Research Article |
Corresponding author: Cheng-Ming Tian ( chengmt@bjfu.edu.cn ) Academic editor: Nalin Wijayawardene
© 2019 Ning Jiang, Hermann Voglmayr, Cheng-Ming Tian.
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:
Jiang N, Voglmayr H, Tian C-M (2019) Morphology and phylogeny reveal two novel Coryneum species from China. MycoKeys 56: 67-80. https://doi.org/10.3897/mycokeys.56.35554
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Coryneum is currently the sole genus of Coryneaceae in Diaporthales, distinguished from other diaporthalean genera by transversely distoseptate brown conidia. However, Coryneum species are presently difficult to identify because of variability and overlap of morphological characters and the lack of sequence data for most described species. During fungal collection trips in China, 13 Coryneum isolates were obtained from cankered branches of Ilex and Quercus. Morphological and phylogenetic analyses (ITS, LSU, TEF1-α and RPB2) revealed that these strains belong to two new species (viz. Coryneum ilicis sp. nov. and C. songshanense sp. nov.), and three known species, C. gigasporum, C. sinense, and C. suttonii. Coryneum ilicis has larger conidia and more distosepta than most Coryneum species. Coryneum songshanense was similar to C. sinense from the same host genus, Quercus, in conidial length, but distinct in conidial width and by molecular data.
Coryneaceae, Diaporthales, systematics, taxonomy
The genus Coryneum Nees is currently the only accepted genus in Coryneaceae and it forms a distinct phylogenetic lineage in Diaporthales (
Most Coryneum species were considered as phytopathogens, which were discovered from cankers and dieback of shoots and twigs (
Coryneum species are generally considered highly host-specific, and 28 species and a variety were accepted in this genus before this study (
Hosts, conidial sizes, and numbers of distosepta of currently accepted Coryneum species.
Species | Host genus | Host family | Host order | Conidial size (μm) | No. of distosepta | References |
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C. arausiacum | Quercus | Fagaceae | Fagales | 42–56 × 13–16 | 4–5 |
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C. betulinum | Betula | Betulaceae | Fagales | 31–36 × 14–17 | 4–5 |
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C. calophylli | Calophyllum | Guttiferae | Parietales | 38–48 × 12.5–14.5 | 5–6 |
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C. carpinicola | Carpinus | Betulaceae | Fagales | 50–68 × 8–11 | 7–11 |
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C. castaneicola | Castanea | Fagaceae | Fagales | 56–80 × 9.5–13 | 5–8 |
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C. cesatii | Aesculus | Hippocastanaceae | Sapindales | 80–90 × 13–15 | 6–7 |
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C. clusiae | Clusia | Clusiaceae | Malpighiales | 30–40 × 20–30 | 3–5 |
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C. compactum | Ulmus | Ulmaceae | Urticales | 40–58 × 15–21 | 4–6 |
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C. depressum | Quercus | Fagaceae | Fagales | 44–53 × 19–23 | 4–6 |
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C. elevatum | Quercus | Fagaceae | Fagales | 56–69 × 24–28 | 5–7 |
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C. gigasporum | Castanea | Fagaceae | Fagales | 88–117 × 18–23 | 7–9 |
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C. gregoryi | Eucalyptus | Myrtaceae | Myrtales | 32.5–43 × 12–16 | 5–9 | Sutton and Sharma (1983) |
C. heveanum | Hevea | Euphorbiaceae | Malpighiales | 40–68 × 14–20 | 4–6 |
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C. ilicis | Ilex | Aquifoliaceae | Sapindales | 82–105 × 9.5–12.5 | 10–11 | This study |
C. japonicum | Quercus | Fagaceae | Fagales | 45–60 × 11–12 | 5–7 |
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C. lanciforme | Betula | Betulaceae | Fagales | 45–53 × 16–18 | 4–6 |
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C. megaspermum | Quercus | Fagaceae | Fagales | 73–97 × 13–16 | 7–11 |
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C. megaspermum var. cylindricum | Quercus | Fagaceae | Fagales | 100–125 × 10–13 | 7–8 |
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C. modonium | Castanea | Fagaceae | Fagales | 50–71 × 14–19 | 5–8 |
