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Research Article
Two new species of Colletotrichum (Glomerellaceae, Glomerellales) causing walnut anthracnose in Beijing
expand article infoLin Zhang‡, Yue-Qi Yin‡, Li-Li Zhao‡, Yu-Qing Xie‡, Jing Han‡, Ying Zhang‡
‡ Beijing Forestry University, Beijing, China

Corresponding author: Ying Zhang ( yzhang@bjfu.edu.cn )

Academic editor: Sajeewa Maharachchikumbura
© 2023 Lin Zhang, Yue-Qi Yin, Li-Li Zhao, Yu-Qing Xie, Jing Han, Ying Zhang.
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: Zhang L, Yin Y-Q, Zhao L-L, Xie Y-Q, Han J, Zhang Y (2023) Two new species of Colletotrichum (Glomerellaceae, Glomerellales) causing walnut anthracnose in Beijing. MycoKeys 99: 131-152. https://doi.org/10.3897/mycokeys.99.106812
Open Access

Abstract

Colletotrichum species are plant pathogens, saprobes and endophytes on various plant hosts. It is regarded as one of the 10 most important genera of plant pathogens in the world. Walnut anthracnose is one of the most severe diseases affecting walnut productivity and quality in China. In this study, 162 isolates were obtained from 30 fruits and 65 leaf samples of walnut collected in Beijing, China. Based on morphological characteristics and DNA sequence analyses of the concatenated loci, namely internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase 1 (CHS-1) and beta-tubulin (TUB2), these isolates were identified as two novel species of Colletotrichum, i.e. C. juglandicola and C. peakense. Koch’s postulates indicated that both C. juglandicola and C. peakense could cause anthracnose in walnut.

Key words

Anthracnose, multi-gene phylogeny, pathogenicity, walnut

Introduction

Walnut (Juglans regia L.), a deciduous tree, is an essential woody nut and oil crop cultivated worldwide (Da Lio et al. 2018). Walnut fruit is rich in linolenic acid and lacking cholesterol and ranks first amongst the world’s “four major dried fruits” (Pei and Lu 2011). Walnut cultivation in China has a history of more than two thousand years and is China’s “woody grain and oil” strategic tree species (Guo 2016; Liu et al. 2021). The walnut productivity in China contributed 47% of the global production in 2017 and ranked first worldwide since 2017 (Liu et al. 2021).

Colletotrichum Corda (Glomerellaceae, Glomerellales, Sordariomycetes) was introduced, based on the morphological feature of the conidiomata with setae and Colletotrichum lineola Corda was assigned as the generic type (Corda 1831). The sexual morph of Colletotrichum was previously known as the genera Gnomoniopsis and Glomerella (Stoneman 1898; von Schrenk and Spaulding 1903; 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). Colletotrichum was characterised 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). More than 1,000 epithets have been accommodated within Colletotrichum (http://www.indexfungorum.org, accessed 20 March 2023), while about 300 species have DNA sequence data to support their taxonomic status within Colletotrichum. Sixteen species complexes have been recognised within Colletotrichum, with C. gloeosporioides species complex as the largest one, which occupies more than 18% of all the recognised taxa of Colletotrichum (Marin-Felix et al. 2017; Damm et al. 2019; Jayawardena et al. 2020; Bhunjun et al. 2021; Mu et al. 2021; Talhinhas and Baroncelli 2021; Alizadeh et al. 2022; Liu et al. 2022; Tsushima and Shirasu 2022; Zheng et al. 2022).

Colletotrichum spp. comprised important plant pathogens, while others are endophytes or saprobes (Cannon et al. 2012; Hyde et al. 2014; Jayawardena et al. 2016). Some Colletotrichum species have been reported causing anthracnose disease on various host plants (Hyde et al. 2014; Nilsson et al. 2014). For instance, the causal agents of ginseng anthracnose were C. lineola and C. panacicola in China (Liu et al. 2020). Anthracnose of Pyrus spp. was caused by 12 Colletotrichum species in China, viz. C. aenigma, C. citricola, C. conoides, C. fioriniae, C. fructicola, C. gloeosporioides, C. karstii, C. plurivorum, C. siamense, C. wuxiense, C. jinshuiense and C. pyrifoliae (Fu et al. 2019). Quite a few species of Collectotrichum have been reported to be causing walnut anthracnose disease, which has resulted in a considerable reduction in walnut production worldwide (Zhu et al. 2014; Wang et al. 2016; Da Lio et al. 2018). For instance, the causal agent of walnut anthracnose identified as Colletotrichum spp. led to 50–70% losses, with some walnut orchards experiencing 100% losses in nut production in France (Giraud and Verhaeghe 2015). Colletotrichum nymphaeae caused anthracnose in walnut in Brazil, destroyed approximately 50% of the fruits and the incidence was higher in rainy and hot summers (Savian et al. 2019).

In China, 12 Colletotrichum species have been reported causing walnut anthracnose. Sever walnut anthracnose occurred in the orchards of Shandong Province, with the causal agents C. gloeosporioides sensu lato, C. siamense, C. fructicola and C. viniferum (Zhu et al. 2014; Wang et al. 2017, 2018; He et al. 2019). The walnut leaf anthracnose caused by C. fioriniae led to severe loss in nut production in Hechi, Guangxi Province (Zhu et al. 2015). In addition, Colletotrichum aenigma caused severe fruit anthracnose in Hebei Province (Wang et al. 2021). Colletotrichum nymphaeae caused walnut branches anthracnose in Gansu Province (Ma et al. 2022). Colletotrichum gloeosporioides, C. kahawae, C. nymphaeae, C. godetiae, C. fioriniae and C. juglandis caused leaf spots of walnut in Hubei Province (Wei et al. 2022). Colletotrichum godetiae caused severe anthracnose of walnut in Shaanxi Province and Yunnan Province with diseased fruits over 60% in the orchard (Wang et al. 2023). Li et al. (2023) collected 900 walnut leaves and 300 fruits samples from seven districts of Beijing and 377 isolates of Colletotrichum spp. were identified into six species, namely C. aenigma, C. fructicola, C. gloeosporioides, C. siamense, C. liaoningense and C. sojae. All of these six species caused anthracnose of walnut and C. gloeosporioides showed the highest virulence.

