Research Article |
Corresponding author: Cheng-Ming Tian ( chengmt@bjfu.edu.cn ) Academic editor: Huzefa Raja
© 2020 Ning Jiang, Ling-Yu Liang, 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, Liang L-Y, Tian C-M (2020) Gnomoniopsis chinensis (Gnomoniaceae, Diaporthales), a new fungus causing canker of Chinese chestnut in Hebei Province, China. MycoKeys 67: 19-32. https://doi.org/10.3897/mycokeys.67.51133
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Chinese chestnut (Castanea mollissima) is an important crop tree species in China. However, branch canker and fruit rot are two kinds of severe diseases, which weaken the host and decrease chestnut production. During our investigations into chestnut diseases in China, several fungi have been confirmed as casual agents in previous studies, namely Aurantiosacculus castaneae, Cryphonectria neoparasitica, Cry. parasitica, Endothia chinensis and Gnomoniopsis daii. In this study, a new canker pathogen is introduced based on morphology, phylogeny and pathogenicity. Typical Gnomoniopsis canker sign of wide, orange tendrils emerging from hosts’ glaucous lenticels were obvious on the diseased trees in the field. Symptomatic branches or bark on stems from different chestnut plantations were sampled and isolated, then strains were identified by comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions as well as morphological features. As a result, these strains appeared different from any known Gnomoniopsis species. Hence, we propose a novel species named Gnomoniopsis chinensis. Pathogenicity was further tested using the ex-type strain (CFCC 52286) and another strain (CFCC 52288) on both detached branches and 3-year-old chestnut seedlings. The inoculation results showed that Gnomoniopsis chinensis is mildly pathogenic to Chinese chestnut. However, further studies are required to confirm its pathogenicity to the other cultivated Castanea species in America, Europe and Japan.
Castanea mollissima, chestnut disease, taxonomy
The Chinese chestnut (Castanea mollissima), as well as the American chestnut (C. dentata), the European chestnut (C. sativa) and the Japanese chestnut (C. crenata), are known as the four main cultivated sweet chestnut species in the world (
The fungal genus Gnomoniopsis (Gnomoniaceae, Diaporthales) includes species all occurring in plant tissues as pathogens, endophytes or saprobes (
Gnomoniopsis smithogilvyi is an important nut rot agent on chestnut nuts, an endophyte in asymptomatic flowers, leaves and stems, and a saprobe on dead burrs and branches (
During 2016 to 2019, investigations were conducted in chestnut plantations of nine provinces/municipalities in China, including Beijing, Fujian, Hebei, Hubei, Hunan, Liaoning, Shandong, Shaanxi and Tianjin. Typical Gnomoniopsis canker symptoms were only observed in Hebei Province (Fig.
Genomic DNA was extracted from mycelium grown on PDA using a CTAB (cetyltrimethylammonium bromide) method (
Species | Country | Host | Strain | GenBank Accession Number | ||
---|---|---|---|---|---|---|
ITS | tub2 | tef1 | ||||
Apiognomonia veneta | France | Platanus occidentalis | CBS 342.86 | DQ313531 | EU219235 | DQ318036 |
Gnomoniopsis alderdunensis | USA | Rubus pedatus | CBS 125679 | GU320826 | GU320788 | GU320813 |
Gnomoniopsis alderdunensis | USA | Rubus parviflorus | CBS 125680 | GU320825 | GU320787 | GU320801 |
Gnomoniopsis alderdunensis | USA | Rubus parviflorus | CBS 125681 | GU320827 | GU320789 | GU320802 |
Gnomoniopsis chamaemori | Finland | Rubus chamaemorus | CBS 804.79 | GU320817 | GU320777 | GU320809 |
Gnomoniopsis chinensis | China | Castanea mollissima | CFCC 52286 | MG866032 | MH545366 | MH545370 |
Gnomoniopsis chinensis | China | Castanea mollissima | CFCC 52287 | MG866033 | MH545367 | MH545371 |
Gnomoniopsis chinensis | China | Castanea mollissima | CFCC 52288 | MG866034 | MH545368 | MH545372 |
Gnomoniopsis chinensis | China | Castanea mollissima | CFCC 52289 | MG866035 | MH545369 | MH545373 |
Gnomoniopsis clavulata | USA | Quercus falcata | CBS 121255 | EU254818 | EU219211 | GU320807 |
Gnomoniopsis comari | Finland | Comarum palustre | CBS 806.79 | EU254821 | EU219156 | GU320810 |
Gnomoniopsis comari | Finland | Comarum palustre | CBS 807.79 | EU254822 | GU320779 | GU320814 |
Gnomoniopsis comari | Switzerland | Comarum palustre | CBS 809.79 | EU254823 | GU320778 | GU320794 |
Gnomoniopsis daii | China | Castanea mollissima | CFCC 54043 | MN598671 | MN605517 | MN605519 |
Gnomoniopsis daii | China | Castanea mollissima | CMF002B | MN598672 | MN605518 | MN605520 |
Gnomoniopsis fructicola | USA | Fragaria vesca | CBS 121226 | EU254824 | EU219144 | GU320792 |
Gnomoniopsis fructicola | France | Fragaria sp. | CBS 208.34 | EU254826 | EU219149 | GU320808 |