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C. neesii | Quercus | Fagaceae | Fagales | 68–82 × 18–22 | 6–8 |
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C. pruni | Prunus | Rosaceae | Rosales | 14–23 × 5.5–9 | 4–5 |
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C. psidii | Psidium | Myrtaceae | Myrtales | 25–40 × 14–17 | 5–6 |
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C. pyricola | Pyrus | Rosaceae | Rosales | 61–70 × 24–32 | 5–7 |
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C. quercinum | Quercus | Fagaceae | Fagales | 45–60 × 14–16 | 6–7 |
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C. sinense | Quercus | Fagaceae | Fagales | 50–76 × 13–17 | 5–7 |
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C. songshanense | Quercus | Fagaceae | Fagales | 51–76 × 9–11.5 | 5–7 | This study |
C. stromatoideum | Tsuga | Pinaceae | Pinales | 105–180 × 16–20 | 9–17 |
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C. suttonii | Castanea | Fagaceae | Fagales | 60–76 × 10–14.5 | 4–5 |
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C. sydowianum | Alnus | Betulaceae | Fagales | 50–58 × 14–17 | 5–6 |
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C. terrophilum | NA | NA | NA | 25–55 × 15–24 | 3–7 | Sutton and Sharma (1983) |
C. umbonatum | Quercus | Fagaceae | Fagales | 57–72 × 13–16 | 5–7 |
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Molecular phylogenies based on multi-gene loci including the internal transcribed spacer (ITS) and the large subunit (LSU) regions of the nuclear rDNA, translation elongation factor-1α (TEF1-α) and the second largest subunit of the RNA polymerase II (RPB2) have been widely used to infer species delimitation within many genera in Diaporthales (
Sample collection trips were conducted in Beijing, Hebei and Shaanxi Provinces of China during June to October in 2017 and 2018, aiming to collect fresh specimens with Coryneum–like taxa. Fagales plants were the main hosts and other hardwoods with rough barks were also investigated. Healthy branches and twigs were covered by green leaves, hence the dying and dead materials were conspicuous during our investigations. Asexual fruiting bodies were easily discovered as black spots on the host barks. Tree tissues with fruiting bodies were cut into small pieces, packed in paper bags and taken to the laboratory for further studies. Isolations were obtained by removing the ascospores or conidial masses from the fruiting bodies on to clean potato dextrose agar (PDA) plates, which were incubated at 25 °C until spores germinated. Single germinating spores were transferred on to new PDA plates, which were kept at 25 °C in the dark. Specimens were deposited at the Museum of the Beijing Forestry University (
Species identification was based on the morphological characters of the sexual and asexual morphs produced on natural substrates. Cross-sections were prepared manually using a double-edged blade under a Leica stereomicroscope (M205 FA). Photomicrographs were captured with a Nikon Eclipse 80i microscope equipped with a Nikon digital sight DS-Ri2 high-definition colour camera, using differential interference contrast (DIC) illumination and the Nikon software, NIS-Elements D Package 3.00. Measurements of ascospores and conidia are reported as the maximum and minimum in parentheses and the range representing the mean ± standard deviation of the number of measurements is given in parentheses (
Recognition and identification of Coryneum species were based on fruiting bodies formed on tree bark, supplied by conidiomata produced on PDA plates. Ascomata and conidiomata from tree bark were sectioned by hand using a double-edged blade, and conidiomata from PDA plates were picked using a needle, which were observed under a dissecting microscope. At least 10 conidiomata/ascomata, 10 asci, and 50 conidia/ascospores were measured to calculate the mean sizes and standard deviation. Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination.
Genomic DNA was extracted from colonies grown on cellophane-covered PDA plates using a modified CTAB method (
Strains used in the phylogenetic tree and their culture accession and GenBank numbers. Strains from this study are in bold.
