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Research Article
Colletotrichum chinense sp. nov. from Yucca gloriosa and C. quercicola sp. nov. from Quercus variabilis in China
expand article infoCheng-Bin Wang, Ning Jiang, Han Xue, Chun-Gen Piao, Yong Li
‡ Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, China
Open Access

Abstract

Colletotrichum is an important plant pathogenic genus causing anthracnose on a wide range of host plants. During 2019 and 2021, Colletotrichum isolates were obtained during surveys of anthracnose on garden plants in China. Multi-gene phylogenetic analyses of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (gapdh), chitin synthase 1 (chs-1), actin (act) and beta-tubulin (tub2) sequences coupled with morphological evidence support the introduction of two novel species namely Colletotrichum chinense sp. nov. from Yucca gloriosa in Beijing and C. quercicola sp. nov. from Quercus variabilis in Shaanxi Province. Phylogenetic inference revealed that two isolates of C. chinense belonged to the agaves species complex and were closely related to C. agaves, and differed from the other species within this species complex by shorter conidia and the host association. Molecular identification showed that two isolates of C. quercicola formed a highly supported lineage close to C. tanaceti in the destructivum species complex, which could be distinguished from C. tanaceti by straighter conidia. In pathogenicity tests, yellow spots and orange conidial masses displayed on the inoculated Y. gloriosa leaves and brown spots appeared on the inoculated Q. variabilis leaves. In addition, C. chinense and C. quercicola were re-isolated from spots of the tested leaves of Y. gloriosa and Q. variabilis.

Keywords

Ascomycota, multigene phylogeny, new species, taxonomy

Introduction

The genus Colletotrichum (Glomerellaceae, Glomerellales, Sordariomycetes) is represented by its type species Colletotrichum lineola (Corda 1831; Damm et al. 2009; Hyde et al. 2020). The sexual morph of Colletotrichum, characterized by solitary or gregarious ascomata, 8-spored asci, and one-celled hyaline ascospores, was previously known as the genera Gnomoniopsis and Glomerella (Stoneman 1898; von Schrenk and Spaulding 1903; Marin-Felix et al. 2017). The asexual morph is characterized by acervular conidiomata, often with setae, producing cylindrical or crescent-shaped conidia, and by the formation of appressoria (Sutton 1992; Marin-Felix et al. 2017). With the implementation of “one fungus one name” nomenclature, Colletotrichum has been chosen to represent this genus based on priority (Réblová et al. 2016).

Previously, species of Colletotrichum were distinguished based on host range and a suite of morphological characteristics, especially the size and shape of conidia, appressoria, and sporulating structures (von Arx 1957; Cai et al. 2009; Hyde et al. 2009a, b). However, many taxonomic problems arose, due to few reliable and often variable morphological characters among species, and uncertain or broad host relationships (Cai et al. 2009; Hyde et al. 2009a; Cannon et al. 2012; Liu et al. 2016). Thus, many species are required taxonomic revision in order to clarify their taxonomic placement (Weir et al. 2012; Damm et al. 2014; Liu et al. 2022).

To establish a stable and natural classification system, Cai et al. (2009) recommended using a polyphasic approach, emphasizing multi-locus phylogeny in conjunction with morphology, geographical and ecological information to characterize and differentiate Colletotrichum species. Subsequently, many Colletotrichum species had been successfully identified and epitypified, resulting in a much better understanding of phylogenetic relationships of this genus (Weir et al. 2012; Damm et al. 2014). Currently, more than 1000 Colletotrichum epithets are listed in Index Fungorum (http://www.indexfungorum.org), and at least 303 species, grouped in 16 species complexes and some singleton species (Mu et al. 2021; Alizadeh et al. 2022; Liu et al. 2022; Zheng et al. 2022).

Many species of Colletotrichum have been identified as plant pathogens causing anthracnose on a wide range of hosts, especially in subtropical and tropical regions, leading to significant economic losses (Hyde et al. 2009a; Cannon et al. 2012; Lima et al. 2013). In addition, Colletotrichum species may occur as endophytes, saprobes, or opportunistic human pathogens, sometimes as latent plant pathogens, which may switch to a pathogenic lifestyle depending on the host plant, Colletotrichum species, and environmental conditions. (Huang et al. 2013; Rai and Agarkar 2014; Crous et al. 2016a; De Silva et al. 2017).

