Research Article |
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
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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.
Anthracnose, multi-gene phylogeny, pathogenicity, walnut
Walnut (Juglans regia L.), a deciduous tree, is an essential woody nut and oil crop cultivated worldwide (
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 (
Colletotrichum spp. comprised important plant pathogens, while others are endophytes or saprobes (
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 (
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.
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 (
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 (
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 (
PCR amplification and sequencing followed the protocols of
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 |
|
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 |
|
Camellia sinensis | China | KJ955081 | KJ954782 | MZ799255 | KJ954363 | KJ955230 |
C. changpingense | MFLUCC 15-0022 = |
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 |
|
Chili pepper | China | KP890168 | KP890162 | KP890156 | KP890144 | KP890174 |
C. cordylinicola | ICMP 18579* | Cordyline fruticosa | Thailand | JX010226 | JX009975 | JX009864 | HM470235 | JX010440 |
C. dimorphum |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
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
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 (
Sequences were analysed using the GCPSR model by performing a pairwise homoplasy index (PHI) test as described by
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 (
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.
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.
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.
Named from “Juglans”, in reference to the host genus.
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.
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.
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).
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 (
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 |
|
C. aenigma | Holotype | Persea americana | Israel | 14.5 × 6.1 | 6.0–10.0 | Not observed |
|
C. dimorphum | Holotype | Ageratina adenophora | China | 14.6 ± 2 × 4.8 ± 0.7 | 5.7–10.6 × 5.0–9.0 | Not observed |
|
C. fructicola | Holotype | Coffea arabica | Thailand | 11.5 ± 1.0 × 3.6 ± 0.3 | 4.3–9.7 × 3.7–7.3 | Not observed |
|
C. gloeosporioides | epitype | Citrus sinensis | Italy | 14.4 × 5.6 | 7.2–8.6 × 4.7–6.0 | 40.0–120.0 |
|
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 |
|
C. mengyinense | Holotype | Rosa chinensis | China | 14.3 ± 1.1 × 5.3 ± 0.4 | Not observed | Not observed |
|
C. nanhuaensis | Holotype | Ageratina adenophora | China | 14.0 ± 1.1 × 5.4 ± 0.4 | 8.0–14.0 × 5.0–8.0 | 25.0 |
|
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 |
|
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) |
Named after Beijing where the fungus was collected.
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.
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.
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.
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.
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 (
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.
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).
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 |
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.
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 (
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) (
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 (
The authors have declared that no competing interests exist.
No ethical statement was reported.
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.
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.
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
All of the data that support the findings of this study are available in the main text.