Research Article |
Corresponding author: Cheng-Ming Tian ( chengmt@bjfu.edu.cn ) Academic editor: Rungtiwa Phookamsak
© 2019 Qin Yang, Wen-Yan Chen, Ning Jiang, 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:
Yang Q, Chen W-Y, Jiang N, Tian C-M (2019) Nectria-related fungi causing dieback and canker diseases in China, with Neothyronectria citri sp. nov. described. MycoKeys 56: 49-66. https://doi.org/10.3897/mycokeys.56.36079
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To clarify phylogenetic relationships amongst Nectria, Neothyronectria and Thyronectria in Nectriaceae, we examined detailed morphological characters and performed phylogenetic analyses of a concatenated dataset, based on the ITS, LSU, tef1 and tub2 DNA sequences of fungal specimens in China. Four species of nectria-related fungi were identified, i.e. Nectria dematiosa, N. pseudotrichia, Neothyronectria citri and Thyronectria pinicola. The newly described species, Neothyronectria citri, is characterised by its ascomatal wall with bright yellow scurf, unitunicate asci, each with 4-spored and ascospores allantoid to short-cylindrical, uniseriate, muriform, hyaline to slightly yellowish-brown. This species has affinities with other one known species of Neothyronectria and can be distinguished by molecular data.
DNA phylogeny, Nectriaceae, Systematic, Taxonomy
Nectriaceae Tul. & C. Tul., typified by the genus Nectria (Fr.) Fr., was established by
Nectria, typified by N. cinnabarina (Tode: Fr.) Fr., was initially established by
During trips to collect forest pathogens in China, several nectria-related fungi associated with canker or dieback diseases were collected. Based on a multi-locus phylogeny (ITS, LSU, tef1 and tub2), we identified four nectria-related species in three genera of Nectriaceae and propose one new species in Neothyronectria.
Fresh specimens were collected from infected branches or twigs of diverse hosts from Beijing, Heilongjiang, Jiangxi, Shaanxi and Xinjiang provinces, China. Strains were isolated from fresh diseased branches and grown from ascospores or conidia by spreading the suspension on the surface of 1.8% potato dextrose agar (PDA), incubated at 25 °C for up to 24 h. Single germinating conidia were removed and transferred to fresh potato dextrose agar (PDA) plate. Specimens and isolates of the new species have been deposited in the Museum of Beijing Forestry University (
Morphological observations of the sexual and asexual morph in the natural environment were based on features of the fruiting bodies produced on infected plant tissues and micromorphology, supplemented by cultural characteristics. Gross morphology of fruiting bodies was recorded using a Leica stereomicroscope (M205 FA). Perithecia, pycnidia, synnemata and stromata were observed and described. To test ascomatal wall reactions, 3% KOH and 100% lactic acid (LA) were used. The micromorphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at 1000× magnification were determined for each isolate using a Leica compound microscope (DM 2500) with differential interference contrast (DIC) optics. Colony characters and pigment production on PDA were noted after 10 d. Colony colours were described according to
Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using a modified CTAB [cetyltrimethylammonium bromide] method (
Gene | PCR primers (forward/reverse) | PCR: thermal cycles: (Annealing temp. in bold) | References of primers used |
ITS | ITS1/ITS4 | (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles |
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LSU | LROR/ LR5 | (95 °C: 45 s, 55 °C: 45 s, 72 °C: 1 min) × 35 cycles |
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tef1 | EF1-728F and EF-1567R | (95 °C: 15 s, 55 °C: 20 s, 72 °C: 1 min) × 35 cycles |
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tub2 | T1/T2 | (95 °C: 30 s, 55 °C: 30 s, 72 °C: 1 min) × 35 cycles |
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The quality of our amplified nucleotide sequences was checked and combined by SeqMan v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), according to recent publications of the family Nectriaceae (
Phylogenetic analyses of the combined gene regions were performed using Maximum Parsimony (MP), Maximum-Likelihood (ML) and Bayesian Inference (BI) methods. The data were edited in AliView version: 1.19-beta1k and the evolutionary model obtained using MrModeltest v. 2.3 (
To reveal the phylogenetic position amongst Nectria, Neothyronectria and Thyronectria in Nectriaceae, a phylogenetic analysis was performed with combined ITS, LSU, tef1 and tub2 sequence data. Sequences of representative species were selected from NCBI (
Strains and GenBank accession numbers of the isolates used in this study.