In the present study, by using a nucleotide basic local alignment search tool (BLASTn) analysis (Boratyn et al. 2013) of the ITS sequences, four Colletotrichum isolates from Yucca gloriosa and Quercus variabilis showed highest similarity lower than 98% with species in the agaves and destructivum species complex, respectively. The agaves species complex, represented by Colletotrichum agaves and four closely related species, occupies a monophyletic clade within this genus (Bhunjun et al. 2021; Talhinhas and Baroncelli 2021). The destructivum species complex is a monophyletic group of C. destructivum and 19 closely related species that are mainly plant pathogens (Damm et al. 2014; Bhunjun et al. 2021; Talhinhas and Baroncelli 2021). Members of this species complex are serious economic pathogens, such as C. destructivum, C. lentis and C. higginsianum (Damm et al. 2014; Bhadauria et al. 2019; Khodaei et al. 2019). They are characterized by conidia that are slightly curved due to their unilaterally tapering ends and by the small inconspicuous acervuli with rather effuse growth that are sometimes difficult to spot on the host plants (Damm et al. 2014).

Recently, we investigated the phylogenetic diversity of Colletotrichum species associated with anthracnose on garden plants in China. Four novel isolates were collected from Y. gloriosa and Q. variabilis in Beijing and Shaanxi, respectively. The aim of this study was to identify these isolates based on phylogenetic data and morphology and to confirm their pathogenicity.

Materials and methods

Sampling and fungal isolation

From 2019 to 2021, symptomatic leaves of garden plants were collected in China. Specimens were transferred to the laboratory in paper bags and stored at 4 °C until further processing. The surface of diseased leaves were sterilized with 70% ethanol and 2% NaClO for 1 min, rinsed three times with sterile water, and then samples were cut into 0.4 × 0.4 cm small pieces excised from the margins of foliar lesions, and placed on potato dextrose agar (PDA; potato extract 20 g, dextrose 20 g, agar 20 g, 1 L distilled water) plates at 25 °C in the dark. After 2–3 days, single colonies growing from the diseased tissue were transferred to new PDA plates. Single-spore cultures were obtained from the pure colonies and examined morphologically. The cultures were deposited in the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).

Morphological and culture characterisation

Agar plugs (6 mm in diameter) were taken from the edge of actively growing cultures on PDA and transferred in triplicate on PDA, synthetic low-nutrient agar (SNA; Nirenberg 1976), and malt extract agar (MEA; malt extract 20 g, agar 20 g, yeast extract 2 g, sucrose 5 g, sterile deionized water 1 L) incubated in the dark at 25 °C. After 7 days, the colony characteristics, colony diameters, and pigment production on the three media were noted. Appressoria were observed on slide cultures according to Weir et al. (2012). Moreover, the shape, color and size of conidia, conidiophores, setae, conidiogenous cells and appressoria were measured and captured at least 20 measurements using a Nikon Eclipse 80i compound microscope with differential interference contrast optics.

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from fungal mycelia using a CTAB DNA extraction protocol (Doyle and Doyle 1990). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (gapdh), chitin synthase 1 (chs-1), actin (act) and beta-tubulin (tub2) 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 (Carbone and Kohn 1999), ACT-512F/ACT-783R (Carbone and Kohn 1999) and T1/Bt2b (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997), respectively. PCR was performed in 20 μL reaction mixtures containing 10 μL 2× Taq polymerase (Tiangen, China), which contains a premix of Taq DNA polymerase (0.1 U), dNTPs (0.5 mM), MgCl2 (3 mM) Tris-HCl (20 mM), KCl (100 mM) and the appropriate buffer system, 7 μL RNase-free water, 1 μL of each primer (0.5 µM) and 1 μL of DNA template (20 ng/μl). The PCR conditions were as follows: initial heat treatment of 5 min at 94 °C, followed by 35 cycles of 30 sec at 94 °C, 30 s at 54 °C (ITS), 60 °C (gapdh), 59 °C (chs-1), 58 °C (act) or 55 °C (tub2), and 1 min at 72 °C, and a final elongation period of 7 min at 72 °C. Amplicons were purified and sequenced by ABI3730XL Gene Analyzer at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

Newly generated sequences from the four isolates in this study were assembled using SeqMan v. 7.1.0, and the closest match using BLASTn analyses. Reference Colletotrichum sequences (Table 1) were downloaded from GenBank, based on recent publication (Liu et al. 2022). Multiple sequences were aligned using the MAFFT v.7.110 online programme (http://mafft.cbrc.jp/alignment/server/, Katoh et al. 2019) by default settings, and adjusted manually in MEGA v.7.0 (Kumar et al. 2016). The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike information criteria (AIC).

Table 1.

Colletotrichum spp. used for phylogenetic analyses in the study.

