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Botryosphaerialean fungi associated with woody oil
plants cultivated in Sichuan Province, China
Wen-Li Li1, Rui-Ru Liang1, Asha J. Dissanayake1, Jian-Kui Liu1
1School of Life Science and Technology, Center for Informational Biology, Electronic Science and Technology
University, Chengdu 611731, China
Corresponding author: Jian-Kui Liu (liujiankui@uestc.edu.cn)
Academic editor: Nalin Wijayawardene|Received 7 March 2023|Accepted 1 May 2023|Published 23 May 2023
Citation: Li W-L, Liang R-R, Dissanayake AJ, Liu J-K (2023) Botryosphaerialean fungi associated with woody oil
plants cultivated in Sichuan Province, China. MycoKeys 97: 71–116. https://doi.org/10.3897/mycokeys.97.103118
Abstract
Woody oil plants are important economic trees which are widely cultivated and distributed throughout
China. Surveys conducted during 2020 and 2021 on several woody oil plantations from ve regions of
Sichuan Province, China, revealed a high diversity of Botryosphaerialean fungi. e identication of
50 botryosphaeriaceous isolates was carried out based on both morphology and multi-gene phyloge-
netic analysis of internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (tef1)
and β-tubulin gene (tub2). is allowed the identication of twelve previously known Botryosphaeriales
species: Aplosporella prunicola, A. ginkgonis, Barriopsis tectonae, Botryosphaeria dothidea, Bo. fabicerciana,
Diplodia mutila, Di. seriata, Dothiorella sarmentorum, Neofusicoccum parvum, Sardiniella guizhouensis,
Sphaeropsis citrigena, and Sp. guizhouensis, and four novel species belonging to the genera Diplodia and
Dothiorella, viz. Di. acerigena, Di. pistaciicola, Do. camelliae and Do. zanthoxyli. e dominant species
isolated across the surveyed regions were Botryosphaeria dothidea, Sardiniella guizhouensis and Diplodia
mutila, representing 20%, 14% and 12% of the total isolates, respectively. In addition, most isolates
were obtained from Pistacia chinensis (14 isolates), followed by Camellia oleifera (10 isolates). e present
study enhances the understanding of Botryosphaeriales species diversity on woody oil plants in Sichuan
Province, China.
Keywords
Botryosphaeriales, diversity, new species, phylogeny, taxonomy
MycoKeys 97: 71–116 (2023)
doi: 10.3897/mycokeys.97.103118
https://mycokeys.pensoft.net
Copyright Wen-Li Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
72
Introduction
Botryosphaeriaceae is a diverse group of fungi that includes endophytes, saprobes and
plant pathogens (Phillips et al. 2013). ey have broad host ranges, and are widely
distributed in tropical and temperate regions (Batista et al. 2021). Botryosphaeriaceae
was introduced by eissen and Sydow (1918) to accommodate three genera Botry-
osphaeria, Dibotryon and Phaeobotryon. Botryosphaeriales was proposed to include
the single family, Botryosphaeriaceae, based on multi-gene phylogeny (Schoch et al.
2006). Up to date, six families and 32 genera are accepted in Botryosphaeriales, while
Botryosphaeriaceae is known to be the largest monophyletic family, including 22
genera and more than 200 species (https://botryosphaeriales.org/, accessed on 15th
April 2023).
e members of Botryosphaeriaceae have been taxonomically characterized
based on both sexual and asexual morphs. e production of large, ovoid to oblong,
typically hyaline, aseptate ascospores, which may become brown and septate with
age, as well as bitunicate asci within unilocular or multilocular botryose ascomata
known as pseudothecia is typical to the sexual state (Sivanesan 1984; Phillips et al.
2005). e asexual states of Botryosphaeriaceae exhibit a wide range of conidial
morphologies; for example, its conidia can be thin-walled and hyaline, or thick-
walled and pigmented, aseptate or 1–2-septate (Phillips et al. 2005). Additonaly,
the spermatial states were also frequently observed in Botryosphaeriaceae species,
which produced unicellular, hyaline, allantoid to rod-shaped spermatia on culture.
Botryosphaeriaceae species are signicantly dierent from other fungi in that the
color of its aerial hyphae, changing from gray to black with age on 2% potato
dextrose agar (PDA), which can be used for the rapid determination of botryospha-
eriaceous fungi.
e geographic distribution and host range of botryosphaeriaceous taxa are di-
verse. Seven genera in Botryosphaeriaceae: Botryosphaeria, Diplodia, Dothiorella,
Lasiodiplodia, Neodeightonia, Neofusicoccum and Phaeobotryon are common and fre-
quently reported from various geographical regions (Batista et al. 2021), while Botryo-
bambusa, Oblongocollomyces, Sakireeta and Sardiniella appear to be limited to a single
region or country (Liu et al. 2012; Crous et al. 2015; Linaldeddu et al. 2016; Yang
et al. 2017; Dissanayake et al. 2021). Many Botryosphaeriaceae species have wide
host ranges (e. g. Botryosphaeria dothidea, Diplodia mutila, Dothiorella sarmentorum,
Lasiodiplodia theobromae and Neofusicoccum parvum), while other species have nar-
rower host ranges (e. g. Diplodia olivarum was reported on olive, oleaster, carob, grape-
vine, almond et al.) (Lazzizera et al. 2008; Granata et al. 2011; Linaldeddu et al. 2015;
Olmo et al. 2016) or even in very specic hosts (e. g. Eutiarosporella darliae was only
reported on infected wheat and wheat-stubble) (ynne et al. 2015; Farr and Ross-
man 2021). Dierent species of Botryosphaeriaceae exhibit dierent environmental
adaptations and host preferences (Braunsdorf et al. 2016). Botryosphaeriaceous taxa
Botryosphaerialean fungi associated with woody oil plants in China 73
with narrow host ranges or limited geographic distribution will be more susceptible to
climatic eects (Slippers et al. 2017; Li et al. 2020).
Woody oil plants are economically important as they are used for the production
of cooking and industrial oil. Recently, many Botryosphaeriaceae species have been
frequently reported on woody oil plants. Diplodia olivarum was rst reported from
rotting olive drupes in Italy (Lazzizera et al. 2008) and later it was reported as as-
sociated with declining Prunus dulcis trees in Spain (Gramaje et al. 2012). Diplodia
insularis was isolated from branch canker of Pistacia lentiscus in Italy (Linaldeddu
et al. 2015). Dothiorella gregaria was isolated from the stems with asymptomatic
of Zanthoxylum bungeanum in China (Li et al. 2016b). Botryosphaeria dothidea,
Diplodia mutila, Di. seriata, Dothiorella iberica, Do. omnivora, Do. sarmentorum,
Lasiodiplodia citricola, L. pseudotheobromae, L. theobromae, Neofusicoccum mediter-
raneum, N. nonquaesitum, N. parvum, N. ribis, N. vitifusiforme, and Neoscytalidium
dimidiatum have been reported as pathogens of English walnut (Juglans regia L.)
in California (Chen et al. 2014), Chile (Jimenez Luna et al. 2022), China (Li et
al. 2016a; Zhang et al. 2017), Iran (Abdollahzadeh et al. 2013; Panahandeh et al.
