Research Article
Research Article
Three new species of Nigrograna (Dothideomycetes, Pleosporales) associated with Arabica coffee from Yunnan Province, China
expand article infoLi Lu§, Samantha C. Karunarathna§, Dong-qin Dai§, Ruvishika S. Jayawardena, Nakarin Suwannarach|, Saowaluck Tibpromma§
‡ Mae Fah Luang University, Chiang Rai, Thailand
§ Qujing Normal University, Qujing, China
| Chiang Mai University, Chiang Mai, Thailand
Open Access


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 (Zhang et al. 2014). To date, about 170 varieties of coffee (Global Biodiversity Information Facility database (GBIF), available at: (accessed on 07 November 2022)) are available in the world, of which Coffea arabica L. is the most popular coffee accounting for 75% of the world’s production, while 25% is provided by C. canephora Pierre ex A. Froehner, and less than 1% by C. liberica W. Bull and other varieties (Sharma 2020). The coffee production in Yunnan Province is approximately 90% of China’s total coffee production (Neilson and Wang 2019), while Pu’er is the largest coffee planting area in Yunnan, in terms of the highest yield and the best quality (Li 2014).

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 (Hawksworth and Lücking 2017; Hyde et al. 2020; Bhunjun et al. 2022). Fungi are important organisms in terrestrial and aquatic ecosystems that are involved in the decomposition and nutrient cycling of dead plant material (Hyde et al. 2020; Bhunjun et al. 2022; Phukhamsakda et al. 2022). Also, saprobic fungi play vital roles in soil food chains, decomposition of plant, and animal materials, and solubilization of phosphorous (Dighton 2003; Pandey et al. 2008). However, coffee saprobic fungi have been poorly investigated (Arias and Abarca 2014; Lu et al. 2022a). Coffee saprobic fungi are distributed in 15 orders, and among them, Pleosporales Luttr. is the most common order (Lu et al. 2022a).

Pleosporales, belonging to Dothideomycetes O.E. Erikss. & Winka, was first proposed by Luttrell (1955), and later it was formally established by Barr (1987). In 2021, it consists of 91 families and 614 genera as the largest order (Hongsanan et al. 2020a; Wijayawardene et al. 2022). They are distributed in terrestrial and aquatic habitats (Zhang et al. 2008; Jiang et al. 2021). The members of Pleosporales are characterized by perithecioid and ostiolar ascomata, with or without periphyses, presence of cellular pseudoparaphyses, bitunicate, with ocular chambers or apical ring asci, various shapes of ascospores, with pigmentation and septation, and sheath present or absent (Zhang et al. 2012; Tennakoon et al. 2021; Yang et al. 2022).

Nigrogranaceae Jaklitsch & Voglmayr (Pleosporales) was proposed as a new family by Jaklitsch and Voglmayr (2016) to accommodate Nigrograna Gruyter, Verkley & Crous as the type genus. Liu et al. (2017) estimated that the divergence time of Nigrogranaceae is around 79 (44–124) Mya in crown age and 131 (86–180) Mya in stem age. Nigrogranaceae is monotypic, and they exist as endophytic, human pathogenic, and saprobic lifestyles (Hongsanan et al. 2020b; Zhang et al. 2020; Boonmee et al. 2021). The sexual morph of Nigrogranaceae is characterized by globose and black, ostiolar, clavate, and fissitunicate ascomata, with a short stipe and asci with a knob-like base, fusoid to narrowly ellipsoid, septate, and smooth or faintly verruculose ascospores (Jaklitsch and Voglmayr 2016). The asexual morph is characterized by pycnidia similar to ascomata, filiform and branched conidiophores, ampulliform or lageniform phialides, rod-like to ellipsoid, and hyaline or sub-hyaline conidia (Jaklitsch and Voglmayr 2016).

Nigrograna was introduced by de Gruyter (2012) with N. mackinnonii (Borelli) Gruyter, Verkley & Crous (basionym: Pyrenochaeta mackinnonii Borelli) as the type species. Pyrenochaeta mackinnonii was reported from a mycetoma patient by Borelli (1976), but it was found to be remote from the generic type species P. nobilis De Not. (de Gruyter et al. 2010, 2013). Since it was not possible to determine which family in Pleosporales P. mackinnonii belongs to, only the new genus Nigrograna was introduced to accommodate P. mackinnonii and named as N. mackinnonii (de Gruyter 2012). Later, Nigrograna was used as a synonym of Biatriospora K.D. Hyde & Borse, as N. mackinnonii is phylogenetically closely related to the type species of Biatriospora (B. marina K.D. Hyde & Borse) (Ahmed et al. 2014), while Hongsanan et al. (2020a) treated Biatriospora and Nigrograna as two separate genera. In 2022, Nigrograna represents 20 epithets listed in Index Fungorum (2022), and the members have been reported as saprobic, human pathogenic, and endophytic worldwide (Kolařík 2018; Zhao et al. 2018), showing a wide range of hosts (marine and terrestrial habitats) (Hyde et al. 2017; Tibpromma et al. 2017; Dayarathne et al. 2020). The sexual morph of Nigrograna is characterized by globose to subglobose and black ascomata, with ostiolar, two-layered peridium, clavate and fissitunicate asci, fusoid to narrowly ellipsoid, straight or curved, septate, and smooth or verruculose ascospores (Jaklitsch and Voglmayr 2016; Zhang et al. 2020). Asexual morph is characterized by globose to subglobose or pyriform pycnidia, filiform and branched conidiophores, hyaline, phialidic, discrete conidiogenous cells, sub-hyaline, aseptate and ellipsoidal conidia (de Gruyter 2012; Jaklitsch and Voglmayr 2016).

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.

Materials and methods

Collection, morphology and isolation

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 ( 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 Senanayake et al. (2020). Herbarium specimens were deposited at Zhongkai University of Agriculture and Engineering (ZHKU), while the living cultures growing on potato dextrose agar (PDA) were deposited at the culture collection of Zhongkai University of Agriculture and Engineering (ZHKUCC). Faces of fungi (FoF) numbers and Index Fungorum (IF) numbers were obtained as explained in Jayasiri et al. (2015) and Index Fungorum (2022).

DNA extraction and PCR amplification

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. Lu et al. (2021) was followed for the Polymerase Chain Reaction (PCR). Five partial gene regions were used in this study viz. the internal transcribed spacer (ITS) region was amplified with the primers ITS4 and ITS5 (White et al. 1990), the 18 s small subunit (SSU) region was amplified by primers NS1 and NS4 (White et al. 1990), the nuclear ribosomal 28 s large subunit (LSU) region was amplified by the primers LROR and LR5 (Vilgalys and Hester 1990), the partial RNA polymerase II subunit (rpb2) region was amplified with the primers RPB2-5F and RPB2-7cR (Liu et al. 1999), and the partial translation elongation factor 1-alpha (tef1-α) gene was amplified with the primers EF1-983F and 2218R (Rehner and Buckley 2005). Lu et al. (2022b) was followed for the amplification reactions of different primers. Amplified PCR products were sent to Sango Biotechnology Co., Ltd. (Shanghai, China) for sequencing. All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.

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

Phylogenetic analyses of the aligned sequences referred to Dissanayake et al. (2020). Newly generated reverse and forward sequences were assembled with Geneious program (9.1.2) and the preliminary identification was done by the BLASTn search in NCBI ( Additional highly similar sequences were downloaded from GenBank ( based on the BLASTn results and recent publications. Single-gene sequence alignments were made in MAFFT v. 7 (, edited in trimAl v1.2 (, and multi-gene alignments were made by Sequence Matrix program (1.7.8) (Vaidya et al. 2011). The sequence datasets used to build the phylogenetic trees are shown in Table 1.

