Research Article |
Corresponding author: Qirui Li ( lqrnd2008@163.com ) Academic editor: Andrew Miller
© 2021 Sihan Long, Lili Liu, Yinhui Pi, Youpeng Wu, Yan Lin, Xu Zhang, Qingde Long, Yingqian Kang, Jichuan Kang, Nalin N. Wijayawardene, Feng Wang, Xiangchun Shen, Qirui 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:
Long S, Liu L, Pi Y, Wu Y, Lin Y, Zhang X, Long Q, Kang Y, Kang J, Wijayawardene NN, Wang F, Shen X, Li Q (2021) New contributions to Diatrypaceae from karst areas in China. MycoKeys 83: 1-37. https://doi.org/10.3897/mycokeys.83.68926
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In this study, fungal specimens of the family Diatrypaceae were collected from karst areas in Guizhou, Hainan and Yunnan Provinces, China. Morpho-molecular analyses confirmed that these new collections comprise a new genus Pseudodiatrype, three new species (Diatrype lancangensis, Diatrypella pseudooregonensis and Eutypa cerasi), a new combination (Diatrypella oregonensis), two new records (Allodiatrype thailandica and Diatrypella vulgaris) from China and two other known species (Neoeutypella baoshanensis and Paraeutypella citricola). The new taxa are introduced, based on multi-gene phylogenetic analyses (ITS, β-tubulin), as well as morphological analyses. The new genus Pseudodiatrype is characterised by its wart-like stromata with 5–20 ascomata immersed in one stroma and the endostroma composed of thin black outer and inner layers of large white cells with thin, powdery, yellowish cells. These characteristics separate this genus from two similar genera Allodiatrype and Diatrype. Based on morphological as well as phylogenetic analyses, Diatrype lancangensis is introduced as a new species of Diatrype. The stromata of Diatrype lancangensis are similar to those of D. subundulata and D. undulate, but the ascospores are larger. Based on phylogenetic analyses, Diatrype oregonensis is transferred to the genus Diatrypella as Diatrypella oregonensis while Diatrypella pseudooregonensis is introduced as a new species of Diatrypella with 8 spores in an ascus. In addition, multi-gene phylogenetic analyses show that Eutypa cerasi is closely related to E. lata, but the ascomata and asci of Eutypa cerasi are smaller. The polyphyletic nature of some genera of Diatrypaceae has led to confusion in the classification of the family, thus we discuss whether the number of ascospores per asci can still be used as a basis for classification.
Five novel taxa, phylogeny, systematics, taxonomy, Xylariales
Diatrypaceae is an important family of higher ascomycetes, belonging to Xylariales (
Members of Diatrypaceae occur on a wide range of hosts in terrestrial and marine environments worldwide, some of which are important plant pathogens (
Thirteen species of Cryptosphaeria and Diatrype were introduced by Vasiljeva and Ma (2014) from north-eastern China, which includes two new species and four new records. China has the largest range of karst distribution in the world. The landform of karst can be found in almost all Provinces of China, with the most extensive distribution in Guizhou and Yunnan Provinces (
In this study, we revisit species of Diatrypaceae collected from karst areas in Guizhou, Hainan and Yunnan Provinces of China. Based on morpho-molecular analyses, one new genus and three new species are introduced; in addition, a new combination and two new records from China are reported. Descriptions and illustrations of new taxa and new records are provided.
Samples of decaying wood were collected from October 2019 to November 2020 in forests and nature reserves of Guizhou, Hainan and Yunnan Provinces in China. The specimens were observed with a stereomicroscope while microscopic images of the samples were taken using a Nikon ECLIPSE Ni compound microscope, with a Canon EOS 700D digital camera. Measurements were taken with Tarosoft (R) Image Frame Work (v.0.9.7). More than 30 asci and ascospores were measured for each specimen examined. Photoplates were arranged and improved by using Adobe Photoshop CS6 software. Isolations of fungi were made by single spore isolation (
Taxa used in the phylogenetic analyses and their corresponding GenBank accession numbers.