Species name Accession no.a GenBank Accession No.
ITS Gapdh chs-1 act tub2
agaves CBS 118190* DQ286221 NA NA NA NA
C. agaves LC0947 MZ595831 MZ664053 MZ799266 MZ664129 MZ673955
C. americae-borealis ATCC 11869 KM105223 KM105578 KM105293 KM105433 KM105503
C. americae-borealis CBS 136232* KM105224 KM105579 KM105294 KM105434 KM105504
C. antirrhinicola CBS 102189* KM105180 KM105531 KM105250 KM105390 KM105460
C. atractylicola SAUCC 1307* KR149280 KR259334 KR259333 KR132243 KU058178
C. atractylicola SAUCC 130801 KU289192 KU289207 KU289202 KU289197 KU289212
C. boninense CBS 123755* JQ005153 JQ005240 JQ005327 JQ005501 JQ005588
C. brasiliense CBS 128501* JQ005235 JQ005322 JQ005409 JQ005583 JQ005669
C. bryoniicola CBS 109849* KM105181 KM105532 KM105251 KM105391 KM105461
C. chinense CFCC 57501* ON692808 ON755050 ON755046 ON755042 ON755054
C. chinense CFCC 57502 ON692809 ON755051 ON755047 ON755043 ON755055
C. destructivum CBS 114801 KM105219 KM105574 KM105289 KM105429 KM105499
C. destructivum CBS 157.83 KM105215 KM105570 KM105285 KM105425 KM105495
C. destructivum IMI 387103 KM105221 KM105576 KM105291 KM105431 KM105501
C. destructivum CBS 136228* KM105207 KM105561 KM105277 KM105417 KM105487
C. euphorbiae CBS 134725* KF777146 KF777131 KF777128 KF777125 KF777247
C. fuscum CBS 133701* KM105174 KM105524 KM105244 KM105384 KM105454
C. fuscum CBS 133702 KM105178 KM105528 KM105248 KM105388 KM105458
C. fuscum CBS 133703 KM105175 KM105525 KM105245 KM105385 KM105455
C. fusiforme MFLUCC 12-0437 KT290266 KT290255 KT290253 KT290251 KT290256
C. higginsianum CPC 19379* KM105184 KM105535 KM105254 KM105394 KM105464
C. higginsianum CPC 19364 KM105185 KM105537 KM105255 KM105395 KM105465
C. higginsianum CPC 19369 KM105188 KM105540 KM105258 KM105398 KM105468
C. higginsianum CPC 19394 KM105193 KM105546 KM105263 KM105403 KM105473
C. ledebouriae CBS 141284* KX228254 NA NA KX228357 NA
C. lentis CBS 127604* JQ005766 KM105597 JQ005787 JQ005829 JQ005850
C. lentis CBS 127605 KM105241 KM105598 KM105311 KM105451 KM105521
C. lini CBS 172.51* JQ005765 KM105581 JQ005786 JQ005828 JQ005849
C. lini CBS 136856 KM105233 KM105589 KM105303 KM105443 KM105513
C. lini CBS 130828 KM105234 KM105590 KM105304 KM105444 KM105514
C. neorubicola CCR144* MK529906 MK547520 MK547526 MK547523 MN186400
C. neorubicola CCR145 MK529908 MK547521 MK547527 MK547524 MN186401
C. neorubicola CCR146 MK529907 MK547522 MK547528 MK547525 MN186402
C. neosansevieriae CBS 139918* KR476747 KR476791 NA KR476790 KR476797
C. ocimi CBS 298.94* KM105222 KM105577 KM105292 KM105432 KM105502
C. panacicola C08048 GU935867 GU935847 NA GU944757 NA
C. panacicola C08061 GU935868 GU935848 NA GU935791 NA
C. panacicola C08087 GU935869 GU935849 NA GU944758 NA
C. pisicola CBS 724.97 * KM105172 KM105522 KM105242 KM105382 KM105452
C. pleopeltidis CBS 147082* MW883412 NA MW890035 MW890024 NA
C. quercicola CFCC 54457* ON692810 ON755052 ON755048 ON755044 ON755056
C. quercicola CFCC 57507 ON692811 ON755053 ON755049 ON755045 ON755057
C. sansevieriae MAFF 239721* LC179806 LC180130 LC180129 LC180127 LC180128
C. sansevieriae BTGN2 MN386823 MN386911 NA NA MN386867
C. shisoi JCM 31818* MH660930 MH660931 MH660929 MH660928 MH660932
C. shisoi MAFF 240106 MH660936 MH660935 MH660934 MH660933 MH660937
C. tabacum CBS 124249 KM105206 KM105560 KM105276 KM105416 KM105486
C. tabacum CBS 161.53 JQ005763 KM105559 JQ005784 JQ005826 JQ005847
C. tabacum CPC 18945* KM105204 KM105557 KM105274 KM105414 KM105484
C. tanaceti BRIP 57316 JX218230 JX218245 JX259270 JX218240 JX218235
C. tanaceti CBS 132693 JX218228 JX218243 JX259268 JX218238 JX218233
C. tanaceti CBS 132818 JX218229 JX218244 JX259269 JX218239 JX218234
C. truncatum IMI 135524 GU227874 GU228266 GU228364 GU227972 GU228168
C. utrechtense CBS 130243* KM105201 KM105554 KM105271 KM105411 KM105481
C. utrechtense CBS 135827 KM105202 KM105555 KM105272 KM105412 KM105482
C. utrechtense CBS 135828 KM105203 KM105556 KM105273 KM105413 KM105483
C. vignae CBS 501.97* KM105183 KM105534 KM105253 KM105393 KM105463
C. vignae CPC 19383 KM105182 KM105533 KM105252 KM105392 KM105462

