The genus Colletotrichum is one of the most important phytopathogenic genera worldwide (Dean et al., 2012). Many Colletotrichum species also exhibit endophytic or saprobic associations with plants (Jayawardena et al., 2021). To date, a large number of species in the genus Colletotrichum have been reported with a wide range of hosts including crops and wild plants, while many others have a narrow host range or host specificity (Jayawardena et al., 2021; Talhinhas and Baroncelli, 2023). Many studies were conducted in the genus Colletotrichum, but the host range of Colletotrichum species is still poorly understood. For instance, C. higginsianum was primarily known to be specific to Brassicaceae as mentioned by Damm et al. (2014), this species was later reported from Polygonaceae (Rumex acetosa) (Zhang et al., 2018), Campanulaceae (Campanula sp.) (Khodaei et al., 2019) and Araliaceae (Panax ginseng) (Thao et al., 2024b). Of the relevant elements, accurate identification of Colletotrichum species is crucial for understanding fungal diversity and their host range, leading to effective disease management, especially in biological control. For example, it helps avoid the use of crops that are infected by the same Colletotrichum species in intercropping systems.
The nuclear ribosomal internal transcribed spacer (ITS) region has been widely used as a primary barcode marker for fungal species identification and it could separate major clades in the Colletotrichum genus, but is insufficient to resolve at the species level (Cannon et al., 2012). Recently, a multi-locus phylogenetic analysis of ITS, glyceraldehyde-3-phosphate dehydrogenase (gapdh), chitin synthase 1 (chs-1), histone-3 (his3), actin (act), and beta-tubulin 2 (tub2) was used to differentiate many species in the genus Colletotrichum (Liu et al., 2022; Zhang et al., 2023). Meanwhile, other makers such as calmodulin (cal) and glutamine synthase (gs) were additionally used for species delimitation in the C. boninense and the C. orbiculare complex, respectively (Damm et al., 2012, 2013). A dataset of ITS; the 5′ end of the DNA lyase gene (apn2); the 3′ end of apn2 and the 5′ end of the mating type gene Mat1-2 (Mat1/Apn2); and manganese-superoxide dismutase (sod2) was used for the C. caudatum and C. graminicola complexes (Crouch, 2014; Crouch et al., 2009).
In our previous studies (Thao et al., 2023, 2024a, 2024b, 2024c), 194 Colletotrichum isolates preserved in KACC were re-identified into 37 known species, one newly described species, and four new species candidates, based on multi-locus sequence analyses. There are 12 species and three new species candidates in the C. gloeosporioides species complex, six species and one new species candidate in the C. acutatum complex, eight species with the neotypification of C. panacicola and one new species (C. kummerowiae) in the C. destructivum complex, four species in the C. orchidearum complex, three species in the C. dematium complex, two species in the C. magnum complex, and two species in the C. orbiculare complex. Of these, 101 isolates were renamed from primary names, and 30 isolates (Colletotrichum sp.) were identified at the species level. Thirty-eight other isolates of Colletotrichum preserved in KACC were collected in Korea since the 1990s. They were originally identified based on host plants and morphological features, or only ITS region by depositors. This could lead to inaccurate or unreliable species names. Therefore, the aim of this study is to (1) re-identify the 38 isolates in KACC that belong to the C. boninense, C. spaethianum species complexes and related groups based on phylogenetic analyses of multiple loci; (2) re-arrange the host plants of identified fungal species in Korea.
Materials and Methods
Fungal isolates
Thirty-eight Colletotrichum isolates in KACC that originated from crops and wild plants in Korea were selected in this study based on the ITS sequence analysis. These isolates have been preserved in liquid nitrogen and were retrieved on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) for DNA extraction. Details of the fungal isolates (host plants, locations, collection years, and deposited names) are listed in Table 1.
DNA extraction, polymerase chain reaction amplification, and sequencing
Total genomic DNAs of fungal isolates were extracted from 5-day-old cultures grown on the PDA medium by the DNeasy plant mini kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions.
