Research Article |
Corresponding author: Nakarin Suwannarach ( suwan.462@gmail.com ) Corresponding author: Saowaluck Tibpromma ( saowaluckfai@gmail.com ) Academic editor: Chitrabhanu Bhunjun
© 2022 Li Lu, Samantha C. Karunarathna, Dong-qin Dai, Ruvishika S. Jayawardena, Nakarin Suwannarach, Saowaluck Tibpromma.
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:
Lu L, Karunarathna SC, Dai D-q, Jayawardena RS, Suwannarach N, Tibpromma S (2022) Three new species of Nigrograna (Dothideomycetes, Pleosporales) associated with Arabica coffee from Yunnan Province, China. MycoKeys 94: 51-71. https://doi.org/10.3897/mycokeys.94.95751
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Coffee is one of the most important cash crops in Yunnan Province, China. Yunnan is ranked as the biggest producer of high-quality coffee in China. During surveys of microfungi from coffee plantations in Yunnan, six fungal strains that resemble Nigrogranaceae were collected. Multi-gene analyses of a combined SSU-LSU-ITS-rpb2-tef1-α sequence data matrix were used to infer the phylogenetic position of the new species in Nigrograna while morphological characteristics were used to deduce the taxonomic position of the new species. Six fungal strains isolated from decaying branches of Coffea arabica represent three new saprobic species in Nigrograna. The three new species, N. asexualis, N. coffeae, and N. puerensis, are described with full (macro and micro characteristics) descriptions, illustrations, and a phylogenetic tree that shows the phylogenetic position of new taxa.
3 new taxa, Coffea arabica, Nigrogranaceae, phylogeny, saprobic fungi, taxonomy
Coffee (Coffea L.) was first planted in Yunnan Province, China in 1982 (
Fungal diversity is highly uncertain; the current estimated numbers are between 1.5 to 12 million, of which about 150,000 species have been named and classified (
Pleosporales, belonging to Dothideomycetes O.E. Erikss. & Winka, was first proposed by
Nigrogranaceae Jaklitsch & Voglmayr (Pleosporales) was proposed as a new family by
Nigrograna was introduced by
In this study, three saprobic Nigrograna were collected from Coffea arabica branches in Yunnan Province, China. One species was isolated as an asexual morph (N. asexualis), while the other two isolated as sexual morphs (N. coffeae, N. puerensis) are illustrated and described as new species based on morphology and multi-gene phylogenetic analyses and are compared with closely related taxa.
Coffee branch samples were collected from coffee plantations in Pu’er and Xishuangbanna, Yunnan Province, China. Specimens were put in plastic bags and taken to the mycology laboratory at Qujing Normal University. The vertical sections of fruiting structures were made for microscope studies and photomicrography. Micro-morphological characteristics were observed using a Leica DM2500 compound microscope and photographed with a Leica DMC4500 camera fitted onto the microscope. Color codes in the manuscript followed colorhexa (https://www.colorhexa.com). The measurements were processed in Tarosoft (R) Image Frame Work v. 0.9.7, and photographic plates were made in Adobe Photoshop CC 2018. Single spore isolation was carried out following
Genomic DNA was extracted from the fresh fungal mycelia which were grown on PDA for about two weeks, using Biospin Fungus Genomic DNA Extraction Kit–BSC14S1 (BioFlux, China) following the manufacturer’s instructions.
Taxa names, strain numbers, and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses. Newly generated sequences in this study are indicated in bold. The type species are noted with T after the species name, while NA indicates the unavailability of data.
