Research Article |
Corresponding author: Wenxiu Sun ( wxsun@yangtzeu.edu.cn ) Corresponding author: Zhuang Li ( junwuxue@126.com ) Academic editor: Rungtiwa Phookamsak
© 2020 Shengting Huang, Jiwen Xia, Xiuguo Zhang, Wenxiu Sun, Zhuang Li.
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:
Huang S, Xia J, Zhang X, Sun W, Li Z (2020) Two new species of Microdochium from Indocalamus longiauritus in south-western China. MycoKeys 72: 93-108. https://doi.org/10.3897/mycokeys.72.55445
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Microdochium species have often been reported as plant pathogens and saprophytes and are commonly isolated from some diseased plant hosts. The primary aim of the present study was to describe and illustrate two new Microdochium species isolated from the leaf spot of Indocalamus longiauritus in Yunnan Province, China, namely Microdochium yunnanense and M. indocalami, spp. nov., based on their morphology and multilocus phylogenetic analyses of the combined ITS, LSU, TUB2, and RPB2. DNA sequence data indicate that six strains represent three independent groups from related and similar species in Microdochium. Microdochium indocalami sp. nov. clustered with M. fisheri, M. lycopodinum, M. rhopalostylidis, and M. phragmitis. Microdochium yunnanense sp. nov. grouped with M. bolleyi. In addition, the strain SAUCC1017 is recorded as an unidentified species in Microdochium. Descriptions and illustrations of the new species in the genus and Microdochium sp. indet. are provided.
Microdochiaceae, multigene phylogeny, new species, taxonomy, Xylariales
Microdochium is a genus in Microdochiaceae (Xylariales), which has been well-studied in recent years by
Monographella was described by
Microdochium included important plant pathogens, particularly on grasses and cereals. Kwasna et al. (2007) newly described M. triticicola which was isolated from roots of wheat in the United Kingdom.
Many taxonomic problems have occurred in Microdochium, and the genus was shown to be polyphyletic (
In this study, we introduce two novel species, M. yunnanense and M. indocalami spp. nov., which were isolated from the leaves of Indocalamus longiauritus in China. These two species are introduced based on both morphological features and molecular sequence data.
The samples were collected from Yunnan Province, China. The strains of Microdochium were isolated from diseased or healthy leaves of Indocalamus longiauritus using single spore and tissue isolation methods (
Following 2–3 weeks of incubation, morphological characters were recorded as by
Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in
PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume, which contained 12.5 μL Green Taq Mix (vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Biosune, Shanghai, China), and 1 μL template genomic DNA in amplifier, and were adjusted with distilled deionized water to a total volume of 25 μL.
PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 10 min. Annealing temperature for each gene were 55 °C for ITS, 51 °C for LSU, 56 °C for RPB2 and 53 °C for TUB2. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA 7.0 (
Novel sequences generated from the six strains in this study, and all reference available sequences of Microdochium species downloaded from GenBank (mostly used in
Specimens and GenBank accession numbers of DNA sequences used in this study.
Species | Voucher | Host/Substrate | Country | GeneBank accession numbers | |||
---|---|---|---|---|---|---|---|
LSU | ITS | TUB2 | RPB2 | ||||
Idriella lunata | CBS 204.56* | Root of Fragaria chiloensis | USA | KP858981 | KP859044 | – | – |
Microdochium albescens | CBS 291.79 | On Oryza sativa | Ivory Coast | KP858932 | KP858996 | KP859059 | KP859105 |
CBS 243.83 | Seed Oryza sativa | Unknown country | KP858930 | KP858994 | KP859057 | KP859103 | |
M. bolleyi | CBS 540.92 | Root of Hordeum vulgare | Syria | KP858946 | KP859010 | KP859073 | KP859119 |
M. citrinidiscum | CBS 109067* | Leaf of Eichhornia crassipes | Peru | KP858939 | KP859003 | KP859066 | KP859112 |
M. chrysanthemoides | LC5363 = CGMCC3.17929* | Unnamed Karst Cave | China | KU746736 | KU746690 | KU746781 | – |
LC5466 = CGMCC3.17930 | Unnamed Karst Cave | China | KU746735 | KU746689 | KU746782 | – | |
M. colombiense | CBS 624.94* | On Musa sapientum | Colombia | KP858935 | KP858999 | KP859062 | KP859108 |
M. fisheri | CBS 242.90* | Stem of Oryza sativa | UK | KP858951 | KP859015 | KP859079 | KP859124 |
M. indocalami | SAUCC1016* | Leaves of Indocalamus longiauritus | China | MT199878 | MT199884 | MT435653 | MT510550 |
M. lycopodinum | CBS 146.68 | Air sample | The Netherlands | KP858929 | KP858993 | KP859056 | KP859102 |
CBS 109397 | On Phragmites australis | Germany | KP858940 | KP859004 | KP859067 | KP859113 | |
CBS 109398 | On Phragmites australis | Germany | KP858941 | KP859005 | KP859068 | KP859114 | |
M. majus | CBS 741.79 | On Triticum aestivum | Germany | KP858937 | KP859001 | KP859064 | KP859110 |
M. musae | CBS 111018 = CPC 5380 | Musa cv. Cavendish | Costa Rica | – | AY293061 | – | – |
CBS 143499 = CPC 32809 | Leaves of Musa sp. | Malaysia | MH107941 | MH107894 | MH108040 | – | |
CBS 143500* = CPC 32689 | Leaves of Musa sp. | Malaysia | MH107942 | MH107895 | MH108041 | MH108003 | |
CPC 11234 | Leaves of Musa sp. | Mauritius | MH107943 | MH107896 | MH108042 | – | |
CPC 11240 | Leaves of Musa sp. | Mauritius | MH107944 | MH107897 | MH108043 | – | |
CPC 16258 | Leaves of Musa sp. | Mexico | MH107945 | MH107898 | MH108044 | – | |
CPC 32681 | Leaves of Musa sp. | Malaysia | MH107946 | MH107899 | – | – | |
M. neoqueenslandicum | CBS 445.95 | On Juncus effusus | The Netherlands | KP858933 | KP858997 | KP859060 | KP859106 |
CBS 108926* | On Agrostis sp. | New Zealand | KP858938 | KP859002 | KP859065 | KP859111 | |
M. nivale | CBS 116205* | Roots of Triticum aestivum | UK | KP858944 | KP859008 | KP859071 | KP859117 |
M. nivale var. nivale | CBS 288.50 | Unknown | Unknown country | MH868135 | MH856626 | – | – |
M. novae-zelandiae | CBS 143847 | From turf leaves (Poaceae) | New Zealand | – | LT990655 | LT990608 | LT990641 |
CPC 29693 | From turf leaves (Poaceae) | New Zealand | – | LT990656 | LT990609 | LT990642 | |
M. paspali | HK-ML-1371 | Paspalum vaginatum | China | – | KJ569509 | KJ569514 | – |
QH-BA-48 | Paspalum vaginatum | China | – | KJ569510 | KJ569515 | – | |
SY-LQG66 | Paspalum vaginatum | China | – | KJ569511 | KJ569516 | – | |
WC-WC-85 | Paspalum vaginatum | China | – | KJ569512 | KJ569517 | – | |
WN-BD-452 | Paspalum vaginatum | China | – | KJ569513 | KJ569518 | – | |
M. phragmitis | CBS 285.71* | On Phragmites australis | Poland | KP858949 | KP859013 | KP859077 | KP859122 |
CBS 423.78 | On Phragmites communis | Germany | KP858948 | KP859012 | KP859076 | KP859121 | |
M. rhopalostylidis | CPC 34449 = CBS 145125* | Rhopalostylis sapida | New Zealand | MK442532 | MK442592 | MK442735 | MK442667 |
M. seminicola | KAS3576 = CBS 139951* | Maize kernels | Switzerland | KP858974 | KP859038 | KP859101 | KP859147 |
KAS1516 = CPC 26001 | On grain | Canada | KP858961 | KP859025 | KP859088 | KP859134 | |
KAS3574 = DAOM 250155 | Maize kernels | Switzerland | KP858973 | KP859037 | KP859100 | KP859146 | |
KAS3158 = DAOM 250161 | On Triticum aestivum | Canada | KP858970 | KP859034 | KP859097 | KP859143 | |
KAS1527 = DAOM 250165 | On grain | Canada | KP858966 | KP859030 | KP859093 | KP859139 | |
KAS1473 = DAOM 250176 | On Triticum aestivum | Canada | KP858955 | KP859019 | KP859082 | KP859128 | |
M. sorghi | CBS 691.96 | Living Sorghum halepense | Cuba | KP858936 | KP859000 | KP859063 | KP859109 |
M. sp. indet. | SAUCC1017 | Leaves of Indocalamus longiauritus | China | MT199879 | MT199885 | MT435654 | – |
M. tainanense | CBS 269.76* | Root of Saccharum officinarum | China, Taiwan | KP858945 | KP859009 | KP859072 | KP859118 |
CBS 270.76 | Root of Saccharum officinarum | China, Taiwan | KP858931 | KP858995 | KP859058 | KP859104 | |
M. trichocladiopsis | CBS 623.77* | Rhizosphere of Triticum aestivum | Unknown country | KP858934 | KP858998 | KP859061 | KP859107 |
M. yunnanense | SAUCC1011* | Leaves of Indocalamus longiauritus | China | MT199875 | MT199881 | MT435650 | MT510547 |
SAUCC1012 | Leaves of Indocalamus longiauritus | China | MT199876 | MT199882 | MT543651 | MT510548 | |
SAUCC1015 | Leaves of Indocalamus longiauritus | China | MT199877 | MT199883 | MT435652 | MT510549 | |
SAUCC1018 | Leaves of Indocalamus longiauritus | China | MT199880 | MT199886 | MT435655 | – |
Six Microdochium strains isolated from plant hosts were sequenced. Microdochium was analysed by using multilocus data (ITS, LSU, TUB2 and RPB2) composed of 50 isolates of Microdochium and Idriella lunata (CBS 204.56) as the outgroup taxon. A total of 3257 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1–572, LSU: 573–1429, TUB2: 1430–2395, RPB2: 2396–3257. Of these characters, 2019 were constant, 219 were variable and parsimony-uninformative, and 1019 were parsimony-informative. For the BI and ML analyses, GTR+I+G for LSU and RPB2, SYM+I+G for ITS, and GTR+G for TUB2 were selected and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig.