Taxa | Strain number | GenBank Accession number | Reference | |
---|---|---|---|---|
ITS | β-tubulin | |||
Allocryptovalsa elaeidis | MFLUCC 15-0707 | MN308410 | MN340296 |
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A. polysporaT | MFLUCC 17-0364 | MF959500 | MG334556 |
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A. rabenhorstii | WA08CB | HQ692619 | HQ692523 |
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Allodiatrype arengaeT | MFLUCC 15-0713 | MN308411 | MN340297 |
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A. elaeidicola | MFLUCC 15-0737a | MN308415 | MN340299 |
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A. elaeidis | MFLUCC 15-0708a | MN308412 | MN340298 |
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A. thailandica | MFLUCC 15-3662 | KU315392 | NA |
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A. thailandica | MFLUCC 15-0711 | MN308414 | NA |
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A. thailandica | GMB0050 | MW797108 | MW814880 | This study |
Anthostoma decipiensT | IPV-FW349 | AM399021 | AM920693 | Unpublished. |
A. decipiensT | JL567 | JN975370 | JN975407 |
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Cryptosphaeria ligniota | CBS 273.87 | KT425233 | KT425168 |
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C. pullmanensis | ATCC 52655 | KT425235 | KT425170 |
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C. subcutanea | DSUB100A | KT425189 | KT425124 |
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C. subcutanea | CBS 240.87 | KT425232 | KT425167 |
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Cryptovalsa ampelina | A001 | GQ293901 | GQ293972 |
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C. ampelina | DRO101 | GQ293902 | GQ293982 |
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Diatrype bullata | UCDDCh400 | DQ006946 | DQ007002 |
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D. disciformisT | GNA14 | KR605644.1 | KY352434.1 |
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D. disciformisT | D21C, CBS 205.87 | AJ302437 | NA |
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D. enteroxantha | HUEFS155114 | KM396617 | KT003700 |
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D. enteroxantha | HUEFS155116 | KM396618 | KT022236 |
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D. lancangensis | GMB0045 | MW797113 | MW814885 | This study |
D. lancangensis | GMB0046 | MW797114 | MW814886 | This study |
D. lancangensis | GMB0047 | MW797116 | MW814887 | This study |
D. palmicola | MFLUCC 11-0020 | KP744438 | NA |
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D. palmicola | MFLUCC 11-0018 | KP744439 | NA |
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D. spilomea | D17C | AJ302433 | NA |
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D. stigma | DCASH200 | GQ293947 | GQ294003 |
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D. undulata | D20C, CBS 271.87 | AJ302436 | NA |
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Diatrypella atlantica | HUEFS 136873 | KM396614 | KR259647 |
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D. banksiae | CPC 29118 | KY173402 | NA |
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D. delonicis | MFLUCC 15-1014 | MH812994 | MH847790 |
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D. delonicis | MFLU 16-1032 | MH812995 | MH847791 |
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D. elaeidis | MFLUCC 15-0279 | MN308417 | MN340300 |
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D. favacea | Islotate 380 | KU320616 | NA |
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D. favacea | DL26C | AJ302440 | NA | Unpublished |
D. frostii | UFMGCB 1917 | HQ377280 | NA |
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D. heveae | MFLUCC 15-0274 | MN308418 | MN340301 |
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D. heveae | MFLUCC 17-0368 | MF959501 | MG334557 |
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D. hubeiensis | CFCC 52413 | MW632937 | NA |
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D. iranensis | KDQ18 | KM245033 | KY352429 |
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D. macrospora | KDQ15 | KR605648 | KY352430 |
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D. oregonensis (Diatrype oregonensis) | DPL200 | GQ293940 | GQ293999 |
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D. oregonensis (Diatrype oregonensis) | CA117 | GQ293934 | GQ293996 |
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D. pseudooregonensis | GMB0039 | MW797115 | MW814888 | This study |
D. pseudooregonensis | GMB0040 | MW797117 | MW814889 | This study |
D. pseudooregonensis | GMB0041 | MW797118 | MW814890 | This study |
D. pseudooregonensis | GMB0042 | MW797119 | MW814891 | This study |
D. pseudooregonensis | GMB0043 | MW797120 | MW814892 | This study |
D. pseudooregonensis | GMB0044 | MW797110 | MW814882 | This study |
D. pulvinata | H048 | FR715523 | FR715495 |
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D. pulvinata | DL29C | AJ302443 | NA | Unpublished |
D. tectonae | MFLUCC 12-0172a | KY283084 | NA |
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D. tectonae | MFLUCC 12-0172b | KY283085 | KY421043 |
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D. verruciformisT | UCROK1467 | JX144793 | JX174093 |
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D. verruciformisT | UCROK754 | JX144783 | JX174083 |
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D. vulgaris | HVFRA02 | HQ692591 | HQ692503 |
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D. vulgaris | HVGRF03 | HQ692590 | HQ692502 |
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D. vulgaris | GMB0051 | MW797107 | MW814879 | This study |
D. yunnanensis | VT01 | MN653008 | MN887112 |
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Eutypa armeniacae | ATCC 28120 | DQ006948 | DQ006975 |
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E. astroidea | E49C, CBS 292.87 | AJ302458 | DQ006966 |
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E. cerasi | GMB0048 | MW797104 | MW814893 | This study |
E. cerasi | GMB0049 | MW797105 | MW814877 | This study |
E. flavovirens | E48C, CBS 272.87 | AJ302457 | DQ006959 |
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E. laevata | E40C CBS 291.87 | AJ302449 | NA |
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E. lataT | CBS290.87 | HM164736 | HM164770 | Trouillas and Gubler (2010) |
E. lataT | EP18 | HQ692611 | HQ692501 |
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E. lataT | RGA01 | HQ692614 | HQ692497 |
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E. leioplaca | CBS 248.87 | DQ006922 | DQ006974 |