2019), South Africa (Cloete et al. 2011), Spain (Gramaje et al. 2012) and USA
(Chen et al. 2014). However, very little is known about the Botryosphaerialean
species occurring on native woody oil plants in China. Hence, the aim of this
study was to gain a more comprehensive understanding of the diversity of Botry-
osphaeriaceae species associated with common woody oil plants grown in Sichuan
Province, China.
Materials and methods
Isolates and morphology
e isolates in this study were collected from the woody oil tree plantations in Sichuan
Province during the period of 2020 and 2021. e hosts include Acer truncatum,
Camellia oleifera, Idesia polycarpa, Olea europaea, Paeonia suruticosa, Pistacia chinensis,
Vernicia fordii and Zanthoxylum bungeanum. e samples were collected from decayed
stems, branches and twigs of woody oil trees. Mature fruiting bodies were selected for
fungal isolation and for morphological observations under stereo microscope Motic
SMZ 168 series. Measurements were made with Tarosoft Image Frame Work program
v. 0.9.7 (Liu et al. 2010). irty conidia/ascospores were measured per isolate, and 10–
30 measurements were taken of other morphological structures. At least 20 conidia/
ascospores were used to calculate the average length/width ratio (L/W). Single spore
isolation was conducted in accordance with the methods described in Chomnunti et
al. (2014). Germinated spores were individually placed on PDA plates and grown at
25 °C in daylight.
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
74
Herbarium specimens were stored in the herbarium of Cryptogams Kunming
Institute of Botany, Academia Sinica (KUN-HKAS) and duplicated at Herbarium,
University of Electronic Science and Technology (HUEST), Chengdu, China. Living
cultures were deposited at China General Microbiological Culture Collection Centre
(CGMCC), Beijing, China and duplicated at the University of Electronic Science and
Technology Culture Collection (UESTCC), Chengdu, China. MycoBank numbers
were registered as outlined in MycoBank (http://www.MycoBank.org. Accessed on
11th November 2022).
DNA extraction, PCR amplification and sequencing
e total genomic DNA was extracted from 7day-old isolates grown on 2% PDA
median at 25 °C, using the EZ geneTM fungal gDNA kit (GD2416), following
the manufacturer’s instructions and protocols. Partial gene sequences were deter-
mined for the internal transcribed spacer 1 and 2 including the intervening 5.8S
nrDNA gene (ITS), the nuclear ribosomal 28s large subunit (LSU), the translation
elongation factor 1-alphagene (tef1), and the β-tubulin gene (tub2). e primers
used for amplification are ITS5/ITS4 for ITS (White et al. 1990), LR0R/LR5
for LSU (Vilgalys and Hester 1990), EF1-728F/EF1-986R for tef1 (Carbone and
Kohn 1999) and Bt2a/Bt2b for tub2 (Glass and Donaldson 1995). Polymerase
chain reaction (PCR) amplication conditions were followed as of Dissanayake et
al. (2021). PCR products were sent for sequencing at Beijing Tsingke Biological
Engineering Technology and Services Co. Ltd. (Beijing, P.R. China). All newly
generated sequences are deposited in GenBank, and the obtained accession num-
bers are listed in Table 1.
Phylogenetic analyses
Sequence data for phylogenetic analyses were obtained from GenBank and from recent
publications regarding Botryosphaeriaceae fungi (Dissanayake et al. 2021; Xiao et al.
2021; Zhang et al. 2021; Rathnayaka et al. 2022) (See Suppl. material 1). e single
gene alignments were performed using MAFFT v.7.429 online service (https://mat.
cbrc.jp/alignment/server/, accessed on 15 October 2022) (Katoh et al. 2019) and am-
biguous regions were excluded using TrimAI with the option “-automated1”, which
trimmed sequences based on similarity statistics (Capella-Gutiérrez et al. 2009). Multi-
gene sequences were concatenated by Sequence matrix software (Vaidya et al. 2011).
Multi-gene phylogenetic analyses were obtained from maximum likelihood (ML) and
Bayesian inference (BI) analyses following Dissanayake et al. (2020).
ML analyses was performed using RAxML (Stamatakis 2006). e tree search
included 1,000 non-parametric bootstrap replicates and the best scoring tree was se-
lected from suboptimal trees under the GTRGAMMA substitution model. Maximum
Botryosphaerialean fungi associated with woody oil plants in China 75
Table 1. All newly generated sequences in this study. Ex-type strains are indicated with *. N/A: Notavailable.
Taxo nStain Number GenBank Accession Number
ITS tef1 tub2
Aplosporella ginkgonis UESTCC 22.0091 OQ190504 OQ241438 N/A
Aplosporella prunicola UESTCC 22.0090 OQ190505 N/A N/A
Barriopsis tectonae UESTCC 22.0092 OQ190506 OQ241439 N/A
Botryosphaeria dothidea UESTCC 22.0111 OQ190507 OQ241440 N/A
UESTCC 22.0109 N/A OQ241441 N/A
UESTCC 22.0112 OQ190508 OQ241442 N/A
UESTCC 22.0113 OQ190509 OQ241443 N/A
UESTCC 22.0108 OQ190510 OQ241444 N/A
UESTCC 22.0116 OQ190511 OQ241445 N/A
UESTCC 22.0114 OQ190512 OQ241446 N/A
UESTCC 22.0115 OQ190513 OQ241447 N/A
UESTCC 22.0110 OQ190514 OQ241448 N/A
UESTCC 22.0107 OQ190515 OQ241449 N/A
Botryosphaeria fabicerciana UESTCC 22.0117 OQ190516 OQ241450 N/A
UESTCC 22.0118 OQ190517 OQ241451 N/A
Diplodia acerigena* CGMCC 3.24157 OQ190518 OQ241452 N/A
Diplodia acerigena UESTCC 22.0074 OQ190519 OQ241453 OQ338163
UESTCC 22.0075 OQ190520 OQ241454 OQ338164
Diplodia mutila UESTCC 22.0064 OQ190521 OQ241455 OQ338165
UESTCC 22.0065 OQ190522 OQ241456 OQ338166
UESTCC 22.0069 OQ190523 OQ241457 OQ338167
UESTCC 22.0068 OQ190524 OQ241458 OQ338168
UESTCC 22.0067 OQ190525 OQ241459 OQ338169
UESTCC 22.0063 OQ190526 OQ241460 OQ338170
Diplodia pistaciicola * CGMCC 3.24156 OQ190527 OQ241461 OQ338171
Diplodia pistaciicola UESTCC 22.0071 OQ190528 OQ241462 OQ275062
Diplodia seriata UESTCC 22.0072 OQ190529 OQ241463 N/A
Dothiorella camelliae UESTCC 22.0080 OQ190530 N/A OQ275063
Dothiorella camelliae * CGMCC 3.24158 OQ190531 OQ241464 OQ275064
Dothiorella camelliae UESTCC 22.0079 OQ190532 OQ241465 OQ275065
UESTCC 22.0078 OQ190533 OQ241466 OQ275066
Dothiorella sarmentorum UESTCC 22.0076 OQ190534 N/A OQ275067
UESTCC 22.0077 OQ190535 OQ241467 OQ275068
Dothiorella zanthoxyli * CGMCC 3.24159 OQ190536 OQ241468 OQ275069
Dothiorella zanthoxyli UESTCC 22.0083 OQ190537 OQ241469 OQ275070
UESTCC 22.0084 OQ190538 OQ241470 OQ275071
Neofusicoccum parvum UESTCC 22.0096 OQ190539 OQ241471 N/A
UESTCC 22.0094 OQ190540 N/A N/A
UESTCC 22.0093 OQ190541 N/A N/A
UESTCC 22.0095 OQ190542 N/A N/A
Sardiniella guizhouensis UESTCC 22.0100 OQ190543 OQ241472 N/A
UESTCC 22.0101 OQ190544 OQ241473 N/A
UESTCC 22.0099 OQ190545 OQ241474 N/A
UESTCC 22.0097 OQ190546 OQ241475 N/A
UESTCC 22.0098 OQ190547 OQ241476 N/A
UESTCC 22.0102 OQ190548 OQ241477 N/A
UESTCC 22.0103 OQ190549 OQ241478 N/A
Sphaeropsis citrigena UESTCC 22.0106 OQ190550 OQ241479 N/A
Sphaeropsis citrigena UESTCC 22.0105 OQ190551 OQ241480 N/A
Sphaeropsis guizhouensis UESTCC 22.0104 OQ190552 OQ241481 N/A
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
76
likelihood bootstrap values equal or greater than 75% are marked near each node of
the phylogenetic tree.