Taxon | Strain numbers | ITS | LSU | rpb2 | SSU | tef1-α |
---|---|---|---|---|---|---|
Cyclothyriella rubronotata (Berk. & Broome) Jaklitsch & Voglmayr T | CBS 141486 | KX650544 | KX650519 | NA | KX650507 | KX650574 |
Cyclothyriella rubronotata | CBS 419.85 | NA | GU349002 | GU301875 | NA | GU371728 |
Nigrograna antibiotica (M. Kolařík & A. Kubátová) M. Kolařík T | CCF 4378 | JX570932 | KF925327 | NA | KF925328 | JX570934 |
Nigrograna antibiotica | CCF 4998 | LT221894 | NA | LT221895 | NA | NA |
Nigrograna aquatica W. Dong, H. Zhang & K.D. Hyde T | MFLUCC 14-1178 | MF399065 | MF415392 | NA | MF415394 | MF498582 |
Nigrograna aquatica | MFLUCC 17-2318 | MT627705 | MN913705 | NA | NA | NA |
Nigrograna asexualis T | ZHKUCC 22-0214 | OP450965 | OP450971 | OP432241 | OP450979 | OP432245 |
Nigrograna asexualis | ZHKUCC 22-0215 | OP450966 | OP450972 | OP432242 | OP450980 | OP432246 |
Nigrograna cangshanensis Z.L. Luo, H.Y. Su & K.D. Hyde T | MFLUCC 15-0253 | KY511063 | KY511064 | NA | KY511065 | NA |
Nigrograna carollii M. Kolařík T | CCF 4484 | LN626657 | LN626682 | LN626662 | LN626674 | LN626668 |
Nigrograna chromolaenae Mapook & K.D. Hyde T | MFLUCC 17-1437 | MT214379 | MT214473 | NA | NA | MT235801 |
Nigrograna coffeae T | ZHKUCC 22-0210 | OP450967 | OP450973 | OP432243 | OP450981 | OP432247 |
Nigrograna coffeae | ZHKUCC 22-0211 | OP450968 | OP450974 | OP432244 | OP450982 | OP432248 |
Nigrograna fuscidula (Sacc.) Jaklitsch & Voglmayr T | CBS 141556 | KX650550 | NA | NA | NA | KX650525 |
Nigrograna fuscidula | CBS 141476 | KX650547 | NA | KX650576 | KX650509 | KX650522 |
Nigrograna fuscidula | MF1a | KX650548 | NA | NA | NA | KX650523 |
Nigrograna fuscidula | MF3 | KX650549 | NA | NA | NA | KX650524 |
Nigrograna hydei J.F. Zhang, J.K. Liu & Z.Y. Liu T | GZCC 19-0050 | MN387225 | MN387227 | NA | NA | MN389249 |
Nigrograna impatientis J.F. Zhang, J.K. Liu & Z.Y. Liu T | GZCC 19-0042 | MN387226 | MN387228 | NA | NA | MN389250 |
Nigrograna jinghongensis Wanas. & K.D. Hyde T | KUMUCC 21-0035 | MZ493303 | MZ493317 | MZ508421 | MZ493289 | MZ508412 |
Nigrograna jinghongensis | KUMUCC 21-0036 | MZ493304 | MZ493318 | MZ508422 | MZ493290 | MZ508413 |
Nigrograna kunmingensis T.Y. Du & Tibpromma T | ZHKUCC 22-0242 | OP456214 | OP456379 | NA | OP456382 | OP471608 |
Nigrograna kunmingensis | ZHKUCC 22-0243 | OP484334 | OP456380 | NA | OP456383 | OP471609 |
Nigrograna locuta-pollinis F. Liu & L. Cai T | CGMCC 3.18784 | MF939601 | MF939583 | MF939610 | NA | MF939613 |
Nigrograna locuta-pollinis | LC11690 | MF939603 | MF939584 | MF939611 | NA | MF939614 |
Nigrograna mackinnonii T | CBS 674.75 | KF015654 | KF015612 | KF015703 | GQ387552 | KF407986 |
Nigrograna mackinnonii | E5202H | JX264157 | KJ605422 | JX264156 | JX264155 | JX264154 |
Nigrograna mackinnonii | E9303e | JN545759 | LN626681 | LN626666 | LN626678 | LN626673 |
Nigrograna magnoliae Wanas. T | MFLUCC 20-0020 | MT159628 | MT159622 | MT159611 | MT159634 | MT159605 |
Nigrograna magnoliae | GZCC 17-0057 | MF399066 | MF415393 | NA | MF415395 | MF498583 |
Nigrograna magnoliae | MFLUCC 20-0021 | MT159629 | MT159623 | MT159612 | MT159635 | MT159606 |
Nigrograna mycophila Jaklitsch, Friebes & Voglmayr T | CBS 141478 | KX650553 | NA | NA | NA | KX650526 |
Nigrograna mycophila | CBS 141483 | KX650555 | NA | KX650577 | KX650510 | KX650528 |
Nigrograna mycophila | MF6 | KX650554 | NA | NA | NA | KX650527 |
Nigrograna norvegica Jaklitsch & Voglmayr T | CBS 141485 | KX650556 | NA | KX650578 | KX650511 | NA |