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E. leptoplaca | CBS 287.87 | DQ006924 | DQ006961 |
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E. maura | CBS 219.87 | DQ006926 | DQ006967 |
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E. microasca | BAFC 51550 | KF964566 | KF964572 |
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E. sparsa | 3802 3b | AY684220 | AY684201 |
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E. tetragona | CBS 284.87 | DQ006923 | DQ006960 |
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Eutypella caricae | EL51C | AJ302460 | NA | Acero (2000) |
E. cerviculataT | M68 | JF340269 | NA |
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E. cerviculataT | EL59C | AJ302468 | NA |
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E. leprosa | EL54C | AJ302463 | NA |
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E. leprosa | Isolate 60 | KU320622 | NA |
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E. microtheca | BCMX01 | KC405563 | KC405560 |
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E. parasitica | CBS 210.39 | DQ118966 | NA |
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E. semicircularis | MP4669 | JQ517314 | NA |
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Halocryptovalsa salicorniae | MFLUCC 15-0185 | MH304410 | MH370274 |
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Halodiatrype avicenniae | MFLUCC 15-0953 | KX573916 | KX573931 |
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H. salinicolaT | MFLUCC 15-1277 | KX573915 | KX573932 |
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Kretzschmaria deusta | CBS 826.72 | KU683767 | KU684190 |
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Monosporascus cannonballus T | CMM3646 | JX971617 | NA | Unpublished |
M. cannonballus T | ATCC 26931 | FJ430598 | NA | Unpublished |
Neoeutypella baoshanensisT | GMB0052 | MW797106 | MW814878 | This study |
N. baoshanensisT | LC 12111 | MH822887 | MH822888 |
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N. baoshanensisT | EL51C, CBS 274.87 | AJ302460 | NA |
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N. baoshanensisT | MFLUCC 16-1002 | MT310662 | NA |
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N. baoshanensisT | GL08362 | JX241652 | NA |
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Paraeutypella citricola | HVVIT07 | HQ692579 | HQ692512 |
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Pa. citricola | HVGRF01 | HQ692589 | HQ692521 |
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Pa. citricola | GMB0053 | MW797109 | MW814881 | This study |
Pa. guizhouensisT | KUMCC 20-0016 | MW039349 | MW239660 |
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Pa. guizhouensisT | KUMCC 20-0017 | MW036141 | MW239661 |
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Pa. vitis | UCD2291AR | HQ288224 | HQ288303 |
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Pa. vitis | UCD2428TX | FJ790851 | GU294726 |
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Pedumispora rhizophoraeT | BCC44877 | KJ888853 | NA |
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Pe. rhizophoraeT | BCC44878 | KJ888854 | NA |
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Peroneutypa alsophila | EL58C, CBS 250.87 | AJ302467 | NA |
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Pe. curvispora | HUEFS 136877 | KM396641 | NA |
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Pe. diminutispora | MFLUCC 17-2144 | MG873479 | NA |
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Pe. mackenziei | MFLUCC 16-0072 | KY283083 | KY706363 |
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Pe. mangrovei | PUFD526 | MG844286 | MH094409 |
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Pseudodiatrype hainanensisT | GMB0054 | MW797111 | MW814883 | This study |
Ps. hainanensisT | GMB0055 | MW797112 | MW814884 | This study |
Quaternaria quaternata | EL60C, CBS 278.87 | AJ302469 | NA |
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Q. quaternata | GNF13 | KR605645 | NA |
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Xylaria hypoxylon | CBS 122620 | AM993141 | KX271279 |
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Genomic DNA was extracted from fungal mycelium following the manufacturer’s protocol of the BIOMIGA Fungal gDNA isolation Kit (BIOMIGA, Hangzhou City, Zhejiang Province, China). Extracts of DNA were stored at –20 °C.
PCR was carried out in a volume of 25 μl containing 9.5 μl of ddH2O, 12.5 μl of 2× Taq PCR Master Mix (2 × Taq Master Mix with dye, TIANGEN, China), 1 μl of DNA extracts and 1 μl of forward and reverse primers (10 μM each) in each reaction. Primers pairs, ITS4 and ITS5, fRPB2-7CR and fRPB2-5f, LROR and LR5, T1 and Bt2b, as well as Bt2a and Bt2b (
PCR profiles for the ITS and LSU are as follows: initially at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 1 minute, annealing at 52 °C for 1 minute, elongation at 72 °C for 1.5 minutes and a final extension at 72 °C for 10 minutes. PCR profile for the RPB2 is as follows: initially at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 95 °C for 1 minute, annealing at 54 °C for 2 minutes, elongation at 72 °C for 1.5 minutes and a final extension at 72 °C for 10 minutes (
Phylogenetic analyses were performed by searching homologous sequence data of the family Diatrypaceae in the GenBank database, selected from NCBI and recently published papers (
Based on RAxML and BYPP analyses, phylogenetic analyses were similar in overall tree topologies and did not differ significantly. The dataset consists of 105 taxa for representative strains of species in Diatrypaceae, including outgroup taxa with 1071 characters, including gaps (ITS: 1–486, β-tubulin: 486–1071). The RAxML analyses resulted in a best scoring likelihood tree selected with a final ML optimisation likelihood value of -15731.506304, which is shown in Fig.
Phylogram generated from Maximum Likelihood (RAxML) analyses, based on ITS-β-tubulin matrix. ML bootstrap supports (≥ 70%) and Bayesian posterior probability (≥ 0.90) are indicated as ML/BYPP. The tree is rooted to Kretzschmaria deusta (CBS 826.72) and Xylaria hypoxylon (CBS 122620). Ex-type strains are in red. Newly generated strains are in black bold.
The phylogenetic tree, based on combining ITS and β-tubulin sequence data, is also shown in Fig.
Clade 1: Diatrypella pseudooregonensis and Diatrypella oregonensis clustered with the species of Diatrypella in Clade 1 with high bootstrap support, Diatrypella pseudooregonensis is introduced as an 8-spored new species of Diatrypella and Diatrype oregonensis is renamed as Diatrypella oregonensis.
