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. 2024 Sep 3;108:147–167. doi: 10.3897/mycokeys.108.125382

Three new species of Colletotrichum (Glomerellales, Glomerellaceae) associated with walnut (Juglansregia) anthracnose from China

Yixuan Li 1, Lu Lin 1, Jing Cao 2, Mingxu Gan 3, Xinlei Fan 1,✉
PMCID: PMC11387834  PMID: 39262404

Abstract

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 Colletotrichumcordae, 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 Colletotrichumgloeosporioides species complex. The current results improve the understanding of Colletotrichum species causing walnut anthracnose in China.

Key words: Glomerellaceae, novel species, systematics, taxonomy

Introduction

Walnut (Juglansregia 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 (Liu et al. 2021). According to the FAO statistics (http://www.fao.org/faostat, accessed on 20 March 2024), China is recognized as the world’s largest walnut producer, with over 390,000 hectares dedicated to walnut cultivation (Liu et al. 2021). Since 2017, China has consistently maintained its leading position in global walnut production (Da Lio et al. 2018; Li et al. 2023). Walnut plantations offer substantial economic, social, and ecological benefits (Nie et al. 2016). However, anthracnose induced by Colletotrichum species remains a major hurdle in walnut production worldwide, causing significant losses in productivity, including total crop failures (Wang et al. 2016). For example, Colletotrichumaenigma, C.fructicola, C.gloeosporioides, C.liaoningense, C.siamense and C.sojae were reported to cause anthracnose of walnut in Beijing Province (Li et al. 2023).

Colletotrichum (Glomerellaceae, Glomerellales, Sordariomycetes) is one of the most important and destructive plant pathogens worldwide (Dean et al. 2012). Traditionally, identifying Colletotrichum species based solely on morphological characteristics and host ranges has been challenging (Walker 1980). Consequently, systematic studies of Colletotrichum species complexes have underscored the importance of a multiphasic approach, integrating locus phylogeny with morphological, geographical, and ecological data to accurately characterize and identify Colletotrichum species (Cai et al. 2009; Damm et al. 2009, 2012a, b; Rojas et al. 2010; Liu et al. 2011; Weir et al. 2012; Marin-Felix et al. 2017; Jayawardena et al. 2020). The current taxonomy of the genus encompasses over 300 species, organized into 16 species complexes, with additional singletons (Marin-Felix et al. 2017; Liu et al. 2022; Talhinhas and Baroncelli 2023; Zapata et al. 2024).

Research on walnut anthracnose in China’s primary walnut-producing regions has identified fourteen Colletotrichum species associated with the disease (Zhang et al. 2023a). Colletotrichumfructicola, C.gloeosporioides, C.siamense and C.viniferum have been reported to be associated with walnut anthracnose in Shandong Province (Zhu et al. 2014; Wang et al. 2017, 2018; He et al. 2019). Moreover, C.aenigma has been implicated in Hebei Province, C.fioriniae in Guangxi Province, and C.nymphaeae in Gansu Province as pathogens of walnut anthracnose (Zhu et al. 2015; Wang et al. 2021; Ma et al. 2022). In Hubei Province, additional species including C.fioriniae, C.gloeosporioides, C.godetiae, C.juglandis, C.kahawae, and C.nymphaeae have been reported in association with the disease (Wei et al. 2022). Colletotrichumgodetiae has been identified as a cause of severe anthracnose in walnuts in Shaanxi and Yunnan Provinces. Additionally, in Beijing, a range of species including C.aenigma, C.fructicola, C.gloeosporioides, C.juglandicola, C.liaoningense, C.peakense, C.siamense, and C.sojae have been reported (Li et al. 2023; Wang et al. 2023; Zhang et al. 2023a). Based on previous studies of walnut anthracnose, it is generally accepted that C.gloeosporioides is the main pathogen of walnut anthracnose in China (Qu et al. 2011; Wang et al. 2016). Furthermore, recent studies have revealed a diverse array of Colletotrichum species associated with walnut anthracnose, such as C.acutatum, C.aenigma, C.fioriniae, C.fructicola, and C.siamense, among others, as noted by Li et al. (2023). Therefore, the exploration of pathogen diversity within walnut anthracnose continues to be an essential field of study. In this study we investigated the phylogenetic diversity of Colletotrichum species associated with walnut anthracnose in Shaanxi and Sichuan Provinces. We aimed to classify the isolates from this study based on phylogenetic analyses and morphological characteristics.