Phylogenetic analyses were conducted with maximum likelihood (ML) and Bayesian inference (BI) algorithms on the CIPRES Science Gateway portal ( (Miller et al. 2012). The ML tree was run with RAxML-HPC v.8 on XSEDE (Stamatakis 2014), and GTRGAMMA substitution model with 1000 bootstrap iterations. The BI tree was run with MrBayes on XSEDE (3.2.7a) (Ronquist et al. 2012). MrModeltest 2.2 (Nylander 2004) and PAUP v. 4.0b10 (Ronquist and Huelsenbeck 2003) were used to evaluate the best models of evolution, the evolutionary model of SYM+I+G substitution model was selected for LSU, HKY+I+G substitution model was selected for SSU, and GTR+I+G substitution model was selected for ITS, rpb2 and tef1-α. Six simultaneous Markov Chains were run for two million generations and trees were sampled at every 200th generation (resulting in 10,000 trees), and these chains stopped when all convergences met and the standard deviation fell below 0.01. All resulting trees were plotted using FigTree v. 1.4.0 (Rambaut 2014) and the layout of the trees was made by Microsoft Office PowerPoint 2020.


Phylogenetic analyses

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 1), and Cyclothyriella rubronotata strains CBS 141486 and CBS 419.85 were used as the outgroup taxa. A total of 4485 characters were fed to the phylogenetic analysis after alignment, 1–1047 (SSU), 1048–1956 (LSU), 1957–2477 (ITS), 2478–3510 (rpb2) and 3511–4485 (tef1-α). The topology of the phylogenetic tree generated by the ML method was highly similar to that by BI, and therefore it was chosen to represent the evolutionary history of Nigrograna.

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. 1.

Figure 1. 

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 (Dayarathne et al. 2020). The maximum-likelihood bootstrap values (ML ≥ 60%, left) and Bayesian Inference Posterior Probability values (BIPP ≥ 0.90, right) are shown above the nodes. Strains derived from the current study are in blue, while type strains are in bold.


Nigrograna coffeae L. Lu & Tibpromma, sp. nov.

Fig. 2


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.

Culture characteristics

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).

Material examined

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. 1), and is phylogenetically related to N. yasuniana and N. jinghongensis. Nigrograna yasuniana was reported as endophytes from Conceveiba guianensis Aubl. in Ecuador, but there were not enough morphological data, the comparison of base pairs in ITS showed 3.4% differences (15/433 bp), LSU showed 1.5% differences (12/812bp), SSU only showed 0.3% differences (3/1028 bp), rpb2 showed 14% differences (117/829 bp), and tef1-α showed 3.2% differences (31/954 bp) (Kolařík et al. 2017). Nigrograna jinghongensis was introduced as a saprobic fungus from woody litter in China, and our new isolate shares a similar size (12–16 × 4–5 μm vs 12–15 × 4–5.5 µm) and color (hyaline to dark brown vs yellowish-brown to brown) of ascospores with N. jinghongensis (Boonmee et al. 2021), but there are some significant differences in the size of the ascomata (90–140 µm high, 140–200 μm wide vs 300–400 µm high 220–300 μm wide) and the shape of ascospores (fusiform, straight or slightly curved vs ellipsoid) (Boonmee et al. 2021). Based on the sequence blast results, ITS, LSU and rpb2 gene sequences were similar to Nigrograna sp., with 97.5% (MZ270683), 98.4% (MK762716), and 86% (MZ508421) respectively, SSU was similar to N. mycophila with 99% (KX650510), and tef1-α was similar to N. yasuniana with 96.6% (LN626670). Therefore, we introduce our new isolate as a new species N. coffeae based on both morphological characteristics and phylogenetic analyses.

Figure 2. 

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).

Nigrograna puerensis L. Lu & Tibpromma, sp. nov.

Fig. 3


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.

Culture characteristics

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).

Material examined

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 (Jaklitsch and Voglmayr 2016; Zhang et al. 2020). Blast search results of ITS, LSU and tef1-α sequence data revealed that our taxon (ZHKUCC 22-0212) is similar to N. mackinnonii (96% MZ270697, 99% KJ605422, and 95% LT797087 respectively), while the similarity of SSU sequence to N. carollii is as high as 99%. Based on nucleotide comparisons, our isolate (ZHKUCC 22-0212) differs from N. carollii (CCF 4484) by 9/490 bp (1.8%) in ITS, 2/222 bp (1%) in LSU, 2/1306 bp (0.2%) in SSU, and 10/530 bp (2%) in tef1-α. Unfortunately, for N. carollii, sufficient morphological data was not available to compare with our novel taxon which was isolated as an endophyte on living sapwood of wild Hevea brasiliensis Müll. Arg., and N. mackinnonii which was isolated as a human pathogen (de Gruyter 2012; Kolařík et al. 2017). In addition, the colony morphology of N. carollii on PDA is described as colonies plane, effuse, and light gray (Kolařík et al. 2017), while N. puerensis colony surface is seen as white aerial mycelium, fluffy, with a serrate edge, and grayish to dark brown from center to edge. Therefore, based on morphological and phylogenetic analyses, we introduce N. puerensis as a distinct new species.

Figure 3. 

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).

Nigrograna asexualis L. Lu & Tibpromma, sp. nov.

Fig. 4


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.

Culture characteristics

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).

Material examined

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. 1). Morphologically, N. asexualis conforms to the morphological characteristics of Nigrograna by having hyaline or subhyaline, long and branched conidiophores, solitary phialides, and aseptate, ellipsoidal or cylindrical conidia (Jaklitsch and Voglmayr 2016; Dayarathne et al. 2020; Wanasinghe et al. 2020). Blast results of the sequences show that ITS is similar to N. fuscidula with 89% (MH856004), and SSU is similar to N. mycophila with 99.8% (KX650510). Nigrograna asexualis is different from N. fuscidula and N. mycophila by its ellipsoidal conidia, but the similarities of these three species are hyaline, 1-celled, smooth-walled conidia forming on philipides (Jaklitsch and Voglmayr 2016). The LSU and rpb2 sequences of our strain blast results are similar to N. obliqua, and the similarities are 98.9% (KX650560) and 87% (KX650579) respectively, but N. obliqua lacks the asexual morph (Jaklitsch and Voglmayr 2016). The tef1-α sequence of our strain is 95.8% (MF939615) similar to N. locuta-pollinis, which was isolated from hive-stored pollen of Brassica campestris L. that lacks morphology (Zhao et al. 2018). Therefore, we introduce N. asexualis as a distinct new species from coffee in China.

Figure 4. 

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 (Mapook et al. 2020), and the hotspots of Nigrograna are reported as Central and South America, where the taxa are also found as human pathogens (Kolařík 2018; Puing et al. 2020). To date, five Nigrograna species viz. N. cangshanensis (decaying wood, Yunnan), N. jinghongensis (dead woody litter, Yunnan), N. kunmingensis (dead twigs of Gleditsia sinensis Lam., Yunnan), N. magnoliae (living branches of Magnolia denudate Desr., Yunnan), and N. locuta-pollinis (hive-stored pollen, Hubei) have been isolated from different hosts in China (Tibpromma et al. 2017; Zhao et al. 2018; Wanasinghe et al. 2020; Boonmee et al. 2021; Zhou et al. 2022). In this study, three new saprobic fungi were isolated from decaying branches of Coffea arabica in Yunnan Province, China, and this is the first report of Nigrograna species from coffee.

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 (Jaklitsch and Voglmayr 2016; Zhang et al. 2020). In our research, we found that N. coffeae and N. puerensis have similar morphology, but in phylogeny, they are distributed differently within Nigrograna. This confirms the view of Jaklitsch and Voglmayr (2016) that the gene sequences are important and crucial for the identification of taxa at the genus and the species level.


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.


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