Species | Isolate No. | Substrate/Host | Country | GenBank Accession No. | |||
---|---|---|---|---|---|---|---|
ITS | LSU | tef1 | tub2 | ||||
Allantonectria miltina | CBS 121121 | Agave americana | Italy | HM484547 | HM484572 | HM484524 | HM484609 |
Emericellopsis glabra | CBS 125295 | Soil | Mexico | HM484860 | GQ505993 | HM484843 | HM484879 |
Hydropisphaera fungicola | CBS 122304 | Decaying leaves on Populus trichocarpa | USA | HM484863 | GQ505995 | HM484845 | HM484877 |
N. antarctica | CBS 115033 | Berberis aquifolium | USA | HM484556 | HM484560 | HM484516 | HM484601 |
N. asiatica | MAFF 241439 | Bark of dead wood | Japan | HM484701 | HM484563 | – | HM484604 |
N. aurantiaca | CBS 308.34 | Ulmus sp. | UK | JF832628 | JF832682 | JF832519 | JF832886 |
N. balansae | CBS 123351 | Coronilla sp. | France | HM484552 | GQ505996 | HM484525 | HM484607 |
N. balansae | CBS 129349 | Twigs | China | JF832653 | JF832711 | JF832522 | JF832908 |
N. berberidicola | CBS 128669 | Berberis vulgaris | France | JF832662 | JF832712 | JF832538 | JF832887 |
N. cinnabarina | CBS 125165 | Dead twigs of Aesculus sp. | France | HM484548 | HM484562 | HM484527 | HM484606 |
N. dematiosa Subclade A | CBS 126570 | Bark | USA | HM484557 | HM484561 | HM484534 | HM484603 |
N. dematiosa Subclade A | CFCC 53585 | Tilia mandshurica | China | MK861084 | MK861075 | MK902792 | MK902801 |
N. dematiosa Subclade A | CFCC 53586 | Betula platyphylla | China | MK861085 | MK861076 | MK902793 | MK902802 |
N. dematiosa Subclade B | CBS 125125 | Dead twigs of Acer macrophyllum | Canada | HM484676 | HM484717 | HM484645 | HM484797 |
N. eustromatica | CBS 121896 | – | – | HM534896 | HM534896 | HM534875 | – |
N. eustromatica | CBS 125578 | – | – | HM534897 | HM534897 | HM534876 | – |
N. magnispora | CBS 129362 | – | Japan | JF832663 | JF832683 | JF832539 | JF832896 |
N. magnispora | CBS 129361 | Twigs | Japan | JF832664 | JF832685 | JF832540 | JF832897 |
N. mariae | CBS 125294 | Buxus sempervirens | France | JF832629 | JF832684 | JF832542 | JF832899 |
N. nigrescens | CBS 125148 | Dead twigs of dicotyledonous tree | USA | HM484707 | HM484720 | HM484672 | HM484806 |
N. nigrescens | CBS 128988 | Elaeagnus angustifolia | USA | JF832630 | JF832687 | – | JF832888 |
N. nigrescens | CBS 129808 | Ulmus pumila | USA | JF832632 | JF832690 | – | JF832894 |
N. polythalama | CBS 128672 | Twigs | New Zealand | JF832638 | JF832695 | JF832523 | JF832900 |
N. pseudocinnabarina | CBS 129366 | Dead wood | Venezuela | JF832642 | JF832697 | JF832533 | – |
N. pseudotrichia | CBS 551.84 | Bark | Japan | HM484554 | GQ506000 | HM484532 | HM484602 |
N. pseudotrichia | MAFF 241452 | Bark | Japan | JF832649 | JF832706 | JF832531 | JF832903 |
N. pseudotrichia | G.J.S. 09-1329 | Dead wood | Venezuela | JF832647 | JF832702 | JF832530 | JF832902 |
N. pseudotrichia | CFCC 53587 | Robinia sp. | China | MK861086 | MK861077 | MK902794 | MK902803 |
N. pseudotrichia | CFCC 53588 | Cinnamomum porrectum | China | MK861087 | MK861078 | MK902795 | MK902804 |
N. pseudotrichia | CFCC 53589 | Rubus corchorifolius | China | MK861088 | MK861079 | MK902796 | MK902805 |
N. sordida | CBS 125119 | Living woody vine | French Guiana | HM484857 | HM484868 | HM484848 | HM484874 |
N. triseptata | HAMS 252485 | On rotten twig | China | KM026503 | KM026504 | KM026506 | KM026501 |
N. ulmicola | CFCC 52117 | Ulmus davidiana var. japonica | China | MG231959 | MG231980 | MG232022 | MG232043 |
N. ulmicola | CFCC 52118 | Ulmus davidiana var. japonica | China | MG231960 | MG231981 | MG232023 | MG232044 |
Nectriopsis exigua | CBS 126110 | Myxomycete | Puerto Rico | HM484865 | GQ506014 | HM484852 | HM484883 |
Neothyronectria citri | CFCC 53590 | Citrus maxima cv. Shatian | China | MK861080 | MK861071 | MK902788 | MK902797 |
N. citri | CFCC 53591 | Citrus maxima cv. Shatian | China | MK861081 | MK861072 | MK902788 | MK902798 |
N. sophorae | CBS 142094 | Sophora microphylla | Zew Zealand | KY173470 | KY173559 | – | KY173619 |
Thyronectria aquifolii | CBS 307.34 | Ilex aquifolium | UK | JF832597 | JF832718 | JF832548 | JF832842 |
The concatenated sequence alignment contained 932 parsimony-informative characters, 259 were variable and parsimony uninformative and 1819 were constant. The parsimony analysis yielded the maximum of 10 equally most parsimonious trees (TL = 5493 steps; CI = 0.386; RI = 0.685; RC = 0.264; HI = 0.614).