The ITS region, gapdh, chs-1, act, his3, tub2, and sod2 genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al., 1990), GDF1/GDR1 (Guerber et al., 2003), CHS-79F/CHS-345R, ACT-512F/ACT-783R (Carbone and Kohn, 1999), CYLH3F/CYLH3R (Crous et al., 2004), T1/Bt2b (Glass and Donaldson, 1995; O’Donnell and Cigelnik, 1997), and SOD625F/SOD625R (Crouch et al., 2006), respectively. The polymerase chain reaction (PCR) amplification was performed in a total volume of 25 μL, using an AllInOneCycler Thermal Block (Bioneer, Daejeon, Korea). The PCR mixture contained 8.5 μL nuclease-free water, 12.5 μL PCR Master Mix (2×), 1 μL (4.5 pMol) of each primer, and 2 μL genomic DNA (100 ng/μL). Conditions for amplification of sod2 were an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and final extension at 72°C for 10 min. PCR conditions of the other loci were set up as described by Thao et al. (2023). The PCR amplicons were visualized by gel electrophoresis, then purified and sequenced by the Macrogen Company (Seoul, Korea).
Phylogenetic analysis
The DNA sequences obtained from forward and reverse primers were paired and assembled in MEGA version 11 (Tamura et al., 2021). The sequences generated in our study were deposited to RDA-GeneBank (http://genebank.rda.go.kr) with accession numbers in Table 1. For each locus, the sequences in this study, related sequences and the outgroup from GenBank (Supplementary Table 1) were aligned using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html) with the G-INS-i option. The alignments were manually edited and then concatenated afterwards in MEGA version 11.
Maximum likelihood (ML) phylogenetic analyses of each locus and concatenated sequence datasets were implemented using IQ-TREE Web Server (http://iqtree.cibiv.univie.ac.at/) with 10,000 ultrafast bootstrap replicates. The phylogenetic trees were viewed in MEGA version 11. The Bayesian inference analyses were performed using BEAST (version 2.7.5) package (Bouckaert et al., 2019). The running parameters were 10,000,000 Markov chain Monte Carlo (MCMC) generations and sampling every 1,000 generation. The first 10% of MCMC generations were discarded as burn-in and the tree was visualized using FigTree v1.4.4.
Re-arrangement of host-fungus associations in Korea
Host-fungus associations in this study were confirmed whether they have been previously reported in Korea or elsewhere, and other hosts reported in Korea were investigated based on literature sources obtained from The List of Plant Diseases in Korea (6.2 Edition, 2024) (https://genebank.rda.go.kr/english/plntDissInfo.do), National List of Species of Korea, 2024 (https://kbr.go.kr/content/view.do?menuKey=799&contentKey=174), USDA Fungal Databases (https://fungi.ars.usda.gov/) and other public databases.
Results
Multi-locus phylogenetic analyses
The individual trees (not shown) did not show conflicts among clades at the ML bootstrap support value ≥ 70% and the Bayesian posterior probability value ≥ 0.9 which allowed the combination of the loci. The concatenated sequence alignment of six loci (ITS, gapdh, chs-1, his3, act, and tub2) included 38 isolates in this study, 78 closely related taxa and an outgroup (C. dematium isolate CBS 125.25) from GenBank. The concatenated alignment contained 2,364 characters, including gaps (gene boundaries of ITS: 1-562; gapdh: 563-897; chs-1: 898-1,148; his3: 1,149-1,534; act: 1,535-1,827; and tub2: 1,828-2,364), of which 991 characters were parsimony-informative, 1,150 variable and 1,144 constant. The topologies of the phylogenetic trees generated by ML and Bayesian analysis were congruent.
The six-locus tree (Fig. 1) showed that 38 KACC isolates were divided into 14 clades, including 12 known species (C. boninense, C. caudasporum, C. coccodes, C. karsti, C. liriopes, C. nigrum, C. sansevieriae, C. spaethianum, C. sublineola, C. sydowii, C. truncatum, and C. zhaoqingense) with high bootstrap support and Bayesian posterior probability values, a novel isolate KACC 42402, and an unidentified isolate KACC 46949. The isolate KACC 46949 was grouped in a clade with C. echinochloae and C. jacksonii in the six-locus tree and was finally identified as C. echinochloae based on an additional locus (sod2) (Supplementary Fig. 1). The isolate KACC 42402 was significantly distinct from all known taxa and was considered as a new species candidate. It was closely related to C. spaethianum CBS 167.49, C. lilii CBS 109214, C. guizhouense CGMCC 3.15112, and C. bicoloratum NN055229, but genetically distinct from these species at ITS (98.12%, 97.69%, 97.84%, and 97.89%, respectively), gapdh (90.08%, 91.67%, 89.6%, and 86.6%), chs-1 (97.21%, 96.81%, 96.81%, and 95.89%), his3 (93.60%, 93.83%, 93.82%, and 91.74%), act (97.90%, 98.32%, 95.67%, and 93.15%), and tub2 (94.32%, 93.31%, 93.12%, and 90.55%).
Host-fungus associations in this study
A total of 29 host-fungus combinations were found in this study. Of these, 13 combinations (C. caudasporum on Imperata cylindrica; C. karsti on Actinidia chinensis, Idesia polycarpa, Lilium lancifolium, Mammillaria sp., Pinus densiflora, Pinus strobus; C. spaethianum on Disporum smilacinum, Hosta capitata, Lilium longiflorum, Muscari armeniacum; C. sydowii on Boehmeria japonica; C. zhaoqingense on Pueraria montana) are first recorded in the world, and three combinations (C. echinochloae on Echinochloa crus-galli, C. karsti on Passiflora edulis, C. truncatum on Vigna angularis) were previously reported worldwide, but represent new records in Korea (Table 2).
Discussion
A total of 38 isolates in this research were re-identified as 13 known species and one new species candidate, belonging to the C. boninense, C. spaethianum species complexes and related taxa based on multi-locus sequence analyses. The species names of 14 isolates, originally assigned by depositors, were changed, and 10 isolates (deposited as Colletotrichum sp.) were identified at the species level. The results revealed three previously unrecorded species in Korea, including C. caudasporum, C. echinochloae, and C. zhaoqingense. This study, along with our earlier studies (Thao et al., 2023, 2024a, 2024b, 2024c) reclassified a comprehensive dataset of 232 Colletotrichum isolates collected from various host plants and geographic regions in Korea since the 1990s into 51 species and five new species candidates. Previously, Colletotrichum species reported more than two decades ago in Korea were identified based only on host species and morphological features (The Korean Society of Plant Pathology, 2024). Therefore, data from this study contributes to a broader understanding of the taxonomic status and host range of Colletotrichum species in Korea.
Colletotrichum boninense s. str. was first described from Crinum asiaticum var. sinicum and other plants (Cattleya sp., Clivia miniata, Cucumis melo, Cymbidium sp., Dendrobium kingianum, Passiflora edulis, and Prunus mume) in Japan by Moriwaki et al. (2003). This species was later recorded as a pathogen or endophyte with a wide range of hosts worldwide (Damm et al., 2012; Liu et al., 2022). In Korea, C. boninense s. str. was reported as an anthracnose disease on spindle tree (Euonymus japonicus) based on its morphological characteristics and ITS sequence by Lee et al. (2005). This combination was re-confirmed in this study using multi-locus sequence analysis.
Colletotrichum caudasporum (syn. C. caudisporum) was originally introduced as an endophyte of Bletilla ochracea (Orchidaceae) in China (Tao et al., 2013), and then reported as a pathogen in the Poaceae family (unknown species) in this country (Liu et al., 2022). In our study, C. caudasporum was newly found on Imperata cylindrica (Poaceae). However, the host range of this fungal species is, to date, poorly understood.
Colletotrichum coccodes and C. nigrum have been reported from many different plant species and frequently from the Solanaceae family (Farr and Rossman, 2024). C. coccodes is morphologically indistinguishable from C. nigrum and these two species are genetically closely related (Liu et al., 2011, 2013). C. coccodes was reported as a causal agent of diseases on Capsicum annunm, Rubus coreanus, Solanum lycopersicum, S. melongena and S. tuberosum, and C. nigrum was from Capsicum annunm in Korea, but these two species were not confirmed using molecular data (Kim, 1998; Kim and Cho, 1997; Kim et al., 1998, 2012; Park and Kim, 1992; The Korean Society of Plant Pathology, 2024). Here, C. coccodes on Solanum lycopersicum, S. melongena and S. tuberosum, and C. nigrum on Capsicum annuum were well distinguished based on a combined analysis of 6 loci.
Colletotrichum echinochloae was originally described as a pathogen on E. crus-galli subsp. utilis (syn. Echinochloa utilis) in Japan (Moriwaki and Tsukiboshi, 2009). Later, this fungal species was found on E. crus-galli in China and was considered as a potential bioherbicide agent to control this grass (Gu et al., 2023). C. echinochloae from E. crus-galli identified here is a new record in Korea. To date, this fungal species is restricted to the host genus Echinochloa.
Colletotrichum karsti has been known as an endophyte or a pathogen with a wide host range and global distribution (Farr and Rossman, 2024; Liu et al., 2022). There were high variabilities in morphology and DNA sequences in the population of C. karsti (Damm et al., 2012). C. karsti was first reported in Korea in 2024, causing anthracnose on Juglans mandshurica (Cho et al., 2024). The present study revealed that C. karsti appeared on six unrecorded host plants (worldwide) and one new host plant in Korea, including economically important crops such as kiwi (Actinidia chinensis) and passion fruit plant (Passiflora edulis). However, the pathogenicity of the fungal species was not confirmed. This species could become an important fungus in Korea.
Colletotrichum liriopes was introduced from Liriope muscari, and then recorded from many different host species (Damm et al., 2009; Farr and Rossman, 2024). In Korea, C. liriopes was previously identified on Rohdea japonica and Liriope muscari using ITS sequence analysis and a combination of act, gapdh, and ITS (Kwon and Kim, 2013; Oo and Oh, 2017). This fungus was genetically closely related to a recently introduced species, C. iris (Liu et al., 2022). The isolate on Liriope muscari was here identified as C. liriopes and differentiated from C. iris based on a six-locus phylogenetic analysis.
Colletotrichum sansevieriae was only reported from the host genus Dracaena (syn. Sansevieria) in many countries, including Korea (Farr and Rossman, 2024). The isolate KACC 46835 from Dracaena trifasciata was previously identified as C. sansevieriae based on ITS sequence and morphological characteristics (Park et al., 2013). This isolate was re-confirmed herein using a combined analysis of ITS, chs-1, his3, act, and tub2.
Colletotrichum spaethianum s. str. has been frequently reported from the host genus Hosta (syn. Funkia), Hymenocallis and Lilium (Farr and Rossman, 2024). In Korea, C. spaethianum was earlier found on Lilium sp. (Damm et al., 2009), Hosta plantaginea (Cheon and Jeon, 2016), Convallaria keiskei (Ahn et al., 2017), Hosta longipes (Choi et al., 2024a), and Polygonatum odoratum var. pluriflorum (Choi et al., 2024b). In KACC, C. spaethianum was re-identified from Hosta longipes, H. plantaginea, and four new host plants, including Disporum smilacinum, Hosta capitata, Lilium longiflorum, and Muscari armeniacum. This indicates that C. spaethianum is a common Colletotrichum species occurring in Korea, especially in the Asparagaceae family.
Colletotrichum sublineola is known as one of the most important pathogens of sorghum (Sorghum bicolor) in many countries in the world (Abreha et al., 2021; Farr and Rossman, 2024). The fungus infected approximately 20% of sorghum leaves observed in two fields in Korea in 2014 (Choi et al., 2021). The isolate identified as C. sublineola in this work is from Poaceae (unknown species) and it was deposited in KACC in 1997 (unknown collection date). The occurrence of this pathogen on Sorghum bicolor as well as in the Poaceae family in Korea should be investigated for early control of the pathogen.