In the course of an ongoing survey of pathogenic fungi of walnuts in China initiated in 2021, the symptoms on the fruits included round brown spots in the early stage that later turned black. As environmental humidity increased, the spots were covered with orange-red conidiomata. Some spots were merged into large necrotic areas, causing the whole fruit to blacken and rot, resulting in fruit drop. The symptoms on the leaves included nearly round or irregular black or brown spots and gradually withering. A total of 162 isolates were obtained from 30 fruits and 65 leaf samples of walnut collected in the suburb area of Beijing. Their taxonomic status was evaluated, based on morphological characteristics and DNA sequence comparisons and pathogenicity were evaluated by proving Koch’s postulates.

Materials and methods

Sample collection and fungal isolation

Thirty fruits and sixty-five leaf samples exhibiting anthracnose were collected from the suburb area of Beijing, China, in August, 2021. Specimens were transferred to the laboratory and kept in a freezer. Fragments (0.5 × 0.5 cm) were cut aseptically from the margin of the disease lesion and surface-sterilised with 75% ethanol for 30 s, rinsed three times with sterile distilled water, dried on sterilised filter paper and incubated on malt extract agar (MEA; 2%) for isolation of fungal strains (Zhao et al. 2019a). Petri dishes were incubated in the dark at 25 °C until the fungal colonies were observed. Hyphal tips resembling Colletotrichum colonies were transferred to Petri dishes with MEA. Isolates grown on MEA in the dark were kept at 25 °C to determine colony characteristics.

Morphological characterisation

To assess the colony characteristics, mycelial plugs (8 mm in diameter) were transferred from the growing edges of 7-day-old colonies on to PDA and MEA and incubated at 25 °C under dark conditions (Liu et al. 2017a; Zhao et al. 2019a). Colony diameters were measured after 7 days’ incubation (Liu et al. 2017b) and were used to calculate hyphal growth rate (Mo et al. 2018). Morphology and colony characteristics were determined following Damm et al. (2009, 2014) and Choi et al. (2011). Appressoria were induced on slide cultures, according to Weir et al. (2012). The shape, colour and size of conidia, conidiophores, setae, conidiogenous cells and appressoria were measured by at least 20 measurements using a microscope (Nikon Eclipse E600) (Zhao et al. 2019b). Fungal isolates and specimens were deposited at Beijing Forestry University, with duplicates at the China General Microbiological Culture Collection Center (CGMCC) and the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS).

DNA extraction, PCR amplification and sequencing

DNA was extracted from mycelia grown on MEA plates with CTAB plant genome DNA fast extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing, China) and stored at -20 °C until further use. Five loci, including the 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), partial actin (ACT), beta-tubulin (TUB2) and chitin synthase 1 (CHS-1), were amplified using the primer pairs ITS1/ITS4 (White et al. 1990; Gardes and Bruns 1993), GDF1/GDR1 (Guerber et al. 2003), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/Bt2b (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997) and CHS-79F/CHS-345R (Carbone and Kohn 1999), respectively.

PCR amplification and sequencing followed the protocols of Liu et al. (2017a). PCR amplicons were purified and sequenced at BGI Tech Solutions (Beijing Liuhe) Co., Limited (Beijing, China). Forward and reverse were assembled to obtain a consensus sequence with DNAMAN (v. 6.0.3.99; Lynnon Biosoft). Sequences generated in this study were deposited in GenBank (Table 1).

Table 1.
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GenBank accession numbers of isolates included in this study (newly-generated sequences are in bold). * = ex-type or authentic culture, (*) = ex-type or authentic culture of synonymised taxon and N/A = not available. Newly-generated sequences are indicated in bold.