Materials and methods

Sample Collection and fungal isolation

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. 1). About 25 mm2 tissue fragments were taken from the margin of tissue lesions and the surfaces were sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite for 60 s, rinsed in sterile distilled water for 60 s, and the samples were dried on aseptic filter paper (Gao et al. 2013; Liu et al. 2015). The sterilized sample was then placed in potato dextrose agar (PDA, 200 g potato, 20 g glucose, 20 g agar and 1 L distilled water) and cultured at 25 °C until mycelium grew from the sample. Then hyphae were picked out of the periphery of the colonies and inoculated onto Oatmeal Agar (OA, 30 g oatmeal, 15 g agar and 1 L distilled water) medium to promote the formation of spores. These leaf specimens are kept at the Museum of Beijing Forestry University (BJFC). The cultures are deposited in the China Forestry Culture Collection Centre (CFCC; http://www.cfcc-caf.org.cn/).

Figure 1.

Figure 1.

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Disease symptoms on Juglansregia L. caused by Colletotrichum species (A–F). Red arrows point to symptoms of branches dieback caused by walnut anthracnose.

Morphological analyses

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 Rayner (1970) the color of the colony was described. The colony diameter was measured after 5 and 14 days.

DNA extraction, PCR amplification and sequencing

Mycelium was collected from isolates grown on PDA agar and genomic DNA extraction was performed using the modified CTAB method (Doyle and Doyle 1990). First, the internal transcribed spacer (ITS) of all isolates was sequenced. The other genes were obtained from five nuclear gene regions: the glyceraldehyde-3-phosphate dehydrogenase gene (gapdh), chitin synthase 1 gene (chs1), actin gene (act), beta-tubulin gene (tub2) and histone H3 gene (his3) by using the primer pairs GDF1/GDR1, CHS-79F/CHS-345R, ACT-512F/ACT-783R, T1/Bt2b and CYLH3F/CYLH3R, respectively. The total volume of the PCR mixture is 20 µL, including 1 µL DNA template, 1 µL each 10 µM primer, 10 µL T5 Super PCR Mix and 7 µL sterile water. The gene fragments and amplification conditions used were in accordance with the details shown in Table 1 (Liu et al. 2022). The PCR products were electrophoresed in 1% agarose gel, and the DNA was sequenced by Sino Geno Max Biotechnology Company Limited (Beijing, China).

Table 1.

Genes used in this study with PCR primers and optimal annealing temperature.

Locus PCR Primers PCR: Thermal Cycles: (Annealing Temp. in Bold) Reference
ITS ITS1/ITS4 (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles White et al. (1990)
act ACT-512F/ACT-783R (95 °C: 45 s, 55 °C: 45 s, 72 °C: 1 min) × 35 cycles Carbone and Kohn (1999)
chs1 CHS-79F/CHS-345R (95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles Carbone and Kohn (1999)
gapdh GDR1/GDF1 (95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles Guerber et al. (2003)
his3 CYLH3F/CYLH3R (95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles Crous et al. (2004)
tub2 T1/Bt2b (95 °C: 30 s, 55 °C: 30 s, 72 °C: 1 min) × 35 cycles Glass and Donaldson (1995)
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Phylogenetic analyses

The resulting DNA sequences were combined with the sequences of reference strains from Genbank (Supplementary Suppl. material 1), and each single-gene dataset was aligned on MAFFT v. 6 separately (Katoh and Standley 2013), with both ends cut. Phylogenetic analysis of C.acutatum and C.boninense species complex was performed by combining six loci (ITS, act, chs1, gapdh, his3 and tub2). Phylogenetic analysis of C.gloeosporioides species complex was performed by combining five loci (ITS, act, chs1, gapdh, and tub2). Colletotrichumbambusicola LC8469 and C. orchidophilum CBS 632.80 were used as the outgroup. Phylogenetic analyses of Maximum Likelihood (ML) and Bayesian Inference (BI) were performed. ML and BI analyses were computed using PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). For BI analysis, the best-fitting evolutionary model for each partitioned locus was estimated using the Markov Chain Monte Carlo algorithm in MrModelTest v. 2.3 (Posada and Crandall 1998). The system diagram is plotted in FigTree v. 1.4.3 (Rambaut and Drummond 2010) (http://tree.bio.ed.ac.uk/software/figtree) and edited in Adobe Illustrator 2019 (https://www.adobe.com/cn/products/illustrator.html). Sequence data were submitted to GenBank (https://www.ncbi.nlm.nih.gov) (Suppl. material 1).