Clade 4: Pseudodiatrype formed a separate branch in a clade (Clade 4) basal to the genus Allodiatrype.
Clade 7: Diatrype lancangensis clusters with the species of Diatrypella and Diatrype in an unresolved clade. However, Diatrype and Diatrypella have previously shown confused classification which is difficult to distinguish, based on phylogenetic aspects alone. Therefore, we introduce Diatrype lancangensis as a new species of Diatrype, based on phylogenetic analyses and morphological differences (Table
The dimensions of the present species and some related species of Diatrype and Allodiatrype.
Species name | Stromata | Asci | Ascospores | Reference | |||||
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Length (mm) | Wide (mm) | Length (μm) | Wide (μm) | Length (μm) | Wide (μm) | ||||
Allodiatrype arengae | 0.69–0.94 | 0.37–0.93 | 54–109 | 6–10 | 7–10 | 2–3 |
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A. elaeidicola | 1.2–2.8 | 0.9–1.66 | 60–91 | 4–7 | 8–10 | 1.5–3 |
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A. elaeidis | 0.47–0.86 | 0.44–0.71 | 56–95 | 9–11 | 8–10 | 1.5–3 |
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A. thailandica | NA | 1–2 | 55–80 | 5–7 | 3.8–6.9 | 1–1.4 |
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Diatrype acericola | 1–2 | 1–1.5 | 23–27 | 5–7 | 7.5–9 | 0.9–1.1 |
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D. albopruinosa | 0.5–1 diam. | 0.5–1 diam | 40–60 | 10–15 | 12–15 | 3.5–4 |
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D. bullata | 2–7 diam. | 2–7 diam | 25–30 | 5–7 | 7.5–9 | Very thin |
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D. disciformis | NA | NA | 75–115 | NA | 5–9 | 1.5–2 |
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D. enteroxantha | NA | 1–3.5 | 18–28.5 | 5–9 | 7–10 | 1.5–2.5 |
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D. hypoxyloides | NA | NA | 20–25 | 4–6 | 4–6 | Very thin |
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D. lancangensis | NA | NA | 90.5–160.5 | 7–15 | 11–18.5 | 2–4 | This study | ||
D. lijiangensis | 1 diam. | 1 diam | 50–90 | 6–9 | 6–8 | 1–2 |
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D. macounii | 1–1.8 diam. | 1–1.8 diam | 25–30 | 4–6 | 4–6 | 0.7–1 |
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D. stigma | NA | NA | 25–30 | 5–7 | 6–8 | 1.5–2 |
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D. subundulata | NA | NA | 35–40 | 5–7 | 7–9 | 1.7–1.9 |
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D. undulata | NA | NA | 25–30 | 3.5–4.5 | 5–7 | 0.9–1.3 |
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D. whitmanensis | NA | NA | 50–82 | 8–15 | 7.5–10 | 1–1.5 | Trouilla et al. 2010 | ||
Pseudodiatrype hainanensis | 2–3.6 | 1.6–3 | 110–155.5 | 6–10 | 8.5–13 | 1.5–2.5 | This study |
Clade 8: Eutypa cerasi forms a distinct lineage which is sister to Eutypa lata (EP18, RGA01) (Fig.
The genus Diatrype was introduced by Fries (1849). The genus is characterised by stromata widely effuse or verrucose, flat or slightly convex, with discoid or sulcate ostioles at the surface, 8-spored and long-stalked asci and hyaline or brownish, allantoid ascospores. In this study, we introduce a new species of Diatrype from China.
GMB0045.
Refers to the name of the location, where the type specimen was collected.
Saprobic on decaying branches of an unidentified plant. Sexual morph: Stromata immersed in bark, aggregated, irregular in shape, widely effused, flat, margin diffuse, surface dark brown to black, with punctiform ostioles scattered at surface, with tissues soft, white between perithecia. Entostroma dark with embedded perithecia in one layer. Perithecium semi-immersed in stroma, globose to subglobose, glabrous, with cylindrical neck, brevicollous or longicollous 283.5–343.5 μm high, 207–290 μm broad (av. = 315.5 × 248.0 μm, n = 10), ovoid, obovoid to oblong, monostichous, aterrimus. Ostiole opening separately, papillate or apapillate, central. Peridium 30–50 μm thick, dark brown to hyaline with textura angularis cell layers. Asci 90.5–160.5 × 7.0–15.0 μm (av. = 129.5 × 10.5 μm n = 30) 8-spored clavate, unitunicate, with rounded apex, apical rings inamyloid. Ascospores 11–18.5 × 2–4 μm (av. = 14.9 × 2.8 μm, n = 30), irregularly arranged, allantoid, slightly curved, brown to dark brown, smooth, aseptate, usually with oil droplets. Asexual morph: undetermined.
Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became luteous, dense but, thinning towards edge, margin rough, white from above, reverse white at margin, pale yellow to luteous at centre, no pigmentation produced on PDA medium, no conidia observed on PDA or on OA media.
