Description of Four Novel Species in Pleosporales Associated with Coffee in Yunnan, China

Abstract

In Yunnan Province, the coffee-growing regions are mainly distributed in Pu’er and Xishuangbanna. During the surveys of microfungi associated with coffee in Yunnan Province, seven taxa were isolated from coffee samples. Based on molecular phylogenetic analyses of combined ITS, LSU, SSU, rpb2, and tef1-α sequence data and morphological characteristics, four new species viz. Deniquelata yunnanensis, Paraconiothyrium yunnanensis, Pseudocoleophoma puerensis, and Pse. Yunnanensis, and three new records viz. Austropleospora keteleeriae, Montagnula thailandica, and Xenocamarosporium acaciae in Pleosporales are introduced. In addition, Paracamarosporium fungicola was transferred back to Paraconiothyrium based on taxonomy and DNA sequences. Full descriptions, illustrations, and phylogenetic trees to show the placement of new and known taxa are provided. In addition, the morphological comparisons of new taxa with closely related taxa are given.

Full text

Citation: Lu, L.; Karunarathna, S.C.;
Dai, D.-Q.; Xiong, Y.-R.;
Suwannarach, N.; Stephenson, S.L.;
Elgorban, A.M.; Al-Rejaie, S.;
Jayawardena, R.S.; Tibpromma, S.
Description of Four Novel Species in
Pleosporales Associated with Coffee in
Yunnan, China. J. Fungi 2022,8, 1113.
https://doi.org/10.3390/jof8101113
Academic Editor: Philippe Silar
Received: 9 September 2022
Accepted: 17 October 2022
Published: 21 October 2022
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4.0/).
Fungi
Journal of
Article
Description of Four Novel Species in Pleosporales Associated
with Coffee in Yunnan, China
Li Lu 1,2,3 , Samantha C. Karunarathna 1, Dong-Qin Dai 1, Yin-Ru Xiong 2,3,4, Nakarin Suwannarach 5,
Steven L. Stephenson 6, Abdallah M. Elgorban 7, Salim Al-Rejaie 8, Ruvishika S. Jayawardena 2
and Saowaluck Tibpromma 1,*
1Center for Yunnan Plateau Biological Resources Protection and Utilization, Yunnan Engineering Research
College of Biological Re-Source and Food Engineering, Qujing Normal University, Qujing 655011, China
2Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
3School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
4Innovative Institute for Plant Health, Zhong Kai University, Guangzhou 510550, China
5
Research Center of Microbial Diversity and Sustainable Utilization, Faculty of Science, Chiang Mai University,
Chiang Mai 50200, Thailand
6Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA
7Department of Botany and Microbiology, College of Science, King Saud University,
Riyadh P.O. Box 145111, Saudi Arabia
8Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University,
Riyadh P.O. Box 145111, Saudi Arabia
*Correspondence: saowaluckfai@gmail.com
Abstract:
In Yunnan Province, the coffee-growing regions are mainly distributed in Pu’er and
Xishuangbanna. During the surveys of microfungi associated with coffee in Yunnan Province,
seven taxa were isolated from coffee samples. Based on molecular phylogenetic analyses of com-
bined ITS, LSU, SSU, rpb2, and tef 1-
α
sequence data and morphological characteristics, four new
species viz. Deniquelata yunnanensis,Paraconiothyrium yunnanensis,Pseudocoleophoma puerensis, and
Pse.yunnanensis, and three new records viz. Austropleospora keteleeriae,Montagnula thailandica, and
Xenocamarosporium acaciae in Pleosporales are introduced. In addition, Paracamarosporium fungicola
was transferred back to Paraconiothyrium based on taxonomy and DNA sequences. Full descriptions,
illustrations, and phylogenetic trees to show the placement of new and known taxa are provided. In
addition, the morphological comparisons of new taxa with closely related taxa are given.
Keywords:
Coffea;Deniquelata; new species; new records; Paraconiothyrium; phylogeny; Pseudo-
coleophoma; taxonomy
1. Introduction
The coffee genus Coffea, belonging to the botanical family Rubiaceae, has about 170
varieties [
1
], and one of the varieties, Coffea arabica, is the most popular coffee variety
around the world [
2
]. Coffee, as the world’s second best-selling beverage and a food
additive, sells all over the world, with many additional advantages such as refreshment,
diuresis, invigorating the stomach, and stimulating appetite [
3
,
4
]. China’s total production
of coffee ranks 12th place in the world, and the annual export volume of coffee beans
reaches 82.7 thousand tons [
5
], making it the fourth largest coffee exporter in Asia after
Vietnam, Indonesia, and India [
5
,
6
]. In 2020, the export and import of coffee in China were
worth $145 million and $310 million, respectively [
7
]. Yunnan Province is the largest coffee
capital in China, and the planting area and total yield account for 95–98% of the total in
China [5,8].
Coffee is susceptible to microfungi during pre-harvest and post-harvest processing, es-
pecially pathogenic fungi, which can affect coffee tree growth, fruit yield, and quality [
9
,
10
].
The most recent coffee fungi review in 2022 counted about 966 coffee-associated microfungi
J. Fungi 2022,8, 1113. https://doi.org/10.3390/jof8101113 https://www.mdpi.com/journal/jof

J. Fungi 2022,8, 1113 2 of 24
records worldwide, belonging to 648 species, of which 295 are pathogenic fungi (the most
common), while saprotrophic fungi are the least common (30 species) [
10
]. Several surveys
of the literature also show that a few studies have been conducted on saprotrophic fungi
associated with coffee [
11
]. Most of the studies focus on pathogens, as pathogenic fungal
infections reduce coffee yield and quality, and thus affect farmers’ income and consumers’
health [
12
,
13
]. Some research has shown that saprotrophic fungus Phialomyces macrosporus
isolated from leaf litter has the potential to be used in the management of coffee halo blight in
seedlings [14]. Laborde et al. [15] demonstrated that volatile or non-volatile compounds pro-
duced by the saprotrophic fungus Phialomyces macrosporus can inhibit the growth, sporulation,
and viability of the causative agent of the coffee brown eye-spot, Cercospora coffeicola. Therefore,
saprotrophic fungi have the potential as a biological control agent to manage diseases. In
China, a very few studies have been carried out on fungi associated with coffee. While most
studies have been carried out on coffee pathogens, and only a few instances of saprotrophic
fungi in coffee have been reported [
3
,
8
,
10
,
16
]. Yunnan is known as the province in China
in which the most novel fungal are species reported [
17
]. In addition, coffee is one of the
most important economic crops; thus, isolation and identification of coffee saprotrophic fungi
based on morphology and multigene phylogeny are useful for future studies on the secondary
metabolites of coffee fungi.
The largest fungal order, Pleosporales, was first proposed by Luttrell in 1955 and
formally established by Barr in 1987, and belongs to the most diverse and qualified class,
Dothideomycetes (Ascomycota) [
18
–
20
]. Pleosporales contains 91 families and 614 genera,
and is distributed in various habitats worldwide (terrestrial and aquatic) [
21
,
22
]. Members
of Pleosporales can be epiphytes, endophytes, or parasites of living leaves or stems. They
can also be hyperparasites on fungi or insects, lichenized, or saprotrophic of dead plant
stems, leaves, or bark [
23
,
24
]. Pleosporales are characterized by perithecioid ascomata,
usually with a papillate apex, ostioles with or without periphyses, presence of cellular
pseudoparaphyses, bitunicate asci, and ascospores of various shapes, pigmentations, and
septations [24,25].
Dictyosporiaceae and Didymosphaeriaceae are two families of Pleosporales.Dictyosporiaceae
was introduced with Dictyosporium as the type genus to accommodate a holomorphic
group of Dothideomycetes by Boonmee et al. [
26
]. Dictyosporium contains saprotrophic
fungi from decaying wood and plant debris in terrestrial and freshwater habitats [
26
,
27
].
Didymosphaeriaceae was introduced by Munk [
28
] and typified by Didymosphaeria. As a
ubiquitous fungal family worldwide, Didymosphaeriaceae includes saprotrophic, endophytic,
and pathogenic species associated with a wide variety of substrates [29].
Since Pu’er coffee and Xishuangbanna coffee are famous due to their strong but
not bitter taste, fragrant but not hard smell, and slightly fruity flavor [
30
]. During our
investigations of coffee saprotrophic fungi in Pu’er and Xishuangbanna in Yunnan Province,
China, some interesting fungal taxa belonging to Pleosporales were found. The purposes
of the current research are to isolate and identify the saprotrophic fungi associated with
coffee trees based on morphological examination and multi-gene phylogeny, to provide
full descriptions and photo plates of micromorphological characteristics, and to provide
phylogenetic trees to show the placements of new and known taxa.
2. Materials and Methods
2.1. Specimen Collection, Morphological Study, and Isolation
Coffee branch samples with black fungal fruiting bodies visible to the naked eye were
collected from coffee plantations in subtropical areas (Pu’er) and tropical areas (Xishuang-
banna) in Yunnan Province in China. Each sample was placed in a separate plastic bag
together with collection details such as collection date, collection site, and global position-
ing system (GPS) information, and then transported to the mycology laboratory at Qujing
Normal University. Micro-morphological characteristics were observed and captured by
differential interference contrast (DIC), using a Leica DM2500 compound microscope with
a Leica DMC4500 camera. Measurements of microstructures were obtained by the Tarosoft

J. Fungi 2022,8, 1113 3 of 24
(R) Image Frame Work program, while further processing was conducted in Adobe Pho-
toshop CC 2018. Senanayake et al. [
31
] was followed for single spore isolation by using
potato dextrose agar (PDA). Specimens were deposited at Zhongkai University of Agri-
culture and Engineering (ZHKU), while living cultures are maintained at the Zhongkai
University of Agriculture and Engineering (ZHKUCC). Faces of fungi (FoF) numbers and
Index Fungorum (IF) numbers were obtained as instructed in Jayasiri et al. [
32
] and Index
Fungorum (2022) [33].
2.2. DNA Extraction, PCR Amplification and Sequencing
DNA extraction was carried out from two-week-old mycelium by using the Biospin
Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux
®
, Beijing, China), following the
manufacturer’s instructions, and the methods of Lu et al. [
3
] were followed for the Poly-
merase Chain Reaction (PCR). The amplification and sequencing were carried out for five
partial gene portions, the internal transcribed spacer (ITS) region was amplified with the
primers ITS4 and ITS5 [
34
], the 18 s small subunit (SSU) region was amplified by primers
NS1 and NS4 [
34
], the nuclear ribosomal 28 s large subunit (LSU) region with primers LR0R
and LR5 [
35
], the partial RNA polymerase II subunit (rpb2) region with primers RPB2-5F
and RPB2-7cR [
36
], and the partial translation elongation factor 1-alpha (tef 1-
α
) gene with
primers EF1-983F and 2218R [
37
]. The PCR mixture contains 8.5
µ
L of double-distilled
water (ddH2O), 12.5
µ
L of 2
×
Power Taq PCR MasterMix (mixture of EasyTaqTM DNA
Polymerase, dNTPs, and optimized buffer, Beijing Bio Teke Corporation (Bio Teke), Beijing,
China), 1
µ
L of each forward and reverse primers, and 2
µ
L of DNA. The conditions for
PCR of ITS, LSU, SSU, and tef 1-
α
genes constituted an initial denaturation step of 3 min
at 94
◦
C, followed by 35 cycles of 45 s at 94
◦
C, 50 s at 55
◦
C, 60 s at 72
◦
C, and a final
denaturation step of 10 min at 72
◦
C. For the rpb2 gene, the conditions constituted an initial
denaturation step of 5 min at 95
◦
C, followed by 40 cycles of 60 s at 95
◦
C, 120 s at 55
◦
C, 90 s
at 72
◦
C, and a final denaturation step of 10 min at 72
◦
C. The amplified PCR products were
sent to Bioer Technology Co., Hangzhou, and Beijing Kinco Biotechnology Co., Kunming
Branch, China. Generated sequences were deposited in GenBank and accession numbers
were obtained (Table S1).
2.3. Sequence Alignment and Phylogenetic Analyses
Raw sequences, both reverse and forward, generated in this study were assembled
with the Geneious program (9.1.2) [
38
]. The newly generated assembled sequences in
this study were used for BLAST searches in GenBank [
39
]. The BLAST search results
and sequences from the latest publications were used to obtain sequence data for the
phylogenetic analyses. Single gene sequence alignments were made with the online pro-
gram MAFFT v.7.110 [
40
]. They moved the uninformative gaps and ambiguous regions
by trimAL v1.2 [
41
] and combined multigene sequencing by Sequence Matrix program
(1.7.8) [
42
]. The fasta files have transferred the format in the AliView program [
43
], PHYLIP
for maximum likelihood analysis (ML), and NEXUS for Bayesian analysis (BYPP).
Dissanayake et al. [
44
] was referenced for the phylogenetic analyses, considering both
maximum likelihood and Bayesian methods. Maximum likelihood analysis was performed
by RAxML-HPC v.8 on the online program CIPRES Science Gateway [
45
] with rapid
bootstrap analysis, followed by 1000 bootstrap replicates, with GTRGAMMA substitution
model. Bayesian analysis was performed by MrBayes v3.1.2, and the best models of
evolution were estimated by MrModeltest 2.2 [
46
] and PAUP v. 4.0b10 [
47
]. The best-fit
model was the GTR + I + G substitution model for each locus under the Akaike Information
Criterion (AIC). Six simultaneous Markov Chains were run for 2 million generations, and
trees were sampled at every 200th generation (resulting in 10,000 trees). Phylogenetic
trees were visualized by FigTree v. 1.4.2 [
48
], the trees were edited in Microsoft Office
PowerPoint 2020, and reliable bootstrap support values from ML and BYPP were inserted.
All the obtained alignments and phylogenetic trees were deposited in Figshare [
49
] (https:

J. Fungi 2022,8, 1113 4 of 24
//doi.org/10.6084/m9.figshare.21260589 (accessed on 19 August 2022).) and TreeBASE
(www.treebase.org (accessed on 19 August 2022), submission number 29752).
3. Results
3.1. Phylogenetic Analyses
Maximum likelihood phylogenetic analysis was conducted from combined SSU +
LSU + ITS + rpb2 + tef 1-
α
sequence data of 156 strains, of which 14 were newly sequenced
strains, while the other 142 strains were obtained from BLAST search (NCBI) and recent
papers [
50
–
54
]. Our phylogenetic trees show similar topologies to the recent published
papers [50–54].
In each gene alignment, the missing data was calculated as 8.9% in the ITS gene,
7% in the LSU gene, 34% in the SSU gene, 65% in the tef 1-
α
gene and 90% in the rpb2
gene. Periconia pseudodigitata strains KT1395 and KT1195A were selected as the outgroup
taxa. The 156 strains comprised 4773 characters (SSU = 1–1098 bp, LSU = 1099–2018 bp,
ITS = 2019–2677 bp,
rpb2 = 2678–3776 bp, tef 1-
α
= 3777–4773 bp) after alignment. The
phylogenic tree from the RAxML analysis had a similar topology to the Bayesian analysis.
The RAxML analysis of the combined dataset yielded the best-scoring tree (Figure 1)
with a final ML optimization likelihood value of
−
36,512.450654. Alignment had 2112
distinct alignment patterns, with 50.81% gaps and completely undetermined characters.
The estimated base frequencies were as follows: A = 0.242121, C = 0.244904, G = 0.272113,
T = 0.240862; substitution rates were AC = 1.320338, AG = 2.546543, AT = 1.5403704,
CG = 0.907448, CT = 6.221253, GT = 1.000000; gamma distribution shape parameter was
α= 0.237001.
J. Fungi 2022, 8, x FOR PEER REVIEW 5 of 26
Figure 1. Cont.

J. Fungi 2022,8, 1113 5 of 24
J. Fungi 2022, 8, x FOR PEER REVIEW 6 of 26
Figure 1. The best-scoring RAxML tree was constructed from a concatenated SSU, LSU, ITS, rpb2,
and tef1-α dataset. The tree is rooted with Periconia pseudodigitata (KT1395, KT1195A). Nodes were
annotated if the maximum likelihood bootstrap support value was ≥ 60% (ML, left) or if the Bayesian
posterior probability was ≥ 0.90 (BYPP, right). The newly described species are in red, new records
are in green, and type strains are in bold. Red stars are used to indicate the two species that have
uncertain placements and are discussed in the discussion.
Figure 1.
The best-scoring RAxML tree was constructed from a concatenated SSU, LSU, ITS, rpb2,
and tef 1-
α
dataset. The tree is rooted with Periconia pseudodigitata (KT1395, KT1195A). Nodes were
annotated if the maximum likelihood bootstrap support value was
≥
60% (ML, left) or if the Bayesian
posterior probability was
≥
0.90 (BYPP, right). The newly described species are in red, new records
are in green, and type strains are in bold. Red stars are used to indicate the two species that have
uncertain placements and are discussed in the discussion.
3.2. Taxonomy
Dictyosporiaceae Boonmee and K.D. Hyde, Fungal Diversity 80: 462 (2016).
Pseudocoleophoma Kaz. Tanaka and K. Hiray., Studies in Mycology 82: 89 (2015).
Notes:Pseudocoleophoma (Pse.) was introduced by Tanaka et al. [55] and was typified by
Pse. calamagrostidis based on the pycnidial asexual morph, which differs from Coleophoma [
55
].
Most of the Pseudocoleophoma species were reported as asexual morphs [
55
,
56
], but only three
species have been reported for both sexual and asexual morphs viz. Pse. bauhiniae,Pse. calam-
agrostidis, and Pse. polygonicola [
55
,
57
]. Sexual morph is characterized by ostiolar ascomata,
brown and polygonal to rectangular cells of peridium, and numerous pseudoparaphyses,
which are cylindrical to clavate and fissitunicate asci, fusiform, and septate ascospores, with

J. Fungi 2022,8, 1113 6 of 24
an apparent sheath [
55
,
57
]. Asexual morph is characterized by pycnidial and subglobose
conidiomata, phialidic and doliiform conidiogenous cells, and cylindrical or oblong, hyaline,
aseptate, smooth-walled conidia [50].
In 2022, eight epithets (seven species) isolated as saprotrophic on different hosts and
substrates from both terrestrial and freshwater habitats are listed in Index Fungorum
(2022) [
55
–
60
]. Pseudocoleophoma clematidis was transferred to Pseudocyclothyriella by phy-
logenetic status and morphological distinctiveness [
55
]. Herein, we introduce two new
species in Pseudocoleophoma, and this is the first report of Pseudocoleophoma from coffee.
Pseudocoleophoma puerensis L. Lu and Tibpromma, sp. nov. (Figure 2)
Index Fungorum number: IF 559421; Faces of Fungi number: FoF 12761
Etymology
: Name reflects the location “Pu’er” City in Yunnan Province, where the holotype
was collected.
Holotype: ZHKU 22-0118.
Saprotrophic on decaying branch of Coffea arabica var. catimor.Ascomata: 150–300
×
170–220
µ
m (
x
= 197
×
199
µ
m, n= 15) (includes ostiolar neck), solitary, immersed to
erumpent, brown to black, globose or subglobose. Peridium: 14–20
µ
m wide (
x
= 17
µ
m,
n= 20), thin, wall composed of 2–4 layers textura angularis cells, brown to dark brown.
Hermathecium: 1.5–3
µ
m wide (
x
= 2.3
µ
m, n= 20) septate, branched, numerous, hyaline,
pseudoparaphyses. Asci: 50–70
×
7–11
µ
m (
x
= 57
×
8.5
µ
m, n= 20), 8-spored, fissitunicate,
cylindrical, rounded at the apex, with a shallow ocular chamber, long-stalked with club-like
pedicel. Ascospores: 10–15
×
3.5–6
µ
m (
x
= 11.6
×
4, n= 20), uniseriate to biseriate, narrowly
ellipsoid or oblong, 1–3 thick and dark eusepta, slightly constricted at the septa, straight or
slightly curved, hyaline and yellowish brown when young, turning brown when mature,
normally 4 guttulate, without sheath. Asexual morph: undetermined.
Culture characteristics
: Colonies on PDA after two months attaining a diam of 40
mm at 25
◦
C, above side: pale yellow, circular, flatted, margin filamentous; reverse side:
center brown, middle off yellowish brown and white at the margin.
Material examined
: China, Yunnan Province, Pu’er City, on a decaying branch of
Coffea arabica var. catimor, (100
◦
56
0
9
00
E, 22
◦
40
0
28
00
N, 1090.5 m), 16 September 2021, LiLu,
Pu’er 3-2 (ZHKU 22-0118, holotype), living culture ZHKUCC 22-0204 = ZHKUCC 22-0205.
GenBank number; ITS: OP297799, LSU: OP297769, SSU: OP297783; tef 1-
α
: OP321568
(ZHKUCC 22-0204, ex-type); ITS: OP297800, LSU: OP297770, SSU: OP297784; tef 1-
α
:
OP321569 (ZHKUCC 22-0205).
Notes
: In the phylogenetic tree constructed based on multigene data, our isolates
clustered within Pseudocoleophoma and formed a distinct branch basal to Pse. typhicola
(MFLUCC 16-0123), with fairly good statistical support (60% ML/1.00 BYPP, Figure 1).
However, Pse. typhicola has only been reported as an asexual morph [
56
]. Based on nu-
cleotide comparisons, our isolate (ZHKUCC 22-0204) differs from Pse. typhicola (MFLUCC
16-0123) by 63/533 bp (11%) in ITS, 18/669 bp (2.6%) in LSU, while it lacks the SSU and
tef 1-
α
genes. The morphology of our isolate is similar to Pseudocoleophoma, which was
described by Tanaka et al. [
55
], although we noticed that the ascospores are 1–3 septate and
brown, with no sheath in ascospores. Therefore, herein we introduce Pse. puerensis as a new
species in Pseudocoleophoma.
Pseudocoleophoma yunnanensis L. Lu and Tibpromma, sp.nov. (Figure 3).
Index Fungorum number: IF 559422; Faces of Fungi number: FoF 12762.
Etymology
: Name reflects the location “Yunnan” Province, where the holotype was col-
lected.
Holotype: ZHKU 22-0116.

J. Fungi 2022,8, 1113 7 of 24
J. Fungi 2022, 8, x FOR PEER REVIEW 8 of 26
septate and brown, with no sheath in ascospores. Therefore, herein we introduce Pse.
puerensis as a new species in Pseudocoleophoma.
Figure 2. Pseudocoleophoma puerensis (ZHKU 22-0118). (a,b) Ascomata on a decayed branch of Coffea
arabica; (c) section of an ascoma; (d) peridium at side; (e) pseudoparaphyses; (f–j) immature and
mature asci (arrows indicate the club-like pedicel); (k–n) ascospores; (o) germinated ascospore; (p)
culture on PDA from above and reverse (60 days). Scale bars: (c,e) = 100 µm; (f–j) = 20 µm; (k–n) =
5 µm; (o) = 10 µm.
Figure 2. Pseudocoleophoma puerensis (ZHKU 22-0118). (a,b) Ascomata on a decayed branch of Coffea
arabica; (
c
) section of an ascoma; (
d
) peridium at side; (
e
) pseudoparaphyses; (
f
–
j
) immature and
mature asci (arrows indicate the club-like pedicel); (
k
–
n
) ascospores; (
o
) germinated ascospore;
(
p
) culture on PDA from above and reverse (60 days). Scale bars: (
c
,
e
) = 100
µ
m; (
f
–
j
) = 20
µ
m;
(k–n)=5µm; (o) = 10 µm.