Phylogenetic analyses using Maximum Likelihood (ML) and Bayesian Inference (BI) were performed. ML analyses were constructed on the RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014) using the GTR+GAMMA model with 1000 bootstrap replicates. BI analyses were also performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.2.6 (Ronquist et al. 2012). The analyses were conducted by running 5,000,000 generations in two independent runs and sampling every 100th generations. The first 25% of the trees of MCMC sampling were discarded as burn-in and posterior probabilities (PP) were determined from the remaining trees. The results were visualized in FigTree 1.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS6.0.

Pathogenicity test

The pathogenicity of two Colletotrichum isolates was assessed on detached healthy Y. gloriosa and Q. variabilis plants in the greenhouse. Leaves were washed in running distilled water, surface-sterilized in 70% ethanol and 2% NaClO for 1 min, then rinsed in sterile distilled water. Spores were harvested from two-week-old PDA plates with 10 ml of sterilized water with spore suspension filtered through two layers of cheesecloth to eliminate debris and mycelium. The conidial suspension was adjusted to a final inoculum concentration of 1 × 106–107 conidia/mL with sterile deionized water. Then 10 µL of conidial suspension was placed in the middle portion of the leaves, and inoculated sterile water in the additional leaves served as control. Each treatment had three replicates (three leaves), and the experiment was carried out twice. The inoculated leaves were placed in transparent plastic bags at 25 °C and over 90% humidity in the dark for 14 days. After appearance of symptoms, fungus isolates were re-isolated from the infected leaves and identified based on the morphological and phylogenetic analyses to fulfill Koch’s postulates.

Results

Phylogenetic analyses

Closest matches in BLASTn searches with the ITS sequences, these isolates were preliminarily identified to be in the agaves and destructivum species complexes. Further, phylogenetic trees were constructed based on combined loci of ITS, gapdh, act, chs-1 and tub2 sequences to identify these isolates to species level.

For the agaves species complex, DNA sequences of five genes were obtained from two isolates from Y. gloriosa in this study, with seven reference strains of the agaves species complex, and C. boninense (CBS 123755, ex-type) and C. brasiliense (CBS 128501, ex-type) as the outgroup taxa. A total of 1649 characters including alignment gaps (578 for ITS, 94 for gapdh, 232 for chs-1, 240 for act and 505 for tub2) were included in the phylogenetic analyses. Of these characters, 1271 were constant, 162 were variable and parsimony-uninformative, and 216 were parsimony-informative. The resulting ML and BI trees had similar topologies; the ML tree (Fig. 1) was selected to represent the phylogeny with ML/BI support values. Two isolates (CFCC 57501 and CFCC 57502) formed a close clade to C. agaves (Fig. 1).

Figure 1. 

Phylogenetic tree obtained by Maximum likelihood analyses using the combined ITS, gapdh, chs-1, act and tub2 sequence alignments of the agaves species complex. Numbers above the branches indicate ML bootstraps (left, MLBS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.7). The tree is rooted with C. boninense (CBS 123755, ex-type) and C. brasiliense (CBS 128501, ex-type).

For the destructivum species complex, DNA sequences of five genes were obtained from two isolates from Q. variabilis in this study, and 44 reference strains of the destructivum species complex, and C. truncatum (IMI 135524) and C. fusiforme (MFLUCC 12-0437) as the outgroup taxa. A total of 1875 characters including gaps (560 for ITS, 236 for gapdh, 280 for chs-1, 274 for act and 525 for tub2) were obtained in the phylogenetic analyses. Of these characters, 1292 were constant, 177 were variable and parsimony-uninformative, and 406 were parsimony-informative. The resulting ML and BI trees had similar topologies; the ML tree (Fig. 2) was selected to represent the phylogeny with ML/BI support values. Two new isolates (CFCC 54457 and CFCC 57507) formed a sister clade to C. tanaceti (Fig. 2).

Figure 2. 

Phylogenetic tree obtained by Maximum likelihood analyses using the combined ITS, gapdh, chs-1, act and tub2 sequence alignments of the destructivum species complex. Numbers above the branches indicate ML bootstraps (left, MLBS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.7). The tree is rooted with C. fusiforme (MFLU 12-0437CC) and C. truncatum (IMI 135524).

Taxonomy

Colletotrichum chinense Ning Jiang & C.B. Wang, sp. nov.