Gnomoniopsis fructicola | USA | Fragaria sp. | CBS 125671 | GU320816 | GU320776 | GU320793 |
Gnomoniopsis guttulata | Bulgaria | Agrimonia eupatoria | MS 0312 | EU254812 | NA | NA |
Gnomoniopsis idaeicola | USA | Rubus sp. | CBS 125672 | GU320823 | GU320781 | GU320797 |
Gnomoniopsis idaeicola | USA | Rubus pedatus | CBS 125673 | GU320824 | GU320782 | GU320798 |
Gnomoniopsis idaeicola | France | Rubus sp. | CBS 125674 | GU320820 | GU320780 | GU320796 |
Gnomoniopsis idaeicola | USA | Rubus procerus | CBS 125675 | GU320822 | GU320783 | GU320799 |
Gnomoniopsis idaeicola | USA | Rubus procerus | CBS 125676 | GU320821 | GU320784 | GU320811 |
Gnomoniopsis macounii | USA | Spiraea sp. | CBS 121468 | EU254762 | EU219126 | GU320804 |
Gnomoniopsis occulta | USA | Potentilla sp. | CBS 125677 | GU320828 | GU320785 | GU320812 |
Gnomoniopsis occulta | USA | Potentilla sp. | CBS 125678 | GU320829 | GU320786 | GU320800 |
Gnomoniopsis paraclavulata | USA | Quercus alba | CBS 123202 | GU320830 | GU320775 | GU320815 |
Gnomoniopsis racemula | USA | Chamerion angustifolium | CBS 121469 | EU254841 | EU219125 | GU320803 |
Gnomoniopsis sanguisorbae | Switzerland | Sanguisorba minor | CBS 858.79 | GU320818 | GU320790 | GU320805 |
Gnomoniopsis smithogilvyi | Australia | Castanea sp. | CBS 130190 | JQ910642 | JQ910639 | KR072534 |
Gnomoniopsis smithogilvyi | Australia | Castanea sp. | CBS 130189 | JQ910644 | JQ910641 | KR072535 |
Gnomoniopsis smithogilvyi | Australia | Castanea sp. | CBS 130188 | JQ910643 | JQ910640 | KR072536 |
Gnomoniopsis smithogilvyi | Italy | Castanea sativa | MUT 401 | HM142946 | KR072532 | KR072537 |
Gnomoniopsis smithogilvyi | New Zealand | Castanea sativa | MUT 411 | HM142948 | KR072533 | KR072538 |
Gnomoniopsis tormentillae | Switzerland | Potentilla sp. | CBS 904.79 | EU254856 | EU219165 | GU320795 |
Sirococcus castaneae | Switzerland | Castanea sativa | CBS 142041 | KX929744 | KX958443 | KX929710 |
Species identification was based on morphological features of the asexual fruiting bodies produced on infected plant tissues, supplemented by cultural characteristics. Hence, cross-sections were prepared by hand using a double-edge blade. Morphological characteristics of the fruiting bodies including: size of conidiomata and locules; size and shape of conidiophores and conidia were determined under a Nikon AZ100 dissecting stereomicroscope. More than 20 fruiting bodies were sectioned, and 50 conidia were selected randomly for measurement using a Leica compound microscope (LM, DM 2500). Cultural characteristics of isolates incubated on PDA in the dark at 25 °C were recorded, including the colony color and pycnidium structures (
Two isolates of Gnomoniopsis chinensis (ex-type strain: CFCC 52286; CFCC 52288) were used for inoculations, and agar plugs were used as the negative control. Isolates were grown on PDA for five days at 25 °C before the tests. Inoculations were performed on detached branches and 3-year-old seedlings of Castanea mollissima, respectively. The detached branches and young seedling were collected from Hebei Province where the disease is emerging. The healthy chestnut branches (2 cm in diameter) were sampled from an adult chestnut tree and cut into pieces of 20 cm length. A total of 30 fresh and healthy branches and 15 seedlings were used for the pathogenicity tests. Ten branches and five seedlings were inoculated with each isolate and the negative control. For incubations, incisions were made on the middle of the detached branches and 1 cm above the midpoint of the seedling stem to expose the cambium using a 5-mm-diameter cork borer. Discs of agar were cut from the actively growing margins of the cultures and these were placed into wounds of the same size on the chestnut barks. Inoculated wounds and ends of inoculated branches were sealed with parafilm to reduce desiccation and the chance of contamination. The tested seedlings and branch segments were maintained in the greenhouse randomly at 25 °C under natural light conditions. Detached branches were inoculated in November 2017, and the young seedlings were tested in July 2019. The results from detached branches were evaluated after one month, and seedlings after three months, by measuring the lengths of the lesions on the cambium. The re-isolations were made from the resultant lesions from all tested branches and seedlings by cutting small pieces of discolored xylem and placing them onto the PDA plates. Re-isolations were identified based on morphology on PDA and ITS sequences. Differences among isolates in lesion length were analyzed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests. Statistical analysis was carried out by R software (version 3.4.3.) and considered as significant at p < 0.05.