Bayesian analyses was performed in MrBayes 3.2.6 (Ronquist et al. 2012). e
program MrModeltest 2.2 (Nylander 2004) was used to determine the best nucleo-
tide substitution model for each data partition. e Markov Chain Monte Carlo
(MCMC) sampling approach was used to calculate the posterior probabilities (PP)
(Rannala and Yang 1996). Bayesian analyses of four simultaneous Markov chains
were run for 10,000,000 generations with trees sampled every 1,000th generations.
e rst 20% of trees, representing the burn-in phase of the analyses, were dis-
carded, and the remaining trees were used for calculating posterior probabilities
(PP) in the majority rule consensus tree. PP values equal or greater than 0.95 are
marked near each node.
Trees were visualized with FigTree v1.4.0 (Rambaut 2006), and the layout was
edited using Adobe Illustrator CS6 software (Adobe Systems, USA).
Results
Phylogenetic analyses
A concatenated dataset of ITS and tef1 was used to determine the phylogenetic posi-
tion of Aplosporellaceae and Botryosphaeriaceae isolates obtained in this study. Com-
bined sequences of ITS and tef1 were used for the analyses of Botryosphaeria, while
ITS, tef1 and tub2 were used for the analyses of Diplodia and Dothiorella isolates. All
details of the alignments are provided in Table 2.
In an overview phylogenetic tree (Fig. 1), sixteen newly obtained isolates were
nested with four genera of Botryosphaeriaceae, representing seven known species
viz. Barriopsis tectonae, Neofusicoccum parvum, Sardiniella guizhouensis, Sphaeropsis
guizhouensis and Sp. citrigena. Two isolates were clustered with the genus Aplosporella
(Aplosporellaceae), and were identied as A. ginkgonis and A. prunicola.
ree individual phylogenetic trees were constructed for the genera Botryosphaeria,
Diplodia and Dothiorella. Twelve isolates belonged to the genus Botryosphaeria and ten
of them were nested with Bo. dothidea, while the remaining two isolates clustered with
Bo. fabicerciana (Fig. 2). Another twelve isolates were treated in Diplodia and seven
isolates were clustered with two known species of Diplodia (Di. mutila and Di. seriata,
Fig. 3). e other ve isolates did not cluster with any previously known Diplodia
species, thus, two novel species were preliminarily identied based on phylogenetic
evidence. Eight isolates were nested within Dothiorella and six isolates of them were
occupied in the basal position of the Dothiorella tree and formed two wellsupported
subclades, representing two new species. e other two isolates were nested within the
Do. sarmentorum isolates (Fig. 4).
Botryosphaerialean fungi associated with woody oil plants in China 77
Figure 1. Phylogram generated from RAxML analysis based on combined ITS and tef1 sequence data of
Botryosphaeriaceae and Aplosporellaceae isolates. e tree was rooted to Lecanosticta acicula (LNPV 252).
e ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from
this study are marked in red and ex-type strains are marked in bold.
Tiarosporellagraminis karooCBS118718
BotryobambusafusicoccumMFLUCC11-0143
BotryobambusaguizhouensisCGMCC3.20348
BarriopsisiranianaIRAN1448C
BarriopsisfuscaCBS174.26
OblongocollomycesvariabileCBS121774
PhaeobotryonmamaneCBS122980
SphaeropsiscitrigenaICMP16812
SphaeropsisporosaCBS110496
SphaeropsisvisciCBS100163
SphaeropsisguizhouensisCGMCC3.20352
SphaeropsiseucalypticolaMFLUCC11-0579
DiplodiacupressiCBS168.87
DiplodiaallocellulaCBS130408
DiplodiaseriataCBS112555
DiplodiamutilaCBS112553
NeodeightoniapalmicolaMFLUCC10-0822
NeodeightoniaphoenicumCBS122528
NeodeightoniasubglobosaCBS448.91
DiplodiacorticolaCBS112549
LasiodiplodiagonubiensisCBS115812
LasiodiplodialignicolaCBS134112
LasiodiplodiaparvaCBS456.78
AlanphillipsiaeuphorbiaeCPC21628
AlanphillipsiaaloeicolaCBS138896
CophinformamamaneCBS117444
CophinformaeucalyptiMFLUCC11-0655
SardiniellaguizhouensisCGMCC3.19222
BotryobambusafusicoccumMFLUCC11-0657
BotryobambusaguizhouensisGZCC19-0734
BarriopsisiranianaIRAN1449C
BarriopsistectonaeMFLUCC12-0381
BarriopsistectonaeUESTCC22.0092
OblongocollomycesvariabileCMW25420
OblongocollomycesvariabileCMW25421
PhaeobotryonmamaneCPC12264
SphaeropsiscitrigenaUESTCC22.0106
SphaeropsiscitrigenaUESTCC22.0105
SphaeropsiscitrigenaICMP16818
SphaeropsisguizhouensisUESTCC22.0104
SphaeropsisguizhouensisGZCC19-0273
SphaeropsiseucalypticolaMFLUCC11-0654
NeoscytalidiumhyalinumCBS145.78
NeoscytalidiumdimidiatumCBS499.66
MacrophominaphaseolinaCBS227.33
SardiniellaguizhouensisUESTCC22.0103
SardiniellaguizhouensisGZCC19-0216
SardiniellaguizhouensisUESTCC22.0097
SardiniellaguizhouensisGZCC19-0094
SardiniellaguizhouensisUESTCC22.0098
SardiniellaguizhouensisGZCC19-0229
SardiniellaguizhouensisUESTCC22.0099
SardiniellaguizhouensisUESTCC22.0100
SardiniellaguizhouensisUESTCC22.0101
--/0.99
--/0.97
100/1
100/0.99
100/1
89
91/1
100/1
99/1 100/1
100
87/1
100/1
97/1
95/1
100/--
100/1
99/1
--/1
77/1
85/1
99/1
85/1
100/1
99/1
92/1
97/1
100/1
100/1
85/1
100/1
100/1
--/0.99
75/1
Botryosphaeriaceae
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
78
Table 2. Alignment details and ML, BI analyses results of each phylogenetic tree constructed in this study.