MycoBank No: 844527 Fig. 3

Etymology

Referring to the country, where the species was first collected.

Description

Sexual morph not observed. Asexual morph developed on PDA. Setae and chlamydospores not observed. Conidiomata acervular, abundant, pulvinate, 200–500 μm diam. Conidiophores smooth-walled, unbranched, septate, sometimes constricted at the septa, hyaline, up to 40 µm long. Conidiogenous cells 6.5–19.5 × 3–8 µm (x– = 12.7 ± 2.7 × 5.3 ± 1.3 µm, n = 20), subglobose to ampulliform, smooth-walled, hyaline. Conidia 9.5–25.5 × 3.5–8.5 µm (x– = 14.8 ± 1.8 × 6 ± 1 μm, n = 50), L/W ratio = 2–2.7, cylindrical, obtuse at the apex, smooth-walled, hyaline, contents granular. Appressoria not observed.

Culture characters

Colonies on PDA, flat, with an entire margin, with sparse aerial mycelium, covered with orange conidial masses, reaching 23–25 mm diam in 7 days at 25 °C. Colonies on MEA, flat, with no aerial mycelium, covered with slimy conidial masses, reaching 15–20 diam in 7 days at 25 °C. Colonies on SNA flat, sparse white hyphae, with an entire margin, reaching 12–15 diam in 7 days at 25 °C.

Specimens examined

China, Beijing City, isolated from leaf spot of Yucca gloriosa L., Cheng-Bin Wang, 15 August 2020 (holotype CAF800056; ex-type living culture: CFCC 57501); Ibid (living culture: CFCC 57502).

Notes

Colletotrichum beeveri of the boninense species complex and C. tofieldiae of the spaethianum species complex have been reported from Yucca before the present study (Liu et al. 2022). Colletotrichum chinense from the present study is similar to C. beeveri in the conidial shape, but differs in conidial size (9.5–25.5 × 3.5–8.5 µm in PDA vs. 12.5–15.5 × 5.5–6.5 µm in SNA) (Damm et al. 2012). In addition, C. tofieldiae differs from C. chinense by the falcate conidia (Damm et al. 2009). Based on phylogenetic analyses using multi-locus sequences (ITS, gapdh, chs-1, act and tub2), C. chinense formed a sister clade to C. agaves in the agaves species complex. The sequence identities between C. chinense CFCC 57501 and C. agaves LC0947 (21/578 ITS, 6/94 gapdh, 6/232 chs-1, 19/240 act and 26/505 tub2), C. euphorbiae CBS 134725 (31/578 ITS, 8/94 gapdh, 7/232 chs-1, 35/240 act and 32/505 tub2), C. ledebouriae CBS 141284 (29/578 ITS, 30/240 act), C. neosansevieriae CBS 139918 (28/578 ITS, 6/94 gapdh, 28/240 act and 27/416 tub2) and C. sansevieriae MAFF 239721 (29/578 ITS, 5/94 gapdh, 9/232 chs-1, 31/240 act and 44/505 tub2). (Nakamura et al. 2006; Crous et al. 2013, 2015, 2016b; Liu et al. 2022) The chs-1 sequence of C. neosansevieriae CBS 139918 and the gapdh, chs-1 and tub2 sequences of C. ledebouriae CBS 141284 were missing. Morphologically, the conidia size of C. chinense are shorter than other species (Table 2).

Table 2.

Morphological comparison of species in the agaves species complex.

Species Type Media for Conidia morph Hosts Distribution Conidia (µm) Appressoria (µm) Reference
C. agaves Epitype PDA Agave spp. Mexico; USA; Netherlands (17.5–)19.0–30.5(–33) × 5–8(–9.5) on Not observed Farr et al. (2006)
C. chinense Holotype PDA Yucca gloriosa China (9.5–)12.5–16.5(–25.5) × (3.5–)6.0–7.0(–8.5) Not observed This study
C. euphorbiae Holotype SNA Euphorbia sp South Africa (17–)23–28(–28.5) × (6–)6.5–7 (6.5–)8.5–14.5(–20.5)
× (5.5–)6–10.5(–16)
Crous et al. (2013)
C. ledebouriae Holotype PNA Ledebouria floribunda South Africa (15–)17–21(–22) × (5–)6 Not observed Crous et al. (2016)
C. neosansevieriae Holotype SNA Sansevieria trifasciata South Africa (16–)18–22(–25) × (4–)5–6 Not observed Crous et al. (2015)
C. sansevieriae Holotype PDA Sansevieria spp. Asia; Australia; USA 12.5–(18.4)–32.5 × 3.8–(6.4)–8.8 PDA 6.3–(7.7)–8.8
× 6.3–(7.3)–7.5
Nakamura et al. (2006)
Figure 3. 