J. Fungi 2022,8, 1113 8 of 24
J. Fungi 2022, 8, x FOR PEER REVIEW 10 of 26
Figure 3. Pseudocoleophoma yunnanensis (ZHKU 22-0116). (a,b) Ascomata on a decayed branch of
Coffea sp.; (c) section of an ascoma; (d) peridium at side; (e) pseudoparaphyses; (f–i) immature and
mature asci (arrows indicate the club-shape pedicel); (j) germinated ascospore; (k–m) ascospores;
(n,o) ascospore stained with Lugol’s iodine; (p,q) ascospore stained with Indian ink; (r) culture on
PDA from above and reverse (60 days). Scale bars: (c) = 100 µm; (d–j) = 20 µm; (k–q) = 10 µm.
Didymosphaeriaceae Munk, Dansk botanisk Arkiv 15 (2): 128 (1953).
Austropleospora R.G. Shivas and L. Morin, Fungal Diversity 40 (1): 70 (2010).
Figure 3.
Pseudocoleophoma yunnanensis (ZHKU 22-0116). (
a
,
b
) Ascomata on a decayed branch of
Coffea sp.; (
c
) section of an ascoma; (
d
) peridium at side; (
e
) pseudoparaphyses; (
f
–
i
) immature and
mature asci (arrows indicate the club-shape pedicel); (
j
) germinated ascospore; (
k
–
m
) ascospores;
(
n
,
o
) ascospore stained with Lugol’s iodine; (
p
,
q
) ascospore stained with Indian ink; (
r
) culture on
PDA from above and reverse (60 days). Scale bars: (c) = 100 µm; (d–j) = 20 µm; (k–q) = 10 µm.

J. Fungi 2022,8, 1113 9 of 24
Saprotrophic on decaying branch of Coffea sp.
Sexual morph
:Ascomata: 160–280
×
200–280
µ
m (
x
= 216
×
243
µ
m; n= 15), semi-immersed to erumpent, solitary or scattered,
coriaceous, subglobose to obpyriform, dark brown to black, with protruding ostiolar neck.
Peridium: 22–27
µ
m wide (
x
= 23; n= 15), outer walls comprising 2–4 layers of textura
angularis cells, hyaline to brown, inner walls hyaline, density, several layers of textura
angularis cells. Hamathecium: 1.5–3
µ
m wide (
x
= 2.1
µ
m; n= 15), hyaline, septate, branched,
pseudoparaphyses numerous. Asci: 65–90
×
8–11
µ
m (
x
= 76
×
9
µ
m; n= 20), 8-spored,
fissitunicate, clavate to cylindrical, some slightly curved, constricted at the upper part
when mature, with an ocular chamber, short-stalked with club-shape pedicel. Ascospores:
16–26
×
4–8 (
x
= 21
×
6
µ
m; n= 20), uniseriate to biseriate, hyaline, fusiform, straight
to slightly curved, 1-septate, 4 guttulate, smooth-walled, with a distinct sheath.
Asexual
morph: undetermined.
Culture characteristics
: Ascospores germinated on PDA within 12 h; colonies on PDA
after two months attaining a diam of 40 mm at 25
◦
C, slightly raised, fluffy, white, circular,
margin filamentous, reverse pale yellow to light brown from edge to center.
Material examined
: China, Yunnan Province, Xishuangbanna, on a decaying branch
of Coffea sp., (1672 m), 12 September 2021, LiLu, JHMH 15 (ZHKU 22-0116, holotype), living
culture ZHKUCC 22-0200 = ZHKUCC 22-0201. GenBank number: ITS: OP297795, LSU:
OP297765, SSU: OP297779, tef 1-
α
: OP321564 (ZHKUCC 22-0200, ex-type); ITS: OP297796,
LSU: OP297766, SSU: OP297780, tef 1-α: OP321565 (ZHKUCC 22-0201).
Notes
: In the phylogenetic tree, our species, Pseudocoleophoma yunnanensis, formed a
well-separated clade clustered with Pse. bauhiniae (MFLUCC 17-2280) with high statistical
support (90% ML/1.00 BYPP, Figure 1). Based on nucleotide comparisons, our isolate
(ZHKUCC 22-0200) is different from Pse. bauhiniae (MFLUCC 17-2280) by 13/489 bp (2.6%)
in ITS, 3/833 bp (0.4%) in LSU, 9/1069 bp (0.8%) in SSU, and 73/878bp (8.3%) in tef 1-
α
. In
addition, morphological features of the sexual morph Pse. yunnanensis can be distinguished
by having constricted asci and the presence of a distinct sheath of ascospores with Pse.
bauhiniae and other species in Pseudocoleophoma [
51
,
55
,
58
]. Therefore, we introduce Pse.
yunnanensis as a new species that was isolated from coffee.
Didymosphaeriaceae Munk, Dansk botanisk Arkiv 15 (2): 128 (1953).
Austropleospora R.G. Shivas and L. Morin, Fungal Diversity 40 (1): 70 (2010).
Notes
:Austropleospora (A.) was introduced by Morin et al. [
61
], with A. osteospermi as
the type species. This was isolated as a pathogen with both sexual and asexual morphs
from stems of Chrysanthemoides monilifera subsp. Rotundata, with dieback symptoms in
Australia, but its pathogenicity has not been accurately confirmed [
61
]. The sexual morphs
are characterized by immersed and ostiolate ascomata, filamentous and septate pseudopa-
raphyses, bitunicate, clavate to cylindrical asci, and ellipsoid, yellowish-brown ascospores.
The asexual morph has coelomycetous, pycnidial, globose conidiomata, brown to reddish-
brown conidiomata walls, and yellowish-brown globose to obovate conidia [
51
,
62
,
63
].
Species of this genus have been reported as pathogenic or saprotrophic in Australia, China,
and Thailand [
56
,
60
,
62
,
63
]. Austropleospora contains four epithets (three species) in Index
Fungorum (2022) viz. A. archidendri,A. keteleeriae,A. ochracea, and A. osteospermi, while A.
archidendri has been synonymized under Paraconiothyrium [
62
]. Thus, we introduce the new
host record Austropleospora keteleeriae from coffee.
Austropleospora keteleeriae
Jayasiri, E.B.G. Jones and K.D. Hyde, Mycosphere 10 (1): 65
(2019) (Figure 4)
Index Fungorum number: IF 555541; Faces of Fungi number: FoF 05244.

J. Fungi 2022,8, 1113 10 of 24
J. Fungi 2022, 8, x FOR PEER REVIEW 12 of 26
Figure 4. Austropleospora keteleeriae (ZHKU 22-0120). (a,b) Appearance of the conidiomata on
decaying branch of Coffea arabica var. catimor; (c) cross sections of a conidioma; (d) pycnidial wall;
(e,f) conidia attached to conidiogenous cells; (g–j) conidia; (h) germinated conidium; (l) culture on
PDA from above and reverse (60 days); scale bars: (c) = 100 µm; (d,e) = 20 µm; (f,k) = 10 µm; (g–j) =
5 µm.
Deniquelata Ariyaw. and K.D. Hyde, Phytotaxa 105 (1): 15 (2013).
Notes: Deniquelata (D.) was introduced by Ariyawansa et al. [52], with D.
barringtoniae as the type species, which was isolated as a pathogen, causing leaf spots of
Barringtonia asiatica (Lecythidaceae). The sexual morph of Deniquelata is characterized by
immersed ascomata, with an ostiolar, textura angularis cell peridium, bitunicate, clavate
to broadly-clavate with short furcate pedicel asci and muriform ascospores, three
transverse septa and 1–2 vertical septa, without a sheath [52,64]. The asexual morph is
characterized by branched and septate mycelium, conidiophores reduced to
conidiogenous cells, pale brown and subcylindrical conidiogenous, subglobose to
pyriform and guttulate conidia, with 1–3 transverse and 0–2 vertical septa [65]. Species of
this genus have been reported as pathogens, saprophytes, and endophytes [52,64–66].
Currently, the genus Deniquelata contains four species in the Index Fungorum (2022) viz.
D. barringtoniae, D. hypolithi, D. quercina, and D. vittalii. In this study, Deniquelata
Figure 4.
Austropleospora keteleeriae (ZHKU 22-0120). (
a
,
b
) Appearance of the conidiomata on decaying
branch of Coffea arabica var. catimor; (
c
) cross sections of a conidioma; (
d
) pycnidial wall; (
e
,
f
) conidia
attached to conidiogenous cells; (
g
–
j
) conidia; (
k
) germinated conidium; (
l
) culture on PDA from
above and reverse (60 days); scale bars: (c) = 100 µm; (d,e) = 20 µm; (f,k) = 10 µm; (g–j)=5µm.
Saprotrophic on a decaying branch of Coffea arabica var. catimor.
Sexual morph
: unde-
termined.
Asexual morph
: Coelomycetous. Conidiomata: 70–150
×
150–250
µ
m (
x
= 104
×
209
µ
m, n= 15), pycnidial, immersed, solitary, globose to obpyriform, or irregular, short
ostiolate after maturity. Conidiomata wall: 12–17 (
x
= 14.5
µ
m, n= 20), composed of 2–3
layers, inner wall composed of 1–2 layers, hyaline cells of textura angularis.Conidiophores:
reduced to conidiogenous cells. Conidiogenous cells: 3–6
×
2–4
µ
m (
x
= 4.3
×
2.5
µ
m,
n= 20
),
enteroblastic, phialidic, globose to doliiform, hyaline, smooth-walled, lining the inner
wall layer of the pycnidium. Conidia: 4–7
×
2.5–4.5
µ
m (
x
= 6
×
3.5
µ
m, n= 30), solitary,
the color changes with different ages, hyaline when young, brown to dark-brown; when
mature, subglobose to obovate, one-celled, thick and smooth-walled, aseptate, with small
oil droplets.
Culture characteristics
: Conidia germinated within 12 h on PDA, growing on PDA
reaching around 40 mm after two months at room temperature (25
◦
C). Above: white,