Character Overview
phylogenetic tree
Botryosphaeria Diplodia Dothiorella
Number of base pairs in each
gene region (including the gaps
after alignment)
ITS (603 bp),
tef1 (320 bp)
ITS (555 bp), tef1
(315 bp)
ITS (537 bp), tef1(311 bp),
tub2 (381 bp)
ITS (523 bp), tef 1 (294 bp),
tub2 (427 bp)
Number of isolates obtained in
this study
17 12 11 9
Number of taxa originated
from GenBank
94 45 64 73
Outgroup taxa Lecanosticta
acicula
(LNPV252)
Barriopsis iraniana
(IRAN1448C) and
Barriopsis iraniana
(IRAN1449C)
Dothiorella dulcispinae
(CMW 36460) and
Dothiorella dulcispinae
(CMW 36462)
Neofusicoccum luteum (CBS
562.92) and Neofusicoccum
luteum (CMW 41365)
BI (model
of each gene
region)
ITS GTR+I+G SYM K80+I+G HKY+I+G
tef1 GTR+I+G HKY+G GTR+G GTR+G
tub2 – – GTR+G GTR+I+G
Figure 1. Continued.
Endomelanconiopsismi crosporaCBS353.97
Endomelanconiopsisendophyt icaCBS120397
NeofusicoccummangiferaeCBS118531
NeofusicoccumparvumCMW9081
NeofusicoccumgrevilleaeCBS129518
DothiorellacasuariniCBS120688
DothiorellasarmentorumIMI63581b
DothiorellairanicaCBS124722
DothiorellacitricolaCBS124729
DothiorellayunnanaCGMCC3.17999
DothiorellaviticolaCBS117009
SardiniellaellipticaCGMCC3.20349
SardiniellaurbanaCBS141580
SardiniellaceltidisMFLUCC17-981
AplosporellahesperidicaCBS732.79
AplosporellasophoraeCPC29688
AplosporellamacropycnidiaCGMCC3.17725
AplosporellaafricanaCBS121777
AplosporellaprunicolaCBS121167
AplosporellayalgorensisMUCC511
AplosporellapapillataCBS121780
AplosporellajaveediiCFCC50054
AplosporellaginkgonisCFCC52442
AplosporellathailandicaMFLU16-0615
AplosporellaartocarpiCPC22791
LecanostictaacicolaLNPV252
NeofusicoccumparvumUESTCC22.0096
NeofusicoccumparvumUESTCC22.0095
NeofusicoccumparvumUESTCC22.0093
NeofusicoccumparvumUESTCC22.0094
SardiniellaellipticaGZCC19-0262
SardiniellaellipticaGZCC19-0245
AplosporellahesperidicaCBS208.37
AplosporellamacropycnidiaCGMCC3.17726
AplosporellaafricanaCBS121778
AplosporellaprunicolaSTEU6326
Aplosporella
UESTCC
22.0090
AplosporellapapillataCBS121781
AplosporellajaveediiCFCC50052
AplosporellaginkgonisUESTCC22.0091
AplosporellaginkgonisCFCC89661
79/1
95/1
100/1
99/1
100/1
100/1
83/1
0.1
--/0.99
94/1
--/0.97
91/1
77/0.99
81/0.96
75/0.99
75/0.99
prunicola
--/0.99
--/0.95
99/1
--/0.97
93/1
75/0.99
100/1
90/0.99
83/0.99
97/1
--/0.99
Outgroup
Aplosporellaceae
Botryosphaerialean fungi associated with woody oil plants in China 79
Figure 2. Phylogram generated from RAxML analysis based on combined ITS and tef1 sequence data of
Botryosphaeria isolates. e tree was rooted to Barriopsis iraniana (IRAN1448C and IRAN1449C). e
ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from this
study are marked in red and ex-type strains are marked in bold.
BotryosphaeriaramosaCBS122069
BotryosphaeriascharifiiIRAN1529C
BotryosphaeriadothideaCMW8000
BotryosphaeriaqingyuanensisCGMCC3.18742
BotryosphaeriacorticisCBS119047
BotryosphaeriakuwatsukaiCBS135219
BotryosphaeriafabicercianaCMW27094
BotryosphaeriaagavesMFLUCC11-0125
BotryosphaeriaramosaCGMCC3.18740
BotryosphaeriaramosaCGMCC3.18739
BotryosphaeriascharifiiIRAN1543C
BotryosphaeriadothideaCGMCC3.18744
BotryosphaeriadothideaCGMCC3.18745
BotryosphaeriadothideaUESTCC22.0109
BotryosphaeriadothideaCGMCC3.17722
BotryosphaeriadothideaCGMCC3.17724
BotryosphaeriadothideaUESTCC22.0107
BotryosphaeriadothideaUESTCC22.0108
BotryosphaeriadothideaUESTCC22.0110
BotryosphaeriadothideaUESTCC22.0111
BotryosphaeriadothideaGZCC16-0013
BotryosphaeriadothideaGZCC16-0014
BotryosphaeriadothideaCBS110302
BotryosphaeriadothideaGZCC19-0186
BotryosphaeriadothideaCGMCC3.20094
BotryosphaeriadothideaGZCC19-0188
BotryosphaeriadothideaUESTCC22.0113
BotryosphaeriadothideaCMW25413
BotryosphaeriadothideaUESTCC22.0112
BotryosphaeriadothideaUESTCC22.0114
BotryosphaeriadothideaUESTCC22.0115
BotryosphaeriadothideaUESTCC22.0116
BotryosphaeriaqingyuanensisCGMCC3.18743
BotryosphaeriacorticisATCC22927
BotryosphaeriakuwatsukaiLSP5
BotryosphaeriakuwatsukaiCGMCC3.18007
BotryosphaeriakuwatsukaiCGMCC3.18008
BotryosphaeriafabicercianaMFLUCC11-0507
BotryosphaeriafabicercianaMFLUCC10-0098
BotryosphaeriafabicercianaUESTCC22.0117
BotryosphaeriafabicercianaUESTCC22.0118
BotryosphaeriafabicercianaCMW27108
BotryosphaeriaagavesMFLUCC10-0051
BarriopsisiranianaIRAN1448C
BarriopsisiranianaIRAN1449C
100/1
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100/1
100/0.99
100/1
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--/0.96
87/--
100/--
95/--
99/--
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100/1
79/0.99
98/--
80/1
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100/1
0.01
Outgroup
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
80
Figure 3. Phylogram generated from RAxML analysis based on combined ITS, tef1 and tub2 sequence
data of Diplodia isolates. e tree was rooted to Dothiorella dulcispinae (CMW 36460 and CMW 36462).
e ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from
this study are marked in red and ex-type strains are marked in bold.