China, Yunnan Province, Baoshan City, Lancang River Nature Reserve (25°1'17.44"N, 99°35'10.05"E) on branches of an unidentified plant, 4 October 2019. Altitude: 2549 m., Y.H. Pi & Qiong Zhang, LC172 (GMB0045, holotype,
China, Yunnan Province, Baoshan City, Lancang River Nature Reserve (25°1'17.44"N, 99°35'10.05"E) on branches of an unidentified plant, 4 October 2019. Altitude: 2549 m., Y.H. Pi and Qiong Zhang, LC173 (GMB0046,
GMB0045 (LSU: MW797057, RPB2: MW81490); GMB00046 (LSU: MW797058); GMB0047 (LSU: MW797060, RPB2: MW814903)
Our new strain, GMBC0045 falls into the unresolved clade (Clade 7) which comprises five Diatrypella and one Diatrype species (Fig.
In the phylogenetic analyses, it can be seen that Clade 7 can be defined as a new genus, but it is difficult to find the common morphological similarities among these species. More specimens and sequence or chemical composition analysis are needed in the future to determine whether Clade 7 can be a new genus. The characteristics of the stromata of Diatrypella spp. in clade 7 are solitary and scattered, which is distinctly different from widely effuse, flat and slightly convex stromata of Diatrype lancangensis and Diatrype palmicola (
Refers to this genus resembling Diatrype in morphology, but it is phylogenetically distinct.
Pseudodiatrype hainanensis S. H. Long & Q.R. Li sp. nov.
Saprobic on decaying branches of an unidentified plant. Sexual morph: Stromata scattered or aggregated on host, wart-like, pustulate, visible as black, rounded to irregular in shape on host surface, erumpent through host bark, 5–20 ascomata immersed in one stroma. Endostroma consists of outer layer of black, small, dense, thin parenchymal cells and inner layer of white, large, loose parenchymal cells, thin, pale yellow, powdery near margin of the black cells. Ostiole opening through host bark and appearing as black spots, separately, papillate or apapillate, central. Perithecium immersed in stroma, globose to subglobose, glabrous, with cylindrical neck, brevicollous or longicollous. Peridium is composed of an outer layer of dark brown to black, thin-walled cells, arranged in textura angularis, the inner layer of hyaline thin-walled cells of textura angularis. Asci 8-spored, unitunicate, clavate, long-stalked, apically rounded, apical rings inamyloid. Ascospores irregularly arranged, allantoid, slightly or moderately curved, smooth, subhyaline, aseptate, usually with two oil droplets. Asexual morph: undetermined.
The genus Pseudodiatrype is introduced to accommodate the new collection made from Hainan Province of China and typified by Pseudodiatrype hainanensis. Pseudodiatrype is monotypic and, morphologically, resembles Diatrype and Allodiatrype Konta & K.D. Hyde. However, Pseudodiatrype can be distinguished from Diatrype by its 5–20 ascomata immersed in a stroma, while the stroma of species of Diatrype is distributed over large areas, sometimes covering the surface of the host (
GMB0054.
Refers to the location of collections, Hainan Province.
Saprobic on decaying branches of an unidentified plant. Sexual morph: Stromata wart-like, pustulate, 2–3.6 mm long and 1.6–3 mm broad (av. = 3.2 × 1.9 mm, n = 30), about 2 mm thick, 5–20 in single stroma, visible as black, rounded to irregular in shape on the host surface, erumpent through host bark, solitary to gregarious. Endostroma composed of an outer layer of dark brown to black, small, tightly packed, thin parenchymatous cells and an inner layer of white, large, loose parenchymal cells with powdery, thin, yellowish tissue. Ostiole opening separately, papillate or apapillate, central. Perithecium immersed in the stroma, globose to subglobose, glabrous, with cylindrical neck, brevicollous or longicollous, 193–347 μm high, 138–206 μm diam. (av. = 278 × 156 μm, n = 10). Peridium 30–50 μm thick, dark brown to hyaline with textura angularis cell layers. Asci 110–155.5 × 6–10 μm (av. = 132 × 8 μm, n = 30), 8-spored, unitunicate, clavate, long-stalked, apically rounded with inamyloid rings. Ascospores 8.5–13 × 1.5–2.5 μm (av. = 10.5 × 2 μm, n = 30), irregularly arranged, allantoid, slightly or moderately curved, smooth, subhyaline, aseptate, usually with two oil droplets. Asexual morph: undetermined.
Pseudodiatrype hainanensis (GMB0054, holotype) A stromata on host substrate B, C stromata on host D transverse section through ascostroma E vertical section through ascostroma F culture on PDA G section through the ascostroma H ostiolar canal I peridium J–M ascospores N–P asci. Scale bars: 40 μm (G); 10 μm (H–P).
Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale brown, dense, but thinning towards edge, fluffy to slightly fluffy, white from above, pale brown from below, no pigmentation produced on PDA medium, no conidia observed on PDAor on OA media.
China, Hainan Province, Wuzhishan City, Wuzhishan Nature Reserve (18°54'21.81"N, 109°40'54.12"E) on branches of unidentified plant, 14 November 2020. Altitude: 775 m. Y.H. Pi & Q.R. Li, WZS59 (GMB0054, holotype,
China, Hainan Province, Wuzhishan City, Wuzhishan Nature Reserve (18°54'21.81"N, 109°40'54.12"E) on branches of an unidentified plant, 14 November 2020. Altitude: 775 m, Y.H. Pi & Q.R. Li, WZS66 (GMB0055, living culture GMBC0055)
GMB0054 (LSU: MW797055, RPB2: MW814900); GMB0055 (LSU: MW797056, RPB2 MW814901).
A peculiar feature of Pseudodiatrype hainanensis is the composition of endostroma. There are black outer layer cells, white inner layer cells and powdery, yellowish cells that are smaller than the white cells at the edge of the endostroma near the black cells in endostroma.
The genus Diatrypella was introduced by Cesati & De Notaris (1863) and was typified with Diatrypella verruciformis (Ehrh.) Nitschke. This genus was characterized by pustule-like stromata erumpent through the host surface, polysporous asci and allantoid ascospores and libertella-like asexual morphs (
GMB0041
Refers to its similar species of Diatrype oregonensis.
Saprobic on decaying branches of unidentified plant. Sexual morph: Stromata pustulate, with groups of 3–16 perithecia, rugose, visible as black, erumpent, scattered, surrounded by a thin, black line in host tissue, solitary to gregarious, 1–3 mm long and 0.5–2 mm broad (av. = 2 × 1.5 mm, n = 30), about 1 mm thick. Endostroma white to light yellow. Ostiole opening separately, papillate or apapillate, central. Perithecium immersed in stroma, globose to subglobose, glabrous, with cylindrical neck, brevicollous or longicollous 218.5–465 μm high, 112–257 μm diam. (av. = 306 × 164 μm, n = 10), globose to subglobose, glabrous, ostioles individual. Peridium: 30–50 μm thick, dark brown to hyaline with textura angularis cell layers. Asci 95–149 × 6.5–11.5 μm (av. = 120 × 10.5 μm, n = 30), 8-spored, unitunicate, clavate or cylindrical, long-stalked, apically rounded, apical rings inamyloid. Ascospores 11–16 × 1.5–3.5 μm (av. = 14 × 2.5 μm, n = 30), irregularly arranged, allantoid, slightly or moderately curved, subhyaline to slightly brown, smooth, aseptate, usually with two oil droplets. Asexual morph: undetermined.
Diatrypella pseudooregonensis (GMB0041, holotype) A stromata on host substrate B, C stromata on host substrate D transverse section through ascostroma E vertical section through ascostroma F culture on PDA G section through the ascostroma H ostiolar canal I, J asci K–N ascospores. Scale bars: 20 μm (G); 10 μm (H–N).
Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale brown, dense, but thinning towards the edge, margin rough, white from above, white at margin and light brown at centre from below, no pigmentation produced on PDA medium, no conidia observed on PDA or on OA media.
China, Yunnan Province, Baoshan City, Lancang River Nature Reserve (25°1'19.88"N, 99°35'30.68"E) on branches of an unidentified plant, 5 October 2019. Altitude: 2677 m, Y.H. Pi & Qiong Zhang, LC323 (GMB0041, holotype,
China, Yunnan Province, Baoshan City, Lancang River Nature Reserve (25°1'13.51"N, 99°35'25.59"E) on branches of an unidentified plant, 6 October 2019. Altitude: 2630 m, Y.H. Pi & Qiong Zhang, LC384 (GMB0043,
Additional sequences. GMB0041 (LSU: MW797062, RPB2: MW814906); GMB0043 (LSU: MW797064, RPB2: MW814907); GMB0040 (LSU: MW797061, RPB2: MW814905); GMB0039 (LSU: MW797059, RPB2: MW814904); GMB0042 (LSU: MW797063); GMLB0044 (LSU: MW979054, RPB2: MW814899).
Note. Morphologically, Diatrype has 8 ascospores in a single ascus, while Diatrypella has more than eight ascospores in each ascus (
Saprobic on decaying branches of an unidentified plant. Sexual morph: Stromata scattered on the host, 0.8–1.5 mm long and 0.8–2 mm broad (av. = 1.2 × 1.3 mm, n = 30) pustulate, visible as black, rounded to irregular in shape on host surface, semi-immersed, erumpent through host bark, with 2–8 ascomata immersed in one stroma. Endostroma consists of outer dark brown, small, dense, thin parenchymal cells and an inner layer of white, large, loose parenchymal cells. Ostiole opening separately, papillate or apapillate, central 710.7–787.2 μm high, 270.2–422 μm diam. (av. = 742 × 363 μm, n = 10). Perithecium immersed in stroma, round to oblong, with cylindrical neck, brevicollous or longicollous. Peridium composed of outer layer of dark brown to black, thin-walled cells, arranged in textura angularis, inner layer of hyaline thin-walled cells of textura angularis. Asci 111.4–152.9 × 10.6–17.5 μm (av. = 124.5 × 15.5 μm, n = 30), polysporous, clavate, long-stalked, apically rounded. Ascospores 8–11 × 1–2 μm (av. = 8.9 × 1.7 μm, n = 30), overlapping, crowded, allantoid, slightly or moderately curved, smooth, subhyaline, yellowish in mass, aseptate, usually with two oil droplets. Asexual morph: undetermined.
Diatrypella vulgaris (GMB0051, new record for China) A stromata on host substrate; B, C close-up of stroma D transverse sections through ascostroma E vertical section through ascostroma F culture on PDA G section through the ascostroma H, I ostiolar canal J, K asci L–O ascospores. Scale bars: 20 μm (G); 10 μm (H–I).
Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale brown, dense, but thinning towards edge, medium dense, white from above, reverse side white at margin, flesh to pale brown at centre, no pigmentation produced on PDA medium, no conidia observed on PDA or on OA media.
