Colletotrichum species are commonly known as phytopathogenic fungi causing anthracnose disease and are also recorded as endophytes, epiphytes, or saprobes (Jayawardena et al., 2021). A total of 340 recognised Colletotrichum species, divided into 20 complexes and 9 singleton species, was recently updated by Talhinhas and Baroncelli (2023). The authors also summarized 3,400 recorded combinations of over 760 host plant species and the Colletotrichum species.
According to the DNA sequence analyses by Liu et al. (2022), the C. dematium species complex contains 18 species. Based on morphological characteristics, C. lineola (C. graminicola species complex) and many other accepted species with curved conidia were previously synonymised as C. dematium by von Arx (1957). This synonymy was followed by Sutton (1980), resulting in 22 recognized species in the genus Colletotrichum. However, a combination of morphological and molecular studies showed that curved conidia are uninformative for species identification of C. dematium and related species (Damm et al., 2009). The C. magnum species complex currently has nine accepted species (Guo et al., 2022; Liu et al., 2022, 2023). The ex-type culture CAUOS2 of C. liaoningense was originally introduced by Diao et al. (2017), but the species was later re-identified as a synonym of C. magnum (Liu et al., 2022). The C. orchidearum species complex includes 11 different species (Liu et al., 2022). Some species (C. cliviicola, C. orchidearum, C. plurivorum, and C. sojae) have been reported on a wide range of host plants (Damm et al., 2019; Liu et al., 2022). Damm et al. (2019) suggested that C. cliviicola, C. musicola, C. cattleyicola, and C. piperis seem to appear on specific plants, but C. cliviicola and C. musicola are recently reported on several different plant families (Boufleur et al., 2020; Dada et al., 2023; Liu et al., 2022; Machado et al., 2022; Vásquez-López et al., 2019; Wang et al., 2022). In some species in the C. orchidearum species complex, different strains within a species could only produce the sexual morph or predominantly form the asexual morph, this sometimes led to the misidentification of Colletotrichum species using morphological features (Damm et al., 2019). Prior to the availability of DNA sequence data, four species C. orbiculare, C. lindemuthianum, C. mavarum, and C. trifolii were considered as divergent forms of C. gloeosporioides because of the morphological similarity and particular host plants (von Arx, 1957). Later, these four species were regarded as the same taxon of C. orbiculare, based on analyses of the internal transcribed spacer 2 of the nuclear ribosomal RNA operon (ITS-2) and the large subunit of the nuclear ribosomal RNA gene (Sherriff et al., 1994). These species and four other species (C. bidentis, C. sidae, C. spinosum, and C. tebeestii) were well identified as eight different species in the C. orbiculare species complex using a multi-locus analysis, and they were restricted to herbaceous plants in the Asteraceae, Cucurbitaceae, Fabaceae, and Malvaceae families (Damm et al., 2013).
Due to the high variability of morphological characteristics and a wide host range of many Colletotrichum species, the multi-locus sequence analysis is nowadays considered as a robust method of species identification in the genus Colletotrichum (Damm et al., 2019; Liu et al., 2022; Talhinhas and Baroncelli, 2023; Weir et al., 2012). The ITS region is an effective maker to distinguish the species complexes, whereas combined DNA sequence analyses of ITS, glyceraldehyde-3-phosphate dehydrogenase (gapdh), chitin synthase 1 (chs-1), histone-3 (his3), actin (act), and beta-tubulin 2 (tub2) well resolved a large number of Colletotrichum species, including members in the C. dematium, C. magnum and C. orchidearum species complexes (Cannon et al., 2012; Damm et al., 2019; Liu et al., 2022). Damm et al. (2013) used a combination of seven loci, consisting of ITS, gapdh, chs-1, his3, act, tub2, and the glutamine synthase gene (gs), to effectively identify species in the C. orbiculare complex.
Colletotrichum species caused anthracnose disease of many economically important crops in Korea such as apple, grape, peach, pepper, strawberry and Japanese plum, and most of the reported species belong to the C. acutatum and C. gloeosporioides complexes (Hassan et al., 2019; Kim et al., 2020; Lee et al., 2020, 2021; Nam et al., 2022; Oo and Oh, 2017; Oo et al., 2017). Colletotrichum dematium in the C. dematium species complex, C. malvarum and C. orbiculare in the C. orbiculare species complex were reported in Korea, based on only morphological characteristics (Han et al., 2004; Kim et al., 2008, 2018b; Park and Kim, 1992; Shim et al., 2013). While C. jinshuiense (syn. C. kakiivorum) in the C. dematium species complex, C. cf. ovatense in the C. magnum species complex, C. plurivorum and C. sojae in the C. orchidearum species complex in Korea were identified using multiple DNA sequence analyses (Hassan et al., 2022; Lee and Jung, 2018).