The final combined ITS-tef1-tub2 matrix of Gnomoniopsis included 35 ingroup and two outgroup taxa, comprising 1364 alignment characters. Of these, 783 characters were constant, 117 variable characters were parsimony-uninformative and 464 characters were parsimony informative. The phylogenetic tree obtained from ML analysis is shown in Figure
Named after the country where it was first collected.
Pathogenic on stems and branches of Castanea mollissima. Conidiomata pseudostromatic, globose to pulvinate, occurring separately, yellow to orange, semi-immersed in bark, 400–1000 µm high, 500–1500 µm diam, unilocular, single ostiolate, forming long, wide orange tendrils, 1500–2000 µm × 400–500 µm. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells oval, hyaline, 1-celled, 6–12 µm. Conidia oval, oblate, fusiform, straight to curved, hyaline, 2–3 guttules, (6.0–)6.5–8.5(–9.0) × (2.2–)2.7–3(–3.5) µm (mean = 7.5 × 2.7 µm).
Colonies on PDA attaining 90 mm after 20 days at 25 °C, flat, velutinous to shortly woolly, dark brown in center, gradually lightening to pale grey at margin; margin diffuse; reverse of almost same colors as surface.
China, Hebei Province, Chengde City, chestnut plantation, 40°24'32.16"N, 117°28'56.24"E, 262 m asl, on stems and branches of Castanea mollissima, Ning Jiang, 11 October 2017 (
Three Gnomoniopsis species have been discovered from the host genus Castanea. They share similar conidial dimension (6.0–9.0 × 2.2–3.5 µm in Gnomoniopsis chinensis vs. 5.0–8.0 × 2.0–3.5 µm in G. daii vs. 6.0–9.5 × 2.0–4.0 µm in G. smithogilvyi) (
One month after inoculation on detached branches, the two Gnomoniopsis chinensis isolates produced lesions in the cambium of detached chestnut branches. In contrast, there was no lesion development in any of the negative control inoculations (Fig.
Three months after inoculation on young seedlings, two isolates Gnomoniopsis chinensis and the negative control, produced minor lesions (Fig.
In the past years, our team focused on the fungi inhabiting Chinese chestnut (Castanea mollissima) trees from their taxonomy and pathogenicity aspects. Several fungi including Aurantiosacculus castaneae, Cryphonectria neoparasitica, Cry. parasitica, Endothia chinensis and Gnomoniopsis daii have been proven to cause branch canker or fruit rot (
Gnomoniopsis species appear host-specific, inhabiting hosts of three families, viz. Betulaceae, Fagaceae, Rosaceae and Onagraceae (
Species | Conidial length (µm) | Conidial width (µm) | Reference |
Gnomoniopsis chinensis | (6.0–)6.5–8.5(–9.0) | (2.2–)2.7–3(–3.5) | This study |
Gnomoniopsis clavulata | (5–)6–6.5(–8) | (2–)2.5–3(–4) |
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Gnomoniopsis daii | (5.0–)5.5–7.0(–8.0) | 2.0–3.5 |
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Gnomoniopsis paraclavulata | (6–)7.5–8(–9.5) | (2–)3–3(–3.5) |
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Gnomoniopsis smithogilvyi | (6.0–)8(–9.5) | (2.0–)2.5(–4.0) |
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This study is financed by National Natural Science Foundation of China (Project No.: 31670647). We are grateful to Chungen Piao, Minwei Guo (China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing.