Research Article |
Corresponding author: Xinlei Fan ( xinleifan@bjfu.edu.cn ) Academic editor: Sajeewa Maharachchikumbura
© 2024 Yixuan Li, Lu Lin, Jing Cao, Mingxu Gan, Xinlei Fan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Li Y, Lin L, Cao J, Gan M, Fan X (2024) Three new species of Colletotrichum (Glomerellales, Glomerellaceae) associated with walnut (Juglans regia) anthracnose from China. MycoKeys 108: 147-167. https://doi.org/10.3897/mycokeys.108.125382
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Colletotrichum species are significant pathogens of various economic plant hosts worldwide. In this study, 45 Colletotrichum isolates were obtained from symptomatic walnut leaves of walnut anthracnose in Shaanxi and Sichuan Provinces. In conjunction with morphological evidence and multi-gene phylogenetic analyses of internal transcribed spacer (ITS), actin (act), chitin synthase 1 (chs1), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and beta-tubulin (tub2) sequences support the introduction of three new species, namely Colletotrichum cordae, C. guangyuanense and C. juglandium. Five species of Colletotrichum were identified to be C. fioriniae of the C. acutatum species complex, C. karsti of the C. boninense species complex, C. gloeosporioides, C. mengyinense and C. siamense of the C. gloeosporioides species complex. The three new species are described and illustrated in this paper and compared with taxa in the Colletotrichum gloeosporioides species complex. The current results improve the understanding of Colletotrichum species causing walnut anthracnose in China.
Glomerellaceae, novel species, systematics, taxonomy
Walnut (Juglans regia L.) is an economically significant woody nut and edible oil tree cultivated globally. It is widely grown across various regions in America, Asia, and Europe (
Colletotrichum (Glomerellaceae, Glomerellales, Sordariomycetes) is one of the most important and destructive plant pathogens worldwide (
Research on walnut anthracnose in China’s primary walnut-producing regions has identified fourteen Colletotrichum species associated with the disease (
A total of 45 isolates were isolated from 25 walnut leaf samples with symptoms of anthracnose and collected in Shaanxi and Sichuan Provinces in China. The walnut anthracnose is characterized by small brown or black dry spots (Fig.
Conidial structure and size were imaged with Leica stereo microscope (M205) (Leica Microsystems, Wetzlar, Germany). Conidia and other microstructures were randomly selected and observed by using Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon digital sight DSRi-2 high-definition color camera with differential interference contrast (DIC). Fifty conidia were selected randomly to measure their lengths and widths. Colony morphology was observed on PDA and OA cultured at 25 °C. According to the color map of
Mycelium was collected from isolates grown on PDA agar and genomic DNA extraction was performed using the modified CTAB method (
Genes used in this study with PCR primers and optimal annealing temperature.
Locus | PCR Primers | PCR: Thermal Cycles: (Annealing Temp. in Bold) | Reference |
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ITS | ITS1/ITS4 | (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles |
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act | ACT-512F/ACT-783R | (95 °C: 45 s, 55 °C: 45 s, 72 °C: 1 min) × 35 cycles |
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chs1 | CHS-79F/CHS-345R | (95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles |
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gapdh | GDR1/GDF1 | (95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles |
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his3 | CYLH3F/CYLH3R | (95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles |
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tub2 | T1/Bt2b | (95 °C: 30 s, 55 °C: 30 s, 72 °C: 1 min) × 35 cycles |
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The resulting DNA sequences were combined with the sequences of reference strains from Genbank (Supplementary Suppl. material
Forty-five strains of Colletotrichum, isolated from leaves of Juglans regia L., were identified based on phylogenetic analyses of six loci. In the phylogenetic analysis of the C. acutatum species complex, a total of 2238 characters, including gaps, were identified (ITS: 549, act: 248, chs1: 282, gapdh: 267, his3: 390 and tub2: 502). Similarly, the phylogenetic analysis of the C. boninense species complex yielded a total of 2639 characters, including gaps (ITS: 592, act: 248, chs1: 300, gapdh: 321, his3: 410 and tub2: 768). An additional analysis of the C. gloeosporioides species complex resulted in 2294 characters, including gaps (ITS: 575, act: 323, chs1: 300, gapdh: 348 and tub2: 748). The GTR+I+G model was proposed for ITS, act and gapdh, and the HKY+I+G model was proposed for chs1, his3 and tub2 (
The phylogenetic tree showed 45 isolates across three species complexes: the C. acutatum species complex with a single isolate (Fig.
Phylogram of Colletotrichum acutatum complex species based on Maximum Likelihood (ML) analysis of the dataset of combined ITS, gapdh, act, tub2, chs1 and his3 genes. ML bootstrap support values above 60% and Bayesian posterior probability above 0.90 are shown near nodes. Ex-type cultures are in bold. Isolates obtained in this study are highlighted with blue colors.