J. Fungi 2022,8, 1113 11 of 24
circular, surface flocculent, with a significant amount of aerial mycelia. Reverse: the color
gradually becomes darker from the edge to the center, white, yellowish to light brown.
Material examined
: China, Yunnan Province, Pu’er City, on a decaying branch of
Coffea arabica var. catimor, (100
◦
56
0
9
00
E, 22
◦
40
0
28
00
N, 1090.5 m), 16 September 2021, LiLu,
Pu’er 3-9 (ZHKU 22-0120), living culture ZHKUCC 22-0208 = ZHKUCC 22-0209. GenBank
number; ITS: OP297801, LSU: OP297771, SSU: OP297785, tef 1-
α
: OP321570 (ZHKUCC
22-0208); ITS: OP297802, LSU: OP297772, SSU: OP297786, tef 1-
α
: OP321571 (ZHKUCC
22-0209).
Notes
:Austropleospora keteleeriae was isolated as saprotrophic on a decaying cone of
Keteleeria fortunei from China [
58
]. Our new isolates form a sister clade to A. keteleeriae
in the phylogenetic analyses, with high statistical support (98 ML/1.00 BYPP, Figure 1).
The morphological characteristics of the new isolates fit with A. keteleeriae by having
conidiomata consisting of textura angularis cells, phialidic, enteroblastic, hyaline, and
smooth conidiogenous cells, and globose to obovate, one-celled, thick and smooth-walled,
hyaline to brown conidia [
58
]. While ITS blast results showed that our strain is 99% similar
to A. archidendri (Paraconiothyrium archidendri) (MZ855427), the results of the LSU blast
showed that it is 100% similar to Paraconiothyrium sp. (JX496165), the results of the SSU
blast showed that it is 99.9% similar to Austropleospora sp. (MT808321), and the tef 1-
α
blast
showed that it is 99.8% similar to A. keteleeriae (MK360045). Therefore, we introduce A.
keteleeriae as a new host record from a decaying branch of Coffea arabica var. catimor in China,
based on morphology and phylogeny.
Deniquelata Ariyaw. and K.D. Hyde, Phytotaxa 105 (1): 15 (2013).
Notes
:Deniquelata (D.) was introduced by Ariyawansa et al. [
52
], with D. barringtoniae
as the type species, which was isolated as a pathogen, causing leaf spots of Barringtonia
asiatica (Lecythidaceae). The sexual morph of Deniquelata is characterized by immersed
ascomata, with an ostiolar, textura angularis cell peridium, bitunicate, clavate to broadly-
clavate with short furcate pedicel asci and muriform ascospores, three transverse septa
and 1–2 vertical septa, without a sheath [
52
,
64
]. The asexual morph is characterized by
branched and septate mycelium, conidiophores reduced to conidiogenous cells, pale brown
and subcylindrical conidiogenous, subglobose to pyriform and guttulate conidia, with
1–3 transverse and 0–2 vertical septa [
65
]. Species of this genus have been reported as
pathogens, saprophytes, and endophytes [
52
,
64
–
66
]. Currently, the genus Deniquelata
contains four species in the Index Fungorum (2022) viz. D. barringtoniae,D. hypolithi,D.
quercina, and D. vittalii. In this study, Deniquelata yunnanensis is introduced from coffee in
Yunnan Province, China, and this is the first report of the genus Deniquelata from coffee.
Deniquelata yunnanensis L. Lu and Tibpromma, sp. nov. (Figure 5),
Index Fungorum number: IF 559423; Faces of Fungi number: FoF 12763.
Etymology
: Name reflects the location, “Yunnan” Province, where the holotype was
collected.
Holotype: ZHKU 22-0115.
Saprotrophic on decaying branch of Coffea sp.
Sexual morph
:Ascomata: 140–240
×
170
−
240
µ
m (
x
= 187
×
209
µ
m, n= 20), semi-immersed, aggregated to solitary, globose to
subglobose, dark brown to black, with ostiole and apex papillate to depressed. Peridium:
12
−
17
µ
m wide (
x
= 14, n = 15), composed of two walls, outer wall light brown to brown,
comprising 2–3 layers, cells of textura angularis, fused with the host cells, inner wall thin,
hyaline cell. Hamathecium: composed of dense, 1.5
−
3
µ
m wide (
x
= 2, n= 20), branched,
hyaline, septate pseudoparaphyses, surrounding the numerous asci and enclosed in a
gelatinous matrix. Asci: 50–85
×
10–17
µ
m (
x
= 68
×
13
µ
m, n= 30), 8-spored, bitunicate,
fissitunicate, clavate, with a short furcate pedicel. Ascospores: 11–16
×
4–8
µ
m (
x
= 14
×
6.6
µ
m, n= 30), 2-seriated ascospores, partially overlapping, muriform, hyaline when
young, yellow to brown at maturity and with distinct guttulate, ellipsoidal to oblong,
with 0–2 longitudinal septate in each cell, 1–3 transverse septate, constricted at the septa,

J. Fungi 2022,8, 1113 12 of 24
slightly curved to straight, apically conical to elliptical, without a sheath.
Asexual morph
:
undetermined.
J. Fungi 2022, 8, x FOR PEER REVIEW 14 of 26
Figure 5. Deniquelata yunnanensis (ZHKU 22-0115). (a,b) Ascomata on a decaying branch of Coffea
sp.; (c) vertical section of an ascoma; (d) peridium; (e) pseudoparaphyses; (f–k) asci (arrows indicate
the short furcate pedicel); (l–n) ascospores; (o) germinated ascospore; (p) culture on PDA from
above and reverse (30 days). Scale bars: (c) = 100 µm; (d–k) = 15 µm; (l–o) = 5 µm.
Montagnula Berl., Icones Fungorum. Pyrenomycetes 2: 68 (1896).
Notes: Montagnula (M.) was introduced in Montagnulaceae by Berlese in 1896 to
accommodate M. gigantean and M. infernalis (the type species) [67,68]. The sexual morph
of the genus is characterized by globose or spherical and immersed ascomata with
clypeus, claviform asci, fusoid, or ellipsoid ascospores, as well as with transverse septa
and one or more longitudinal septa [55,69], while the asexual morph remains
Figure 5.
Deniquelata yunnanensis (ZHKU 22-0115). (
a
,
b
) Ascomata on a decaying branch of Coffea sp.;
(
c
) vertical section of an ascoma; (
d
) peridium; (
e
) pseudoparaphyses; (
f
–
k
) asci (arrows indicate the
short furcate pedicel); (
l
–
n
) ascospores; (
o
) germinated ascospore; (
p
) culture on PDA from above
and reverse (30 days). Scale bars: (c) = 100 µm; (d–k) = 15 µm; (l–o)=5µm.
Culture characteristics
: Ascospores germinated within 12 h on PDA, growing on PDA
and reaching around 50 mm after one month at room temperature (25
◦
C). The surface

J. Fungi 2022,8, 1113 13 of 24
is white to light grey, circular, cottony and fluffy, slightly raised in the middle, margin
filamentous, reverse yellowish to light brown.
Material examined
: China, Yunnan Province, Xishuangbanna, on a decaying branch
of Coffea sp., (1672 m), 12 September 2021, LiLu, JHMH 10 (ZHKU 22-0115, holotype),
living culture ZHKUCC 22-0198 = ZHKUCC 22-0199. GenBank number; ITS: OP297803,
LSU: OP297773, SSU: OP297787, tef 1-
α
: OP321572, rpb2: OP321562 (ZHKUCC 22-0198,
ex-type); ITS: OP297804, LSU: OP297774, SSU: OP297788, tef 1-
α
: OP321573, rpb2: OP321563
(ZHKUCC 22-0199).
Notes
: In the phylogenetic analyses, Deniquelata yunnanensis formed a distinct sister
clade to Deniquelata with strong statistical support (100 ML/1.00 BYPP, Figure 1). In the NCBI
blast results of sequences, ITS was 92.8% similar to D. barringtoniae (MH141242), LSU/tef1-
α
/rpb2 was similar to D. hypolithi (NG_076735, 97.8%), (MZ078250, 97%), and (MZ078201,
94%), respectively, while SSU highly overlapped with Deniquelata sp. (MH316155) at 99%.
Morphologically, our strains well fit with the generic characteristics of Deniquelata [
52
,
64
]. An
asexual morph of D. hypolithi was reported [
65
], while the sexual morph of D. yunnanensis
can be distinguished from D. barringtoniae by the color of the ascospores, which are yellow
to brown at maturity and have a distinct guttulate as well as 1–3 transverse septa and
0–2 longitudinal septa in each ascospore, while ascospores in D. barringtoniae are reddish-
brown, with three transverse septa and 1
−
2 vertical septa in each ascospore [
63
]. Both of the
phylogenetic analyses and morphological characteristics supported our species as a distinct
new species in Deniquelata.
Montagnula Berl., Icones Fungorum. Pyrenomycetes 2: 68 (1896).
Notes
:Montagnula (M.) was introduced in Montagnulaceae by Berlese in 1896 to ac-
commodate M. gigantean and M. infernalis (the type species) [
67
,
68
]. The sexual morph of
the genus is characterized by globose or spherical and immersed ascomata with clypeus,
claviform asci, fusoid, or ellipsoid ascospores, as well as with transverse septa and one
or more longitudinal septa [
55
,
69
], while the asexual morph remains undetermined [
70
].
According to the multi-gene phylogeny inferred from the combined dataset, Didymosphaeri-
aceae incorporates members of Montagnulaceae, so the genus Montagnula was moved to
Didymosphaeriaceae [
70
]. The genus comprises saprotrophic fungi growing on dead wood,
branches, stems, bark, and leaves, which play an important role [
70
–
73
]. A total of 47
epithets (43 species) are listed in Index Fungorum (2022). The species of Montagnula are
distributed over 29 countries and 65 host species [
54
,
74
]. In this paper, we introduce a new
host and country record in Montagnula from coffee in China.
Montagnula thailandica
Mapook and K.D. Hyde, Fungal Diversity 101: 35 (2020) (Figure 6)
Index Fungorum number: IF 557299; Faces of Fungi number: FoF 07792.
Saprotrophic on a decaying branch of Coffea arabica var. catimor.
Sexual morph
:As-
comata: 300–500
×
280–350
µ
m (
x
= 26
µ
m, n= 10), semi-immersed to erumpent, brown
to black, spherical to obpyriform, solitary or scattered, with papillate ostiole. Peridium:
20–30
µ
m wide (
x
= 377
×
313
µ
m, n= 10), comprising several layers of thin-walled, hya-
line to brown cells of textura angularis.Hamathecium: 1.5–2.5
µ
m wide, hyaline, cellular,
branched, septate, numerous pseudoparaphyses. Asci: 80–110
×
9–13
µ
m (
x
= 93
×
11
µ
m, n= 25), 6–8-spored, bitunicate, elongate-clavate, long-stalked with club-shape pedicel,
slightly curved. Ascospores: 12–16
×
5.5–6.5
µ
m (
x
= 14
×
5.8
µ
m, n= 25), overlapping 1–2-
seriate, hyaline to yellowish-brown when immature, brown to reddish-brown when mature,
broadly fusiform to ellipsoid, 1-septate, constricted at the septum, slightly wider upper
cell and tapering towards ends, straight or slightly curved, with 2–4 guttulate, without
terminal appendages or a sheath. Asexual morph: undetermined.

J. Fungi 2022,8, 1113 14 of 24
J. Fungi 2022, 8, x FOR PEER REVIEW 16 of 26
Figure 6. Montagnula thailandica (ZHKU 22-0119). (a,b) Ascomata on a decaying branch of Coffea
arabica; (c) vertical section of an ascoma; (d) peridium; (e) pseudoparaphyses; (f–j) asci (arrows
indicate the club-shape pedicel); (k–n) ascospores; (o) germinated ascospore; (p) culture on PDA
from above and reverse (30 days). Scale bars: (c) = 100 µm; (d–j) = 30 µm; (k–n) = 5 µm; (o) = 10 µm.
Paraconiothyrium Verkley, Studies in Mycology 50 (2): 327 (2004).
Figure 6.
Montagnula thailandica (ZHKU 22-0119). (
a
,
b
) Ascomata on a decaying branch of Coffea
arabica; (
c
) vertical section of an ascoma; (
d
) peridium; (
e
) pseudoparaphyses; (
f
–
j
) asci (arrows
indicate the club-shape pedicel); (
k
–
n
) ascospores; (
o
) germinated ascospore; (
p
) culture on PDA
from above and reverse (30 days). Scale bars: (
c
) = 100
µ
m; (
d
–
j
) = 30
µ
m; (
k
–
n
)=5
µ
m; (
o
) = 10
µ
m.