The phylogeny, resulting from the MP analysis of combined gene sequence data, is shown in Fig.
Maximum parsimony phylogenetic tree generated from analysis of a combined ITS, LSU, tef1 and tub2 sequence dataset for 59 taxa of Allantonectria, Nectria, Neothyronectria and Thyronectria. Emericellopsis glabra (CBS 125295), Hydropisphaera fungicola (CSB 122304), Nectriopsis exigua (CBS 126110) and Verrucostoma freycinetiae (MAFF 240100) as outgroup taxa. Values above the branches indicate maximum parsimony and maximum likelihood bootstrap (left, MP BP ≥ 50%; right, ML BP ≥ 50%). The branches with significant BIPP values (≥ 0.95) in the BI analysis are thickened. Scale bar = 80 nucleotide substitutions. Strains in current study are in blue. Ex-type strains are indicated in bold.
Nectria cinnabarina (Tode) Fr., Summa veg. Scand., Sectio Post. (Stockholm): 388, 1849.
Members of Nectria are typically weak parasites of woody plants and occur on hardwood trees and shrubs throughout the temperate zone of the northern hemisphere (
See
CHINA. Heilongjiang Province, Liangshui Nature Reserve, 47°10'50.64"N, 128°53'41.03"E, on twigs or branches of Tilia mandshurica Rmpr.et Maxim., 29 July 2016, Q. Yang (
Nectria dematiosa has a broad host range and is widely distributed in China, occurring as the most commonly Nectria species (
See
CHINA. Shaanxi Province, Ankang City, 32°40'32.85"N, 109°18'57.38"E, on twigs or branches of Robinia sp., 29 July 2016, N. Jiang (
Nectria pseudotrichia is one of the common tropical fungi in the genus Nectria and is distinguished in the genus by having muriform ascospores and a synnematous asexual morph.
Neothyronectria sophorae Crous & Thangavel, Persoonia 37: 329, 2016.
The genus Neothyronectria was described by Crous & Thangavel (2016) based on the only species, N. sophorae, which is known from a pycnidial asexual morph. Neothyronectria is characterised by pycnidial conidiomata that exude a creamy mucoid conidial mass and hyaline, ampulliform to subcylindrical conidia. In this study, we collected and illustrated here one additional taxon in Neothyronectria.
Neothyronectria citri differs from its closest phylogenetic neighbour Neothyronectria sophorae in ITS, LSU and tub2 loci, based on the alignments deposited in TreeBASE.
CHINA. Jiangxi Province: Ganzhou city, 25°51'27.87"N, 114°58'18.95"E, on symptomatic branches of Citrus maxima (Burm.) Merr. cv. Shatian Yu, 11 May 2018, Q. Yang, Y.M. Liang & Y. Liu (holotype
Named after the host genus on which it was collected, Citrus.