Colletotrichum chinense (CFCC 57501; ex-type) A colony on PDA B colony on MEA C colony on SNA D conidiomata formed in PDA E conidiophores from the host F, G conidiophores H, I conidia F–I from PDA. Scale bars: 200 µm (D); 50 µm (E); 20 µm (F, G); 10 µm (H, I).

Colletotrichum quercicola Ning Jiang & C.B. Wang, sp. nov.

MycoBank No: 844528 Fig. 4

Etymology

Referring to the host genus, Quercus.

Description

Sexual morph not observed. Asexual morph developed on PDA. Chlamydospores not observed. Conidiomata acervular, abundant, globose to pulvinate, 200–400 μm diam. Conidiophores, hyaline, branched, smooth-walled, up to 50 μm long. Setae medium brown, smooth-walled, 60–145 μm long, 1–3-septate. Conidiogenous cells 6–18 × 3–7 µm (x– = 7.9 ± 3.6 × 4 ± 1.2 µm, n = 20), hyaline, smooth-walled, cylindrical to elongate ampulliform. Conidia 14.5–23 × 3–5 µm (x– = 17 ± 1.7 × 3.9 ± 0.5 μm, n = 50), L/W ratio =4–5, hyaline, smooth-walled, fusiform, straight to slightly curved with both ends rounded or one end round and the other truncate. Appressoria 6–11 × 4–8 µm (x– = 8.4 ± 1.4× 5 ± 1 μm, n = 50), L/W ratio = 1.5–2, single, medium brown, smooth-walled, subglobose, ovate to broadly elliptical in outline.

Culture characters

Colonies on PDA flat, with moderate aerial mycelium, margin white to light gray, gray to brown in the center, reaching 46–50 mm diam in 7 days at 25 °C. Colonies on MEA flat, covered by white aerial mycelium, white margin and light orange in the center, reaching 30–35 mm diam after 7 days at 25 °C. Colonies on SNA flat, with entire margin, covered by sparse white aerial mycelium, reaching 20 mm diam after 7 days at 25 °C.

Specimens examined

China, Shaanxi Province, Foping County, Dongshan Park, isolated from leaf spot of Quercus variabilis Bl., Yong Li, 11 September 2019 (holotype CAF800057; ex-type living culture: CFCC 54457); Ibid (living culture: CFCC 57507).

Notes

Four Colletotrichum species are presently known to occur on Quercus hosts, viz. C. clidemiae, C. gloeosporioides, C. karstii and C. theobromicola (Weir et al. 2012; Liu et al. 2021). Colletotrichum quercicola can be distinguished from those species based on any of the loci (ITS, gapdh, chs-1, act and tub2) and the fusiform conidia. Colletotrichum quercicola is a member of the destructivum species complex and near to C. tanaceti. Phylogenetically, this species can be distinguished from C. tanaceti CBS 132693 by 88 nucleotide differences in concatenated alignment (20/560 in ITS, 14/274 in act, 2/280 in chs-1, 17/236 in gapdh, and 33/525 in tub2) (Damm et al. 2014). Morphologically, C. quercicola CFCC 54457 conidia are straight to slightly curved, differing from distinctly curved conidia in C. tanaceti CBS 132693 (Damm et al. 2014).

Figure 4. 

Colletotrichum quercicola (CFCC 54457; ex-type) A colony on PDA B colony on MEA C colony on SNA D conidiomata formed in PDA E, F conidiophores G, H conidia I appressoria were producing using a slide culture technique E–H from PDA. Scale bars: 200 µm (D); 50 µm (E); 20 µm (F); 10 µm (G–I).

Pathogenicity

Pathogenicity tests were conducted to confirm Koch’s postulates on Q. variabilis leaves for C. quercicola, and on Y. gloriosa leaves for C. chinense. After 14 days of inoculation, necrotic lesions and typical orange conidial masses were observed from the inoculated site of Y. gloriosa leaves, and Q. variabilis leaves showed brown spot from the inoculated site, whereas all control leaves remained healthy (Fig. 5). Furthermore, Colletotrichum isolates could consistently be re-isolated from symptomatic lesions, but never from control leaves. And these isolates were identified as material used for inoculations based on multigene phylogenetic analyses and morphological characters, fulfilling Koch’s postulates.

Figure 5. 

Typical field symptoms of disease and artificial inoculation results A–E Yucca gloriosa leaves F–J Quercus variabilis leaves A, F Anthracnose field symptoms B–D Symptoms resulting from Colletotrichum chinense (CFCC 57501; ex-type) after 14 days G–I symptoms resulting from Colletotrichum quercicola (CFCC 54457; ex-type) after 14 days E, J symptoms resulting from sterile deionized water after 14 days.