Results

Phylogenetic analyses

Forty-five strains of Colletotrichum, isolated from leaves of Juglansregia 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 (Ronquist and Huelsenbeck 2003). The best-fit models used the statistics of ML trees are shown in Suppl. material 2. Both Maximum Likelihood (ML) and Bayesian Inference (BI) methods were employed in these analyses. The topology of Bayesian analysis of cascading datasets is almost the same as ML consistency tree.

The phylogenetic tree showed 45 isolates across three species complexes: the C.acutatum species complex with a single isolate (Fig. 2), the C.boninense species complex with a single isolate (Fig. 3), and the C.gloeosporioides species complex with 43 isolates (Fig. 4). In the C.acutatum species complex, one isolate clustered with four reference isolates of C.fioriniae. In the C.boninense species complex, one isolate clustered with seven reference isolates of C.karsti. In the C.gloeosporioides species complex, 14 isolates clustered together with C.mengyinense, 17 isolates clustered with C.gloeosporioides and four isolates clustered with C.siamense, eight isolates formed three separate clades with high support (Fig. 4).

Figure 2.

Figure 2.

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Phylogram of Colletotrichumacutatum 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.

Figure 3.

Figure 3.

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Phylogram of Colletotrichumboninense 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.

Figure 4.

Figure 4.

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Phylogram of Colletotrichumgloeosporioides 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.

Taxonomy

. Colletotrichum cordae

Y.X. Li & X.L. Fan sp. nov.

F2FB6293-7524-5424-942D-35A322C964FF

852127

Fig. 5

Figure 5.

Figure 5.

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Colletotrichumcordae (ex-holotype culture CFCC 59618) A colonies on OA media above and below after 5 days at 25 °C B conidiomata on OA C, D conidiophores and conidia E, F conidia. Scale bars: 200 µm (B); 10 µm (C–F).

Etymology.

Named after Corda who established the genus Colletotrichum.

Typification.

China, Sichuan Province, Guangyuan City, Chaotian District, Longmen Valley Leisure Villa, 32°39'08"N, 105°55'11"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (holotype BJFC-S2250, ex-holotype culture CFCC 59618).

Description.

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.

Cultural characteristics.

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.

Additional material examined.

China. Sichuan Province, Guangyuan City, Chaotian District, Longmen Valley Leisure Villa, 32°39'08"N, 105°55'11"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2251, living culture CFCC 70160).

Notes.

Two strains of Colletotrichumcordae constitute a distinct clade within the C.gloeosporioides species complex, as revealed by multi-locus phylogenetic analysis. Colletotrichumcordae 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) (Sharma et al. 2017). Morphologically, C.cordae can be differentiated from C.perseae by the presence of conidia with obtuse or rounded apices.

. Colletotrichum fioriniae

(Marcelino & Gouli) R.G. Shivas & Y.P. Tan, Fungal Divers. 39: 117, 2009

FB7FD827-6948-528D-87F2-3993B03D967B

Material examined.

China. Shaanxi Province, Shangluo City, Lonan County, Red kernel walnut base, 34°03'10"N, 110°14'11'′E, from leaf of Juglansregia L., 14 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2253, living culture CFCC 59932).

Notes.

Colletotrichumfioriniae, a worldwide fungus with a wide range of host, is associated with walnut anthracnose disease (Shivas and Tan 2009; Zhu et al. 2015). Relative to other species within the Colletotrichum genus, C.fioriniae is generally considered less prevalent and less virulent, as noted by Talhinhas and Baroncelli (2021). In our research, the strain CFCC 59932 clusters robustly with C.fioriniae on both Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic trees, indicating strong statistical support (ML/BI = 100/1).