J. Fungi 2022,8, 1113 15 of 24
Culture characteristics
: Ascospores germinated on PDA within 24 h at room tempera-
ture (25
◦
C). One month after growing on PDA, it reached 60 mm. The obverse mycelium is
fluffy, slightly raised, white to light brown, circular, filamentous at the margin; the reverse
is yellowish to dark brown from edge to center.
Material examined
: China, Yunnan Province, Pu’er City, on a decaying branch of
Coffea arabica var. catimor, (100
◦
56
0
9
00
E, 22
◦
40
0
28
00
N, 1090.5 m), 16 September 2021, LiLu,
Pu’er 3-4 (ZHKU 22-0119), ZHKUCC 22-0206 = ZHKUCC 22-0207. GenBank number; ITS:
OP297807, LSU: OP297777, SSU: OP297791, tef 1-
α
: OP321576 (ZHKUCC 22-0206); ITS:
OP297808, LSU: OP297778, SSU: OP297792, tef 1-α: OP321577 (ZHKUCC 22-0207).
Notes
:Montagnula thailandica was introduced as a new species from dead stems of
Chromolaena odorata from Thailand by Mapook et al. [
73
], based on the morphology of the
sexual morph and the phylogenetic analyses. In this paper, our new isolates clustered with
the ex-type strain of M. thailandica (MFLUCC 17-1508) with fairly good bootstrap support
(ML/BI = 74/-). The morphology of the new isolate (ZHKU 22-0119) is very similar to
the holotype M. thailandica (MFLU 20-0325) in size and color of asci and ascospores [
73
].
Based on blast results, ITS and tef 1-
α
are 99.6% similar to M. thailandica (MFLUCC 17-1508),
and LSU and SSU are 100% similar to M. thailandica (MFLUCC 17-1508). Therefore, the
new isolate (ZHKU 22-0119) is identified as M. thailandica, and this is a new host and new
country record of M. thailandica, isolated from a decaying branch of Coffea arabica in China.
Paraconiothyrium Verkley, Studies in Mycology 50 (2): 327 (2004).
Notes
:Paraconiothyrium (Paraco.) was proposed to accommodate four new species,
Paraco. estuarinum,Paraco. brasiliense,Paraco. cyclothyrioides, and Paraco. fungicola by Verkley
et al. [
75
]. The genus is reported as a phytopathogen, saprophyte, and endophyte in a wide
range of hosts and substrates worldwide [
69
,
75
,
76
]. The asexual morph characteristics of
Paraconiothyrium are eustromatic conidiomata, phialidic conidiogenous cells and aseptate,
sometimes 1-septate, thin-walled, smooth or minutely warted, and hyaline to brown
conidia [
75
]. Sexual morph characteristics are globose or subglobose ascomata, clavate
or cylindrical asci, and fusiform to ellipsoidal ascospores [
76
]. The genus comprises 29
epithets (21 species) in the Index Fungorum (2022), but some species have already been
moved to other genera. The taxonomic affiliation of the Paraconiothyrium species is still
confusing, with contrasting differences at the phylogenetic and morphological levels [
77
].
In our study, we introduce a new species of Paraconiothyrium from coffee.
Paraconiothyrium yunnanensis L. Lu and Tibpromma, sp. nov. (Figure 7),
Index Fungorum number: IF 559424; Faces of Fungi number: FoF 12764.
Etymology
: Name reflects the location, “Yunnan” Province, where the holotype was
collected.
Holotype: ZHKU 22-0114.
Saprotrophic on a decaying branch of Coffea sp.
Sexual morph
:Ascomata: 150–250
×
160–260
µ
m (
x
= 193
×
214
µ
m, n= 20), solitary, scattered, immersed to semi-immersed,
brown to black, globose or subglobose, coriaceous, small papilla, with small ostiole, os-
tiolar canal lined without hyaline periphyses. Peridium: 16–28
µ
m (
x
= 22
µ
m, n= 20)
wide, thick, composed of 4–6 layers of textura prismatica cells, yellowish to brown. Hamath-
ecium:
1.5–3 µm
wide (
x
= 2.2
µ
m, n = 20), hyaline, branched and septate, numerous
pseudoparaphyses. Asci: 60–110
×
8–11
µ
m (
x
= 79
×
9.3
µ
m, n= 30), 8-spored, bitunicate,
fissitunicate, oblong or cylindrical, short-stalked, some with club-like pedicels, slightly
curved. Ascospores: 15–18
×
4–5
µ
m (
x
= 16
×
4.6, n= 30), biseriate or partially overlapping,
fusiform or ellipsoidal, yellowish to light brown, 2–4-septate, smooth-walled, penultimate
cell enlarged, without a sheath or guttulate. Asexual morph: undetermined.

J. Fungi 2022,8, 1113 16 of 24
J. Fungi 2022, 8, x FOR PEER REVIEW 18 of 26
Figure 7. Paraconiothyrium yunnanensis. (ZHKU 22-0114). (a,b) Ascomata on a decaying branch of
Coffea sp.; (c) vertical section of an ascoma; (d) peridium; (e) pseudoparaphyses; (f–i) asci (arrows
indicate the club-like pedicel); (j) germinated ascospore; (k–o) ascospores; (p) culture on PDA from
above and reverse (30 days). Scale bars: (c) = 100 µm; (d–i) = 15 µm; (j–o) = 10 µm.
Xenocamarosporium Crous and M.J. Wingf., Persoonia 34: 185 (2015)
Notes: Xenocamarosporium (X.) was first proposed to includethe Camarosporium
complex by Crous et al. [78], and the type species X. acaciae was an asexual morph isolated
from leaf spots of Acacia mangium in Malaysia [78]. Later, the sexual morph of X. acaciae
was introduced by Jayasiri et al. [57] from a decaying pod of Leucaena sp. as a saprotrophic
fungus in Thailand. The asexual characteristics of this genus are brown and globose
Figure 7.
Paraconiothyrium yunnanensis (ZHKU 22-0114). (
a
,
b
) Ascomata on a decaying branch of
Coffea sp.; (
c
) vertical section of an ascoma; (
d
) peridium; (
e
) pseudoparaphyses; (
f
–
i
) asci (arrows
indicate the club-like pedicel); (
j
) germinated ascospore; (
k
–
o
) ascospores; (
p
) culture on PDA from
above and reverse (30 days). Scale bars: (c) = 100 µm; (d–i) = 15 µm; (j–o) = 10 µm.
Culture characteristics
: Ascospores germinated within 12 h on PDA, growing on PDA
and reaching around 40 mm after one month at room temperature (25
◦
C). Colonies were
circular, filiform, dark green, with the mycelium raised and a lot of aerial hyphae in the
edges; the center of the reverse was brown with dark green edges.
Material examined
: China, Yunnan Province, Xishuangbanna, on a decaying branch
of Coffea sp., (1672 m), 12 September 2021, LiLu, JHMH 7 (ZHKU 22-0114, holotype),

J. Fungi 2022,8, 1113 17 of 24
living culture ZHKUCC 22-0196 = ZHKUCC 22-0197. GenBank number; ITS: OP297797,
LSU: OP297767, SSU: OP297781, tef 1-
α
: OP321566, rpb2: OP321560 (ZHKUCC 22-0196,
ex-type); ITS: OP297798, LSU: OP297768, SSU: OP297782, tef 1-
α
: OP321567, rpb2: OP321561
(ZHKUCC 22-0197).
Notes
: Phylogenetic analyses show that Paraconiothyrium yunnanensis is well-separated
from Paraco. fungicola, with 100% ML, 1.00 BYPP statistical support (Figure 1). Based on
BLAST search results of sequence data, ITS and LSU are closely related to Paraco. fungicola,
with similarity rates of 98.9% (MK619287) and 99.8% (JX496133). SSU is 99.8% similar
to Paraco. variabile (KM096136), rpb2 is 88% (MT473955) similar to Paraconiothyrium sp.,
and tef 1-
α
is 97% (LT797134) similar to Paraco. cyclothyrioides. In terms of morphological
characteristics, our new species is similar to Paraco. magnoliae in that it has ellipsoidal,
yellowish to light brown, and septate ascospores [
68
]. However, the difference between our
new species and Paraco. magnoliae is that the ascospores have 2 or 4 septa, without a sheath,
and the penultimate cell is enlarged, while Paraco. magnoliae ascospores have three septa,
a sheath, and a distinct guttule. Therefore, our isolate is described as a new species from
Coffea sp. in China.
Xenocamarosporium Crous and M.J. Wingf., Persoonia 34: 185 (2015)
Notes
:Xenocamarosporium (X.) was first proposed to includethe Camarosporium com-
plex by Crous et al. [
78
], and the type species X. acaciae was an asexual morph isolated
from leaf spots of Acacia mangium in Malaysia [
78
]. Later, the sexual morph of X. acaciae
was introduced by Jayasiri et al. [
57
] from a decaying pod of Leucaena sp. as a saprotrophic
fungus in Thailand. The asexual characteristics of this genus are brown and globose conid-
iomata, brown textura angularis cells of the peridium, hyaline and smooth conidiogenous
cells lining the inner conidiomatal cavity, and ellipsoidal to subcylindrical, hyaline to
golden-brown, verruculose, septate conidia [
78
]. The sexual characteristics of this genus
are brown and immersed ascomata, textura angularis cells of the peridium, filiform and
septate hamathecium, bitunicate, cylindrical to cylindric-clavate asci, and hyaline to brown,
cylindrical, septate ascospores, which are often enlarged at the fourth cell [
57
]. To date, this
genus consists of only one species [
22
,
79
]. Here, we introduce one new host and country
record in Xenocamarosporium for coffee in China.
Xenocamarosporium acaciae Crous and M.J. Wingf., Persoonia 34: 185 (2015) (Figure 8).
Index Fungorum number: IF 812423; Faces of Fungi number: FoF 05248.
Saprotrophic on decaying branch of Coffea sp.
Sexual morph
:Ascomata: 100–200
×
110–
200
µ
m (
x
= 177
×
169
µ
m, n= 15), immersed, subglobose, scattered, solitary, uniloculate,
with short ostiole penetrating through host surface and becoming brown to black spots on
the substrate. Peridium: 14–19
µ
m wide (
x
= 16.9
µ
m; n= 20), unequal thickness, composed
of 3–4 layers of hyaline to light brown, pseudoparenchymatous cells of textura angularis.
Hamathecium: 1.5–3
µ
m wide (
x
= 2.1
µ
m, n= 20), sparse, filamentous, numerous, with a
distinct constricted septum. Asci: 60–80
×
9–11
µ
m (
x
= 71.5
×
10
µ
m; n= 20), bitunicate,
8-spored, cylindrical to cylindric-clavate, with short or absent pedicellate, apically rounded
with an indistinct ocular chamber. Ascospores: 13–19
×
4–5.5
µ
m (
x
= 15.8
×
4.7
µ
m;
n= 30
),
uniseriate or overlapping, hyaline to brown, cylindrical, narrower and longer at the lower
cell, 2–5-septate, often enlarged at the fourth cell, conical at the top.
Asexual morph
:
undetermined.
Culture characteristics
: Ascospores germinated in PDA within 12 h. Colonies reached
50 mm diameter after two months at 25
◦
C, surfaces were yellow-gray to white, had
margins with aerial mycelium, and were circular and flatted. The reverse side was brown
to light yellow.
Material examined
: China, Yunnan Province, Xishuangbanna, on a decaying branch
of Coffea sp., (101
◦
2
0
44
00
E, 22
◦
31
0
18
00
N, 856.9 m), 15th September 2021, Li Lu, JHPW 9
(ZHKU 22-0117), ZHKUCC 22-0202 = ZHKUCC 22-0203. GenBank number; ITS: OP297805,
LSU: OP297775, SSU: OP297789, tef 1-
α
: OP321574, (ZHKUCC 22-0202); ITS: OP297806,
LSU: OP297776, SSU: OP297790, tef 1-α: OP321575, (ZHKUCC 22-0203).