Nigrograna obliqua Jaklitsch & Voglmayr T | CBS 141477 | KX650560 | NA | KX650580 | NA | KX650531 |
Nigrograna obliqua | CBS 141475 | KX650558 | NA | KX650579 | KX650512 | KX650530 |
Nigrograna obliqua | MRP | KX650561 | NA | KX650581 | NA | KX650532 |
Nigrograna peruviensis (M. Kolařík & R. Gazis) M. Kolařík T | CCF 4485 | LN626658 | LN626683 | LN626665 | LN626677 | LN626671 |
Nigrograna puerensis T | ZHKUCC 22-0212 | OP450969 | OP450975 | NA | OP450983 | OP432249 |
Nigrograna puerensis | ZHKUCC 22-0213 | OP450970 | OP450976 | NA | OP450984 | OP432250 |
Nigrograna rhizophorae Dayar., E.B.G. Jones & K.D. Hyde T | MFLUCC 18-0397 | MN047085 | NA | MN431489 | NA | MN077064 |
Nigrograna rhizophorae | MFLU 19-1234 | NA | MN017845 | MN431490 | NA | MN077063 |
Nigrograna samueliana Devadatha, V.V. Sarma & E.B.G. Jones T | NFCCI-4383 | MK358817 | MK358812 | MK330939 | MK358810 | MK330937 |
Nigrograna thymi Mapook, Camporesi & K.D. Hyde T | MFLUCC 14-1096 | KY775576 | KY775573 | NA | KY775574 | KY775578 |
Nigrograna yasuniana M. Kolařík T | YU.101026 | HQ108005 | LN626684 | LN626664 | LN626676 | LN626670 |
Occultibambusa bambusae D.Q. Dai & K.D. Hyde T | MFLUCC 13-0855 | KU940123 | KU863112 | KU940170 | NA | KU940193 |
Occultibambusa fusispora Phookamsak, D.Q. Dai & K.D. Hyde | MFLUCC 11-0127 | MZ329036 | MZ325466 | MZ329032 | MZ329028 | MZ325469 |
Occultibambusa pustula D.Q. Dai & K.D. Hyde T | MFLUCC 11-0502 | KU940126 | KU863115 | NA | NA | NA |
Paradictyoarthrinium diffractum Matsush. | MFLUCC13-0466 | KP744455 | NA | KP744498 | NA | NA |
Paradictyoarthrinium tectonicola Doilom & K.D. Hyde T | MFLUCC 13-0465 | KP744456 | NA | KP744500 | KP753961 | KX437763 |
Seriascoma didymosporum Phookamsak, D.Q. Dai, Karun. & K.D. Hyde T | MFLUCC 11-0179 | KU940127 | KU940196 | KU863116 | NA | KU940173 |
Seriascoma honghense H.B. Jiang, Phookamsak & K.D. Hyde T | KUMCC 21-0021 | MZ329039 | MZ325468 | MZ329035 | NA | MZ325470 |
Versicolorisporium triseptatum Sat. Hatak., Kaz. Tanaka & Y. Harada T | HHUF 28815 | NR_119392 | NA | NG_042318 | NG_060995 | NA |
Phylogenetic analyses of the aligned sequences referred to
Phylogenetic analyses were conducted with maximum likelihood (ML) and Bayesian inference (BI) algorithms on the CIPRES Science Gateway portal (https://www.phylo.org/) (
Three new species formed a distinct clade in Nigrograna with strong statistical support (N. coffeae and N. puerensis ML = 100%, BIPP = 1.00, and N. asexualis ML = 68%, BIPP = 0.97). Multi-locus data (SSU, LSU, ITS, rpb2 and tef1-α) composed of 54 strains (Table
The ML analysis of the combined dataset yielded a best-scoring tree with a final ML optimization likelihood value of -23091.568105. The alignment has 1495 distinct alignment patterns, with 33.58% completely undetermined characters and gaps. Parameters for the GTR + I + G model of the combined SSU, LSU, ITS, rpb2 and tef1-α were as follows: estimated base frequencies A = 0.247145, C = 0.250645, G = 0.263985, T = 0.238225; substitution rates AC = 1.810004, AG = 4.475190, AT = 1.758134, CG = 1.340389, CT = 10.583215, GT = 1.000; gamma distribution shape parameter α = 0.167006. The phylogenetic tree resulting from RAxML analysis is shown in Fig.
The maximum-likelihood phylogram of Nigrograna based on a combined SSU, LSU, ITS, rpb2 and tef1-α sequence dataset with Cyclothyriella rubronotata CBS 141486 and CBS 419.85 as the outgroup taxa (
Species epithet refers to the host genus “Coffea” where the fungus was isolated.
ZHKU 22-0121.
