representative sample was selected (CNC-NP3) and deposited in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. Colony on PDA was white with sparse aerial mycelium, and the center was dark grey with abundant acervuli. Conidia (n = 100) were hyaline, aseptate, cylindrical with rounded apex, 13.5 to 15.2 × 4.3 to 5.1 μm. Appressoria (n = 20) were terminal or lateral, obovoid to clavate and some with slightly lobed, 9.9 to 11.6 × 5.3 to 6.6 μm. Based on the morphology, the isolate was identified as belonging to the Colletotrichum gloeosporioides species complex (Jayawardena et al. 2016). The representative isolate CNC-NP3 was identified by multilocus phylogenetic analysis using nucleotide sequences of internal transcribed spacer (ITS), actin (ACT), β-tubulin (TUB2), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (Jayawardena et al. 2016). The sequences were deposited in GenBank (accessions nos. OQ861102 (ITS), OQ870548 (ACT), OQ870549 (TUB2), OQ870550 (GAPDH). The phylogenetic analysis was carried out by Maximum likelihood method using concatenated sequences of ITS, ACT, TUB2 and GAPDH genes (Kozlov et al. 2019). The multilocus phylogenetic analysis revelated clearly the isolate CNC-NP3 as Colletotrichum chrysophilum. To confirm pathogenicity of CNC-NP3, 30 healthy fruits were inoculated. Fifteen disinfected nance with wounds and fifteen nance without wounds were inoculate with 10 µL of conidial suspension (1×105 spores/mL) from 7-day old culture. And controls were inoculated using sterile distilled water. Fruits were placed in a moist chamber covered with plastic bag at 25 °C for 48 h to maintain high humidity. After 4 days the inoculation sites development symptoms that were identical to those initially observed in the field, whereas the control group remained symptomless. The pathogenicity test was performed twice, with the same results. The pathogen was re-isolated from the lesion to fulfill Koch's postulates. Currently, Colletotrichum chrysophilum has been reported causing anthracnose disease in several crops: apple in New York (Khodadadi et al. 2020), papaya in Mexico (Pacheco- Esteva et al.2022), Blueberry (Soares et al. 2022) and banana in Brazil (Astolfi et al. 2022). To our knowledge, this is the first report of anthracnose in Byrsonima crassifolia caused by Colletotrichum chrysophilum in Oaxaca, Mexico.

Diversity and Characterization of Resistance to Pyraclostrobin in Colletotrichum spp. from Strawberry

Strawberry crown rot poses a significant menace to strawberry production during the seedling stage, and the main pathogen is Colletotrichum spp. Pyraclostrobin is one of the main fungicides that have been registered to control anthracnose caused by Colletotrichum spp. The diversity of pathogens and the risk of fungicide resistance may change from year to year. In order to explore the diversity of pathogens causing crown rot and evaluate the resistance risk of pathogens to pyraclostrobin in different years, crown rot samples were collected in Jiande, Zhejiang Province in 2019 and 2021, and the pathogens were identified. Based on the morphological identification and phylogenetic analysis based on ACT, CAL, CHS, GAPDH, and ITS, all 55 strains were identified as C. gloeosporioides species complexes, including 23 C. siamense isolates and 2 C. fructicola isolates in 2019, and all isolates were identified as C. siamense in 2021. C. siamense was the dominant pathogen of strawberry crown rot in 2019 and 2021. The resistance frequencies of the isolates collected in 2019 and 2021 to pyraclostrobin were 69.57% and 100%, respectively. In general, compared to that in 2019, the resistance frequencies of the pathogen to pyraclostrobin increased in 2021. In terms of fitness, there was no significant difference between resistant strains and sensitive strains in the mycelium growth rate, sporulation and spore germination rate. In addition, the resistant mutants exhibited positive cross-resistance to kresoxim-methyl and azoxystrobin. A sequential analysis of cytochrome b gene showed that C. siamense resistance to pyraclostrobin is linked to the G143A point mutation. Our study indicated that the risk of resistance a fungicide gradually increases with the increase in use years, and in order to reduce the emergence and spread of resistant populations, we should choose fungicides of different mechanisms of action for rotation to reduce the risk of resistance development.

Antifungal efficacy of biogenic waste derived colloidal/nanobiochar against Colletotrichum gloeosporioides species complex

Anthracnose caused by Colletotrichum spp. usually resulting in significant postharvest losses in the banana production chain. This study investigated the inhibitory effect of corn cob colloidal/nanobiochar (CCN) and Gliricidia sepium wood colloidal/nanobiochar (GCN) on the Colletotrichum gloeosporioides species complex. The CCN and GCN materials were synthesized and thoroughly characterized using various techniques, including UV–Vis and Fluorescence spectroscopy. Then after the fungal growth was examined on Potato Dextrose Agar (PDA) media supplemented with different CCN and GCN concentrations of 0.4 – 20 g/L and CCN and GCN with zeolite at various weight percentages of 10% to 50% w/w. Results from the characterization revealed that CCN exhibited a strong UV absorbance peak value of 0.630 at 203 nm, while GCN had a value of 0.305 at 204 nm. In terms of fluorescence emission, CCN displayed a strong peak intensity of 16,371 at 412 nm, whereas GCN exhibited a strong peak intensity of 32,691 at 411 nm. Both CCN and GCN, at concentrations ranging from 1 to 8 and 0.4 – 20 g/L, respectively, displayed notable reductions in mycelial densities and inhibited fungal growth compared to the control. Zeolite incorporation further enhanced the antifungal effect. To the best of our knowledge, this is the first study to demonstrate the promising potential of colloidal/nanobiochar in effectively controlling anthracnose disease. The synthesized CCN and GCN demonstrate promising antifungal potential against Colletotrichum gloeosporioides species complex, offering the potential for the development of novel and effective antifungal strategies for controlling anthracnose disease in Musa spp.

Characterization of Colletotrichum Species Infecting Litchi in Hainan, China.

Litchi (Litchi chinensis) is an evergreen fruit tree grown in subtropical and tropical countries. China accounts for 71.5% of the total litchi cultivated area in the world. Anthracnose disease caused by Colletotrichum species is one of the most important diseases of litchi in China. In this study, the causal pathogens of litchi anthracnose in Hainan, China, were determined using phylogenetic and morphological analyses. The results identified eight Colletotrichum species from four species complexes, including a proposed new species. These were C. karsti from the C. boninense species complex; C. gigasporum and the proposed new species C. danzhouense from the C. gigasporum species complex; C. arecicola, C. fructicola species complex; C. arecicola, C. fructicola and C. siamense from the C. gloeosporioides species complex; and C. musicola and C. plurivorum from the C. orchidearum species complex. Pathogenicity tests showed that all eight species could infect litchi leaves using a wound inoculation method, although the pathogenicity was different in different species. To the best of our knowledge, the present study is the first report that identifies C. arecicola, C. danzhouense, C. gigasporum and C. musicola as etiological agents of litchi anthracnose.

First report of Anthracnose caused by Colletotrichum theobromicola on Cyclamen persicum in South Carolina, USA.

Florist's cyclamen (Cyclamen persicum) is an herbaceous perennial native to the Mediterranean region and has become an increasingly popular plant around the world. Leaves of these plants are cordate-shaped with varying green and silver patterns. Flowers vary in color from white through different shades of pink, lavender, and red. In September 2022, symptoms of anthracnose including leaf spots and chlorosis, wilting, dieback, and crown and bulb rot were observed on 20 to 30% of approximately 1,000 cyclamen plants in an ornamental production nursery in Sumter County, SC. Tissue samples surrounding the necrotic crowns were excised and sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto acidified potato dextrose agar (APDA), and incubated at 25°C with 24-h photoperiod. A total of five Colletotrichum isolates, 22-0729-A, 22-0729-B, 22-0729-C, 22-0729-D, and 22-0729-E were obtained by transferring hyphal tips to new plates. The morphology of these five isolates was identical, observed as gray and black with aerial gray-white mycelia and orange-colored spore masses. Conidia (n=50) measured 19.4 ± 5.1 mm (11.7 to 27.1 mm) in length and 5.1 ± 0.8 mm (3.7 to 7.9 mm) in width. Conidia were tapered with rounded ends. Setae and irregular appressoria were infrequently observed in aged cultures (> 60-day-old). These morphological features resembled those of members of the Colletotrichum gloeosporioides species complex (Rojas et al. 2010; Weir et al. 2012). Sequence of the internal transcript spacer (ITS) region of a representative isolate 22-0729-E (GenBank accession No. OQ413075) is 99.8% (532 / 533 nt) and 100% (533 / 533 nt) identical to those of the ex-neotype of Co. theobromicola CBS124945 (JX010294) and the ex-epitype of Co. fragariae (= Co. theobromicola) CBS 142.31 (JX010286), respectively. Its glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequence is 99.6% (272 / 273 nt) identical to those of CBS124945 (JX010006) and CBS 142.31 (JX010024). Its actin (ACT) gene sequence shares 99.7% (281 / 282 nt) and 100% (282 / 282 nt) identities with those of CBS124945 (JX009444) and CBS 142.31 (JX009516), respectively. Lastly, its beta-tubulin 2 (TUB2) gene sequence is 99.6% (704 / 707 nt) and 100% (707 / 707 nt) identical to those of CBS124945 (JX010447) and CBS 142.31 (JX010373), respectively. The causal agent causing anthracnose on cyclamen in SC was identified as Co. theobromicola. To confirm the pathogenicity, cyclamen 'Verano Red' plants grown in 2.5-inch pots were used in two pathogenicity assays using different inoculation methods. In the first assay, three plants were inoculated by spraying a conidial suspension (1 × 106 conidia per ml; 30 ml per plant) of isolate 22-0729-E onto the foliage. Three non-inoculated control plants were sprayed with distilled water. All six plants were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 7 days to maintain humidity. Early symptoms including small spots, marginal necrosis, and chlorosis were observed on leaves and flowers 8 days after inoculation (DAI) and the entire aboveground tissues of inoculated plants were blighted 13 to 21 DAI. Non-inoculated plants remained asymptomatic. In the second assay, sterile toothpicks were used to slightly wound the crown and bulb surface of three plants and secure a mycelial APDA plug of isolate 22-0729-E (5×5 mm2) onto each wound (three wounds per plant). Three control plants were wounded in the same manner, while sterile APDA plugs were used in place of mycelial plugs. All six plants were maintained in the same manner as in the first assay. Apparent leaf yellowing and wilting symptoms appeared as early as 13 DAI. On 21 to 28 DAI, severe crown rot on inoculated plants caused the entire foliage to collapse. At least one third of the inner crown and bulb tissues of each inoculated plant were rotten, while those of non-inoculated plants appeared healthy. Each assay was repeated once. Colletotrichum isolates resembling morphological characters of 22-0729-E were recovered from leaves and inner crown tissues of all inoculated plants in both assays, respectively, but not from non-inoculated control plants. Anthracnose diseases on Cyclamen persicum caused by Co. theobromicola (syn. Co. fragariae) have been reported in NC, USA (Lui et al. 2011) and Israel (Sharma et al. 2016). This is the first report of anthracnose on cyclamen in SC, USA. Colletotrichum gloeosporioides (teleomorph Glomerella cingulate) species complex on cyclamen has also been reported in Argentina (Wright et al. 2006), South Africa, and several other U.S. states (Farr and Rossman 2022). However, it remains unknown whether these previous reports in fact attributed to Co. theobromicola due to lack of molecular identification (Weir et al. 2012). Colletotrichum theobromicola can cause diseases on at least 30 other agricultural and horticultural crops such as strawberry, cacao, and boxwood (Farr and Rossman 2022). It may pose a threat to cyclamen in greenhouse and nursery productions. Therefore, management strategies are warranted in the future.