Diplodiapistaciicola CGMCC 3.24156
Diplodia seriataUESTC22.0072
Diplodiaacerigena
DiplodiabulgaricaCBS124254
DiplodiarosulataCBS116470
DiplodiafraxiniCBS136010
DiplodiaolivarumCBS121887
DiplodiamalorumCBS124130
DiplodiaafricanaCBS120835
DiplodianeojuniperiCPC22753
DiplodiamutilaCBS112553
DiplodiasubglobosaCBS124133
DiplodiascrobiculataCBS118110
DiplodiacrataegicolaMFLU15-1311
DiplodiagaliicolaMFLU15-1310
DiplodiasapineaCBS393.84
DiplodiaseriataCBS112555
DiplodiaallocellulaCBS130408
DiplodiaeriobotryicolaCBS140851
DiplodiapseudoseriataCBS124906
DiplodiaarengaeMFLU17-2769
DiplodiaafrocarpiCBS131681
DiplodiacorticolaCBS112549
DiplodiaquercivoraCBS133852
DiplodiatsugaeCBS418.64
DiplodiacupressiCBS168.87
DiplodiabulgaricaCBS124135
DiplodiarosulataCBS116472
DiplodiafraxiniCBS136011
DiplodiaolivarumIMI390972
DiplodiamalorumBN 37
DiplodiaafricanaSTE U5946
DiplodianeojuniperiCPC22754
DiplodiasubglobosaCBS124132
Diplodiafici septicaeNCYUCC19-0007
Diplodiafici septicaeMFLUCC20-0037
DiplodiaagrifoliaCBS124.3
DiplodiaparvaKNU16-007
DiplodiasapineaCBS109726
DiplodiaalanphillipsiiIRAN1574C
DiplodiaalanphillipsiiIRAN1509C
DiplodiaalanphillipsiiIRAN1508C
DiplodiacitricarpaCBS124715
DiplodiaestuarinaCMW41230
DiplodiaestuarinaCMW41363
DiplodiaallocellulaCMW36470
DiplodiacorticolaCBS112546
DiplodiaquercivoraMEAN 1017
DiplodiagallaeCBS212.25
DiplodiagallaeCBS211.25
Diplodiasp.1CBS678.88
Diplodiasp.2UCD1275So
DiplodiacupressiCBS261.85
DothiorelladulcispinaeCMW36460
DothiorelladulcispinaeCMW36462
DiplodiamutilaUESTC22.0068
DiplodiamutilaUESTCC 22.0067
DiplodiamutilaUESTC22.0069
DiplodiamutilaUESTCC 22.0064
DiplodiamutilaUESTCC 22.0065
DiplodiamutilaUESTCC 22.0063
Diplodiapistaciicola UESTC 22.0071
Diplodiaacerigena UESTCC 22.0075
Diplodiaacerigena UESTCC 22.0074
100/1
100/1
100/1
100/1
100/1
100/1
96/1
100/1
94/1
86/1
96/1
100/1
100/1
95/1
95/1
94/1
-/1
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91/-
79/1
100/1
92/1
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86/1
98/1
94/1
75/1
100/1
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-/0.98
100/1
87/-
88/-
-/1 100/1
100/1
0.01
Outgroup
CGMCC 3.24157
Botryosphaerialean fungi associated with woody oil plants in China 81
Figure 4. Phylogram generated from RAxML analysis based on combined ITS, tef1 and tub2 sequence
data of Dothiorella isolates. e tree was rooted to Neofusicoccum luteum (CBS 562.92 and CMW 41365).
e ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from
this study are marked in red and ex-type strains are marked in bold.
Dothiorella camelliae
Dothiorella zanthoxyl
Dothiorella uruguayensis CBS 12490 8
Dothiorella striata DAR 80992
Dothiorella obovata MFLUCC 22−0058
Dothiorella brevicollis CMW 36463
Dothiorella heterophyllae CMW 46458
Dothiorella viticola WA10NO01
Dothiorella mangifericola CBS 121760
Dothiorella vineagemmae B116 3
Dothiorella dulcispinae CMW 25407
Dothiorella thailandica MFLUCC 11−0438
Dothiorella albiziae MFLUCC 22−0057
Dothiorella italica MFLUCC 17−0951
Dothiorella iberica CBS 115041
Dothiorella omnivora CBS 140349
Dothiorella vidmadera DAR 78992
Dothiorella parva CBS 124720
Dothiorella guttulata MFLUCC 17−0242
Dothiorella symphoricarposicola MFLUCC 13−0497
Dothiorella sarmentorum IMI 63581b
Dothiorella americana UCD 2272MO
Dothiorella rhamni CBS 140852
Dothiorella pretoriensis CMW 36480
Dothiorella moneti MUCC 505
Dothiorella santali MUCC 509
Dothiorella koae CMW 48017
Dothiorella acacicola CPC 26349
Neofusicoccum luteum CMW 41365
Dothiorella striata ICMP 16819
Dothiorella tectonae MFLUCC 12−0382
Dothiorella obovata MFLU 22−0094
Dothiorella lampangensis MFLUCC 18−0232
Dothiorella diospyricola CBS 145972
Dothiorella longicollis CBS 122068
Dothiorella viticola IRNBS 28
Dothiorella viticola MFLUCC 22−0059
Dothiorella viticola WA10NO02
Dothiorella acericola KUMCC 18−0137
Dothiorella citricola ICMP 16828
Dothiorella mangifericola IRAN 1584C
Dothiorella magnoliae CFCC 51563
Dothiorella alpina CGMCC 3.18001
Dothiorella yunnana CGMCC 3.18000
Dothiorella plurivora IRAN 1557C
Dothiorella rosacear MFLUCC 23-0038
Dothiorella dulcispinae CMW 36460
Dothiorella MFLUCC 23-0035
Dothiorella septata MFLUCC
23-0039
Dothiorella albiziae MFLU 22−0093
Dothiorella prunicola CAP 187
Dothiorella iberica CBS 113189
Dothiorella juglandis CBS 188.87
Dothiorella vidmadera CBS 621.74
Dothiorella vidmadera CBS 725.79
Dothiorella sempervirentis IRAN 1583C
Dothiorella sempervirentis IRAN 1581C
Dothiorella parva CBS 124721
Dothiorella symphoricarposicola MFLUCC 13−0498
Dothiorella sarmentorum CBS 115038
Dothiorella americana UCD 2252MO
Dothiorella thripsita BRIP 51876
Dothiorella casuarini CBS 120688
Dothiorella capriamiss CBS 121763
Dothiorella iranica IRAN 1587C
Neofusicoccum luteum CBS 562.92
Dothiorella camelliae UESTC 22.0078
Dothiorella camelliae
Dothiorella camelliae UESTC 22.0079
Dothiorella zanthoxyl
UESTC
22.0084
Dothiorella zanthoxyl
UESTC
22.0083
Dothiorella sarmentorum UESTC 22.0076
Dothiorella sarmentorum UESTC 22.0077
100/1
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93/1
87/0.98
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100/1
99/1
87/1
96/1
100/1
96/1
96/1
95/1
91/1
98/1
99/0.98
100/--
96/0.84
92/1
82/--
100/1
94/1
100/1
97/1
86/1
100/1
0.01
_
_
Dothiorella sarmentorum
UESTC22.0081
_
CGMCC 3.24159
CGMCC 3.24158
i
i
i
Outgroup
ovata
um
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82
Taxonomy
Aplosporella ginkgonis C.M. Tian, Z. Du & K.D. Hyde. Mycosphere 8(2):
1249 (2017).