Taxon Isolate designation Host Location GenBank accession number(s)
ITS GAPDH CHS-1 ACT TUB2
Colletotrichum aenigma ICMP 18608* Persea americana Israel JX010244 JX010044 JX009774 JX009443 JX010389
C. aeschynomenes ICMP 17673* Aeschynomene virginica USA JX010176 JX009930 JX009799 JX009483 JX010392
C. alatae CBS 304.67* Dioscorea alata India JX010190 JX010190 JX009837 JX009471 JX010383
C. alienum ICMP 12071* Malus domestica New Zealand JX010251 JX010028 JX009882 JX009572 JX010411
C. aotearoa ICMP 18537* Coprosma sp. New Zealand JX010205 JX010005 JX009853 JX009564 JX010420
C. arecicola CGMCC 3.19667* Areca catechu China MK914635 MK935455 MK935541 MK935374 MK935498
C. artocarpicola MFLUCC 18-1167* Artocarpus heterophyllus Thailand MN415991 MN435568 MN435569 MN435570 MN435567
C. asianum ICMP 18580* Coffea arabica Thailand FJ972612 JX010053 JX009867 JX009584 JX010406
C. australianum VPRI 43075* Citrus sinensis Australia MG572138 MG572127 MW091987 MN442109 MG572149
C. boninense MAFF 305972* = CBS 123755 Crinum asiaticum var. sinicum Japan JQ005153 JQ005240 JQ005327 JQ005501 JQ005588
C. camelliae CGMCC 3.14925 Camellia sinensis China KJ955081 KJ954782 MZ799255 KJ954363 KJ955230
C. changpingense MFLUCC 15-0022 = CGMCC 3.17582* Rhizome of Fragaria × ananass China KP683152 MZ664048 KP852449 KP683093 MZ673952
C. chiangmaiense MFLUCC 18-0945* Magnolia garrettii Thailand MW346499 MW548592 MW623653 MW655578 N/A
C. chrysophilum CMM 4268* Musa sp. Brazil KX094252 KX094183 KX094083 KX093982 KX094285
C. cigarro ICMP 18539* Olea europaea Australia JX010230 JX009966 JX009800 JX009523 JX010434
C. citrulli CAASZT54 Citrullus lanatus China MZ475134 OL456686 OL901154 OL449284 OL456645
CAASZT52 Citrullus lanatus China MZ475133 OL456685 OL901153 OL449283 OL456644
C. clidemiae ICMP 18658* Clidemia hirta USA, Hawaii JX010265 JX009989 JX009877 JX009537 JX010438
C. cobbittiense BRIP 66219* Cordyline stricta × C. australis Australia MH087016 MH094133 MH094135 MH094134 MH094137
C. conoides CGMCC 3.17615 Chili pepper China KP890168 KP890162 KP890156 KP890144 KP890174
C. cordylinicola ICMP 18579* Cordyline fruticosa Thailand JX010226 JX009975 JX009864 HM470235 JX010440
C. dimorphum CGMCC 3.16083* Ageratina adenophora China OK030867 OK513670 OK513566 OK513606 OK513636
YMF 1.07303 Ageratina adenophora China OK030866 OK513669 OK513565 OK513605 OK513635
C. dracaenigenum MFLUCC 19-0430* Dracaena sp. Thailand MN921250 MT215577 MT215575 MT313686 N/A
C. endophyticum MFLUCC 13-0418* Pennisetum purpureum Thailand KC633854 KC832854 MZ799261 KF306258 MZ673954
C. fici-septicae MFLU 19-27708* Capsicum annuum China KP145441 KP145413 KP145385 KP145329 KP145469
C. fructicola ICMP 18581* Coffea arabica Thailand JX010165 JX010033 JX009866 FJ907426 JX010405
C. fructivorum CBS 133125* Vaccinium macrocarpon Burlington JX145145 MZ664047 MZ799259 MZ664126 JX145196
C. gloeosporioides IMI 356878* = ICMP 17821 Citrus sinensis Italy JX010152 JX010056 JX009818 JX009531 JX010445
CBS 273.51(*) = ICMP 19121 Citrus limon Italy JX010148 JX010054 JX009903 JX009558 N/A
DAR 76936 = ICMP 18738 Carya illinoinensis Australia JX010151 JX009976 JX009797 JX009542 N/A
ICMP12939 Citrus sp. New Zealand JX010149 JX009931 JX009747 JX009462 N/A
CBS 119204 = ICMP 18678 Pueraria lobata USA JX010150 JX010013 JX009790 JX009502 N/A
ICMP 12066 Ficus sp. New Zealand JX010158 JX009955 JX009888 JX009550 N/A
ICMP 18730 Citrus sp. New Zealand JX010157 JX009981 JX009861 JX009548 N/A
C. gloeosporioides ICMP 12938 Citrus sinensis New Zealand JX010147 JX009935 JX009746 JX009560 N/A
ICMP 18694 Mangifera indica South Africa JX010155 JX009980 JX009796 JX009481 N/A
ICMP 18695 Citrus sp. USA JX010153 JX009979 JX009779 JX009494 N/A
ICMP 18697 Vitis vinifera USA JX010154 JX009987 JX009780 JX009557 N/A
C. grevilleae CBS 132879* Grevillea sp. Italy KC297078 KC297010 KC296987 KC296941 KC297102
C. grossum CGMCC 3.17614* Chili pepper China KP890165 KP890159 KP890153 KP890141 KP890171
C. hebeiense MFLUCC 13-0726* Vitis vinifera China KF156863 KF377495 KF289008 KF377532 KF288975
C. hederiicola MFLU 15-0689* Hedera helix Italy MN631384 N/A MN635794 MN635795 N/A
C. helleniense CBS 142418* Poncirus trifoliata Greece, Arta KY856446 KY856270 KY856186 KY856019 KY856528
C. henanense CGMCC 3.17354* Camellia sinensis China KJ955109 KJ954810 MZ799256 KM023257 KJ955257
C. horii NBRC 7478* Diospyros kaki Japan GQ329690 GQ329681 JX009752 JX009438 JX010450
C. hystricis CBS 142411* Citrus hystrix Italy, Catania KY856450 KY856274 KY856190 KY856023 KY856532
C. jiangxiense CGMCC 3.17361* Camellia sinensis China KJ955149 KJ954850 MZ799257 KJ954427 OK236389
C. kahawae IMI 319418* Coffea arabica Kenya JX010231 JX010012 JX009813 JX009452 JX010444
C. ledongense CGMCC3.18888* Quercus palustris China MG242008 MG242016 MG242018 MG242014 MG242010
C. makassarense CBS 143664* Capsicum annuum Indonesia MH728812 MH728820 MH805850 MH781480 MH846563
C. mengyinense SAUCC0702* Rosa chinensis China MW786742 MW846240 MW883686 MW883695 MW888970
C. musae CBS 116870* Musa sp. USA JX010146 JX010050 JX009896 JX009433 HQ596280
C. nanhuaensis CGMCC 3.18962* Ageratina adenophora China OK030870 OK513673 OK513569 OK513609 OK513639
YMF 1.04990 Ageratina adenophora China OK030871 OK513674 OK513570 OK513610 OK513640
C. nupharicola CBS 470.96* Nuphar lutea subsp. polysepala USA JX010187 JX009972 JX009835 JX009437 JX010398
C. pandanicola MFLUCC 17-0571* Pandanaceae Thailand MG646967 MG646934 MG646931 MG646938 MG646926
C. perseae CBS 141365* Avocado Israel KX620308 KX620242 MZ799260 KX620145 KX620341
C. proteae CBS 132882* Protea sp. South Africa KC297079 KC297009 KC296986 KC296940 KC297101
C. pseudotheobromicola MFLUCC 18-1602* Prunus avium China MH817395 MH853675 MH853678 MH853681 MH853684
C. psidii CBS 145.29* Psidium sp. Italy JX010219 JX009967 JX009901 JX009515 JX010443
C. queenslandicum ICMP 1778* Carica papaya Australia JX010276 JX009934 JX009899 JX009447 JX010414
C. rhexiae CBS 133134* Rhexia virginica Sussex JX145128 MZ664046 MZ799258 MZ664127 JX145179
C. salsolae ICMP 19051* Salsola tragus Hungary JX010242 JX009916 JX009863 JX009562 JX010403
C. siamense ICMP 18578* Coffea arabica Thailand JX010171 JX009924 JX009865 FJ907423 JX010404
C. syzygicola MFLUCC 10-0624* Syzygium samarangense Thailand KF242094 KF242156 N/A KF157801 KF254880
C. tainanense CBS 143666* Capsicum annuum Taiwan MH728818 MH728823 MH805845 MH781475 MH846558
C. temperatum CBS 133122* Vaccinium macrocarpon Bronx JX145159 MZ664045 MZ799254 MZ664125 JX145211
C. tengchongense YMF 1.04950 Isoetes sinensis China OL842169 OL981264 OL981290 OL981238 N/A
C. theobromicola CBS 124945* Theobroma cacao Panama JX010294 JX010006 JX009869 JX009444 JX010447
C. ti ICMP 4832* Cordyline sp. New Zealand JX010269 JX009952 JX009898 JX009520 JX010442
C. tropicale CBS 124949* Theobroma cacao Panama JX010264 JX010007 JX009870 JX009489 JX010407
C. viniferum GZAAS 5.08601* Vitis vinifera cv. Shuijing China JN412804 JN412798 N/A JN412795 N/A
C. vulgaris YMF 1.04940 Hippuris vulgaris China OL842170 OL981265 OL981291 OL981239 N/A
C. wuxiense CGMCC 3.17894* Camellia sinensis China KU251591 KU252045 KU251939 KU251672 KU252200
C. xanthorrhoeae BRIP 45094* Xanthorrhoea preissii Australia JX010261 JX009927 JX009823 JX009478 JX010448
C. xishuangbannaense MFLUCC 19-0107* Magnolia liliifera China MW346469 MW537586 MW660832 MW652294 N/A
C. yulongense CFCC 50818* Vaccinium dunalianum var. urophyllum China MH751507 MK108986 MH793605 MH777394 MK108987
C. yunanjiangensis CGMCC 3.18964* Ageratina adenophora China OK030885 OK513686 OK513583 OK513620 OK513649
C. peakense CGMCC3.24308* Juglans regia China OQ263017 OQ282975 OR004795 OQ282968 OQ282982
CGMCC3.24307 Juglans regia China OQ263016 OQ282974 OR004794 OQ282967 OQ282981
C. juglandicola CGMCC3.24312* Juglans regia China OQ263015 OQ282973 OR004793 OQ282966 OQ282980
CGMCC3.24313 Juglans regia China OQ263018 OQ282977 OR004797 OQ282970 OQ282984
CGMCC3.24310 Juglans regia China OQ263020 OQ282979 OR004799 OQ282972 OQ282986
CGMCC3.24309 Juglans regia China OQ263021 OQ282978 OR004798 OQ282971 OQ282985
CGMCC3.24311 Juglans regia China OQ263019 OQ282976 OR004796 OQ282969 OQ282983