China, Guizhou Province, Guiyang City, Gaopo Township (26°29'72.02"N, 106°29'55.57"E), on branches of unidentified plant, 30 October 2020. Altitude: 1589 m, S.H. Long, GP02 (GMB0051,
GMB0051 (LSU: MW797051, RPB2: MW814897).
The comparison of ITS sequences in NCBI showed that this isolate is 100% similar to the strain of Diatrypella vulgaris (HVGRF03), isolated from holotype specimens. Morphologically, GMB0051 shows the same features as Diatrypella vulgaris. The stromata of these specimens are similar, but ascospores of GMB0051 are thinner than those of the HVGRF03 (8–10 × 2–2.5 μm) and, when compared with the ascospores of strain MFLUCC 17-0128 (4.5–7.5 × 1–2 μm), they are shorter than GMB0051 (
≡ Eutypella oregonensis Wehm. Pap. Mich. Acad. Sci. 11: 163 (1930)
≡ Diatrype oregonensis (Wehm.) Rappaz, Mycol. helv. 2(3): 420 (1987)
See
The strains of Diatrype oregonensis (DPL200, CA117) generated from
The genus Allodiatrype was introduced by
≡ Diatrype thailandica R.H. Pereraet al., Fungal Diversity 78: 1–237, [105] (2016)
Saprobic on decaying branches of unidentified plant. Sexual morph: Stromata wart-like, pustulate, 0.5–1.8 mm long and 0.8–2.2 mm broad (av. = 1.2 × 1.3 mm, n = 30), about 1 mm thick, 1–18 in a single stroma, visible as black, rounded to irregular in shape on the host surface, erumpent through host bark, solitary to gregarious. Endostroma composed of an outer layer of dark brown to black, small, tightly packed, thin parenchymatous cells and an inner layer of white to yellow, large, loose parenchymal cells. Ostiole opening separately, papillate or apapillate, central. Perithecium immersed in stroma, globose to subglobose, glabrous, with cylindrical short neck, 377–447 μm high, 191–264 μm diam. (av. = 406 × 221 μm, n = 10). Peridium hyaline to dark brown with textura angularis cell layers. Asci 80–113.5 × 6.9–10 μm (av. = 109.3 × 8.5 μm, n = 30), 8-spored, unitunicate, clavate, long-stalked, upper part inflated, apically rounded to truncate, apical rings inamyloid. Ascospores 6–11 × 2–2.5 μm (av. = 8.9× 2.3 μm, n = 30), irregularly arranged, allantoid, slightly curved, smooth, subhyaline, aseptate, usually with two oil droplets. Asexual morph: undetermined.
Allodiatrype thailandica (GMB0050, new record for China) A stromata on host substrate B, C close-up of stromata D transverse section through ascostroma E vertical section through ascostroma F culture on PDA G section through the ascostroma H ostiolar canal I–K ascospores L–N asci. Scale bars: 20 μm (G); 10 μm (H–N).
Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale yellow, irregular in shape, medium dense, flat or effuse, slightly raised, with edge fimbriate, fluffy to fairly fluffy, white from above, reverse side white at margin, pale brown at centre, no pigmentation produced on PDA medium, no conidia observed on PDA or on OA media.
China, Yunnan Province, Baoshan City, Lancang River Nature Reserve (24°57'25.35"N, 99°44'22.82"E), on branches of unidentified plant, 2 October 2019. Altitude: 1317 m, Y.H. Pi & Qiong. Zhang, LC103 (GMB0050,
GMB0050 (LSU: MW797052).
The ITS sequence data were subjected to BLAST in NCBI and the results showed that it is 100% similar to Allodiatrype thailandica. Additionally, based on morphological and phylogenetic analyses, this strain was identified as the A. thailandica. The stromata are similar, but the ascospores of GMB0050 are longer and wider than the ascospores of strain MFLUCC 15-3662 (3.8–6.9 × 1–1.4 μm) isolated from the holotype specimen, but it is similar to the strain MFLU 17-0735 (6.5–10.7 × 1.6–2.7 μm) (
The genus Neoeutypella was introduced by
see
China, Guizhou Province, Guiyang City, Gaopo Township (26°29'72.37"N, 106°29'59.33"E), on branches of unidentified plant, 30 November 2020. Altitude: 1589 m, S.H. Long, GP01 (GMB0052,
GMB0052 (LSU: MW797050, RPB2: MW814896).
The morphological characteristics of this specimen are consistent with those of N. baoshanensis a species described by
Neoeutypella baoshanensis (GMB0052) A stromata on host substrate B close-up of stromata C transverse section through ascostroma D vertical section through ascostroma E pigments in KOH F culture on PDA G section through the ascostroma H ostiolar canal I, J ascospores K–M asci. Scale bars: 20 μm (G); 10 μm (H–M).
Tulasne & Tulasne (1863) introduced the genus Eutypa with Eutypa lata as the type species. This genus includes several phytopathogens, such as E. lata (Pers.) Tul. & C. Tul. and E. leptoplaca (Durieu & Mont.) Rappaz (Moyo et al. 2017). The morphological characteristics of this genus are black, rounded to irregular-shaped stromata on the host surface, erumpent through host epidermis, solitary to gregarious, entostromatic region, consisting of white pseudoparenchymatous cells and thin black pseudoparenchymatous tissue around the white entostroma, 8-spored, spindle-shaped asci and hyaline, oblong to allantoid ascospores (
GMB0048.