MycoBank No: 552938
Fig. 5
Description. Saprobic on decaying branches of Zanthoxylum bungeanum. Sexual
morph: Not observed. Asexual morph: Coelomycetous, Conidiomata 558–
657 × 216–241 µm (x
– = 235.5 × 228.5 µm, n = 10), immersed, partially erumpent
when mature, multilocular, locules separated by pale brown cells of textura angula-
ris. Peridium 65–106 µm wide, wall 6–10 cell-layers thick, outer layers composed
of 3–4 layers of pale brown cells of textura globulosa, intermediate layers composed
of dark brown cells of textura angularis, becoming pale brown towards the inner
region. Ostiole 138–171 µm diam., central. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells 12–13 × 7.5–8 µm (x
– = 12.5 × 8 µm, n = 20), holoblas-
tic, hyaline, cylindrical to doliiform, smooth-walled. Conidia 17–20 × 6.5–7.5 µm
(x
– = 18.5 × 7 µm, n = 30), L/W ratio = 2.5, ellipsoidal to subcylindrical, with both
ends rounded, initially hyaline, becoming dark brown, aseptate.
Culture characteristics. Colonies on PDA developing dense aerial mycelium with
age, becoming white to gray-brown at the surface, and whitish to yellowish brown at
the reverse, producing a brown pigment, with sinuate edges.
Material examined. C, Sichuan Province, Yaan City, Hanyuan County,
29°16'51"N, 102°37'48"E, elevation 1,689 m, on dead branches of Zanthoxylum
bungeanum, 30th October 2021, W.L. Li, HJ 511 (HUEST 22.0092), living culture
UESTCC 22.0091.
Notes. Aplosporella ginkgonis was introduced by Du et al. (2017) and isolated
from diseased branches of Ginkgo biloba and Morus alba from Gansu Province in
China. One isolate (UESTCC 22.0091) obtained in this study from Zanthoxylum
bungeanum is morphologically similar to the original description of Aplosporella gink-
gonis, and the sequences data are identical to the previous data (99%–100%). We,
thus, identify the new collection as Aplosporella ginkgonis and this is the rst report
from Zanthoxylum bungeanum.
Aplosporella prunicola Damm & Crous Fungal Diversity 27: 39 (2007).
MycoBank No: 504373
Fig. 6
Description. Saprobic on decaying branches of Zanthoxylum bungeanum. Sexual
morph: Not observed. Asexual morph: Coelomycetous, Conidiomata 355–
408 × 568.5–599 µm (x
– = 381.5 × 584 µm, n = 10), immersed, partially erumpent
when mature, multilocular, locules divided by pale brown cells of textura angularis.
Botryosphaerialean fungi associated with woody oil plants in China 83
Peridium 107–122 µm wide, composed of 3–5 layers of pale brown cells of textura
globulosa. Ostiole 70–88 µm diam., central. Conidiophores reduced to conidiogenous
cells. Conidiogenous cells 6.5–10 × 2–3 µm (x
– = 8 × 2.5 µm, n = 20), holoblastic, hya-
Figure 5. Aplosporella ginkgonis (HUEST 22.0092, new host record) a–c appearance of conidiomata on
natural substrate d vertical section of conidioma e section of peridium f conidiogenous cells and develop-
ing conidia g–k brown aseptate conidia l upper view of the colony on PDA after 14 d m reverse view of
the colony on PDA after 14 d. Scale bars: 100 µm (d); 40 µm (e); 10 µm (f–k).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
84
line, cylindrical, smooth-walled. Conidia 20–23.5 × 12–13.5 µm (x
– = 21.5 × 13 µm,
n = 30), L/W ratio = 1.6, ellipsoidal to subcylindrical, with both ends broadly round-
ed, initially hyaline, becoming dark brown, aseptate, smooth.
Culture characteristics. Colonies on PDA after 7 d, becoming pale olivaceous-
gray to olivaceous-black at the surface, and olivaceous black at the reverse, with ir-
regular edges.
Figure 6. Aplosporella prunicola (HUEST 22.0091, new host record) a, b appearance of conidiomata on
natural substrate c vertical section of multiloculate conidioma d, e section of peridium f–i conidiogenous
cells and developing conidia j–n brown aseptate conidia. Scale bars: 100 µm (c); 20 µm (d, e ); 10 µm (f–n).
Botryosphaerialean fungi associated with woody oil plants in China 85
Material examined. C, Sichuan Province, Yaan City, Hanyuan County,
29°16'51"N, 102°37'48"E, elevation 1,689 m, on dead branches of Zanthoxylum
bungeanum, 30th October 2021, W.L Li, HJ 509 (HUEST 22.0091), living culture
UESTCC 22.0090.
Notes. Our isolate UESTCC 22.0090 morphologically lines up with the descrip-
tion of Aplosporella prunicola provided by Damm et al. (2007) in having immersed to
erumpent, multilocular conidiomata and brown, smoothwalled, ovoid to oblong co-
nidia. e strain UESTCC 22.0090 is phylogenetically and morphologically similar to
A. yalgorensis and A. prunicola, however, A. yalgorensis can be distinguished from other
Aplosporella species by its pitted conidial walls. us, the strain UESTCC 22.0090 was
identied as A. prunicola based on current evidence. is is the rst time A. prunicola
is reported from Zanthoxylum bungeanum in China.
Diplodia acerigena L.W. Li & Jian K. Liu, sp. nov.
MycoBank No: 847163
Figs 7, 8
Etymology. e epithet ‘‘acerigena’’ refers to the host genus Acer, on which the holo-
type was collected.
Holotype. HKAS 125891.