. Colletotrichum gloeosporioides

(Penz.) Penz. & Sacc., Atti Reale Ist. Veneto Sci. Lett. Arti., ser. 6, 2: 670. 1884

A3ADA95E-B723-545D-9E3C-9CDB3B4B517D

Materials examined.

China. • Sichuan Province, Guangyuan City, Chaotian District, Walnut Cultural Square, 32°40'58"N, 106°02'08"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2239, living culture CFCC 59613; BJFC-S2240, living culture CFCC 59621; BJFC-S2242, living culture CFCC 59634); • Chaotian District, Mianguang Expressway, 32°40'50"N, 105°59'19"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2246, living culture CFCC 59611; BJFC-S2247, living culture CFCC 59615; BJFC-S2244, living culture CFCC 59632); • Chaotian District, Zhongzi Town, 32°41'34"N, 106°02'23"E, from leaf of Juglansregia L, 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2230, living culture CFCC 59633; BJFC-S2228, living culture CFCC 59907; BJFC-S2252, living culture CFCC 59913; BJFC-S2254, living culture CFCC 59933). China. • Shaanxi Province, Shangluo City, Danfeng County, Dihua Ancient Town, 33°44'23"N, 110°12'07"E, from leaf of Juglansregia L., 13 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2257, living culture CFCC 59938; BJFC-S2258, living culture 59939); • Danfeng County, Walnut Theme Park, 33°44'33"N, 110°11'55"E, from leaf of Juglansregia L., 13 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2263, living culture CFCC 59925; BJFC-S2264, living culture CFCC 70189; BJFC-S2259, living culture CFCC 59940; BJFC-S2267, living culture CFCC 59949; BJFC-S2261, living culture CFCC 70190).

Notes.

Colletotrichumgloeosporioides was originally described as Vermiculariagloeosporioides and collected from Citrus sp. in Italy. The current name Colletotrichumgloeosporioides was proposed by Penzig (1882). Colletotrichumgloeosporioides is a worldwide fungus that inhabits a wide range of host plants. In our study, 17 strains and three species (i.e., C.citrulli, C.juglandicola and C.peakense) were robustly grouped with C.gloeosporioides, supported by high Maximum Likelihood (ML) and Bayesian Inference (BI) confidence values (ML/BI = 88/0.90). Zhang et al. (2023b) reduced C.dimorphum and C.nanhuaense as synonyms of C.gloeosporioides. Further research is needed to confirm the taxonomic status of C.citrulli, C.juglandicola and C.peakense. The morphology of the strains in our study closely resembles the type specimen of C.gloeosporioides, as described by Cannon et al. (2008). Thus, we propose the identification of our strains as C.gloeosporioides, based on both morphological characteristics and phylogenetic analyses. The result proves that walnut anthracnose has been attributed to C.gloeosporioides (Wang et al. 2020a; Mu et al. 2021; Yang et al. 2021; Li et al. 2023).

. Colletotrichum guangyuanense

Y.X. Li & X.L. Fan sp. nov.

2A654579-9F22-5F0B-97E3-74AA4912115F

852126

Fig. 6

Figure 6.

Figure 6.

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Colletotrichumguangyuanense (ex-holotype culture CFCC 59902) A symptom caused by ColletotrichumguangyuanenseB colonies on OA media above and below after 5 days at 25 °C C conidiomata on OA D conidiophores and conidia E, F conidia. Scale bars: 200 µm (C); 10 µm (D–F).

Etymology.

Named after the location where the fungal was first collected, which is Guangyuan City.

Typification.

China, Sichuan Province, Guangyuan City, Lizhou District, Shuiwo Village, 32°23'55"N, 105°39'24"E, from leaf of Juglansregia L., 11 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (holotype BJFC-S2225, ex-holotype culture CFCC 59902).

Description.

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.

Culture characteristics.

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.

Additional materials examined.

China. • Sichuan Province, Guangyuan City, Lizhou District, Shuiwo Village, 32°23'31"N, 105°39'22"E, from leaf of Juglansregia L., 11 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2226, living culture CFCC 70249). China. • Sichuan Province, Guangyuan City, Chaotian District, Longmen Valley Leisure Villa, 32°39'08"N, 105°58'17"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2248, living culture CFCC 59912); • Chaotian District, Longmen Valley Leisure Villa, 32°39'11"N, 105°55'26"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2249, living culture CFCC 70161).