Discussion

In the present study, we collected garden plants with anthracnose symptoms or leaf spots in China. From these samples, the obtained Colletotrichum isolates were identified based on morphological features of the asexual morph obtained in culture and five combined loci (ITS, gapdh, chs-1, act and tub2) phylogenies. The phylogenetic analyses revealed two novel species, C. chinense from Y. gloriosa in Beijing, and C. quercicola from Q. variabilis in the Shaanxi Province, and morphological characters can distinguish these isolates from related species. Pathogenicity test revealed C. chinense appearing as a causal agent of Y. gloriosa anthracnose and C. quercicola as a pathogen of Q. variabilis anthracnose.

ITS is evaluated as a universal DNA barcode marker for fungi (Schoch et al. 2012). However, most Colletotrichum species could not be distinguished based on ITS only (Cai et al. 2009; Jayawardena et al. 2016). Further, multi-locus DNA sequences, including ITS combined with supplementary barcodes, for which including some of act, the intergenic region between DNA lyase and the mating-type (mat1-2) gene (apMat), DNA lyase (apn2), calmodulin (cal), chs-1, gapdh, glutamine synthetase (gs), superoxide dismutase (sod2) or tub2 genes for species delimitation (Cannon et al. 2012; Silva et al. 2012a, b; Weir et al. 2012; Vieira et al. 2020). Generally, ITS, act, chs-1, gapdh and tub2 gene regions have provided adequate resolution to differentiate species within this genus (Bhunjun et al. 2021; Jayawardena et al. 2021; Talhinhas and Baroncelli 2021). In this study, phylogenetic analyses based on five combined loci (ITS, gapdh, chs-1, act and tub2) supported that these isolates clustered in a well-supported clade in the agaves and destructivum species complexes with high confidence.

The agaves species complex groups Colletotrichum agaves, and four related species, C. ledebouriae, C. neosansevieriae, C. euphorbiae and C. sansevieriae (Bhunjun et al. 2021; Talhinhas and Baroncelli 2021). They are unable to be distinguished based on conidial dimensions alone (Table 2). Members of this species complex were assumed to have host specificity (Nakamura et al. 2006; Jayawardena et al. 2021; Talhinhas and Baroncelli 2021). However, three species (C. ledebouriae, C. neosansevieriae and C. euphorbiae) were found only once from its type strain (Crous et al. 2013, 2015, 2016b). Four species, C. agaves on Agave spp., C. sansevieriae on Sansevieria sp., C. ledebouriae on Ledebouria floridunda, C. neosansevieriae on Sansevieria trifasciata, have only been recorded from Asparagaceae (Talhinhas and Baroncelli 2021). In this study, C. chinense was isolated from symptomatic leaves of Y. gloriosa, belonging to the family Asparagaceae.

Species in the destructivum species complex are serious pathogens undergoing a hemibiotrophic lifestyle and have been associated with 49 plant species belonging to 41 genera (Damm et al. 2014; Jayawardena et al. 2021; Talhinhas and Baroncelli 2021). Many species appear to have a wide host range, while some species may affect single host species or genera (Damm et al. 2014; Talhinhas and Baroncelli 2021). Typical characteristics of species in this species complex are characterized by the presence of straight or slightly curved conidia with obtuse apices (Bhunjun et al. 2021; Jayawardena et al. 2021). Morphological differences in the size of conidia and appressoria were observed between this species complex (Table 3). The morphological approach alone makes it difficult to distinguish in this complex due to few and variable morphological characteristics.

Table 3.

Morphological comparison of species in the destructivum species complex.