Phylogram of Colletotrichum boninense complex species based on Maximum Likelihood (ML) analysis of the dataset of combined ITS, gapdh, act, tub2, chs1 and his3 genes. ML bootstrap support values above 60% and Bayesian posterior probability above 0.90 are shown near nodes. Ex-type cultures are in bold. Isolates obtained in this study are highlighted with blue colors.
Phylogram of Colletotrichum gloeosporioides complex species based on Maximum Likelihood (ML) analysis of the dataset of combined ITS, gapdh, act, tub2 and chs1 genes. ML bootstrap support values above 60% and Bayesian posterior probability above 0.90 are shown near nodes. Ex-type cultures are in bold. Isolates obtained in this study are highlighted with blue colors.
Named after Corda who established the genus Colletotrichum.
China, Sichuan Province, Guangyuan City, Chaotian District, Longmen Valley Leisure Villa, 32°39'08"N, 105°55'11"E, from leaf of Juglans regia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (holotype
Sexual morph not observed. Asexual morph developed on OA. Conidiomata acervular, color ranged from peach to light brown. Appressoria and Setae not observed on OA. Conidiophores directly formed on hyphae, usually degenerated into conidiogenous cells. Conidiophores hyaline, unbranched, approximately cylindrical, 16.1–28.2 × 2.5–4.5, mean ± SD = 21.0 ± 2.9 × 3.5 ± 0.5 µm, n = 50. Conidiogenous cells transparent, cylindrical, formed at the end or side of the hyphae. Conidia straight, hyaline, cylindrical, obtuse at the base, rounded at the apex, with smooth walls and granular contents, 11.8–17.7 × 4.5–6.5 µm, mean ± SD = 14.5 ± 1.1 × 5.5 ± 0.5 µm, L/W radio = 2.6, n = 50.
Colonies on OA initially white, rapidly growing to 4 cm after 3 d at 25 °C, and completely covering a 6-centimeter Petri dish after 7 d. The aerial mycelium white or gray, with a flocculent cotton-like appearance, edge white, center iron gray.
China. Sichuan Province, Guangyuan City, Chaotian District, Longmen Valley Leisure Villa, 32°39'08"N, 105°55'11"E, from leaf of Juglans regia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
Two strains of Colletotrichum cordae constitute a distinct clade within the C. gloeosporioides species complex, as revealed by multi-locus phylogenetic analysis. Colletotrichum cordae is phylogenetically near to C. perseae CBS 141365, but differs by 17 nucleotide differences in concatenated alignment (7/573 in ITS, 5/281 in act, 1/269 in chs1, 1/317 in gapdh, and 3/452 in tub2) (
China. Shaanxi Province, Shangluo City, Lonan County, Red kernel walnut base, 34°03'10"N, 110°14'11'′E, from leaf of Juglans regia L., 14 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
Colletotrichum fioriniae, a worldwide fungus with a wide range of host, is associated with walnut anthracnose disease (
China. • Sichuan Province, Guangyuan City, Chaotian District, Walnut Cultural Square, 32°40'58"N, 106°02'08"E, from leaf of Juglans regia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
Colletotrichum gloeosporioides was originally described as Vermicularia gloeosporioides and collected from Citrus sp. in Italy. The current name Colletotrichum gloeosporioides was proposed by
Named after the location where the fungal was first collected, which is Guangyuan City.
China, Sichuan Province, Guangyuan City, Lizhou District, Shuiwo Village, 32°23'55"N, 105°39'24"E, from leaf of Juglans regia L., 11 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (holotype
Sexual morph not observed. Asexual morph developed on OA. Conidiomata acervular, color ranged from peach to light brown. Appressoria and Setae not observed on OA. Conidiophores hyaline, unbranched, approximately cylindrical, 22.2–35.1 × 3.2–5.3, mean ± SD = 27.4 ± 3.1 × 3.8 ± 0.5 µm, n = 50. Conidiogenous cells transparent, cylindrical, formed at the end or side of the hyphae. Conidia straight, hyaline, cylindrical, obtuse at the base, rounded at the apex, with smooth walls and granular contents, 9.7–17.7 × 3.9–6.9 µm, mean ± SD = 14.7 ± 1.9 × 5.5 ± 0.6 µm, L/W radio = 2.7, n = 100.
Colonies on OA initially white, rapidly growing to 5 cm after 3 d at 25 °C, and completely covering a 6 cm Petri dish after 7 d. The aerial mycelium white or gray, with a flocculent cotton like appearance, edge white, center pale greenish grey.
China. • Sichuan Province, Guangyuan City, Lizhou District, Shuiwo Village, 32°23'31"N, 105°39'22"E, from leaf of Juglans regia L., 11 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
In phylogenetic analyses, Colletotrichum guangyuanense forms a distinct clade within the C. gloeosporioides species complex, closely related to C. changpingense. Genetic differences between C. guangyuanense and the type strain of C. changpingense are observed at several loci: 4 bp in the ITS region, 3 bp in the act gene, 8 bp in the chs1 gene, 6 bp in the gapdh gene, and 1 bp in the tub2 gene (
Named after the host genus on which it was collected, Juglans regia L.