Notes.

In phylogenetic analyses, Colletotrichumguangyuanense 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 (Jayawardena et al. 2016a). Morphologically, C.guangyuanense is distinguishable from C.changpingense by the absence of a distinct opaque region in the center of the conidia.

. Colletotrichum juglandium

Y.X. Li & X.L. Fan sp. nov.

922AE37C-8E8D-52FC-AC1C-F69BFE928562

852125

Fig. 7

Figure 7.

Figure 7.

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Colletotrichumjuglandium (ex-holotype culture CFCC 59974) A symptoms caused by ColletotrichumjuglandiumB colonies on OA media above and below after 5 days at 25 °C C conidiomata D, E conidiophores and conidia F, G conidia. Scale bars: 200 µm (C); 10 µm (D–G).

Etymology.

Named after the host genus on which it was collected, Juglansregia L.

Typification.

China. Sichuan Province, Guangyuan City, Chaotian District, Mianguang Expressway, 32°40'50"N, 105°59'19"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (holotype BJFC-S2243, ex-holotype culture CFCC 59974).

Description.

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.

Culture characteristics.

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.

Additional material examined.

China. Sichuan Province, Guangyuan City, Chaotian District, Mianguang Expressway, 32°40'36"N, 105°59'26"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2245, living culture CFCC 70165).

Notes.

Colletotrichumjuglandium form a solitary clade the C.gloeosporioides species complex (Fig. 3). Colletotrichumjuglandium is closely related to C.citrulli, C.gloeosporioides, C.juglandicola and C.peakense (Zhang et al. 2023a). Sequence identity comparisons reveal that C.juglandiumCFCC 59974 differs from other species at various loci: C.citrulli CGMCC3.20769 (3/544 in ITS, 0/244 in act, 0/228 in chs1, 10/297 in gapdh, and 0/324 in tub2), C.gloeosporioides IMI 356878 (2/544 in ITS, 0/289 in act, 0/236 in chs1, 18/341 in gapdh, and 0/324 in tub2), C.juglandicola CGMCC 3.24312 (2/544 in ITS, 0/279 in act, 1/249 in chs1, 18/341 in gapdh, and 1/324 in tub2), and C.peakense CGMCC 3.24308 (2/544 in ITS, 0/279 in act, 1/249 in chs1, 13/341 in gapdh, and 1/324 in tub2) (Guo et al. 2022; Yu et al. 2022; Zhang et al. 2023a). Morphologically, C.juglandium differs from C.gloeosporioides by having narrower conidia (L/W ratio: 3.1 vs. 2.6) and differs from C.juglandicola and C.peakense by having wider conidia (L/W ratio: 3.1 vs. 3.3).

. Colletotrichum karsti

Y.L. Yang, Z.Y. Liu, K.D. Hyde & L. Cai, Cryptog. Mycol. 32: 241. 2011

39216FDC-8F10-5D0B-9131-701F5644AC45

Material examined.

China. Sichuan Province, Guangyuan City, Lizhou District, Shuiwo Village, 32°23'38"N, 105°39'22"E, from leaf of Juglansregia L., 11 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2224, living culture CFCC 59901).

Notes.

Colletotrichumkarsti 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 (Yang et al. 2011; Damm et al. 2012a; Jayawardena et al. 2016b). Zhang et al. (2022) synonymized C.wuxuhaiense with C.karsti. In this study, the strain CFCC 59901 is confirmed to be C.karsti based on morphological characteristics and DNA sequence data. Furthermore, this represents a new host record for C.karsti on walnut trees.

. Colletotrichum mengyinense

T.C. Mu, J.W. Xia, X.G. Zhang & Z. Li, MycoKeys 85: 66 (2021)

1BF9EECA-1541-5E88-90BF-B0CE7F243B17

Materials examined.