Colletotrichum isolates preserved in the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Sciences have been collected from different plants and geographic locations in Korea since the 1990s. Most of them had been identified based on morphological characteristics and host plants by depositors. In our previous studies, 64 Colletotrichum isolates in KACC were assigned to six known species and one new species candidate in the C. acutatum species complex, based on multi-locus sequence analysis, of these 48 isolates changed their species names (Thao et al., 2023). While another 71 KACC isolates in the C. gloeosporioides species complex were identified into 12 accepted species and three new species candidates, the species names of 43 isolates in this complex changed (Thao et al., 2024). In the preliminary analysis of ITS sequences (unpublished data), 27 Colletotrichum isolates in KACC had been identified as C. dematium sensu lato (s.l.), C. magnum s.l., C. orchidearum s.l., and C. orbiculare s.l. Therefore, in this work, we aimed to: (1) re-identify 27 Colletotrichum isolates in KACC in the C. dematium, C. magnum, C. orchidearum and C. orbiculare species complexes using combined sequence analyses of ITS, gapdh, chs-1, his3, act, tub2, and gs; (2) investigate the host diversity of identified fungal species.
Materials and Methods
Fungal isolates
Cultures of Colletotrichum in KACC were retrieved on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) from liquid nitrogen preservation. Primary analysis based on ITS sequences resulted in 27 Colletotrichum isolates belonging to the C. dematium, C. magnum, C. orchidearum, and C. orbiculare species complexes. These isolates, with details of the host plant, location, collection year and deposited name in Table 1, were used for species identification based on the multi-locus DNA sequence analyses in this study.
DNA extraction, polymerase chain reaction amplification and sequencing
The genomic DNA of each fungal isolate was extracted from a 5-day-old culture on a PDA medium, using the DNeasy plant mini kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions.
The ITS region and six protein-encoding genes including gapdh, gs, chs-1, act, his3, and tub2 were amplified by the primer pairs ITS1/ITS4 (White et al., 1990), GDF1/GDR1, GSF1/GSR1 (Guerber et al., 2003), CHS-79F/CHS-345R, ACT-512F/ACT-783R (Carbone and Kohn, 1999), CYLH3F/CYLH3R (Crous et al., 2004), and T1/BT2b (Glass and Donaldson, 1995; O’Donnell and Cigelnik, 1997), respectively. The polymerase chain reaction (PCR) amplification was performed in an AllInOneCycler Thermal Block (Bioneer, Daejeon, Korea) in a total volume of 25 μl, consisting of 12.5 μl PCR Master Mix (2×), 8.5 μl nuclease-free water, 1 μl (4.5 pMol) of each primer, and 2 μl genomic DNA (100 ng/μl). PCR conditions of primer pair GSF1/GSR1 started with the initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The conditions for PCR of the other primer pairs were set up as described by Thao et al. (2023). The PCR products were visualized by gel electrophoresis and purified using the QIAquick PCR Purification Kit (Qiagen). The purified PCR products were sent to the Macrogen Company (Seoul, Korea) for sequencing with the amplification primers.
Phylogenetic analysis
The DNA sequences from forward and reverse primers were paired, and consensus sequences were calculated using MEGA 11 software (Tamura et al., 2021). Assemble sequences were deposited to RDA-GeneBank (http://genebank.rda.go.kr) with accession numbers in Table 1. Reference sequences of closely related fungal species and outgroups were obtained from NCBI’s GenBank by BLASTn searches (Supplementary Table 1). The dataset of each locus, including sequences in this study and reference sequences, was separately aligned using MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/) with the G-INS-1 option. The alignments were manually edited and concatenated afterwards in MEGA 11. Maximum likelihood (ML) phylogenetic trees of the C. dematium, C. magnum and C. orchidearum species complexes were generated using a concatenated alignment of six loci (ITS, gapdh, chs-1, his3, act, and tub2), while a ML phylogenetic tree of the C. orbiculare species complex was based on a concatenated alignment of seven loci (ITS, gapdh, chs-1, his3, act, tub2, and gs). Phylogenetic trees were inferred by IQ-TREE Web Server (http://iqtree.cibiv.univie.ac.at/) with 1,000 ultrafast bootstrap replicates. The phylogenetic trees were viewed in MEGA11 and depicted in Adobe Illustrator.