Description. Saprobic on decaying branches of Acer truncatum. Sexual morph:
Ascomata 304.5–321 × 217–260 (x
– = 313 × 238.5 µm, n = 20), more or less subglo-
bose, solitary or gregarious, semi-immersed, medium brown to dark brown, unilocular,
papillate, ostiolate. Ostiole 101–115 µm diam., conical or circular, central, papillate,
periphysate. Peridium 23–29 µm wide, composed of 3–5 layers of dark brown cells
of textura angularis. Pseudoparaphyses 3.5–5 µm wide, hyaline, branched, septate.
Asci 98–120 × 24–32.5 µm (x
– = 109 × 28 µm, n = 30), (4–)8-spored, clavate, stipi-
tate, irregularly bitunicate, apex rounded with an ocular chamber. Ascospores 24.5–
31.5 × 13.5–16 µm (x
– = 28 × 14.5 µm, n = 30), L/W ratio = 2, biseriate, broadly fusi-
form to oval, widest in the middle, both ends obtuse, hyaline, moderately thick-walled,
smooth, becoming brown and 2-septate when aged. Asexual morph: Coelomycetous,
pycnidia produced on mycelium in PDA. Conidiomata stromatic, mostly solitary,
gray to black, globose to subglobose. Paraphyses 2–3.5 µm wide, hyaline, subcylindri-
cal, branched, septate. Conidiophores absent. Conidiogenous cells 9–12 × 3.5–5 µm
(x
– = 10.5 × 4.5 µm, n = 20), holoblastic, hyaline, cylindrical. Conidia 21–24 × 10–
11 µm (x
– = 22.5 × 10.5 µm, n = 30), L/W ratio = 2, aseptate, thick-walled, wall exter-
nally smooth, roughened on the inner surface, initially hyaline becoming dark brown,
obovoid to ellipsoid, both ends broadly rounded. Spermatogenous cells 7–9.5 × 2.5–
3.5 µm (x
– = 8 × 3 µm, n = 20), discrete or integrated, hyaline, smooth, cylindri-
cal, holoblastic or proliferating via. determinate phialides with periclinal thickening.
Spermatia 7–11.5 × 3–4 µm (x
– = 9 × 3.5 µm, n = 30), hyaline, smooth, aseptate,
rod-shaped with rounded ends.
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
86
Figure 7. e sexual morph of Diplodia acerigena (HKAS 125891, holotype) a, b appearance of as-
comata on natural substrate c vertical section of ascoma d ostiole e section of peridium f–h asci with
hyaline ascospores i asci with brown 2-sepatate ascospores j, k hyaline immature aseptate ascospores
l–n mature brown 2-septate ascospores o germinated ascospore p upper view of the colony on PDA
after 14 d q reverse view of the colony on PDA after 14 d. Scale bars: 100 µm (c); 10 µm (d, e, j–o);
20 µm (f–i).
Botryosphaerialean fungi associated with woody oil plants in China 87
Culture characteristics. Ascospores germinating on PDA within 12 h. Colonies
growing on PDA, reaching a diam. of 4 cm after ve days at 25 °C, euse, velvety, with
entire to slightly undulate edge. Surface initially white and later turning dark oliva-
ceous from the surrounding of the colony and dark gray in reverse.
Material examined. C, Sichuan Province, Chengdu City, Pidu District,
30°19'57"N, 103°59'47"E, elevation 442 m, on dead branches of Acer truncatum
(Anacardiaceae), 19th March 2021, W.L Li, YBF 96 (HKAS 125891, holotype), ex-
Figure 8. e asexual morph of Diplodia acerigena (HKAS 125891, holotype) a–d appearance of conidi-
omata on PDA e–i conidiogenous cells and developing conidia j, k hyaline immature conidia l–o mature
brown aseptate conidia p–t Spermatogenous cells and Spermatia. Scale bars: 10 µm (e–o, t); 5 µm (p–s).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
88
type living culture UESTCC 22.0073 = CGMCC 3.24157; ibid., YBF103 (HUEST
22.0075, paratype), living culture UESTCC 22.0074. Additional sequences: LSU:
OQ164827 (CGMCC 3.24157), OQ164828 (UESTCC 22.0074).
Notes. ree isolates of Diplodia acerigena clustered closer to Di. pseudoseriata
(CBS 124906) with high bootstrap support (ML/BI 100%/1). e asexual morph
of Diplodia pseudoseriata was introduced by Pérez et al. (2010), collected and isolat-
ed from the Blepharocalyx salicifolius in Uruguay and its sexual morph has not been
reported. e asexual morph of Diplodia acerigena diers from Di. pseudoseriata in
having conidia which become 1-septate when aged. Diplodia acerigena shares similar
sexual morph characters as of other Diplodia species by having immersed to semi-
immersed pseudothecia, clavate asci, broadly fusiform to ovoid and hyaline ascospores.
However, conidia of Diplodia acerigena become brown and septate when aged, which
is rarely observed in any other sexual morph species of this genus.
Diplodia mutila (Fr.) Mont., Ann. Sci. nat., sér. 2, 1: 302. 1834.
MycoBank No: 201741
Fig. 9
Sphaeria mutila Fr., Syst. Mycol. (Lundae) 2: 424. 1823. Basionym.
≡ Physalospora mutila (Fr.) N.E. Stevens, Mycologia 28: 333. 1936.
= Botryosphaeria stevensii Shoemaker, Canad. J. Bot. 42: 1299. 1964.
Description. Saprobic on decaying branches of Camellia oleifera. Sexual morph: Not
observed. Asexual morph: Coelomycetous, Conidiomata 330–394 × 215–230 µm
(x
– = 362 × 223 µm, n = 10), immersed, erumpent, gregarious, dark brown to black,
subglobose, unilocular. Ostiole 48.5–67 µm diam., central. Peridium 29–38 µm wide,
thick-walled, outer and inner layers composed of 1–2 layers dark brown textura angu-
laris, intermediate layers composed of 3–5 layers of hyaline cells of textura angularis.
Conidiophores reduced to conidiogenous cells. Conidiogenous cells 8.5–12 × 3–5 µm
(x
– = 10 × 4 µm, n = 20), cylindrical, thin-walled, hyaline, holoblastic, indetermi-
nate, proliferating at the same level to produce periclinal thickenings, or proliferating
percurrently giving rise to 2–3 indistinct annellations. Conidia 19–21 × 9.5–11 µm
(x
– = 20 × 10.5 µm, n = 30), L/W ratio = 2, oblong, with broadly rounded apex and
truncate base, thick-walled, wall externally smooth, roughened on the inner surface,
hyaline, aseptate, becoming dark brown when aged.
Culture characteristics. Colonies on PDA initially olivaceous bu in the center
of the colony and white at the edge, becoming olivaceous within 7 d on the surface,
with smooth edge.