Species | Strains | GenBank numbers | |||
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ITS | LSU | TEF1-α | RPB2 | ||
Coryneum castaneicola | CFCC 52315 | MH683551 | MH683559 | MH685731 | MH685723 |
Coryneum castaneicola | CFCC 52316 | MH683552 | MH683560 | MH685732 | MH685724 |
Coryneum depressum | D202 | MH674330 | MH674330 | MH674338 | MH674334 |
Coryneum heveanum | MFLUCC 17-0369 | MH778707 | MH778703 | MH780881 | NA |
Coryneum heveanum | MFLUCC 17-0376 | MH778708 | MH778704 | NA | NA |
Coryneum gigasporum | CFCC 52319 | MH683557 | MH683565 | MH685737 | MH685729 |
Coryneum gigasporum | CFCC 52320 | MH683558 | MH683566 | MH685738 | MH685730 |
Coryneum gigasporum | G14 | MK799957 | MK799944 | MK799830 | MK799820 |
Coryneum gigasporum | G15 | MK799958 | MK799945 | MK799831 | MK799821 |
Coryneum ilicis | CFCC 52994 | MK799948 | MK799935 | NA | NA |
Coryneum ilicis | CFCC 52995 | MK799949 | MK799936 | NA | NA |
Coryneum ilicis | CFCC 52996 | MK799950 | MK799937 | NA | NA |
Coryneum lanciforme | D215 | MH674332 | MH674332 | MH674340 | MH674336 |
Coryneum modonium | D203 | MH674331 | MH674331 | MH674339 | MH674335 |
Coryneum modonium | CBS 130.25 | MH854812 | MH866313 | NA | NA |
Coryneum sinense | CFCC 52452 | MH683553 | MH683561 | MH685733 | MH685725 |
Coryneum sinense | CFCC 52453 | MH683554 | MH683562 | MH685734 | MH685726 |
Coryneum sinense | X20 | MK799952 | MK799939 | MK799825 | MK799815 |
Coryneum sinense | X23 | MK799953 | MK799940 | MK799826 | MK799816 |
Coryneum sinense | X60 | MK799951 | MK799938 | MK799824 | MK799814 |
Coryneum songshanense | CFCC 52997 | MK799946 | MK799933 | MK799822 | MK799812 |
Coryneum songshanense | CFCC 52998 | MK799947 | MK799934 | MK799823 | MK799813 |
Coryneum suttonii | CFCC 52317 | MH683555 | MH683563 | MH685735 | MH685727 |
Coryneum suttonii | CFCC 52318 | MH683556 | MH683564 | MH685736 | MH685728 |
Coryneum suttonii | Z15-1 | MK799954 | MK799941 | MK799827 | MK799817 |
Coryneum suttonii | Z17 | MK799955 | MK799942 | MK799828 | MK799818 |
Coryneum suttonii | Z86 | MK799956 | MK799943 | MK799829 | MK799819 |
Coryneum umbonatum | D201 | MH674329 | MH674329 | MH674337 | MH674333 |
Sequences generated from the above primers of the different genomic regions (ITS, LSU, TEF1-α and RPB2) were analysed in comparison to known species, Stilbospora macrosperma (CBS 115073) and Stegonsporium pyriforme (CBS 120522) were used as the outgroup taxa (
A partition homogeneity test with heuristic search and 1000 replicates was performed using PAUP v. 4.0b10 to assess incongruence among the ITS, LSU, TEF1-α, and RPB2 sequence datasets in reconstructing phylogenetic trees. MP analysis was run using a heuristic search option of 1000 search replicates with random-addition of sequences with a tree bisection and reconnection (TBR) algorithm; branches of zero length were collapsed (collapse = minbrlen), and all equally most parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC). ML analysis was performed using a GTR site substitution model, including a gamma-distributed rate heterogeneity and a proportion of invariant sites (
The alignment based on the combined sequence dataset (ITS, LSU, TEF1-α, and RPB2) included 30 ingroup taxa and two outgroup taxa (Stilbospora macrosperma and Stegonsporium pyriforme), comprising 3544 characters in the aligned matrix. Of these, 2570 characters were constant, 267 variable characters were parsimony-uninformative and 706 characters were parsimony informative. The partition homogeneity test resulted in an insignificant value (level 95%), indicating that ITS, LSU, TEF1-α and RPB2 sequence dataset could be combined. The MP analysis resulted in 2 equally most parsimonious trees; the first tree (TL = 1624, CI = 0.784, RI = 0.822, RC = 0.645) is shown in Fig.
Coryneum ilicis is characterised by its host, Ilex pernyi, and large conidia with 10–11 distosepta.
CHINA. Shaanxi Province: Zhashui County, on branches of Ilex pernyi, 12 August 2017, N. Jiang (holotype:
Named after the host genus on which it was collected, Ilex.
Associated with canker on branches of Ilex pernyi. Sexual morph: Pseudostromata 0.5–1.5 mm diam., typically distinct, circular, without perithecial bumps, containing 1 or 2 perithecia embedded in a well-developed entostroma. Central column and entostroma grey. Ostioles inconspicuous and often invisible at the surface of the ectostromatic disc. Perithecia (350–)500–700(–850) μm diam. (n = 20), globular, somewhat flattened at the base. Asci 110–155 × 13–20 μm, 8-spored, unitunicate, clavate, shortly pedicellate, apically rounded, with a conspicuous apical ring. Ascospores (26.2–)29.7–35.5(–36.2) × (11.0–)11.8–14.3(–15.2) μm, l/w = (1.9–)2.2–2.9(–3.2) (n = 50), 1-seriate, fusiform, ends pointed, uniseptate, constricted at the septa, hyaline, guttulate, smooth-walled. Asexual morph: Conidiomata acervular, 0.2–1 mm wide, 0.2–1.2 mm high, solitary, erumpent through the outer periderm layers of the host, scattered, surface tissues above slightly domed. Conidiophores 40–85 μm long, 3–7 μm wide, branched, cylindrical, septate, hyaline at the apex, pale brown at the base. Conidiogenous cells holoblastic, integrated, indeterminate, cylindrical, expanding towards the apices, pale brown, smooth, with 0–1 percurrent extensions. Conidia (82–)87–95(–105) × (9.5–)10.5–11.5(–12.5) μm, l/w = (7.4–)7.7–9.1(–9.3) (n = 50), variable in shape, curved, broadly fusiform to fusiform, cylindrical or clavate, dark brown, smooth-walled, 10–11-distoseptate, apical cell with a hyaline tip, truncate and black at the base.