Phylogram of Microdochium based on combined ITS, LSU, TUB2 and RPB2 genes. The ML and BI bootstrap support values above 75% and 0.95 BYPP are shown at the first and second position, respectively. Strains marked with “*” are ex-type or ex-epitype. Strains from the current study are in red. Some branches were shortened to fit them to the page – these are indicated by two diagonal lines with the number of times a branch was shortened indicated next to the lines.
ML bootstrap support values (≥ 75%) and Bayesian posterior probability (≥ 0.95) are shown as first and second position above nodes, respectively. The 50 strains were assigned to 23 species clades based on the four gene loci phylogeny (Fig.
Name refers to the genus of the host plant Indocalamus longiauritus.
Characterised by the size of conidia and the number of septa of conidia.
China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Indocalamus longiauritus. 16 April 2019, S.T. Huang, HSAUP1016, holotype, ex-type living culture SAUCC1016.
Colonies on PDA attaining 46.1–51.2 mm in diameter after 7 days, formed a conspicuous concentric circle, periphery of aerial mycelium cottony, centre with scarce aerial mycelium, white initially, then becoming greyish sepia after 25 days. Some aerial hyphae aggregated and form a sporodochium within 15 days or longer. Mycelium composed of hyaline, immersed and superficial, smooth, branched, septate, 2.0–3.0 μm wide hyphae. Due to the soluble pigment secreted, reverse white to salmon. Conidiophores straight or slightly curved, aseptate, aggregated in the aerial mycelium, often reduced to conidiogenous cells borne directly from the hyphae. Conidiogenous cells terminal or intercalary, mono-or polyblastic, denticulate, smooth, hyaline, cylindrical, straight or bent, 11.0–28.3 × 1.5–2.9 μm. Conidia cylindrical, clavate to obovoid, 1–3-septate, 13.0–15.5 × 3.5–5.5 μm, base usually flattened 0.5–1.0 μm. Sometimes borne directly from the mycelial hyphae. Sexual morph: unknown.
Colonies on OA 62.0–64.0 mm in diameter after 7 days, centre with aerial mycelium cottony, periphery with scarce aerial mycelium. Mycelium mostly immersed, hyphae hyaline, septate, smooth, exudate and soluble pigment produced, reverse white initially, then becoming pale mouse-grey in periphery and mouse-grey in center. Sporodochia formed on agar surface. Colonies on MEA 50.8–52.7 mm in diameter after 7 days, aerial mycelium abundant, with concentric rings, white to pale pink, periphery with cottony aerial mycelium, centre with scarce aerial mycelium, exudate absent. Reverse white to pale pink with age.
Isolated from leaves of Indocalamus longiauritus in China.
Microdochium and allied genera were revised by
Named after Yunnan Province, where the fungus was collected.
Microdochium yunnanense (SAUCC1011) a leaves of host plant b sporodochia on media surface c–e surface of colony after 15 days on PDA (c), OA (d), MEA (e) f–h reverse of colony after 15 days on PDA (f), OA (g), MEA (h) i sporodochial conidiophores and conidiogenous cells with conidia j conidiogenous cells k–l conidia. Scale bars: 10μm (i–l).
Characterised by conidiomata sporodochium-like and the size of conidia.