J. Fungi 2022,8, 1113 18 of 24
J. Fungi 2022, 8, x FOR PEER REVIEW 20 of 26
Figure 8. Xenocamarosporium acaciae (ZHKU 22-0117). (a,b) Appearance of ascomata on a decaying
branch of Coffea sp.; (c,d) vertical section of an ascoma; (e) peridium; (f) pseudoparaphyses; (g–j)
asci; (k) germinated ascospore; (l–p) ascospores; (q) culture on PDA from above and reverse (60
days). Scale bars: (d) = 100 µm; (e–f) = 15 µm; (g–k) = 20 µm; (l–p) = 5 µm.
Paraconiothyrium fungicola Verkley and Wicklow, in Verkley, da Silva, Wicklow and
Crous, Stud. Mycol. 50(2): 331 (2004).
Index Fungorum number: IF 500084
Figure 8.
Xenocamarosporium acaciae (ZHKU 22-0117). (
a
,
b
) Appearance of ascomata on a decaying
branch of Coffea sp.; (
c
,
d
) vertical section of an ascoma; (
e
) peridium; (
f
) pseudoparaphyses; (
g
–
j
) asci;
(
k
) germinated ascospore; (
l
–
p
) ascospores; (
q
) culture on PDA from above and reverse (60 days).
Scale bars: (d) = 100 µm; (e–f) = 15 µm; (g–k) = 20 µm; (l–p)=5µm.
Notes
: In phylogeny, our strains form a clade with Xenocamarosporium acaciae, and
the BLAST results of ITS, LSU, SSU, and tef 1-
α
of our strain give 100% (MK347766),

J. Fungi 2022,8, 1113 19 of 24
99% (MK347983), 99.9% (MK360093), and 99.9% (MK347873) similarities with X. acaciae,
respectively. Furthermore, the comparison of morphological characteristics of asci and
ascospores shows that our isolate is highly consistent with the sexual morph of X. acaciae
morphologically [
57
]. Therefore, in this study, X. acaciae (ZHKU 22-0117) is reported as a
new host record and a country record from coffee in China.
Paraconiothyrium fungicola
Verkley and Wicklow, in Verkley, da Silva, Wicklow and
Crous, Stud. Mycol. 50(2): 331 (2004).
Index Fungorum number: IF 500084
Synonyms: Paracamarosporium fungicola (Verkley and Wicklow) Wijayawardene and K.D.
Hyde.
Notes
: Wijayawardene et al. [
80
] transferred Paraconiothyrium fungicola (CBS 113269) to
Paracamarosporium (Paraca.) as a new combination of Paraca. fungicola, since it formed a clade
with Paraca. psoraleae (CPC 21632) in the combined gene phylogeny of ITS, LSU, and SSU.
However, the phylogenetic tree of Wijayawardene et al. [
80
] only contained two species
of Paraconiothyrium viz. Paraco. estuarinum (CBS 109850) and Paraco. cyclothyrioides (CBS
432.75). However, our combined gene phylogenetic analyses of ITS, LSU, SSU, rpb2, and
tef 1-
α
included all Paraconiothyrium and Paracamarosporium species, showing that Paraco.
fungicola still clusters within Paraconiothyrium and is closely related to Paraco. magnoliae
(MFLUCC 10-0278). Our results are consistent with Wang et al. [
76
]. Based on nucleotide
comparisons, Paraco. fungicola (CBS 113269) is different from Paraca. psoraleae (CPC 21632)
by 38/552 bp (6.8%) of the ITS and 3/896 bp (0.3%) of the LSU, while it is different from
Paraco. magnoliae (MFLUCC 10-0278) in 33/524 bp (6.2%) of the ITS, and 0/899 bp (0%) of
the LSU. In addition, the morphology fits with the characteristics of Paraconiothyrium [
71
,
73
].
Therefore, we recommend that Paraco. fungicola should be transferred back to the genus
Paraconiothyrium, as first reported by Verkley et al. [75].
4. Discussion
In this study, four new taxa and three new records isolated from coffee are introduced
based on morphological and phylogenetic analyses. In addition, a known species was trans-
ferred to Paraconiothyrium based on our phylogenetic analyses and the study of Wang et al. [
76
].
Dictyosporiaceae contains 17 genera and 125 species [
22
], but none belong to coffee-
associated fungi. Herein, we introduce two new species in the genus of Pseudocoleophoma
viz. Pse. puerensis and Pse. yunnanensis, from Yunnan Province, China. This is the first
report of coffee-associated fungi in Dictyosporiaceae [10,74].
Didymosphaeriaceae contains 33 genera and 255 species [
22
]. To date, only five species
have been reported as coffee-associated fungi in Didymosphaeriaceae viz. Didymosphaeria
sp., Montagnula donacina,Paraconiothyrium brasiliense,Phaeodothis winteri, and Spegazzinia
meliolae [
10
,
74
]. In this study, we introduce two new species viz. Deniquelata yunnanensis
and Paraconiothyrium yunnanensis, and three new records viz. Austropleospora keteleeriae,
Montagnula thailandica, and Xenocamarosporium acaciae in Didymosphaeriaceae. This is the first
report of fungi belonging to Austropleospora,Deniquelata, and Xenocamarosporium associated
with coffee.
In our multigene phylogeny, Paraconiothyrium is polyphyletic and paraphyletic within
Didymosphaeriaceae, which repeats the results of previous studies [
74
,
76
,
77
,
81
]. Most species
of Paraconiothyrium that were published earlier were mainly based on morphological
taxonomy, especially asexual morphology [
76
,
82
]. In subsequent studies, some species were
classified into other genera. In our phylogenetic tree, we also found the same taxonomic
problems in this genus as Phukhamsakda et al. [
83
]. Paraconiothyrium nelloi and Paraco.
fuscomaculans clustered in the genus Kalmusia, while Paraco.nelloi clustered with K.italica
with high statistical support (ML/BYPP = 96/1.00). Paraconiothyrium nelloi (MFLU 14-
0813) shows few nucleotide differences compared with K.italica (MFLUCC 13-0066), by
1/502 bp (0.2%) of the ITS, 3/857 bp (0.35%) of the LSU, and 2/995 bp (0.2%) of the SSU.
The asexual morph of Paraco.nelloi was isolated from a dead twig, while K.italica was
observed from the culture of PDA. Conidia of both species differ in size but are similar

J. Fungi 2022,8, 1113 20 of 24
in shape and color [
84
]. Paraconiothyrium fuscomaculans (CBS 116.16), grouped with K.
longispora, showed good statistical support (ML/BYPP = 82/0.90). Based on nucleotide
comparisons, Paraco.fuscomaculans (CBS116.16) is slightly different from K.longispora (CBS
582.83) by 0/532 bp (0%) of the ITS and 1/901 bp (0.1%) of the LSU. Morphologically, Paraco.
fuscomaculans and K.longispora have septate conidiogenous cells and almost the same size
conidia [
58
,
85
]. In 2020, Gonçalves et al. [
77
] also reported Paraco.nelloi (MFLU 14-0813)
and Paraco. fuscomaculans (CBS 116.16) that fit in the genus Kalmusia, and mentioned that
morphological analyses of Didymosphaeriaceae are needed to evaluate the redisposition of
Paraconiothyrium-like species. Therefore, in future research, we strongly recommend that
these two species should be re-collected and re-identified with more collections in order
to demonstrate their taxonomic positions. When studying the species of Paraconiothyrium,
both morphology and multigene phylogeny are important, and all the Paraconiothyrium
taxa should be included in the phylogenetic analyses. In our study, we introduce a new
species of Paraconiothyrium based on multigene phylogeny and morphology.
We excluded the type species Austropleospora osteospermi in our phylogenetic analyses
due to the lack of multi-gene sequence data [
61
]. In ITS phylogenetic analysis (not shown),
A. osteospermi was not grouped within Austropleospora, thus, we believe that only the ITS
gene is insufficient to distinguish intra-species in the phylogeny. The sexual morph of
A. osteospermi is similar to A. ochracea [
61
,
63
]; therefore, we suggest adding multi-gene
sequence data for A. osteospermi in future studies.
In this research, we mainly focused on coffee-associated saprotrophic fungi in Yun-
nan Province, China, which is one of the biodiversity hotspots in the Greater Mekong
Subregion [
86
,
87
]. Saprotrophic fungi are considered one of the most active plant litter
decomposers that play an important role in the cycling of carbon, nitrogen, and soil nutri-
ents [
88
]. Some saprotrophic fungi can also release various chemical compounds to increase
plant resistance in harsh environments [
89
]. In addition, some saprotrophic fungi have
been investigated for plant disease control [
14
]. Including the fungi that are reported in this
study, to date, about 40 saprotrophic fungi have been recorded from coffee [
3
,
8
,
10
]. Even
though coffee is one of the important economic crops worldwide, very few saprotrophic
fungi studies on coffee have been carried out in China, and thus, it is important to study
coffee saprophytic fungi and their contribution to coffee plants. Our research increases
the knowledge of coffee saprophytic fungi and provides a basis for future research on the
applied aspects of coffee saprotrophic fungi.
5. Conclusions
In conclusion, four novel taxa belonging to Pleosporales that are associated with coffee
were discovered. Pseudocoleophoma puerensis and Pse. yunnanensis are introduced as new
species in Dictyosporiaceae, while Deniquelata yunnanensis and Paraconiothyrium yunnanensis
are introduced as new species in Didymosphaeriaceae. The phylogenetic relationships among
species of these three genera were also updated in this study. In addition, Austropleospora
keteleeriae as a new host record for coffee, as well as Montagnula thailandica, and Xenoca-
marosporium acaciae as the new host and country records for coffee in China were reported.
Furthermore, based on taxonomy and phylogenetic analyses, Paracamarosporium fungicola
was transferred back to Paraconiothyrium.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/jof8101113/s1, Table S1: Taxa names, collection numbers, and
corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses. Newly
generated sequences in this study are indicated in black bold. The type species are noted with
T
after
the species name, while NA indicates the unavailability of data.
Author Contributions:
Conceptualization, S.C.K. and S.T.; data curation, L.L.; formal analysis,
L.L. and Y.-R.X.; funding acquisition, S.C.K., D.-Q.D., N.S., A.M.E., S.A.-R. and S.T.; investigation,
L.L.; methodology, L.L. and Y.-R.X.; resources, D.-Q.D.; software, L.L.; supervision, S.C.K. and S.T.;
validation, S.C.K., D.-Q.D., N.S., S.L.S., A.M.E., S.A.-R., R.S.J. and S.T.; writing—original draft, L.L.;