Refers to its host, Prunus cerasus.
Saprobic on decaying branches of Prunus cerasus. Sexual morph: Stromata immersed in bark, covering surface of host, irregular in shape, widely effused, flat, margin diffuse, surface dark brown to black, with punctiform ostioles scattered at surface. Endostroma consists of an outer layer of black, small, dense, thin parenchymal cells and an inner layer of white, large, loose parenchymal cells. Perithecium semi-immersed in stroma, globose to subglobose, glabrous, with cylindrical neck, brevicollous 203–304 μm high, 346–477 μm diam. (av. = 408 × 250 μm, n = 10), ovoid, obovoid to oblong. Ostiole opening separately, papillate or apapillate, central. Peridium 30–50 μm thick, dark brown to hyaline with textura angularis cell layers. Asci 83.2–120 × 5.1–8.2 μm (av. = 104.4 × 6.3 μm n = 30) 8–spored clavate, unitunicate, rounded to truncate apex, apical rings inamyloid. Ascospores 7.3–9.9 × 1.4–2 μm (av. = 8.5 × 1.7 μm, n = 30), overlapping, allantoid, slightly curved, subhyaline, smooth, aseptate, usually with oil droplets. Asexual morph: undetermined.
Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale yellow, irregular in shape, medium dense, flat or effuse, white from above, reverse white at margin, pale yellow at centre, no pigmentation produced on PDA medium, no conidia observed on PDA or on OA media.
China, Guizhou Province, Guiyang City, Aha Lake National Wetland Park (26°32'50.21"N, 106°40'15.78"E), on branches of Prunus cerasus, 12 August 2020. Altitude: 1089 m, S.H. Long, AH4 (GMB0048, holotype,
China, Guizhou Province, Guiyang City, Aha Lake National Wetland Park (26°32'47.79"N, 106°40'21.09"E), on branches of Cerasus sp., 12 August 2020. Altitude: 1089 m, S.H. Long, AH40 (GMB0049,
GMB0048 (LSU: MW797048, RPB2: MW814894); GMB0049 (LSU: MW797049, RPB2: MW814895).
Eutypa lata is an important pathogen that has a wide range of hosts. However, the classification of E. lata is confusing because there are many variants in previous studies; now all are classified as E. lata (
Paraeutypella was introduced by
≡ Eutypella citricola Speg., Anal. Mus. nac. Hist. nat. B. Aires 6: 245 (1898)
For description, see
China, Guizhou Province, Guiyang City: Aha Lake National Wetland Park (26°20'37.28"N, 108°21'4.34"E), on branches of unidentified plant, 30 August 2020. Altitude: 802 m, S.H. Long, LGS147 (GMB0053,
GMB0053 (LSU: 797053, RPB2: MW814898).
The ITS sequence data were compared by using NCBI and the result showed that it is 100% similar to the ex-type strain (HVVIT07) of P. citricola. The morphological features of the new collection are consistent with those described by
Paraeutypella citricola (GMB0053) A stromata on host substrate B, C stromata on host D transverse section through ascostroma E vertical section through ascostroma F culture on PDA G section through the ascostroma H ostiolar canal I peridium J–K ascospores L–O asci. Scale bars: 40 μm (G); 10 μm (H–O).
In this study, one new genus, three new species, two new records from China, a novel combination and two known species were reported from karst areas of China. We used molecular data to delimit the species of Diatrypaceae. The new genus Pseudodiatrype is morphologically similar to Allodiatrype and Diatrype, but distinct in the size of stromata, number of ascomata and colour of endostroma; it also formed a distinct branch in the phylogenetic analyses (Fig.
Our phylogenetic analyses, based on ITS and β-tubulin, agree with the previous studies (
Diatrype and Diatrypella have morphologically similar verruculose stromata and allantoid ascospores and the polysporous or 8-spored ascus serve as a basis for distinguishing the two genera. However, in phylogenetic analyses, species of these two genera overlap. In this study, we used the phylogenetic analyses as the main basis for classification following
The phylogenetic tree shows that the classification of Diatrypaceae is confusing. Members of Diatrypella (D. favacea, D. hubeiensis, D. pulvinata and D. yunnanensis) cluster with Diatrype palmicola and Diatrype lancangensis.Maybe this clade should be identified as a new genus. We will discuss its classification status after more strains, more gene sequences and new taxonomic features are collected. Some species of Diatrypella (D. iranensis and D. macrospora) which have polysporous ascus are placed between species of Diatrype, and they are transferred to Diatrype iranensis and Diatrype macrospora by Zhu et al. (
This research was supported by the National Natural Science Foundation of China (32000009 and 31960005); the Fund of the Science and Technology Foundation of Guizhou Province ([2020]1Y059); Guiyang Science and Technology Planning Project No. (2017)30-19; Guizhou Province Ordinary Colleges and Universities Youth Science and Technology Talent Growth Project [2021]154. Nalin N. Wijayawardene would like to thank the National Natural Science Foundation of China (No. NSFC 31950410558), the State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medicial University (No. FAMP201906K) and High-Level Talent Recruitment Plan of Yunnan Provinces (“Young Talents” Program and “High-End Foreign Experts” Program); the Fund of High-Level Innovation Talents [No. 2015-4029], the Base of International Scientific and Technological Cooperation of Guizhou Province [No. [2017]5802].