Coryneum ilex from Ilex pernyi (
On PDA at 25 °C, colonies growing slowly and unevenly, reaching 70 mm diam. within 25 d, gradually becoming brownish dark grey in colour with scant cottony aerial mycelium, asexual morphs developed after 35 d.
CHINA. Shaanxi Province: Zhashui County, on branches of Ilex pernyi, 12 August 2017, N. Jiang (isotype:
Coryneum ilicis is the sole species known from the host genus Ilex; it can be easily recognised by host association and phylogeny (Fig.
Coryneum songshanense can be distinguished from the morphologically similar C. sinense by its narrower conidia.
CHINA. Beijing City: Songshan Mountain, on dead twigs of Quercus dentata, 15 June 2018, N. Jiang & C.M. Tian (holotype:
Named after the mountain on which it was collected, Songshan Mountain.
Associated with canker on twigs of Quercus dentata. Sexual morph: Pseudostromata 0.3–1 mm diam., typically distinct, circular, without perithecial bumps, containing up to 6 perithecia embedded in a well-developed entostroma. Ectostromatic disc distinct, circular, black, 0.3–0.5 mm diam. Central column and entostroma grey. Ostioles inconspicuous and often invisible at the surface of the ectostromatic disc. Perithecia (150–)200–450(–550) μm diam. (n = 20), globular, somewhat flattened at the base with black short neck. Asci 75–145 × 17–23 μm, 8-spored, unitunicate, clavate, shortly pedicellate, apically rounded, with an inconspicuous apical ring. Ascospores (24.1–)25.5–35.4(–38.2) × (7.5–)7.9–9.8(–10.6) μm, l/w = (3.0–)3.3–3.8(–4.2) (n = 50), 2-seriate, fusiform, ends pointed, uniseptate or aseptate, not constricted at the septa, hyaline, guttulate, smooth-walled. Asexual morph: Conidiomata acervular, 0.2–0.6 mm wide, 0.2–0.5 mm high, solitary, erumpent through the outer periderm layers of the host, scattered, surface tissues above slightly domed. Conidiophores 15–35 μm long, 4–7 μm wide, unbranched, cylindrical, septate, hyaline at the apex, pale brown at the base. Conidiogenous cells holoblastic, integrated, indeterminate, cylindrical, expanding towards the apices, pale brown, smooth, with 0–1 percurrent extensions. Conidia (51–)56–67(–76) × (9–)10–11(–11.5) μm, l/w = (5.2–)5.5–6.9(–8.1) (n = 50), variable in shape, curved, broadly fusiform to fusiform, cylindrical or clavate, dark brown, smooth-walled, 5–7-distoseptate, apical cell with a hyaline tip, truncate and black at the base.
Coryneum songshanense from Quercus dentata (
On PDA at 25 °C, colonies growing slowly and unevenly, reaching 70 mm diam. within 30 d, gradually becoming brownish dark grey in colour with scant cottony aerial mycelium, asexual morphs developed after 40 d.
CHINA. Beijing City: Songshan Mountain, on dead twigs of Quercus dentata, 15 June 2018, N. Jiang & C.M. Tian (isotype:
So far, ten species and one variety have been described from Quercus branches, and they can be distinguished by conidial characteristics (
In this study, fresh Coryneum specimens were collected in China and identified based on combined morphological amd molecular data. Additional accessions of three recently described Coryneum species, C. gigasporum, C. sinense, and C. suttonii (
However, most of the Coryneum species are lacking DNA sequences, thus species identification based on DNA sequence analyses is presently difficult. Hence, polyphasic approach, i.e. incorporating morphological characters (such as conidial sizes and numbers of distosepta), as well as host associations are important for species identification (
This study was financed by the National Natural Science Foundation of China (Project No.: 31670647) and the Short-term International Student Program for Postgraduates of Forestry First-Class Discipline (2019XKJS0501). Financial support from the Austrian Science Fund (FWF; project P27645-B16) to H. Voglmayr is gratefully acknowledged. We are grateful to Chungen Piao and Minwei Guo (China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing) for support with strain preservation during this study.