J. Fungi 2022,8, 1113 21 of 24
writing—review and editing, S.C.K., D.-Q.D., N.S., S.L.S., A.M.E., S.A.-R., R.S.J. and S.T. All authors
have read and agreed to the published version of the manuscript.
Funding:
This research was funded by “National Natural Science Foundation of China, grant number
NSFC 31760013, 31950410558”, “High-Level Talent Recruitment Plan of Yunnan Provinces (“Young
Talents” Program)”, Chiangmai University and project number RSP-2021/200 of the King Saud
University, Riyadh, Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All the phylogenetic alignments and trees obtained are available in
Figshare (doi:10.6084/m9.figshare.21260589) and TreeBASE (number 29752).
Acknowledgments:
Li Lu thanks Mae Fah Luang University for the award of fee-less scholarship.
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological
Resource and Food Engineering, Qujing Normal University; and Center of Excellence in Fungal
Research are thanked by Li Lu for the facilities provided for research work.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Coffea L. Available online: https://www.gbif.org/species/2895315 (accessed on 2 March 2022).
2.
Herrera, J.C.; Lambot, C. The coffee tree—Genetic diversity and origin. In The Craft and Science of Coffee; Elsevier: Tours, France,
2017; pp. 1–16.
3.
Lu, L.; Tibpromma, S.; Karunarathna, S.; Thiyagaraja, V.; Xu, J.; Jayawardena, R.S.; Lumyong, S.; Hyde, K.D. Taxonomic and
phylogenic appraisal of a novel species and a new record of Stictidaceae from coffee in Yunnan Province, China. Phytotaxa
2021
,
528, 111–124. [CrossRef]
4.
Zhang, S.; Liu, X.; Li, R.; Wang, X.; Cheng, J.; Yang, Q.; Kong, H. AHP-GIS and MaxEnt for delineation of potential distribution of
Arabica coffee plantation under future climate in Yunnan, China. Ecol. Indic. 2021,132, 108339. [CrossRef]
5.
Zhang, S.; Liu, X.; Wang, X.; Gao, Y.; Yang, Q. Evaluation of coffee ecological adaptability using Fuzzy, AHP, and GIS in Yunnan
Province, China. Arab. J. Geosci. 2021,14, 1366. [CrossRef]
6. Liu, Y.Z.; Jiao, D. Influence of free trade zone construction on coffee industry in Yunnan, China. World Agric. 2017,3, 181–184.
7.
Coffee in China. Available online: https://oec.world/en/profile/bilateral-product/coffee/reporter/chn (accessed on 2 March 2022).
8.
Lu, L.; Karunarathna, S.C.; Hyde, K.D.; Bhat, D.J.; Dai, D.-Q.; Jayawardena, R.S.; Tibpromma, S. Crassiparies yunnanensis sp. nov.
(Neohendersoniaceae,Pleosporales) from dead twigs of Coffea arabica in China. Phytotaxa 2022,543, 244–254. [CrossRef]
9. Waller, J. Control of coffee diseases. In Coffee; Springer: Boston, MA, USA, 1985; pp. 219–229.
10.
Lu, L.; Tibpromma, S.; Karunarathna, S.C.; Jayawardena, R.S.; Lumyong, S.; Xu, J.; Hyde, K.D. Comprehensive Review of Fungi
on Coffee. Pathogens 2022,11, 411. [CrossRef]
11.
Arias, R.M.; Abarca, G.H. Fungal diversity in coffee plantation systems and in a tropical montane cloud forest in Veracruz, Mexico.
Agrofor. Syst. 2014,88, 921–933. [CrossRef]
12.
Pimenta, C.J.; Angélico, C.L.; Chalfoun, S.M. Challengs in coffee quality: Cultural, chemical and microbiological aspects. Ciênc.
Agrotecnol. 2018,42, 337–349. [CrossRef]
13.
Dos Santos, D.G.; Coelho, C.C.d.S.; Ferreira, A.B.R.; Freitas-Silva, O. Brazilian coffee production and the future microbiome and
mycotoxin profile considering the climate change Scenario. Microorganisms 2021,9, 858. [CrossRef]
14.
Botrel, D.A.; Laborde, M.C.F.; Medeiros, F.H.V.d.; Resende, M.L.V.d.; Ribeiro Júnio, P.M.; Pascholati, S.F.; Gusmão, L.F.P. Saprobic
fungi as biocontrol agents of halo blight (Pseudomonas syringae pv. garcae) in coffee clones. Coffee Sci.
2018
,13, 283–291. [CrossRef]
15.
Laborde, M.C.F.; Botelho, D.M.d.S.; Rodríguez, G.A.A.; Resende, M.L.V.d.; Queiroz, M.V.d.; Batista, A.D.; Cardoso, P.G.; Pascholati,
S.F.; Gusmão, L.F.P.; Martins, S.J. Phialomyces macrosporus reduces Cercospora coffeicola survival on symptomatic coffee leaves.
Coffee Sci. 2019,14, 1–11. [CrossRef]
16.
Zeng, M.W. Study on Identification of Coffee Root Exudates and Allelopathic Effects of Coffee Root Exudates on Coffee Seedlings.
Master’s Thesis, Huazhong Agricultural University, Wuhan, China, 2015.
17.
Wang, K.; Chen, S.L.; Dai, Y.C.; Jia, Z.F.; Li, T.H.; Liu, T.Z.; Phurbu, D.; Mamut, R.; Sun, G.Y.; Bau, T. Overview of China’s
nomenclature novelties of fungi in the new century (2000–2020). Mycosystema 2021,40, 822–833.
18. Luttrell, E.S. The ascostromatic ascomycetes. Mycologia 1955,47, 511–532. [CrossRef]
19. Barr, M. Prodromus to Class Loculoascomycetes; University of Massachusetts: Amherst, MA, USA, 1987; 168p.
20.
Pem, D.; Jeewon, R.; Chethana, K.W.T.; Hongsanan, S.; Doilom, M.; Suwannarach, N.; Hyde, K.D. Species concepts of Doth-
ideomycetes: Classification, phylogenetic inconsistencies and taxonomic standardization. Fungal Divers.
2021
,109, 283–319.
[CrossRef]

J. Fungi 2022,8, 1113 22 of 24
21.
Yang, E.-F.; Tibpromma, S.; Karunarathna, S.C.; Phookamsak, R.; Xu, J.-C.; Zhao, Z.-X.; Karunanayake, C.; Promputtha, I.
Taxonomy and phylogeny of novel and extant taxa in Pleosporales associated with Mangifera indica from Yunnan, China (Series I).
J. Fungi 2022,8, 152. [CrossRef]
22.
Wijayawardene, N.; Hyde, K.; Dai, D.; Sánchez-García, M.; Goto, B.; Saxena, R.; Erdogdu, M.; Selçuk, F.; Rajeshkumar, K.; Aptroot,
A. Outline of Fungi and fungus-like taxa-2021. Mycosphere 2022,13, 53–453. [CrossRef]
23.
Kruys, Å.; Eriksson, O.E.; Wedin, M. Phylogenetic relationships of coprophilous Pleosporales (Dothideomycetes, Ascomycota),
and the classification of some bitunicate taxa of unknown position. Mycol. Res. 2006,110, 527–536. [CrossRef]
24. Zhang, Y.; Crous, P.W.; Schoch, C.L.; Hyde, K.D. Pleosporales. Fungal Divers. 2012,53, 1–221. [CrossRef]
25.
Tennakoon, D.S.; Kuo, C.-H.; Maharachchikumbura, S.S.; Thambugala, K.M.; Gentekaki, E.; Phillips, A.J.; Bhat, D.J.; Wanasinghe,
D.N.; de Silva, N.I.; Promputtha, I. Taxonomic and phylogenetic contributions to Celtis formosana,Ficus ampelas,F. septica,
Macaranga tanarius and Morus australis leaf litter inhabiting microfungi. Fungal Divers. 2021,108, 1–215. [CrossRef]
26.
Boonmee, S.; D’souza, M.J.; Luo, Z.; Pinruan, U.; Tanaka, K.; Su, H.; Bhat, D.J.; McKenzie, E.H.; Jones, E.; Taylor, J.E. Dictyosporiaceae
fam. nov. Fungal Divers. 2016,80, 457–482. [CrossRef]
27.
Wanasinghe, D.N.; Mortimer, P.E.; Bezerra, J.D.P. Fungal Systematics and Biogeography. Front. Microbiol.
2022
,12, 827725.
[CrossRef]
28. Munk, A. The system of the Pyrenomycetes. Dansk Bot. Ark. 1953,15, 1–163.
29.
Ariyawansa, H.A.; Tanaka, K.; Thambugala, K.M.; Phookamsak, R.; Tian, Q.; Camporesi, E.; Hongsanan, S.; Monkai, J.;
Wanasinghe, D.N.; Mapook, A. A molecular phylogenetic reappraisal of the Didymosphaeriaceae (=Montagnulaceae). Fungal Divers.
2014,68, 69–104. [CrossRef]
30.
Zhang, M.D.; Wang, R.; Li, Y.; Hu, X.Q.; Li, M.; Zhang, M.; Duan, C.C. Ecological suitability zoning of Coffea arabica L. in Yunnan
Province. Chin. J. Eco-Agric. 2020,28, 168–178.
31.
Senanayake, I.; Rathnayaka, A.; Marasinghe, D.; Calabon, M.; Gentekaki, E.; Lee, H.; Hurdeal, V.; Pem, D.; Dissanayake, L.;
Wijesinghe, S. Morphological approaches in studying fungi: Collection, examination, isolation, sporulation and preservation.
Mycosphere 2020,11, 2678–2754. [CrossRef]
32.
Jayasiri, S.C.; Hyde, K.D.; Ariyawansa, H.A.; Bhat, J.; Buyck, B.; Cai, L.; Dai, Y.-C.; Abd-Elsalam, K.A.; Ertz, D.; Hidayat, I. The
Faces of Fungi database: Fungal names linked with morphology, phylogeny and human impacts. Fungal Divers.
2015
,74, 3–18.
[CrossRef]
33.
Index Fungorum. Available online: http://www.indexfungorum.org/Names/IndexFungorumPartnership.htm (accessed on 2
March 2022).
34.
White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In
PCR Protocols: A Guide to Methods and Applications; Academic Press, Inc.: Cambridge, MA, USA, 1990; Volume 18, pp. 315–322.
35.
Vilgalys, R.; Hester, M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several
Cryptococcus species. J. Bacteriol. 1990,172, 4238–4246. [CrossRef]
36.
Liu, Y.J.; Whelen, S.; Hall, B.D. Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerse II subunit. Mol.
Biol. Evol. 1999,16, 1799–1808. [CrossRef]
37.
Rehner, S.A.; Buckley, E. A Beauveria phylogeny inferred from nuclear ITS and EF1-
α
sequences: Evidence for cryptic diversifica-
tion and links to Cordyceps teleomorphs. Mycologia 2005,97, 84–98. [CrossRef]
38. Geneious. Available online: https://www.geneious.com/ (accessed on 2 March 2022).
39. GenBank. Available online: http://www.ncbi.nlm.nih.gov (accessed on 2 March 2022).
40. Mafft. Available online: https://mafft.cbrc.jp/alignment/server/ (accessed on 2 March 2022).
41. trimAl. Available online: http://trimal.cgenomics.org (accessed on 2 March 2022).
42.
Vaidya, G.; Lohman, D.J.; Meier, R. SequenceMatrix: Concatenation software for the fast assembly of multi-gene datasets with
character set and codon information. Cladistics 2011,27, 171–180. [CrossRef]
43.
Larsson, A. AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics
2014
,30, 3276–3278.
[CrossRef] [PubMed]
44.
Dissanayake, A.; Bhunjun, C.; Maharachchikumbura, S.; Liu, J. Applied aspects of methods to infer phylogenetic relationships
amongst fungi. Mycosphere 2020,11, 2652–2676. [CrossRef]
45.
Miller, M.; Pfeiffer, W. Schwartz Terri. In Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees; Gateway
Computing Environments Workshop (GCE): New Orleans, LA, USA, 2010; pp. 1–8.
46. Nylander, J. MrModeltest v25; Evolutionary Biology Centre, Uppsala University: Uppsala, Sweden, 2004.
47.
Ronquist, F.; Huelsenbeck, J.P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics
2003
,19, 1572–1574.
[CrossRef] [PubMed]
48. Rambaut, A. FigTree 1.4.2 Software; Institute of Evolutionary Biology, University of Edinburgh: Edinburgh, UK, 2014.
49. Figshare. Available online: https://figshare.com/account/home#/data (accessed on 2 September 2022).
50.
Jiang, H.-B.; Jeewon, R.; Karunarathna, S.C.; Phukhamsakda, C.; Doilom, M.; Kakumyan, P.; Suwannarach, N.; Phookam-
sak, R.; Lumyong, S. Reappraisal of Immotthia in Dictyosporiaceae,Pleosporales: Introducing Immotthia bambusae sp. nov. and
Pseudocyclothyriella clematidis comb. et gen. nov. based on morphology and phylogeny. Front. Microbiol.
2021
,12, 656235.
[CrossRef]