Phylogenetic analysis

DNA sequences of concatenated ACT, CHS-1, GAPDH, ITS and TUB2 loci were analysed to investigate the phylogenetic relationships amongst Colletotrichum species with DNA sequences available from GenBank (http://www.ncbi.nlm.nih.gov/genbank/), as well as the sequences generated herein (Table 1). Multiple sequences were aligned using the MAFFT v.7.110 (http://mafft.cbrc.jp/alignment/server/) and adjusted manually in MEGA v.7.0 (Kumar et al. 2016). Gaps were manually adjusted to optimise the alignment (Tamura et al. 2013).

Phylogenetic analyses of Maximum Likelihood (ML), Bayesian Inference (BI) and Maximum Parsimony (MP) 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. The Bayesian phylogenetic analysis was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.2.6 (Ronquist et al. 2012). Four MCMC chains were run from random trees for 2,000,000 generations and trees were sampled by each 1,000th generation. The first 25% of the trees of MCMC sampling were discarded as burn-in and posterior probabilities (PP) were determined from the remaining trees. Maximum Parsimony (MP) analysis, based on the concatenated dataset, was conducted in PAUP* v. 4.0b10 with the default options (Swofford 2002). Ambiguous regions in the alignment were excluded and gaps were treated as missing data. Clade stability was evaluated in a bootstrap analysis with 1,000 replicates with Maxtrees set to 1,000 and other default parameters implemented in PAUP* (Hillis and Bull 1993). Other measurements calculated parsimony scores including consistency index (CI), rescaled consistency (RC), homoplasy index (HI) and retention index (RI). The phylogenetic trees were configured in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited using Adobe Illustrator CC2020 (Adobe Systems Inc., USA).