Mycelium not visible around ascomata or on the host. Stromata erumpent through epidermis, up to 0.6 mm high and 1 mm diam., pseudoparenchymatous, cells forming textura angularis to t. globulosa, intergrading with ascomatal wall. Ascomata superficial on well-developed stromata, scattered to aggregated in groups of 3–10, subglobose to globose, 200–270 μm diam., rarely slightly cupulate upon drying, sometimes with only a depressed apical region, yellowish-brown to grey, apical region slightly darker, no colour change in KOH or LA, sometimes surface scurfy or scaly, bright yellow to greenish-yellow. Ascomatal surface cells forming textura globulosa or t. angularis, sometimes including bright yellow scurf, 9–15 μm diam., walls pigmented, uniformly about 1.5 μm thick. Ascomatal wall 27–46 μm thick, of two regions: outer region 22–35 μm thick, intergrading with stroma, cells forming textura globulosa or t. angularis, walls pigmented, about 1.5 μm thick; inner region 9–15 μm thick, of elongate, thin-walled, hyaline cells, forming textura prismatica. Asci clavate, unitunicate, 53.5–65 × 8.5–11 μm, with inconspicuous ring at apex, 4-spored. Ascospores allantoid to short-cylindrical, uniseriate, rounded at both ends, (17–)18–21(–23.5) × 8–9(–10) μm (n = 20), muriform, hyaline to slightly yellowish-brown.
Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming pale yellowish due to pigment formation, conidiomata absent.
CHINA. Jiangxi Province: Ganzhou City, 25°51'27.87"N, 114°58'18.95"E, on symptomatic branches of Citrus maxima (Burm.) Merr. cv. Shatian Yu, 11 May 2018, Q. Yang, Y.M. Liang & Y. Liu (
Neothyronectria citri, as described here, is known from an ascomatal sexual morph phylogenetically allied to species of Allantonectria and Thyronectria (Fig.
Thyronectria rhodochlora (Mont.) Seeler, J. Arnold Arbor. 21: 455, 1940.
Thyronectria Sacc. was established by Saccardo (1875) to include nectria-like fungi with immersed ascomata and muriform ascospores and characterised by well-developed erumpent stromata which are often covered with yellow-green amorphous scurf and ascospores that sometimes bud in the ascus to produce ascoconidia (
Pleonectria pinicola Kirschst., Abh. Bot. Ver. Prov. Brandenburg 48: 59, 1906.
Stromata erumpent through epidermis, orange to red. Pycnidia solitary or aggregated in groups of 3–6, superficial on stroma or rarely immersed at base, subglobose, smooth to slightly roughened, cerebriformis or slightly cupulate upon drying, 225–400 μm high, 240–440 μm diam., red to bay, KOH+ slightly darker, LA+ slightly yellow. Pycnidial wall 16–40 μm thick, of two regions: outer region 11–15 μm thick, intergrading with stroma, cells forming textura globulosa or t. angularis, walls pigmented, about 1.5 μm thick; inner region 10–24 μm thick, of elongate, thin-walled, hyaline cells, forming textura prismatica. Conidiophores densely branched, generally with 1–3 branches, 8.5–24 μm long, 1.3–1.5 μm wide. Conidiogenous cells cylindrical monophialides on aerial, submerged or repent hyphae. Conidia formed abundantly on slimy heads, ellipsoidal to oblong, hyaline, straight, rounded at both ends, non-septate, (2–)3–3.5 × 0.7–1.0 μm (n = 20), smooth-walled.
Cultures incubated on PDA at 25 °C in darkness. Colony surface cottony with aerial mycelium, becoming yellowish-brown due to pigment formation, small reddish-brown sporodochial conidial masses produced after 3–4 wk.
CHINA. Beijing: Chaoyang District, 40°00'35.31"N, 116°47'55.32"E, on symptomatic branches of Pinus sylvestris var. mongolica Litv., 11 June 2018, Q. Yang & N. Jiang (
The hosts of Thyronectria pinicola, synonymised with Pleonectria pinicola, are restricted to Pinus. Members of the genus distributed in Asia (China, Japan, Pakistan), Australia, Europe (Germany, Russia), North America (USA) and South America (Chile) (
In this investigation of nectria-related fungi in China, we identified four species in three genera (Nectria, Neothyronectria and Thyronectria) of Nectriaceae, based on four combined loci (ITS, LSU, tef1 and tub2), as well as morphological characters. It includes Nectria dematiosa, N. pseudotrichia, and Thyronectria pinicola as well as one new species named Neothyronectria citri. The new species is characterised by well-developed erumpent stromata that are often covered with yellow-green amorphous scurf; asci unitunicate, clavate, with inconspicuous ring at apex, each with 4-spored; ascospores allantoid to short-cylindrical, uniseriate, muriform, hyaline to slightly yellowish.
Species revised by
In the taxonomy of hypocrealean fungi, the reaction of the perithecial wall to KOH is considered as an important character (
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.