Results
Multi-locus phylogenetic analysis
The C. dematium species complex
A concatenated sequence alignment of six loci from five KACC isolates, 22 closely related reference isolates in the C. dematium species complex and an outgroup (C. destructivum isolate CBS 136228) in GenBank contained 2,191 characters including gaps (gen boundaries of act: 1-269; chs-1: 270-520; gapdh: 521-779; his3: 780-1,155; ITS: 1,156-1,678; and tub2: 1,679-2,191), of which 450 characters were parsimony-informative, 685 variable and 1,441 constant. The best-fit model TN+F+G4 was selected for ML phylogenetic analysis.
The multi-locus analysis divided five KACC isolates into three different clades. The isolate KACC 47859 was clustered with C. hemerocallidis (CDLG5, ex-type strain), supported by a ML bootstrap value of 100%. The KACC 47849 and KACC44634 isolates formed a clade with C. jinshuiense (PAFQ26, ex-type strain) and four isolates from NCBI (CGMCC 3.15172, BCTJB4, QSY1005-28.3, and KCTC 46679), representing a ML bootstrap value of 100%. The other isolates KACC 46709 and KACC 48110 were grouped with C. spinaciae (CBS 128.57, no available sequences from ex-type strain) with a ML bootstrap value of 100% (Fig. 1).
The C. magnum and C. orchidearum species complexes
The multi-locus alignment of six loci from 11 KACC isolates, 20 closely related reference isolates in the C. magnum and C. orchidearum species complexes and an outgroup (C. diversisporum isolate NN072578) in GenBank comprised 2,076 characters including gaps (gen boundaries of act: 1-239; chs-1: 240-487; gapdh: 488-697; his3: 698-1,074; ITS: 1,075-1,595; and tub2: 1,596-2,076), of which 262 characters were parsimony-informative, 474 variable and 1,591 constant. The best-fit model TN+F+I+G4 was selected for ML phylogenetic analysis.
The phylogenetic tree (Fig. 2) showed that 11 KACC isolates were separated into four different clades in the C. orchidearum species complex and two different clades in the C. magnum species complex. Three isolates (KACC 40895, KACC 49843, and KACC 40011) clustered with C. plurivorum (CBS 125474, ex-type strain), supported by a ML bootstrap value of 99%. The isolate KACC 46943 fell in a clade with C. reniforme (LC8230, ex-type strain), showing a ML bootstrap value of 100%. Four isolates (KACC 47826, KACC 48699, KACC 49845, and KACC 48237) were in a clade with C. sojae (ATCC 62257, ex-type strain), supported by a ML bootstrap value of 100%. The isolate KACC 40693 clustered with C. cattleyicola (CBS 170.49, ex-type strain), with a ML bootstrap value of 97%. The isolate KACC 49789 formed an individual clade. It was genetically distinct from all other taxa and was regarded as C. cf. ovatense. The isolate KACC 42457 clustered with C. kaifengense (CAASZK33, ex-type strain), supported by a ML bootstrap value of 99%.
The C. orbiculare species complex
The dataset of seven loci for the C. orbiculare species complex consisted of 11 KACC isolates, seven closely related reference isolates in the C. orbiculare species complex and an outgroup (C. magnum isolate CBS 519.97) in GenBank. A concatenated sequence alignment included 3,047 characters including gaps (gen boundaries of act: 1-238; chs-1: 239-489; gapdh: 490-707; gs: 708-1,648; his3: 1,649-2,024; ITS: 2,025-2,567; and tub2: 2,567-3,047), of which 149 characters were parsimony-informative, 604 variable and 2,393 constant. The best-fit model TIM2+F+G4 was applied for ML phylogenetic analysis.
The phylogenetic analysis resolved 11 KACC isolates into two different clades. The first clade was formed by seven KACC isolates (KACC 46934, KACC 46942, KACC 47122, KACC 46935, KACC 40903, KACC 40808, and KACC40809) and an ex-type strain (CBS 570.97) of C. orbiculare, with a ML bootstrap value of 96%. A combination of six loci (ITS, gapdh, chs-1, his3, act, and tub2) did not distinguish the species C. orbiculare from the related species C. sidae (data not shown), but these two species could be separated by the gs gene. The second clade was generated by four KACC isolates (KACC 44642, KACC 43025, KACC 47852, and KACC 47874) and an ex-type strain (CBS 521.97) of C. malvarum, with a ML bootstrap value of 97% (Fig. 3).
Host plants of fungal species in this study
A total of 19 host-fungus combinations were found in this study (Table 2). Ten of them are newly reported worldwide, including C. cattleyicola on Limonium sinuatum; C. jinshuiense on Hemerocallis fulva and Viola rossii; C. kaifengense on Cucumis melo; C. malvarum on Malva cavanillesiana; C. plurivorum on Perilla frutescens; C. reniforme on Ficus carica; C. sojae on Actinidia chinensis var. deliciosa, Althaea officinalis, and Camellia japonica. Colletotrichum hemerocallidis on Hemerocallis fulva and C. plurivorum on Capsicum annuum are new reports in Korea.
Discussion
The phylogenetic analyses showed that 27 KACC isolates belong to the C. dematium (five isolates, three known species), C. magnum (two isolates, two known species), C. orchidearum (nine isolates, four known species), and C. orbiculare (11 isolates, two known species) species complexes. Of 11 species, four (C. cattleyicola, C. hemerocallidis, C. kaifengense, and C. reniforme) are unrecorded species in Korea as far as we know.
Colletotrichum hemerocallidis was first identified as Colletotrichum sp. (CBS 125338) (Damm et al., 2009) on Hemerocallis fulva in Canada, the isolate CBS 125338 and the other isolates collected from Hemerocallis fulva and H. fulva var. kwanso in China were later re-identified by Yang et al. (2012) as new species namely C. hemerocallidis. This fungal species, as far as we know, has been found only on Hemerocallis spp. The species name C. jinshuiense was first published online on 24 July 2018 by Fu et al. (2019) causing anthracnose of pear (Pyrus pyrifolia) in China. Later, on 30 July 2018, C. kakiivorum was published online by Lee and Jung (2018) as a causal agent of anthracnose of persimmon (Diospyros kaki) in Korea. However, these two species shared high sequence similarity at act, chs-1, gapdh, his3, ITS, and tub2 (99.8%) and C. kakiivorum was regarded as a synonym of C. jinshuiense (Liu et al., 2022). The fungal isolate CGMCC 3.15172 was originally introduced as an endophytic species (Colletotrichum sp.) from Bletilla ochracea by Tao et al. (2013) in China. Fu et al. (2019) demonstrated that the isolate CGMCC 3.15172 was genetically distinct from C. jinshuiense, based on sequence analyses of four loci (ITS, gapdh, act, and tub2), and they are different in gapdh (94.98% similarity) and tub2 (98.12% similarity). However, we re-conducted BLASTn searches and the results indicated that gapdh, and tub2 sequences of ex-type strain PAFQ26 of C. jinshuiense have similarities of 96.4% and 99.39% with those of CGMCC 3.15172, respectively. In addition, ITS, gapdh, chs-1, act, and tub2 sequences of the closest isolate of CGMCC 3.15172 (KACC 44634) have similarities of 100%, 97.05%, 98.37%, 99.03%, and 99.39% those of C. jinshuiense (PAFQ26). These results and the phylogenetic tree (Fig. 1) suggest that CGMCC 3.15172, KACC 44634 (from Viola rossii), KACC 47849 (Hemerocallis fulva), BCTJB (from Paris sp.) and QSY1005-28.3 (from leaves of ginseng) are regarded as C. jinshuiense. This result indicates that there is a high genetic diversity within isolates of C. jinshuiense and the fungal species has a wide range of host plants. Colletotrichum spinaciae was reported with a broad host range including Beta vulgaris (Chikuo et al., 1984), Chenopodium album, Medicago sativa, Portulaca oleracea, Spinacia oleracea, Spinacia sp. (Damm et al., 2009), Vicia sativa (Wang et al., 2019), Astragalus membranaceus (Jin et al., 2021), and Chenopodium quinoa (Wei et al., 2023). The fungal species is an important pathogen of Spinacia oleracea worldwide (Correll et al., 1994; Uysal and Kurt, 2017). In Korea, Colletotrichum spinaciae was formerly reported on Spinacia oleracea, but without molecular confirmation (The Korean Society of Plant Pathology, 2022), and this was authentically confirmed by a DNA sequence analysis herein.