Saprobic on decaying branch of Coffea arabica. Sexual morph: Ascomata 90–140 µm high, 140–200 μm wide (x̄ = 115 × 168 μm, n = 10), immersed, solitary, black spots on substrate, subglobose to oval, sometimes obpyriform, some with ostiolate. Peridium 10–15 µm wide, composed of 3–5 layers, hyaline to brown (#937463) cells of textura angularis. Hamathecium 1.5–3 μm wide, composed of numerous, hyaline, filamentous, septate, branched, pseudoparaphyses. Asci 50–70 × 7–11 μm (x̄ = 58 × 9 μm, n = 20), 8-spored, bitunicate, fissitunicate, clavate to cylindric-clavate, short stalked, some with club-shape pedicel, apically rounded, with a small ocular chamber. Ascospores 12–16 × 4–5 μm, (x̄ = 14.4 × 4.6 μm, n = 30), overlapping uni- to bi-seriately arranged, fusiform, straight or slightly curved, hyaline when immature and become pale brown (#e1af33) to dark-brown (#6e5031) when mature, mostly 1-septate, few 2 or 3-septate, constricted at each septum, with obviously guttulate. Asexual morph: Undetermined.
Ascospores germinated on PDA within 24 h and germ tubes arising from both ends. Colonies on PDA, reaching 4.5 cm diam. after two months of incubation at room temperature (22–26 °C), initially white (#f2f3f4) becoming grey (#bbbeb2) to dark brown (#6e5031) at maturity, dense, circular, slightly raised, smooth surface, radially fimbriate at the edge, reverse dark green (#3a4543) to brown (#937463).
Pu’wen Town, Xishuangbanna, Yunnan Province, China, on a decaying branch of Coffea arabica, (22°31'18"N, 101°2'44"E, 856.89 m), 15 September 2021, LiLu, JHPW16 (ZHKU 22-0121, holotype), ZHKUCC 22-0210 = ZHKUCC 22-0211. GenBank number; ITS: OP450967, LSU: OP450973, rpb2: OP432243, SSU: OP450981, tef1-α: OP432247 (ZHKUCC 22-0210, ex-type); ITS: OP450968, LSU: OP450974, rpb2: OP432244, SSU: OP450982, tef1-α: OP432248 (ZHKUCC 22-0211).
Our phylogenetic analyses showed that Nigrograna coffeae forms an independent clade (100% ML, 1.00 BIPP, Fig.
Nigrograna coffeae (ZHKU 22-0121, holotype) a, b ascomata on the host substrate c a vertical section through an ascoma d peridium e hamathecium f–k asci l germinated ascospore m culture on pda from above and reverse n–s ascospores (arrows indicate the septa). Scale bars: 50 μm (c); 10 μm (d–l); 5 μm (n–s).
The specific epithet “puerensis” refers to the location Pu’er City, where the type species was collected.
ZHKU 22-0122.
Saprobic on decaying branch of Coffea arabica. Sexual morph: Ascomata 90–180 µm high, 90–150 μm wide (x̄ = 138 × 115 μm, n = 10), immersed, with only ostiolar necks visible on the host surface or erumpent, solitary, subglobose to ellipsoid, dark brown (#6e5031). Peridium 10–15 μm wide (x̄ = 13 μm, n = 15), outer layer consists of 2–3 layers of textura prismatica, brown (#937463) and thick-walled cells, inner layer hyaline with thin-walled cells. Hamathecium composed of numerous, 1.5–2 µm wide (x̄ = 1.8 μm, n = 20), filamentous, hyaline, septate, pseudoparaphyse. Asci 50–80 × 8–11 μm (x̄ = 66 × 9.5 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical to clavate, short pedicellate, apically rounded, with poorly developed ocular chamber. Ascospores 15–18 × 4–5 μm, (x̄ = 16 × 4.5 μm, n = 30), uni- to bi-seriately arranged, fusoid, apical cell and basal cell acute, and apical cell slightly wider than basal cell, straight or slightly curved, 1-septate, constricted at septum, guttulate, hyaline to yellow-brownish (#daceb8) when young, brownish (#937463) when mature. Asexual morph: Undetermined.
On PDA, colonies reached up to 4 cm diam. after two months at room temperature (22–26 °C). Colony dense, circular, slightly raised at the center, surface with white aerial mycelium, fluffy, with a serrate edge, grayish (#c9bfb3) to dark brown (#6e5031) from center to edge, reverse dark green (#3a4543) to dark brown (#6e5031).
Pu’er City, Yunnan Province, China, on a decaying branch of Coffea arabica, (22°36'2"N, 101°0'59"E, 1016.43 m), 16 September 2021, LiLu, Puer 1-4 (ZHKU 22-0122, holotype), ZHKUCC 22-0212 = ZHKUCC 22-0213. GenBank number; ITS: OP450969, LSU: OP450975, SSU: OP450983, tef1-α: OP432249 (ZHKUCC 22-0212, ex-type); ITS: OP450970, LSU: OP450976, SSU: OP450984, tef1-α: OP432250 (ZHKUCC 22-0213).