Species Type Media for Conidia morph Hosts Distribution Conidia (µm) Appressoria (µm) Reference
C. americae-borealis Holotype SNA Medicago sativa; Glycyrrhiza uralensis America; China (13.5–)15.5–18(–19) × 3.5–4 (4.5–)6–10.5(–13) × (3.5–)4–7(–10) Damm et al. (2014)
C. antirrhinicola Holotype SNA Antirrhinum majus New Zealand; Japan (14.5–)15.5–19(–23.5) × (3.5–) 4–4.5(–5) (9–)9.5–12(–13.5) × (5–)6–8(–10) Damm et al. (2014)
C. atractylodicola Holotype PDA Atractylodes lancea China 13.5–19 × 4–6.5 7.5–14 × 7–10.5 Xu et al. (2018)
C. bryoniicola Holotype SNA genera of Asteraceae, Convolvulaceae, and Fabaceae; etc Netherlands; Italy (13.5–)15–18.5(–22) × 4–5(–5.5) (3.5–) 4–10(–18) × (2.5–)3.5–6.5(–7.5) Damm et al. (2014)
C. destructivum Epitype SNA Trifolium spp.; Bletilla ochracea; Phragmites sp.; etc worldwide (14–)14.5–16.5(–18) × 3.5–4(–4.5) (6.5–)10–15.5(–20.5) × (4.5–)5–8(–10.5 Damm et al. (2014)
C. fuscum Epitype SNA Digitalis spp.; Heracleum sp.; Coreopsis lanceolata Germany; Italy; Netherlands (16–)16.5–20(–34) × (3.5–)4–4.5(–5.5) (6–)8.5–14.5(–19) × (6.5–)7–10(–11.5) Damm et al. (2014)
C. higginsianum Epitype SNA Brassicaceae; Campanula sp.; Rumex acetosa Italy; Japan, Korea; Trinidad; Tobago; America (17–)19–20.5(–21) × (3–)3.5–4(–4.5) (5.5–)10–20(–28.5) × (3.5–) 5–9(–12) Damm et al. (2014)
C. lentis Holotype SNA Lens culinaris; Vicia sativa Canada; China; Romania (13–)16–20(–26) × 3–4(–5) (5–)5.5–7.5(–9) × (3.5–)4.5–6(–6.5) Damm et al. (2014)
C. lini Epitype SNA Linum sp.; Nigella sp.; Taraxacum sp.; etc France; Germany; America; Ireland; Tunisia;Netherlands (13–)15–18(–22.5) × (3–)3.5–4(–4.5) (5–)6.5–10(–12.5) × (4–)4.5–6(–7) Damm et al. (2014)
C. neorubicola Holotype PDA Rubus idaeus China (14.8–)21.5–22.7(–23.5) × (4–)4.9–5.1(–5.6) (4–)8.2–10.5(–17.5)× (3.6–)5.6–6.8(–11.7) Liu et al. (2020)
C. ocimi Holotype SNA Ocimum basilicum Italy; Australia 14.5–15.5(–16.5) × (3.5–)4–4.5 (6.5–)7–13(–15.5) × (4–)4.5–7.5(–9) Damm et al. (2014)
C. quercicola Holotype PDA Quercus variabilis China (14–)14.5–17.5(–21.5) × (3–)3.3–4.3(–4.7) (5.7–)6.8–9.7(–10) × (3.2–)4–6(–8) This study
C. panacicola Panax sp. Eastern Asia 17.0–22.1 × 3.4–5.1 14–8 Takimoto (1919)
C. pleopeltidis Holotype SNA Pleopeltis sp. South Africa (15–)19–23(–25) × (5–)5.5(–6) Not described Crous et al. (2021)
C. pisicola Holotype SNA Pisum sp. America (11–)15–21(–29.5) × (3–)3.5–4 (5.5–)7–11.5(–13.5) × (4–)4.5–6(–6.5) Damm et al. (2014)
C. shisoi Holotype PDA Perilla frutescens Japan (15.0–)17–19(–27.0) × (3.0–)4.0(–5.0) (7.0–)9.0–10.0(–11.0) × (5.0–)7.0–8.0 Gan et al. (2019)
C. tabacum Neotype SNA Nicotiana spp., Centella asiatica France; India; Germany; Madagascar; Zimbabwe (11·5–) 19–20 (–27) × (3–) 5·5–5·8 (–7·6) (10–) 11·5–12·5 (–14·5) × (6·5–) 8·5–9·5 (–11·5) Damm et al. (2014)
C. tanaceti Holotype SNA Tanacetum cinerariifolium Australia (13–)14.5–17.5(–19) × (3–)3.5–4(–4.5) (5–)6.5–12(–14.6)× (3.5–)4.5–7(–10) Damm et al. (2014)
C. utrechtense Holotype PDA Trifolium pratense Netherlands 17.5–20.5(–23) × 3.5–4(–4.5) (7–)10–14.5(–15) × (5–)6.5–9.5(–10) Barimani et al. (2013)
C. vignae Holotype SNA Vigna unguiculata Nigeria (12–)14–17.5(–18.5) × (3–)3.5–4(–4.5) (4–)4.5–8.5(–12.4)× (3.5–)4–5(–6.5) Damm et al. (2014)

Although morphological characters may not prove taxonomically informative for species differentiation within species complex, they are considered as a basis to taxonomic segregation for distinguishing species between different species complexes (Cannon et al. 2012; Liu et al. 2022). A polyphasic approach, emphasizing multi-gene phylogenetic analyses combined with analyses of ecological, geographical and morphological data was essential to the identification of Colletotrichum species (Cai et al. 2009; Jayawardena et al. 2021; Talhinhas and Baroncelli 2021). In recent years, the classification and species concepts in Colletotrichum was changed according to this ideal polyphasic approach (Jayawardena et al. 2021; Talhinhas and Baroncelli 2021; Liu et al. 2022). In the present study, we described two novel species based on molecular sequence analyses and morphological characters, confirming their pathological characterization. To our knowledge, this is the first report of anthracnose on Y. gloriosa and Q. variabilis. These results may provide an important basis for the prevention and control of this disease.

Acknowledgements

This research was funded by the National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (NMRC-2021-7).

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