Sequences were analysed using the GCPSR model by performing a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. (2014) for the phylogenetically close, but not clearly delimited species. The PHI test was performed in SplitsTree v. 4.19.1 (Huson and Bryant 2006) to determine the recombination level within phylogenetically closely-related species using a five-locus concatenated dataset (ACT, CHS-1, GAPDH, ITS and TUB2). If the resulting pairwise homoplasy index was below a 0.05 threshold (Фw < 0.05), it was indicative of significant recombination in the dataset. The relationship between closely-related species was visualised by constructing a split graph (Chethana et al. 2021; Jayawardena et al. 2021; Peng et al. 2023).

Pathogenicity tests and virulence on walnut tissues

All isolated species were tested for their pathogenicity on walnut fruits and leaves. Isolates of all species were incubated on MEA plates for 7 days prior to inoculation. Spore suspension of isolates of Colletotrichum juglandicola (CGMCC3.24312) and Colletotrichum peakense (CGMCC3.24308) obtained in this study were used for pathogenicity testing.

The pathogenicity test was performed on detached living walnut fruits and leaves. Briefly, fruits and leaves were washed with sterilised water and surface sterilised with 75% ethanol for 1 min. The fruits and leaves were inoculated using the spore suspension and non-wound inoculation methods (Fu et al. 2019; Zhao et al. 2019b). For the spore suspension and non-wound method, an aliquot of 20 μl of spore suspension (1.0 × 106 conidia per ml) was inoculated on to fruits and leaves without wounding them. Eight replicates were used for each isolate. Sterilised water was used as the negative control. The inoculated detached fruits and leaves were incubated under 25 °C with 12/12 h light/dark photoperiod. Pathogenicity was determined by measuring the lesion length of fruits and leaves after 10 days’ incubation. Mean comparisons were conducted using Tukey’s honest significant difference (HSD) test (α = 0.05) in R (Version 3.2.2, R Inc. Auckland, NZL).To fulfil Koch’s postulates, small pieces of infected tissue were plated on to MEA to re-isolate the fungal isolates, which were identified, based on morphology and DNA sequences.

Results

Phylogenetic analyses

The concatenated ACT, CHS-1, GAPDH, ITS and TUB2 dataset (1,948 characters with 369 parsimony-informative characters) from 79 in-group isolates of Colletotrichum gloeosporioides species complex was used for phylogenetic analysis. The outgroup taxon was C. boninense CBS 123755. The heuristic search with random addition of taxa (1,000 replicates) generated 5,000 most parsimonious trees (Length = 1,313, CI = 0.673, HI = 0.327, RI = 0.854, RC = 0.575). The topologies obtained from the Maximum Parsimony, Maximum Likelihood and Bayesian analysis were comparable. In three analyses (ML, BI and MP), Colletotrichum juglandicola and Colletotrichum peakense are consistently sibling to all other species of C. gloeosporioides species complex (95/1/89 and 100/0.98/58) (Fig. 1). Additionally, only the ML tree is presented here, with ML, BI and MP values plotted on the branches (Fig. 1).

Figure 1. 

Phylogenetic tree of Maximum Likelihood analyses of 86 isolates in the C. gloeosporioides species complex. The species C. boninense (CBS 123755) was selected as an outgroup. The tree was built using concatenated sequences of ACT, CHS-1, GAPDH, ITS and TUB2 genes. RAxML bootstrap support values (ML ≥ 50%), Bayesian posterior probability (PP ≥ 0.90) and MP bootstrap support values (ML ≥ 50%) are shown at the nodes (ML/PP/MP).

To exclude the possibility that species delimitation might be interfered by recombination amongst the genes used for phylogenetic analyses, the multi-locus (ACT, CHS-1, GAPDH, ITS and TUB2) concatenated datasets were subjected to two PHI tests (Fig. 2) to determine the recombination level within phylogenetically closely-related species. The results showed that no significant recombination events were observed between Colletotrichum juglandicola and phylogenetically related isolates or species (C. gloeosporioides and C. dimorphum) (Fig. 2A) and between C. peakense and phylogenetically related species (C. citrulli, C. gloeosporioides and C. dimorphum) (Fig. 2B).

Figure 2. 

The result of the pairwise homoplasy index (PHI) tests of closely-related species using both LogDet transformation and splits decomposition A, B the PHI of C. juglandicola (A) or C. peakense (B) and their phylogenetically related isolates or species, respectively. PHI test value (Φw) < 0.05 indicate significant recombination within the dataset.

Taxonomy

Colletotrichum juglandicola Y. Zhang ter. & L. Zhang, sp. nov.

MycoBank No: MycoBank No: 848731
Fig. 3

Etymology

Named from “Juglans”, in reference to the host genus.

Description

Sexual morph not observed. Asexual morph developed on MEA. Conidiomata acervular, yellow to light brown, bearing conidial masses. Conidiophores hyaline, smooth-walled, septate, branched. Setae medium to dark brown, smooth to finely verruculose close to the tip, the tip rounded, 1–3 aseptate, 60–107.2 μm long. Conidiogenous cells 19.5–38.9 × 2.8–3.9 μm (mean SD = 28.6 ± 1.2 × 3.3 ± 0.1 μm, n = 20), subcylindrical, straight to curved. Conidia 14.6–20.0 × 4.2–6.6 μm (mean SD = 17.1 ± 1.0 × 5.2 ± 0.4 μm, L/W radio = 3.3, n = 100), hyaline, smooth-walled, subcylindrical, both ends round, 1–3-guttulate, contents granular. Appressoria 5–8.3 × 3.3–6.7 μm (mean SD = 6.3 ± 0.2 × 5.2 ± 0.2 μm, L/W radio = 1.2, n = 20), medium to dark brown, variable in shape, often smooth-walled, subglobose, ovate to broadly elliptical in outline.

Culture characteristics

Colonies on MEA, flat, with entire margin, hyaline, 65–72 mm diam. in 7 d. The colonies are round, white, the edges are flat and the aerial hyphae are lush. Myxospores are orange. The colony diameter reached 63–65 mm on PDA. The colonies are round, green-grey, the edges are flat and the aerial hyphae are lush.

Additional specimens examined

China, Beijing, Changping District, Heishanzhai Village, from leaf of Juglans regia L., Y. Zhang and L. Zhang, 26 August 2021 (holotype HSG826-P5; ex-type living culture: CGMCC3.24312). CHINA, Beijing, Huairou District, Shuichangcheng Village, from leaf of Juglans regia L., Aug 2021, Y. Zhang and L. Zhang (Paratype SCCY826-22; living culture: CGMCC3.24313). CHINA, Beijing, Haidian District, Jiufeng Village, from fruit of Juglans regia L., Aug 2021, Y. Zhang and L. Zhang (Paratype JFG826-P4; living culture CGMCC3.24311). China, Beijing, Changping District, Yanshou Village, from fruit of Juglans regia L., Aug 2021, Y. Zhang and L. Zhang (Paratype YSG826-R1; living culture CGMCC3.24309). CHINA, Beijing, Changping District, Yanshou Village, from leaf of Juglans regia L., Aug 2021, Y. Zhang and L. Zhang (Paratype YSY826-2: living culture CGMCC3.24310).

Figure 3. 

Colletotrichum juglandicola (from ex-type CGMCC3.24312) A, B colonies and reverse after 7 days on PDA medium C, D colonies and reverse after 7 days on MEA medium E conidiomata F, G conidia H conidiophores I, J setae K–N appressoria. Scale bars: 500 μm (E); 10 μm (F–N).

Notes

Phylogenetic analysis of a concatenated five loci dataset indicated that the clade of Colletotrichum juglandicola nested in the clade of C. gloeosporioides species complex and was closely related, but independent to C. citrulli, C. dimorphum, C. gloeosporioides and C. nanhuaensis (Cannon et al. 2008; Guo et al. 2022; Yu et al. 2022). Colletotrichum citrulli was reported from Citrullus lanatus (Cucurbitaceae) in China (Guo et al. 2022). Morphologically, C. juglandicola differed from C. citrulli by having longer conidia and setae and smaller appressoria (Table 2) (Guo et al. 2022). Colletotrichum dimorphum was reported from Ageratina adenophora (Asteraceae) in China (Guo et al. 2022). Morphologically, C. juglandicola differed from C. dimorphum by having setae, shorter appressoria and longer conidia (Yu et al. 2022) (Table 2). Morphologically, C. juglandicola differed from C. gloeosporioides or C. nanhuaensis by having longer conidia (Cannon et al. 2008; Guo et al. 2022) (Table 2). Colletotrichum nanhuaensis was reported from Ageratina adenophora (Asteraceae) in China (Guo et al. 2022) (Table 2). The PHI test (Φw = 1.0) detected no significant recombination between related isolates or species (Fig. 2A).

Table 2.
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Morphological comparison of species in the gloeosporioides species complex.

Species Type Hosts Distribution Conidia (Mean ± SD) (μm) Appressoria (μm) Setae (μm) Reference
Colletotrichum citrulli Holotype Citrullus lanatus China 16.2 ± 0.9 × 5.6 ± 0.5 8.0–12.0 × 6.0–10.0 42.0–79.0 Guo et al. (2022)
C. aenigma Holotype Persea americana Israel 14.5 × 6.1 6.0–10.0 Not observed Weir et al. (2012)
C. dimorphum Holotype Ageratina adenophora China 14.6 ± 2 × 4.8 ± 0.7 5.7–10.6 × 5.0–9.0 Not observed Yu et al. (2022)
C. fructicola Holotype Coffea arabica Thailand 11.5 ± 1.0 × 3.6 ± 0.3 4.3–9.7 × 3.7–7.3 Not observed Prihastuti et al. (2009)
C. gloeosporioides epitype Citrus sinensis Italy 14.4 × 5.6 7.2–8.6 × 4.7–6.0 40.0–120.0 Cannon et al. (2008)
C. juglandicola Holotype Juglans regia L. China 17.1 ± 1.0 × 5.2 ± 0.4 5–8.3 × 3.3–6.7 60.0–107.2 This study
C. kahawae Holotype Coffeae arabicae Kenya 12.5–19.0 × 4.0 8.0–9.5 × 5.5–6.5 Not observed Waller et al. (1993)
C. mengyinense Holotype Rosa chinensis China 14.3 ± 1.1 × 5.3 ± 0.4 Not observed Not observed Mu et al. (2021)
C. nanhuaensis Holotype Ageratina adenophora China 14.0 ± 1.1 × 5.4 ± 0.4 8.0–14.0 × 5.0–8.0 25.0 Yu et al. (2022)
C. peakense Holotype Juglans regia L. China 16.4 ± 1.4 × 4.9 ± 0.5 5.6–8.4 × 3.9–6.1 57.2–152.9 This study
C. siamense Holotype Coffea arabica Thailand 10.2 ± 1.7 × 3.5 ± 0.4 4.7–8.3 × 3.5–5.0 Not observed Prihastuti et al. (2009)
C. viniferum Holotype Vitis vinifera China 13.8 ± 1.0 × 5.4 ± 0.4 6.5–10.5 × 4.8–6.3 Not observed Peng et al. (2013)

Colletotrichum peakense Y. Zhang ter. & L. Zhang, sp. nov.