China. Sichuan Province, Guangyuan City, Chaotian District, Mianguang Expressway, 32°40'50"N, 105°59'19"E, from leaf of Juglans regia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (holotype
Sexual morph not observed. Asexual morph on OA. Conidiomata acervular, color ranged from peach to light brown. Appressoria and Setae not observed on OA. Conidiophores hyaline, unbranched, approximately cylindrical, 16.0–27.6 × 2.2–4.7, mean ± SD = 20.1 ± 3.2 × 3.2 ± 0.6 µm, n = 30. Conidiogenous cells transparent, cylindrical, formed at the end or side of the hyphae. Conidia straight, hyaline, cylindrical, obtuse at the base, rounded at the apex, with smooth walls and granular contents, 13.2–22.4 × 4.4–6.3 µm, mean ± SD = 16.8 ± 1.8 × 5.4 ± 0.4 µm, L/W radio = 3.1, n = 50.
Colonies on OA initially white, rapidly growing to 5 cm after 3 d at 25 °C, and completely covering a Petri dish after 7 days. The aerial mycelium white or gray, with a flocculent cotton like, edge white, center mouse grey.
China. Sichuan Province, Guangyuan City, Chaotian District, Mianguang Expressway, 32°40'36"N, 105°59'26"E, from leaf of Juglans regia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
Colletotrichum juglandium form a solitary clade the C. gloeosporioides species complex (Fig.
China. Sichuan Province, Guangyuan City, Lizhou District, Shuiwo Village, 32°23'38"N, 105°39'22"E, from leaf of Juglans regia L., 11 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
Colletotrichum karsti was identified as a pathogen on Vanda species, causing ellipsoid lesions on leaves, and was also found as an endophyte in roots in Guizhou Province, China. This species is recognized as the most prevalent and geographically widespread within the C. boninense species complex, with a broad host range (
China. • Sichuan Province, Guangyuan City, Chaotian District, Walnut Cultural Square, 32°40'58"N, 106°02'08"E, from leaf of Juglans regia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
Colletotrichum mengyinense was isolated originally on diseased leaves of Rosa chinensis (
China. • Sichuan Province, Guangyuan City, Chaotian District, Zhongzi Town, 32°41'05"N, 106°02'08"E, from leaf of Juglans regia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (
Colletotrichum siamense was first described as a species in association with Coffea arabica by
The genus Colletotrichum comprises significant plant pathogens that impact many economically important crops globally. Despite notable advancements in the taxonomy of Colletotrichum, ongoing debates regarding its taxonomic relationships warrant further research (
The genus Colletotrichum exhibits considerable species diversity in infections of walnut hosts. In China, a variety of Colletotrichum species have been reported on walnut, including those from the C. acutatum species complex (e.g., C. acutatum, C. fioriniae, C. godetiae, C. juglandicola, C. juglandis, C. nymphaeae), the C. gloeosporioides species complex (e.g., C. aenigma, C. fructicola, C. gloeosporioides, C. kahawae, C. mengyinense, C. peakense, C. siamense, C. viniferum), the C. magnum species complex (e.g., C. liaoningense), and the C. orchidearum species complex (e.g., C. sojae). This list is based on numerous studies conducted over the years (
In the current study, five species (i.e., C. citrulli, C. dimorphum, C. juglandicola, C. nanhuaense and C. peakense) along with 17 isolates form part of the C. gloeosporioides clade in both Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic trees. However, these species and isolates were unable to form clear branches in the phylogenetic tree. They also have overlapping morphological characters (
The authors have declared that no competing interests exist.
No ethical statement was reported.
This research was funded by the National Key R & D Program of China (2023YFD1401302), National Science and Technology Fundamental Resources Investigation Program of China (2021FY100900).
Conceptualization: XF, YL. Formal analysis: YL, LL. Funding acquisition: XF. Investigation: XF, YL, LL, JC, MG. Methodology: YL. Collection: YL, XF, LL, JC, MG. Resources: YL, LL, XF. Software: YL, XF. Supervision: XF. Validation: LL. Visualization: YL. Writing - original draft: YL. Writing - review and editing: LL, XF.
Yixuan Li https://orcid.org/0009-0007-6862-9605
Lu Lin https://orcid.org/0000-0003-3456-3760
Xinlei Fan https://orcid.org/0000-0002-4946-4442
All of the data that support the findings of this study are available in the main text or Supplementary Information.
Strains of the Colletotrichum species with details of host, location and GenBank accessions of the sequences
Data type: docx
The statistics of ML trees in this study
Data type: docx