J. Fungi 2022,8, 1113 23 of 24
51.
Verkley, G.; Dukik, K.; Renfurm, R.; Göker, M.; Stielow, J. Novel genera and species of coniothyrium-like fungi in Montagnulaceae
(Ascomycota). Pers.-Mol. Phylogeny Evol. Fungi 2014,32, 25–51. [CrossRef]
52.
Ariyawansa, H.A.; Maharachchikumbura, S.S.; Karunarathne, S.C.; Chukeatirote, E.; Bahkali, A.H.; Kang, J.; Bhat, D.; Hyde, K.D.
Deniquelata barringtoniae gen. et sp. nov., associated with leaf spots of Barringtonia asiatica.Phytotaxa
2013
,105, 11–20. [CrossRef]
53.
Du, T.; Hyde, K.D.; Mapook, A.; Mortimer, P.E.; Xu, J.; Karunarathna, S.C.; Tibpromma, S. Morphology and phylogenetic analyses
reveal Montagnula puerensis sp. nov.(Didymosphaeriaceae,Pleosporales) from southwest China. Phytotaxa
2021
,514, 1–25. [CrossRef]
54.
Ariyawansa, H.A.; Tsai, I.; Thambugala, K.M.; Chuang, W.-Y.; Lin, S.-R.; Hozzein, W.N.; Cheewangkoon, R. Species diversity of
Pleosporalean taxa associated with Camellia sinensis (L.) Kuntze in Taiwan. Sci. Rep. 2020,10, 12762. [CrossRef]
55.
Tanaka, K.; Hirayama, K.; Yonezawa, H.; Sato, G.; Toriyabe, A.; Kudo, H.; Hashimoto, A.; Matsumura, M.; Harada, Y.; Kurihara, Y.
Revision of the Massarineae (Pleosporales, Dothideomycetes). Stud. Mycol. 2015,82, 75–136. [CrossRef]
56.
Hyde, K.; Hongsanan, S.; Jeewon, R.; Bhat, D.; McKenzie, E.; Jones, E.; Phookamsa, R.; Ariyawansa, H.; Boonmee, S.; Zhao,
Q. Fungal diversity notes 367–490: Taxonomic and phylogenetic contributions to fungal taxa. Fungal Divers.
2016
,80, 1–270.
[CrossRef]
57.
Tennakoon, D.; Bhat, D.; Kuo, C.; Hyde, K. Leaf litter saprobic Dictyosporiaceae (Pleosporales, Dothideomycetes): Pseudocoleophoma
zingiberacearum sp. nov. from Hedychium coronarium.Kavaka 2019,53, 1–7. [CrossRef]
58.
Jayasiri, S.; Hyde, K.; Jones, E.; McKenzie, E.; Jeewon, R.; Phillips, A.; Bhat, D.; Wanasinghe, D.; Liu, J.; Lu, Y. Diversity,
morphology and molecular phylogeny of Dothideomycetes on decaying wild seed pods and fruits. Mycosphere
2019
,10, 1–186.
[CrossRef]
59.
Li, W.-J.; McKenzie, E.H.; Liu, J.-K.J.; Bhat, D.J.; Dai, D.-Q.; Camporesi, E.; Tian, Q.; Maharachchikumbura, S.S.; Luo, Z.-L.; Shang,
Q.-J. Taxonomy and phylogeny of hyaline-spored coelomycetes. Fungal Divers. 2020,100, 279–801. [CrossRef]
60.
Phukhamsakda, C.; McKenzie, E.H.; Phillips, A.J.; Gareth Jones, E.; Jayarama Bhat, D.; Stadler, M.; Bhunjun, C.S.; Wanasinghe,
D.N.; Thongbai, B.; Camporesi, E. Microfungi associated with Clematis (Ranunculaceae) with an integrated approach to delimiting
species boundaries. Fungal Divers. 2020,102, 1–203. [CrossRef]
61.
Morin, L.; Shivas, R.; Piper, M.; Tan, Y. Austropleospora osteospermi gen. et sp. nov. and its host specificity and distribution on
Chrysanthemoides monilifera ssp. rotundata in Australia. Fungal Divers. 2010,40, 65–74. [CrossRef]
62.
Ariyawansa, H.A.; Hyde, K.D.; Jayasiri, S.C.; Buyck, B.; Chethana, K.; Dai, D.Q.; Dai, Y.C.; Daranagama, D.A.; Jayawardena, R.S.;
Lücking, R. Fungal diversity notes 111–252—Taxonomic and phylogenetic contributions to fungal taxa. Fungal Divers.
2015
,75,
27–274. [CrossRef]
63.
Dissanayake, L.S.; Wijayawardene, N.N.; Samarakoon, M.C.; Hyde, K.D.; Kang, J.-C. The taxonomy and phylogeny of Austro-
pleospora ochracea sp. nov.(Didymosphaeriaceae) from Guizhou, China. Phytotaxa 2021,491, 217–229. [CrossRef]
64.
Alidadi, A.; Kowsari, M.; Javan-Nikkhah, M.; Karami, S.; Ariyawansa, H.A.; Jouzani, G.S. Deniquelata quercina sp. nov., a new
endophyte species from Persian oak in Iran. Phytotaxa 2019,405, 187–194. [CrossRef]
65.
Crous, P.; Cowan, D.; Maggs-Kölling, G.; Yilmaz, N.; Thangavel, R.; Wingfield, M.; Noordeloos, M.; Dima, B.; Brandrud, T.; Jansen,
G. Fungal planet description sheets: 1182–1283. Pers. Mol. Phylogeny Evol. Fungi 2021,46, 313–528. [CrossRef]
66.
Devadatha, B.; Sarma, V.V.; Ariyawansa, H.; Jones, E.G. Deniquelata vittalii sp. nov., a novel Indian saprobic marine fungus on
Suaeda monoica and two new records of marine fungi from Muthupet mangroves, East coast of India. Mycosphere
2018
,9, 565–582.
[CrossRef]
67. Berlese, A.N. Icones Fungorum, Pyrenomycetes; Kessinger Publishing, LLC: Whitefish, MT, USA, 1896; Volume 2, pp. 1–216.
68.
Tennakoon, D.; Hyde, K.; Wanasinghe, D.; Bahkali, A.; Camporesi, E.; Khan, S.; Phookamsak, R. Taxonomy and phylogenetic
appraisal of Montagnula jonesii sp. nov.(Didymosphaeriaceae,Pleosporales). Mycosphere 2016,7, 1346–1356. [CrossRef]
69.
Barr, M.E. Some Dictyosporous Genera and Species of Pleosporales in North America; The New York Botanical Garden: New York, NY,
USA, 1990; Volume 62, pp. 1–92.
70.
Ariyawansa, H.A.; Camporesi, E.; Thambugala, K.M.; Mapook, A.; Kang, J.-C.; Alias, S.A.; Chukeatirote, E.; Thines, M.; Mckenzie,
E.H.; Hyde, K.D. Confusion surrounding Didymosphaeria—Phylogenetic and morphological evidence suggest Didymosphaeriaceae
is not a distinct family. Phytotaxa 2014,176, 102–119. [CrossRef]
71.
Tibpromma, S.; Hyde, K.D.; McKenzie, E.H.; Bhat, D.J.; Phillips, A.J.; Wanasinghe, D.N.; Samarakoon, M.C.; Jayawardena, R.S.;
Dissanayake, A.J.; Tennakoon, D.S. Fungal diversity notes 840–928: Micro-fungi associated with Pandanaceae.Fungal Divers.
2018
,
93, 1–160. [CrossRef]
72.
Hongsanan, S.; Hyde, K.; Phookamsak, R.; Wanasinghe, D.; McKenzie, E.; Sarma, V.; Boonmee, S.; Lücking, R.; Bhat, D.; Liu, N.
Refined families of Dothideomycetes: Orders and families incertae sets in Dothideomycetidae. Mycosphere J. Fungal Biol.
2020
,11,
1553–2107. [CrossRef]
73.
Mapook, A.; Hyde, K.D.; McKenzie, E.H.; Jones, E.; Bhat, D.J.; Jeewon, R.; Stadler, M.; Samarakoon, M.C.; Malaithong, M.;
Tanunchai, B. Taxonomic and phylogenetic contributions to fungi associated with the invasive weed Chromolaena odorata (Siam
weed). Fungal Divers. 2020,101, 1–175. [CrossRef]
74.
U.S. National Fungus Collections Fungal Database. Available online: https://nt.ars-grin.gov/fungaldatabases (accessed on 2
March 2022).
75.
Verkley, G.J.; da Silva, M.; Wicklow, D.T.; Crous, P.W. Paraconiothyrium, a new genus to accommodate the mycoparasite
Coniothyrium minitans, anamorphs of Paraphaeosphaeria, and four new species. Stud. Mycol. 2004,50, 323–335.

J. Fungi 2022,8, 1113 24 of 24
76.
Wang, J.; Shao, S.; Liu, C.; Song, Z.; Liu, S.; Wu, S. The genus Paraconiothyrium: Species concepts, biological functions, and
secondary metabolites. Crit. Rev. Microbiol. 2021,47, 781–810. [CrossRef]
77.
Gonçalves, M.F.; Esteves, A.C.; Alves, A. Revealing the hidden diversity of marine fungi in Portugal with the description of two
novel species, Neoascochyta fuci sp. nov. and Paraconiothyrium salinum sp. nov. Int. J. Syst. Evol. Microbiol.
2020
,70, 5337–5354.
[CrossRef]
78.
Crous, P.; Wingfield, M.; Guarro, J.; Hernández-Restrepo, M.; Sutton, D.; Acharya, K.; Barber, P.; Boekhout, T.; Dimitrov, R.;
Dueñas, M. Fungal planet description sheets: 320–370. Pers.-Mol. Phylogeny Evol. Fungi 2015,34, 167–266. [CrossRef]
79.
Wijayawardene, N.; Hyde,K.; Al-Ani, L.K.T.; Tedersoo, L.; Haelewaters, D.; Becerra, A.G.; Schnittler, M.; Shchepin, O.; Novozhilov,
Y.; Silva-Filho, A. Outline of Fungi and fungus-like taxa. Mycosphere 2020,11, 1060–1456. [CrossRef]
80.
Wijayawardene, N.; Tibpromma, S.; Hyde, K.; An, Y.; Camporesi, E.; Wang, Y. Additions to Pseudocamarosporium; two new species
from Italy. Stud. Fungi 2016,1, 1–10. [CrossRef]
81.
Khodaei, S.; Arzanlou, M.; Babai-Ahari, A.; Rota-Stabelli, O.; Pertot, I. Phylogeny and evolution of Didymosphaeriaceae (Pleosporales):
New Iranian samples and hosts, first divergence estimates, and multiple evidences of species mis-identifications. Phytotaxa
2019
,
424, 131–146. [CrossRef]
82.
Hyde, K.D.; Abd-Elsalam, K.; Cai, L. Morphology: Still essential in a molecular world. Mycotaxon
2010
,114, 439–451. [CrossRef]
83.
Phukhamsakda, C.; Nilsson, R.H.; Bhunjun, C.S.; de Farias, A.R.G.; Sun, Y.-R.; Wijesinghe, S.N.; Raza, M.; Bao, D.-F.; Lu, L.;
Tibpromma, S. The numbers of fungi: Contributions from traditional taxonomic studies and challenges of metabarcoding. Fungal
Divers. 2022,114, 327–386. [CrossRef]
84.
Liu, J.K.; Hyde, K.D.; Jones, E.; Ariyawansa, H.A.; Bhat, D.J.; Boonmee, S.; Maharachchikumbura, S.S.; McKenzie, E.H.;
Phookamsak, R.; Phukhamsakda, C. Fungal diversity notes 1–110: Taxonomic and phylogenetic contributions to fungal species.
Fungal Divers. 2015,72, 1–197. [CrossRef]
85.
Boerema, G.; Loerakker, W. Contributions towards a monograph of Phoma (Coelomycetes)—III. 2. Misapplications of the type
species name and the generic synonyms of section Plenodomus (Excluded species). Pers. Mol. Phylogeny Evol. Fungi
1996
,16,
141–189.
86.
Chaiwan, N.; Gomdola, D.; Wang, S.; Monkai, J.; Tibpromma, S.; Doilom, M.; Wanasinghe, D.; Mortimer, P.; Lumyong, S.; Hyde, K.
https://gmsmicrofungi.org: An online database providing updated information of microfungi in the Greater Mekong Subregion.
Mycosphere 2021,12, 1409–1422. [CrossRef]
87.
Wang, K.; Cai, L.; Yao, Y. Overview of nomenclature novelties of fungi in the world and China (2020). Biodivers. Sci.
2021
,29, 1064.
[CrossRef]
88. Reverchon, F.; María del Ortega-Larrocea, P.; Pérez-Moreno, J. Saprophytic fungal communities change in diversity and species
composition across a volcanic soil chronosequence at Sierra del Chichinautzin, Mexico. Ann. Microbiol.
2010
,60, 217–226.
[CrossRef]
89.
Purwanto, B.; Sumadi, S.; Nuraini, A.; Setiawati, M.R. Effect of water content on conidia of Trichoderma spp., indole acetic acid
content, electrical conductivity, and pH. Biodivers. J. Biol. Divers. 2022,23, 2553–2560. [CrossRef]