MycoBank No: MycoBank No: 848730
Fig. 4

Etymology

Named after Beijing where the fungus was collected.

Description

Sexual morph not observed. Asexual morph developed on MEA. Conidiomata acervular, yellow, bearing conidial masses. Conidiophores hyaline, smooth-walled, septate and branched. Setae medium to dark brown, smooth to finely verruculose close to the tip, the tip rounded, 1–3 aseptate, 57.2–152.9 μm long. Conidiogenous cells 20–35.6 × 2.8–3.9 μm (mean SD = 26.1 ± 0.9 × 3.0 ± 0.1 μm, n = 20), subcylindrical, straight to curved. Conidia 13.5–20.5 × 3.1–5.9 μm (mean SD = 16.4 ± 1.4 × 4.9 ± 0.5 μm, L/W radio = 3.3, n = 100), hyaline, smooth-walled, subcylindrical, both ends round, 1–3-guttulate, contents granular. Appressoria 5.6–8.4 × 3.9–6.1 μm (mean SD = 6.7 ± 0.2 × 5.1 ± 0.1 μm, L/W radio = 1.3, n = 20), medium to dark brown, variable in shape, often smooth-walled, subglobose, ovate to broadly elliptical in outline.

Figure 4. 

Colletotrichum peakense (from ex-type CGMCC3.24308) A, B colonies and reverse after 7 days on PDA medium C, D colonies and reverse after 7 days on MEA medium E conidiomata F, G conidia H, I conidiophores J setae K–N appressoria. Scale bars: 500 μm (E); 10 μm (F–N).

Asexual morph developed on PDA. Conidia 14.7–22.2 × 4.1–6.3 μm (mean SD = 17.4 ± 1.6 × 5.2 ± 1.6 μm, L/W radio = 3.3, n = 50), hyaline, smooth-walled, subcylindrical, both ends round, 1–3-guttulate, contents granular.

Culture characteristics

Colonies on MEA, flat, with entire margin, hyaline, 68–78 mm diam. in 7 d. The colonies are round, aerial mycelium white or grey, floccose cottony; surface and reverse grey in the centre and white margin. Myxospores are orange. The colony diameter reached 76–80 mm on PDA. The colonies are round, aerial mycelium white or grey, floccose cottony; surface and reverse grey in the centre and white margin.

Additional specimens examined

China, Beijing, Changping District, Heishanzhai Village, from leaf of Juglans regia L., 26 Aug 2021, Y. Zhang and L. Zhang (holotype HSY826-18; ex-type living culture, CGMCC3.24308. China, Beijing, Changping District, Heishanzhai Village, from leaf of Juglans regia L., 26 Aug 2021, Y. Zhang and L. Zhang (Paratype HSY826-18): living culture, CGMCC3.24307.

Notes

Phylogenetic analysis of a concatenated five loci dataset indicated that the clade of Colletotrichum peakense nested in the clade of C. gloeosporioides species complex and was closely related, but independent to C. citrulli, C. dimorphum, C. gloeosporioides and C. nanhuaensis (Cannon et al. 2008; Guo et al. 2022; Yu et al. 2022). Morphologically, Colletotrichum peakense was distinguishable from C. citrulli by having longer setae and smaller appressoria (Guo et al. 2022) (Table 2), while from C. dimorphum by having longer conidia and longer setae (Yu et al. 2022) (Table 2), from C. gloeosporioides by having longer conidia (Cannon et al. 2008) (Table 2) and from C. nanhuaensis by having longer conidia and shorter appressoria (Guo et al. 2022) (Table 2). The PHI test (Φw = 1.0) detected no significant recombination between related isolates or species-related species (Fig. 2B).

Pathogenicity tests on walnut tissues

Pathogenicity tests were conducted to confirm Koch’s postulates on fruits and leaves of walnut for C. juglandicola and C. peakense. The symptom of circular, necrotic, sunken lesions on fruits and as circular, necrotic lesions on leaves after 10 days of inoculation with typical orange conidial masses were observed from the inoculated site, whereas all control fruits and leaves remained healthy (Fig. 5). For spore suspension and non-wound methods, both on fruits and leaves, the lesion diameter of C. peakense was significantly higher than C. juglandicola (P < 0.05) (Table 3). Furthermore, Colletotrichum isolates could consistently be re-isolated from symptomatic lesions, but never from control. Koch’s postulates were performed by successful pathogen re-isolation from all the necrotic fruits and leaves. The morphology and DNA sequences of these new isolates were consistent with the initial inoculation.

Figure 5. 

Anthracnose symptoms on walnut fruits and leaves caused by C. peakense and C. juglandicola A anthracnose caused by C. juglandicola on leaf B anthracnose caused by C. peakense on leaf C anthracnose fruits caused by C. juglandicola D, G symptoms of C. juglandicola (CGMCC3.24312) using spore suspension and non-wound inoculation methods after 10 days inoculation on walnut fruit (D) and leaf (G) E, H symptoms of C. peakense (CGMCC3.24308) using spore suspension and non-wound inoculation methods after 10 days inoculation on walnut fruit (E) and leaf (H) F, I symptoms resulting from sterilised water and non-wound inoculation methods after 10 days inoculation on walnut fruit (F) and leaf (I).

Table 3.
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Pathogenicity of Colletotrichum juglandicola (CGMCC3.24312) and Colletotrichum peakense (CGMCC3.24308) on walnut fruits and leaves using spore suspension as inoculum 10 days after inoculation.