China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Indocalamus longiauritus. 16 April 2019, S.T. Huang, HSAUP1011 holotype, ex-type living culture SAUCC1011.
Colonies on PDA attaining 48.0–61.5 mm in diameter after 15 days, felty, compact, erose or dentate, initially white, then becoming yellowish with age. Mycelium superficial, consisting of hyaline, smooth, branched, septate, 1.0–2.5 μm wide hyphae. Conidiomata sporodochium-like, appeared within 15 days or longer, formed in aerial mycelium or on agar surface, slimy, hyaline or orange, semi-submerged. Exudate occasionally appeared on old sporodochia. Reverse colorless to yellowish, due to the soluble pigment secreted. Conidiophores formed terminal or lateral with sympodial proliferation, solitary or aggregated. Most conidiophores tightly aggregated in a sporodochium, inconspicuous flat-tipped loci, irregularly branched, or borne directly on mycelial hyphae, straight or slightly curved, aseptate, guttulate, smooth-walled, apex subobtuse, base truncate. Conidiogenous cells of two types: some polyblastic, ampulliform, lageniform, with percurrent proliferations, 6.5–10.0 × 2.5–3.4 μm, neck up to 4.5 μm long, 1.0–1.5 μm wide, others straight or bent, smooth, cylindrical up to 10.0–11.5 μm long, 1.0–2.0 μm wide. Conidia aseptate, mostly lunate, a few ellipsoid and cylindrical, hyaline, straight or curved, obtuse, 0–2 guttulate in mature conidia, 6.8–10.0 × 2.4–3.5 μm. Chlamydospores was not observed. Sexual morph: unknown.
Colonies on OA 58.1–61.5 mm in diameter after 15 days, entire, flat, white, lobate and radially margin, aerial mycelia cottony or sparse. Less exudate. Reverse white. Vegetative hyphae hyaline, abundant, branched, septate, thin-walled. Colonies on MEA 39.5–48.2 mm in diameter after 15 days, dense, initially white, becoming pale yellow, the centre of aerial mycelium cottony, periphery with scarce aerial mycelium, aerial mycelium formed a protuberance at center of colony.
Isolated from leaves of Indocalamus longiauritus in China.
China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Indocalamus longiauritus. 16 April 2019, S.T. Huang, HSAUP1012, HSAUP1015, and HSAUP1018 paratype; living culture SAUCC1012, SAUCC1015, and SAUCC1018.
Strains SAUCC1011, SAUCC1012, SAUCC1015, and SAUCC1018 belong to a single species as they have similar morphological features including culture characteristics, sporodochium, and conidia, the nearly identical sequence data, and cluster in a separate branch with a good support (ML-BS: 100% and BYPP: 1.00). The species is most phylogenetically close to M. bolleyi, and their branch lengths are slightly different. Nevertheless, the morphology of M. yunnanense and M. bolleyi (
Colonies on PDA attaining 73.9–80.4 mm in diameter after 15 days, felty to cottony, flat, margin entire or dentate, white, aerial mycelium abundant. Mycelium superficial, hyphae hyaline, septate, branched, smooth-walled. Reverse white to pale yellow, with yellow pigment produced with aging. Aerial hyphae aggregated to form numerous chlamydospores on agar surface. Chlamydospores thick-walled, terminal or intercalary, more frequently arranged in chains than clusters. Conidiophores not observed. Colonies on OA attaining 79.9–81.7 mm in diameter after 15 days, fluffy, margin entire, white. Reverse white. Colonies on MEA attaining 73.2–78.4 mm in diameter after 15 days, flat, with pale pink inconspicuous concentric circle near the centre, margin entire and white, aerial mycelium abundant.
China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Indocalamus longiauritus. 16 April 2019, S.T. Huang, HSAUP1017, living culture SAUCC1017.
Strain SAUCC1017 failed to produce conidia and lacks a complete morphological description. It formed a conspicuous independent lineage from other Microdochium species in the tree. ITS sequence BLASTn search of SAUCC1017 showed many different species with 97% identity. BLASTn searches with LSU (GenBank MH869857) sequences result in 99% identity with M. bolleyi (CBS 172.63) and TUB2 (GenBank AB625368) sequences result in 99% identity with Xylaria cubensis (strain BCC 18758). Thus, here we listed it as an unidentified species.
Previous studies placed Microdochium in Amphisphaeriaceae (
Three sections were widely accepted in Microdochium based on the type of conidiogenous cells and conidia by
From the previous molecular studies of Microdochium (
This work was jointly supported by the National Natural Science Foundation of China (no. 31770016, 31750001, and 31900014) and the China Postdoctoral Science Foundation (no. 2018M632699).