Colletotrichum sydowii was reported from Sambucus sp. in Taiwan (Marin-Felix et al., 2017), Saraca dives and monocotyledon plant in China (Liu et al., 2022), and leaf of Albizia julibrissin in Korea (Cha et al., 2023). C. sydowii was significantly distinct from all known species in the phylogeny and did not belong to any Colletotrichum species complexes. In this study, the fungus is, as far as we know, the first report on Boehmeria japonica (eudicot plant).
Colletotrichum truncatum s. str. was an important pathogen causing anthracnose of numerous plant species with a wide distribution (Damm et al., 2009; Jayawardena et al., 2016). This fungus was reported as a pathogen on important crops in Korea, including Capsicum annuum, Carica papaya, Glycine max, Raphanus raphanistrum subsp. sativus (syn. Raphanus sativus), and Vigna radiata (Aktaruzzaman et al., 2018; Choi et al., 2019; Han and Lee, 1995; Kim et al., 2002; Oo and Oh, 2020). Of which, C. truncatum was previously identified from Capsicum max and Vigna radiata based on only morphological characteristics. The isolates of C. truncatum from Glycine max and a new host Vigna angularis in Korea were re-identified herein using a sequence analysis of 6 loci.
Colletotrichum zhaoqingense was first described by Liu et al. (2022) on palm (Arecaceae), Carica papaya and Musa sp. in China, then recorded on Camellia spp. and Cinnamomum camphora in the country (Sui et al., 2024). The fungal species was newly found in Korea on a common weed Pueraria montana. This data supports that C. zhaoqingense could have a wide range of hosts and the impact of this fungus is still poorly understood.
The isolate KACC 42402 isolated from Vicia venosa was considered as a new species candidate based on molecular data. This taxon is genetically closely related to C. guizhouense, C. lilii, C. spaethianum, and C. bicoloratum. To confirm the taxonomic delimitation, more isolates of this taxon need to be collected.
Multi-locus sequence analyses of fungal species conducted in this study confirmed some previously reported confusing combinations, where fungal species were identified using only morphological characteristics such as C. boninense on Euonymus japonicus; C. coccodes on Solanum lycopersicum, S. melongena and S. tuberosum; C. nigrum on Capsicum annuum; C. truncatum on Capsicum annuum, Glycine max, and Vigna angularis (Kim, 1998; Kim and Cho, 1997; Kim et al., 1998, 2002; Lee et al., 2005; Oo and Oh, 2020; Sharma and Kaushal, 1999; The Korean Society of Plant Pathology, 2024). The pathogenicity of most fungal isolates in the current study was not confirmed, except for KACC 46835 (C. sansevieriae), which was earlier tested as a pathogen of Dracaena trifasciata (Park et al., 2013). They could be pathogens, endophytes or saprobes. However, all accepted fungal species in Table 2 were previously known as plant pathogens, including C. caudasporum, C. sydowii, and C. zhaoqingense (Liu et al., 2022). Some of them were also known as endophytes (C. boninense, C. caudasporum, C. liriopes, and C. sydowii) and saprobes (C. liriopes and C. zhaoqingense) (Jayawardena et al., 2021; Liu et al., 2022; Yang et al., 2011). Endophytic and saprobic lifecycles could play important roles in the ecological adaptation of phytopathogenic fungi to different host plants (Jayawardena et al., 2021; Peres et al., 2005). Some species could switch their lifestyle from endophytic to pathogenic within the same host plants (Photita et al., 2001, 2004). Therefore, the identification of Colletotrichum species associated with their host plants is important in biodiversity research and agricultural farming practices, especially in the biological control of anthracnose pathogens. For instance, understanding the diversity of fungal species on a host plant is crucial for resistance breeding programs, and the host range of fungal species will help optimize crop arrangements in intercropping systems to reduce disease infection among crops.
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