Species Walnut fruits inoculated with Spore suspension and non-wound ± SD (mm) Walnut leaves inoculated with Spore suspension and non-wound ± SD (mm)
Colletotrichum juglandicola 5.80 ± 1.27 b 8.90 ± 2.28 b
C. peakense 9.50 ± 1.0 a 16.79 ± 2.58 a
Non-inoculated control 0 ± 0 c 0 ± 0 c

Note: Data followed by different letters in each column are significantly different, based on HSD tests at the P < 0.05 level.

Discussion

Phylogenetic analyses, based on five concatenated loci (ACT, CHS-1, GAPDH, ITS and TUB2), indicated that either Colletotrichum juglandicola or C. peakense formed a distinct clade within the C. gloeosporioides complex, while sibling to other species (Fig. 1). On the phylogenetic tree, C. juglandicola is closely related to C. citrulli, C. dimorphum, C. gloeosporioides and C. nanhuaensis (Fig. 3). Morphologically, C. juglandicola can be readily distinguished from C. citrulli, C. dimorphum, C. gloeosporioides and C. nanhuaensis, based on its longer conidial size, presence or absence of setae and appressoria size (Table 2). Phylogenetically, C. peakense is closely related to C. citrulli, C. dimorphum, C. gloeosporioides and C. nanhuaensis (Fig. 4). Colletotrichum peakense can be distinguishable from C. citrulli, C. dimorphum, C. gloeosporioides and C. nanhuaensis by its longer setae and smaller appressorial size (Table 2).

Thus far, 14 species of Colletotrichum have been reported from Juglans regia L., namely C. acutatum, C. fioriniae, C. godetiae, C. juglandis and C. nymphaeae of the Acutatum species complex, C. aenigma, C. fructicola, C. gloeosporioides, C. kahawae, C. mengyinense, C. siamense and C. viniferum of Gloeosporioides species complex, C. liaoningense of Magnum species complex and C. sojae of Orchidearum species complex (Simmonds 1966; Alvarez 1976; Gorter 1977; Pennycook 1989; Liu et al. 1995; Crous et al. 2000; Chen 2003; Cho and Shin 2004; Gadgil et al. 2005; Juhásová et al. 2005; Sreenivasaprasad and Talhinhas 2005; Kobayashi 2007; Qu et al. 2011; Damm et al. 2012; Zhu et al. 2014; Zhu et al. 2015; Wang et al. 2017, 2018; Da Lio et al. 2018; He et al. 2019; Savian et al. 2019; Wang et al. 2020; Wang et al. 2021, 2023; Luongo et al. 2022; Ma et al. 2022; Wei et al. 2022; Li et al. 2023), of which, Colletotrichum acutatum, C. fioriniae, C. godetiae, C. juglandis and C. nymphaeae differed from C. juglandicola and C. peakense by their acute-ended conidia (Damm et al. 2012). Colletotrichum aenigma, C. fructicola, C. gloeosporioides, C. kahawae, C. mengyinense, C. siamense and C. viniferum differed from C. juglandicola and C. peakense, by the size of conidia (Table 2). The conidia shape of Colletotrichum juglandicola and C. peakense was comparable to C. liaoningense, while the longer conidia and longer appressoria size were distinguishable from the latter (Diao et al. 2017) (Table 2). Colletotrichum juglandicola and C. peakense were distinguishable from C. sojae by their shorter appressoria (Damm et al. 2019) (Table 2).

Pathogenicity tests indicated that both Colletotrichum juglandicola and C. peakense cause anthracnose disease in walnut fruits and leaves. Both on fruits and leaves, the virulence of C. peakense was more severe than C. juglandicola (P < 0.05). Colletotrichum gloeosporioides had been reported more severe than most other species in Beijing, which was supported by the current study in that C. gloeosporioides was more severe than C. juglandicola (12.33 ± 0.29 mm in 4 days vs. 8.90 ± 2.28 mm in 10 days) (Li et al. 2023).

Both Colletotrichum juglandicola and C. peakense belong to the C. gloeosporioides species complex, which has been reported as one of the most important pathogens worldwide and has infected at least 1,000 plant species (Phoulivong et al. 2010). Colletotrichum gloeosporioides species complexes could be either broad or narrow host ranges (Crouch et al. 2014; Liu et al. 2022). It appears that some species of Colletotrichum, such as C. horii (on persimmon) and C. kahawae (on coffee) may be restricted to certain hosts genera or families, while some others may have a wide range of hosts. For instance, C. fructicola has been reported from walnut, coffee, chilli, longan, shine muscat, papaya and tea (Prihastuti et al. 2009; Yang et al. 2009; Sharma and Shenoy 2014; Wang et al. 2018; Lim et al. 2020; Lin et al. 2021). To summarise, in Beijing, C. juglandicola and C. peakense, two species new to science, were the causal agents of walnut anthracnose.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the National Natural Science Foundation of China (General Program) under grant nos. 31971658, 31770015 and 31370063; and the National Natural Science Foundation of China Projects of International Cooperation and Exchanges under grant no. 3155461143028.

Author contributions

YZ designed the experiments. YZ, LZ and LLZ prepared the samples, conducted the molecular experiments, and analyzed the data. LZ drafted the manuscript. YZ, LZ, YQY, LLZ, YQX and JH revised the manuscript. All authors contributed to the article and approved the submitted version.

Author ORCIDs

Lin Zhang https://orcid.org/0009-0002-6325-1440

Yue-Qi Yin https://orcid.org/0009-0009-0756-5075

Li-Li Zhao https://orcid.org/0000-0003-1451-3301

Yu-Qing Xie https://orcid.org/0009-0009-8720-3276

Ying Zhang https://orcid.org/0000-0001-8817-6032

Data availability

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

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