Nigrograna puerensis clusters with N. carollii with significant statistical support from ML 100% and BIPP 1.00. In morphology, our new strains best fit Nigrograna by having immersed ascomata, clavate and short pedicellate asci, and pale to brown, fusoid to narrowly ellipsoid, and septate ascospores (
Nigrograna puerensis (ZHKU 22-0122, holotype) a, b ascomata observed on host substrate c a vertical section through an ascoma d peridium e hamathecium f–j asci k germinated ascospore l–p ascospores q culture on PDA from above and reverse. Scale bars: 50 μm (c); 30 μm (d); 15 μm (e–k); 5 μm (l–p).
The species epithet ‘asexualis’ refers to the asexual morph.
ZHKU 22-0123.
Saprobic on decaying branch of Coffea arabica. Sexual morph: Undetermined. Asexual morph: Coelomycetous. Pycnidia 100–230 µm high, 120–180 µm wide (x̄ = 156 × 144 µm, n = 10), globose to subglobose, or pyriform, immersed, solitary, unilocular, dark brown, papillate ostiole, appearing as black spots on host surface. Pycnidial wall 11–16 µm wide (x̄ = 14 µm, n = 15), brown (#937463), the wall with pseudoparenchymatous cells. Conidiophores arising from the pycnidial wall, up to 46 µm long and 3–4.4 µm wide (x̄ = 3.4 µm, n = 25), filiform, septate, hyaline, simple to sparsely branched, with pegs along one or two sides and solitary phialides terminally. Phialides 3–6 × 1–2 µm (x̄ = 4.5 × 1.5 µm, n = 15), variable in shape, phialidic, discrete, ampulliform-lageniform-subcylindrical. Conidia 5–6.5 × 3–4 µm (x̄ = 5.5 × 3.7 µm, n = 30), ellipsoidal, unicellular, aseptate with 1–2 granules, subhyaline, smooth-walled.
Conidium germinated on PDA within 24 h. Colonies growing on PDA reaching 5 cm diam. after two months at room temperature (22–26 °C). Colony dense, circular, surface sparsely hairy, radially striate, with a fimbriate edge, yellowish (#eabf83) to pale brown (#e1af33) at the center and dark brown (#6e5031) at the margin, reverse dark brown (#6e5031).
Pu’er City, Yunnan Province, China, on a decaying branch of Coffea arabica, (22°36'2"N, 101°0'59"E, 1016.43 m), 16 September 2021, LiLu, Puer 1-14 (ZHKU 22-0123, holotype), ZHKUCC 22-0214 = ZHKUCC 22-0215. GenBank number; ITS: OP450965, LSU: OP450971, rpb2: OP432241, SSU: OP450979, tef1-α: OP432245 (ZHKUCC 22-0214, ex-type); ITS: OP450966, LSU: OP450972, rpb2: OP432242, SSU: OP450980, tef1-α: OP432246 (ZHKUCC 22-0215).
In multi-gene phylogeny, Nigrograna asexualis formed a separate (68% ML, 0.97 BIPP) and distinct clade within Nigrograna (Fig.
Nigrograna asexualis (ZHKU 22-0123, holotype) a, b conidiomata on the host substrate c, d vertical sections of a conidioma e peridium f, g conidiophores with phialides h conidia i culture on PDA from above and reverse. Scale bars: 100 μm (c); 50 μm (d); 15 μm (e); 30 μm (f); 20 μm (g); 10 μm (h).
Members of Nigrograna are distributed worldwide in soil, wood, and other plant debris (
Species of Nigrograna are morphologically very similar and overlapping, hence can be interpreted as cryptic species. Therefore, it is difficult to delimit the species based only on their morphological characteristics (
Li Lu thanks Mae Fah Luang University for the award of a fee-less scholarship. The Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University is thanked for the facilities provided for the research work. Dr. Shaun Pennycook is thanked for his advice on new fungi names. Dai Dong-qin thanks the National Natural Science Foundation of China (No. NSFC 31760013, 31950410558), and High-Level Talent Recruitment Plan of Yunnan Provinces (“Young Talents” Program). Samantha C. Karunarathna thanks the National Natural Science Foundation of China grant number 32260004 for the support. Nakarin Suwannarach thanks Chiang Mai University, Thailand for financial support. Saowaluck Tibpromma thanks “the most cited article award” for allowing one free publication in MycoKeys.