China. • Sichuan Province, Guangyuan City, Chaotian District, Walnut Cultural Square, 32°40'58"N, 106°02'08"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2241, living culture CFCC 59604; BJFC-S2236, living culture CFCC 59605; BJFC-S2237, living culture CFCC 59608; BJFC-S2238, living culture CFCC 59910); • Chaotian District, Cypress Bridge, 32°41'16"N, 106°02'22"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2232, living culture CFCC 59614); • Chaotian District, Zhongzi Town, 32°41'34"N, 106°02'23"E, from leaf of Juglansregia L., 10 Oct. 2023,Y.X. Li, L. Lin & X.L. Fan (BJFC-S2229, living culture CFCC 59908); • Lizhou District, Tulongzi, 32°30'36"N, 105°36'51"E, from leaf of Juglansregia L., 11 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2227, living culture CFCC 59903). China. • Shaanxi Province, Shangluo City, Danfeng County, Walnut Theme Park, 33°44'33"N, 110°11'55"E, from leaf of Juglansregia L., 13 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2260, living culture CFCC 59923; BJFC-S2262, living culture CFCC 59943; BJFC-S2265, living culture CFCC 59944; BJFC-S2266, living culture CFCC 59945; BJFC-S2268, living culture CFCC 59950); • Shangzhou District, United Village, 33°52'03"N, 109°51'01"E, from leaf of Juglansregia L., 14 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2256, living culture CFCC 59935; BJFC-S2255, living culture CFCC 70187).

Notes.

Colletotrichummengyinense was isolated originally on diseased leaves of Rosachinensis (Mu et al. 2021). Additionally, the current 14 isolates are morphologically not significantly different from C.mengyinense and aggregated together with C.mengyinense with high support (ML/BI = 89/1.00) on the phylogenetic tree. Therefore, they are identified as Colletotrichummengyinense.

. Colletotrichum siamense

Prihastuti, L. Cai & K.D. Hyde, Fungal Divers. 39: 98 (2009)

EE1B963D-283B-5E18-A125-5CB4A340083F

Materials examined.

China. • Sichuan Province, Guangyuan City, Chaotian District, Zhongzi Town, 32°41'05"N, 106°02'08"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (BJFC-S2231, living culture CFCC 59601; BJFC-S2233, living culture CFCC 59625; BJFC-S2235, living culture CFCC 59964); • 32°41'34"N, 106°02'23"E, from leaf of Juglansregia L., 10 Oct. 2023, Y.X. Li, L. Lin & X.L. Fan (holotype BJFC-S2234, living culture CFCC 59909)

Notes.

Colletotrichumsiamense was first described as a species in association with Coffeaarabica by Prihastuti et al. (2009). The broader concept of C.siamensesensu lato has been a subject of considerable debate, as noted by Weir et al. (2012) and Sharma et al. (2015), due to the application of the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) approach. Liu et al. (2016) concluded that C.siamense is a single species, not a species complex. Based on phylogenetic evidence, Zhang et al. (2022) proposed that C.menglaense, C.pandanicola, and C.parvisporum are synonyms of C.siamense. More recent studies have further synonymized the closely related species C.rhizophorae and C.thailandica with C.siamense, considering morphological characteristics, phylogenetic analyses, and GCPSR (Aumentado et al. 2024). In this study, C.siamense was isolated from walnut leaf spots affected by walnut anthracnose. The four isolates our study examined while forming a distinct lineage were found within the C.siamense clade in our phylogenetic analysis. Moreover, our isolate (CFCC 59909) is similar to the holotype of C.siamense (ICMP 18578). While the conidia of our isolate (CFCC 59909) are wider than strain ICMP 18578 (12.3–14.5 × 4.9–6.4 vs. 7–18.3 × 3–4.3 μm) (Prihastuti et al. 2009). Based on this evidence, we identify our isolate as C.siamense.

Discussion

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 (Cannon et al. 2008, 2012; Cai et al. 2009; Liu et al. 2013, 2016). This study focused on walnut anthracnose in Sichuan and Shaanxi Provinces of China, conducting phylogenetic analyses using DNA sequence data. Five known species (C.fioriniae, C.gloeosporioides, C.karsti, C.mengyinense and C.siamense) and three new species (C.cordae, C.guangyuanense and C.juglandium) associated with walnut anthracnose were identified in the current study.