The species in the C. orchidearum complex have straight conidia similar to those in the C. gloeosporioides complex (Weir et al., 2012). Therefore, several KACC isolates in the C. orchidearum complex were originally identified as C. gloeosporioides or Glomerella cingulate based on morphological characteristics by depositors. In the C. orchidearum species complex, C. plurivorum has been reported from many different plant families, causing anthracnose of economically important crops such as Capsicum annuum and Glycine max (Boufleur et al., 2021; Damm et al., 2019; de Silva et al., 2019; Zaw et al., 2020). In the study of Hassan et al. (2022), C. plurivorum was found as one of the causative agents of anthracnose of Atractylodes ovata in Korea. In this study, the fungal species is newly reported on Capsicum annuum in Korea, but it is not a dominant species on the plant. The dominant species on Capsicum annuum is C. scovillei (Thao et al., 2023). Colletotrichum reniforme is a recently accepted species and was first described by Liu et al. (2022) on Smilax cocculoides (Smilacaceae family) and Paederia foetida (Rubiaceae family) in China. This species was herein isolated from a new host family Moraceae (Ficus carica), suggesting that the fungus would be associated with a broad range of plants. Colletotrichum sojae was previously reported on Arctium lappa, Bletilla ochracea, Capsicum sp., Glycine max, Medicago sativa, Phaseolus vulgaris, Vigna unguiculata, Panax quinquefolium, and Atractylodes ovata, with global distribution (Boufleur et al., 2021; Damm et al., 2019; Guan et al., 2021; Hassan et al., 2022). In the present study, this fungus was newly associated with anthracnose of kiwi (Actinidia chinensis var. deliciosa). While kiwi (Actinidia spp.) is also a host of C. nymphaeae, C. fioriniae and C. theobromicola in Korea (Kim et al., 2018a; Thao et al., 2023, 2024). This supports that a high diversity of Colletotrichum species is occurring on kiwi in the country. Colletotrichum cattleyicola has been reported from only the Orchidaceae family in the previous studies, it was originally identified as C. orchidearum and C. gloeosporioides on Cattleya sp. in Belgium and Japan, respectively (Damm et al., 2019; Sato et al., 2012), then reported on Cymbidium sp. in Thailand (Liu et al. 2022). The species was newly found in the second family Plumbaginaceae (Limonium sinuatum) in our study. The isolate KACC 49789 (ex-type strain) was previously named as a new species, C. ovataense, and the fungus is one of the causal agents of anthracnose of Atractylodes ovata in Korea (Hassan et al., 2022). However, to date, C. ovataense is an orthographical variant of C. ovatense (MycoBank: 842879, invalid). In this study, we re-confirmed the taxonomic position of the isolate KACC 49789 using a multi-locus analysis of six loci. The result showed that the isolate KACC 49789 was genetically distinct from all other taxa and it was treated as C. cf. ovatense.
Colletotrichum orbiculare (syn. C. lagenarium) has been known as a common pathogen of cucurbit plants in Korea and worldwide (Farr et al., 2021). In Korea, the fungus was only reported on the family Cucurbitaceae, including Citrullus lanatus, Cucumis melo, Cucurbita moschata, Cucumis sativus, and Lagenaria siceraria (syn. Lagenaria leucantha), but without molecular confirmation (Kim et al., 2018b; Lee, 1983; Shim et al., 2013; The Korean Society of Plant Pathology, 2022). In this study, C. orbiculare isolates from Citrullus lanatus, Cucumis melo, and Cucumis sativus were authentically identified based on DNA sequence data. However, one other isolate from Cucumis melo previously regarding as C. orbiculare by the depositor was re-identified as C. kaifengense herein. Colletotrichum kaifengense (a recently accepted species) and 14 other species (C. aenigma, C. citrulli, C. chlorophyti, C. fructicola, C. jiangxiense, C. karstii, C. magnum, C. nymphaeae, C. nigrum, C. orbiculare, C. plurivorum, C. qilinense, C. sojae, and C. truncatum) caused anthracnose disease of Citrullus lanatus in China (Guo et al., 2022). Of these, C. fructicola and C. nymphaeae are known as the prevalent species on apples and many other host plants in Korea (Thao et al., 2023, 2024). These suggest that may be not only C. orbiculare associated with the family Cucurbitaceae, but other Colletotrichum species could be occurring on cucurbit plants in Korea. To date, C. malvarum is restricted to the Malvaceae family and has a global distribution (Damm et al., 2013; Farr et al., 2021). Kim et al. (2008) demonstrated that C. malvarum caused 30% of anthracnose disease incidence of Malva verticillata in the 12 greenhouses investigated in Korea (Kim et al., 2008). Isolates in the present study identified as C. malvarum are not only from Malva verticillata, but also from a new host Malva cavanillesiana. This result also supports the host specificity of C. malvarum to the Malvaceae family.
This study revealed that 12 host-fungus combinations are first reported in Korea and 10 of them are newly reported worldwide. Of which, several new combinations are from economically important crops, including: C. kaifengense on Cucumis melo, C. sojae on Actinidia chinensis var. deliciosa (new reports worldwide), and C. plurivorum on Capsicum annuum (new report in Korea). These are important information for understanding disease epidemiology and biological control. Although known species identified in this study have been formerly published as phytopathogenic fungi, we did not investigate the host-fungus relationships via pathogenicity tests, hence the combinations found herein could be pathogenic, endophytic, epiphytic, or saprobic interactions.
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