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 (Simmonds 1966; Alvarez 1976; Gorter 1977; Pennycook 1989; Liu et al. 1995; Chen 2003; Gadgil et al. 2005; Juhásová et al. 2005; Sreenivasaprasad and Talhinhas 2005; Kobayashi 2007; Qu et al. 2011; Damm et al. 2012b; Zhu et al. 2014, 2015; Wang et al. 2017, 2018; Da Lio et al. 2018; He et al. 2019; Savian et al. 2019; Wang et al. 2020b, 2021; Luongo et al. 2022; Ma et al. 2022; Wei et al. 2022; Li et al. 2023; Wang et al. 2023; Zhang et al. 2023a). Pathogenicity tests have demonstrated that numerous Colletotrichum species are responsible for anthracnose disease on walnut fruits and leaves, as Li et al. (2023) and Zhang et al. (2023a) reported. Wang et al. (2020a) revealed that the virulence of the pathogen of walnut anthracnose to walnut fruits was different. In this study, the pathogenicity of two established species, C.mengyinense and C.karsti, and three newly identified species, requires further investigation.

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 (Guo et al. 2022; Yu et al. 2022; Zhang et al. 2023a). Consequently, the 17 isolates in question have been identified as C.gloeosporioides. Liu et al. (2022) demonstrated that the boundaries between C.gloeosporioides and its closely related species are unclear. Zhang et al. (2023b) synonymized C.dimorphum and C.nanhuaense with C.gloeosporioides. Thus, we propose that C.citrulli, C.juglandicola, and C.peakense be considered as synonyms of C.gloeosporioides. Further genome-wide data studies are needed to clarify the species boundaries in this large clade (Liu et al. 2015).

Supplementary Material

XML Treatment for Colletotrichum cordae
XML Treatment for Colletotrichum fioriniae
XML Treatment for Colletotrichum gloeosporioides
XML Treatment for Colletotrichum guangyuanense
XML Treatment for Colletotrichum juglandium
XML Treatment for Colletotrichum karsti
XML Treatment for Colletotrichum mengyinense
XML Treatment for Colletotrichum siamense

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

Funding Statement

National Key R & D Program of China (2023YFD1401302),National Science and Technology Fundamental Resources Investigation Program of China (2021FY100900).

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

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).

Author contributions

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.

Author ORCIDs

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

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

Supplementary materials

Supplementary material 1

Strains of the Colletotrichum species with details of host, location and GenBank accessions of the sequences

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Yixuan Li, Lu Lin, Jing Cao, Mingxu Gan, Xinlei Fan

Data type

docx

Supplementary material 2

The statistics of ML trees in this study

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Yixuan Li, Lu Lin, Jing Cao, Mingxu Gan, Xinlei Fan

Data type

docx

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Colletotrichum cordae
mycokeys.108.125382-treatment1.xml (12.1KB, xml)
XML Treatment for Colletotrichum fioriniae
mycokeys.108.125382-treatment2.xml (9.1KB, xml)
XML Treatment for Colletotrichum gloeosporioides
mycokeys.108.125382-treatment3.xml (27.4KB, xml)
XML Treatment for Colletotrichum guangyuanense
mycokeys.108.125382-treatment4.xml (15KB, xml)
XML Treatment for Colletotrichum juglandium
mycokeys.108.125382-treatment5.xml (22.8KB, xml)
XML Treatment for Colletotrichum karsti
mycokeys.108.125382-treatment6.xml (11.3KB, xml)
XML Treatment for Colletotrichum mengyinense
mycokeys.108.125382-treatment7.xml (15.8KB, xml)
XML Treatment for Colletotrichum siamense
mycokeys.108.125382-treatment8.xml (17.7KB, xml)
Supplementary material 1

Strains of the Colletotrichum species with details of host, location and GenBank accessions of the sequences

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Yixuan Li, Lu Lin, Jing Cao, Mingxu Gan, Xinlei Fan

Data type

docx

mycokeys-108-147-s001.docx (130KB, docx)
Supplementary material 2

The statistics of ML trees in this study

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Yixuan Li, Lu Lin, Jing Cao, Mingxu Gan, Xinlei Fan

Data type

docx

mycokeys-108-147-s002.docx (18.4KB, docx)

Data Availability Statement

All of the data that support the findings of this study are available in the main text or Supplementary Information.

Articles from MycoKeys are provided here courtesy of Pensoft Publishers

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