Materials examined. C, Sichuan Province, Jiangyou City, Shuanghe County,
31°54'10"N, 104°55'57"E, elevation 657 m, on dead branches of Camellia oleifera,
Botryosphaerialean fungi associated with woody oil plants in China 89
Figure 9. Diplodia mutila (HUEST 22.0069, new host record) a, b appearance of conidiomata on
natural substrate c vertical section of conidioma d ostiole e section of peridium f–i conidiogenous cells
and developing conidia j hyaline immature conidium k–n mature brown conidia. Scale bars: 40 µm (c);
20 µm (d, e); 10 µm (f–n).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
90
11th July 2021, W.L Li, 286 (HUEST 22.0069), living culture UESTCC 22.0068;
ibid., 289 (HUEST 22.0068), living culture UESTCC 22.0067; ibid., Guangyu-
an city, Qingchuan County, 32°40'38"N, 105°28'57"E, elevation 634 m, on dead
branches of Olea europaea, 20th April 2021, W.L Li, 188 (HUEST 22.0065), living
culture UESTCC 22.0064; ibid., 257 (HUEST 22.0070), living culture UESTCC
22.0069; ibid., on dead branches of Vernicia fordii, 20th April 2021, W.L Li, 238
(HUEST 22.0066), living culture UESTCC 22.0065; ibid., Chengdu City, Pidu
District, 30°49'27"N, 103°47'42"E, elevation 442 m, on dead branches of Pistacia
chinensis, 5th March 2021, W.L Li, A61 (HUEST 22.0064), living culture UESTCC
22.0063. Additional sequences: LSU: OQ164832 (UESTCC 22.0063), OQ164830
(UESTCC 22.0064), OQ164831 (UESTCC 22.0065).
Notes. e phylogenetic tree show that six strains isolated from Camellia oleifera,
Olea europaea and Vernicia fordii nested with Diplodia mutila (CBS 112553) with a
moderate bootstrap support (ML/BI 86%/1). Diplodia mutila, the type of the genus,
is a well-known and most commonly reported species. It has been recorded mainly
from woody substrates, and it is known from more than 50 hosts (Batista et al. 2021).
Morphologically, one of the isolates obtained in this study UESTCC 22.0068 shares
similar conidia shape and size with Di. mutila, but hardly observed the mature conidia
with septa. We identify these taxa as Di. mutila based on morphology and phylogeny
evidences. is is the rst report of Di. mutila, isolated from Camellia oleifera, Olea
europaea and Vernicia fordii.
Diplodia pistaciicola L.W. Li & Jian K. Liu, sp. nov.
MycoBank No: 847166
Fig. 10
Etymology. e epithet ‘‘pistaciicola’’ refers to the host genus Pistacia, on which the
holotype was collected.
Holotype. HKAS 125890.
Description. Saprobic on decaying branches of Pistacia chinensis. Sexual morph:
Not observed. Asexual morph: Coelomycetous, Conidiomata 353–441 × 274.5–
316 µm (x
– = 397 × 295 µm, n = 10), immersed, forming split-like opening on the
host, solitary or gregarious, globose to subglobose, dark brown to black, unilocular,
papillate, ostiolate. Ostiole 38–49.5 µm diam., conical or circular, centrically located.
Peridium 42–60 µm wide, composed of thick walled, dark brown to hyaline cells
of textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous
cells 10–14 × 3–4 µm (x
– = 12 × 3.5 µm, n = 20), holoblastic, discrete, cylindrical, hya-
line, smooth, indeterminate, arising from the inner cavity of the conidiomata. Conidia
24.5–27 × 11–13 µm (x
– = 25.5 × 12 µm, n = 30), L/W ratio = 2.2, ellipsoid to obo-
void, aseptate, hyaline, thick-walled, guttulate.
Culture characteristics. Conidia germinate on PDA within 12 h. Colonies grow-
ing on PDA, reaching a diameter of 4 cm after ve days at 25 °C, euse, velvety, with
Botryosphaerialean fungi associated with woody oil plants in China 91
entire to slightly undulate edge. e early stage of the colony is white, later turning
dark olivaceous and dark gray in reverse.
Material examined. C, Sichuan Province, Chengdu City, Pidu District,
30°49'27"N, 103°47'42"E, elevation 442 m, on dead branches of Pistacia chinensis
(Anacardiaceae), 5th March 2021, W.L Li, 049 (HKAS 125890, holotype), ex-type
Figure 10. Diplodia pistaciicola (HKAS 125890, holotype) a–c appearance of conidiomata on natural
substrate d , e vertical section of conidiomata/conidioma f ostiole g section of peridium h–l conidiog-
enous cells and developing conidia m–q hyaline aseptate conidia. Scale bars: 200 µm (d); 50 µm (e);
20 µm (f, g); 10 µm (h–q).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
92
living culture UESTCC 22.0070 = CGMCC 3.24156; ibid., 049B (HUEST 22.0072
isotype), ex-isotype living culture UESTCC 22.0071. Additional sequence: LSU:
OQ164833 (CGMCC 3.24156).
Notes. Phylogenetic analyses showed that two strains of Diplodia pistaciicola isolat-
ed from Pistacia chinensis are distinct but closely related to Di. agrifolia (CBS 124.30).
e comparison of ITS, tef1 and tub2 of these two species indicate 5 bp (502), 3bp
(224), 9 bp (425) dierances, respectively. Morphologically, Di. agrifolia diers from
Di. pistaciicola in producing two to three times larger ascomata than that of Di. pistaci-
icola (721–836 vs. 274.5–316 µm) and possessing smaller conidia (27–36.5 × 14.5–
17.8 µm vs. 24.5–27 × 11–13 µm). In addition, conidia of Di. pistaciicola are hya-
line, aseptate, rarely becoming pale brown and uniseptate with age, whereas conidia
of Di. agrifolia are mostly dark brown and uniseptate before discharge from pycnidia.
Diplodia seriata De Not., Micr. Ital. Dec. 4: 6. (1942).
MycoBank No: 180468
Fig. 11
Description. Saprobic on decaying branches of Camellia oleifera. Sexual morph:
Ascomata 301–343 × 293–340 (x
– = 322 × 316 µm, n = 10), more or less subglo-
bose, solitary or gregarious, semi-immersed, medium brown to dark brown, unilocular,
papillate, ostiolate. Ostiole 72–78 µm diam., conical or circular, central, papillate, pe-
riphysate. Peridium 33–44 µm wide, composed of dark brown, 4–6 layers of textura
angularis. Pseudoparaphyses 2–2.5 µm wide, hyaline, branched, septate. Asci 112–
141 × 27.5–30 µm (x
– = 126 × 28.5 µm, n = 30), clavate, stipitate, bitunicate, contain-
ing eight, biseriate ascospores. Ascospores 31.5–32.5 × 12–13.5 µm (x
– = 32 × 13 µm,
n = 30), L/W ratio = 2.5, broadly fusiform to oval, widest in the middle, both ends ob-
tuse, hyaline, moderately thick-walled, smooth, becoming brown when aged. Asexual
morph: Not observed.
Culture characteristics. Ascospores germinate on PDA within 12 h. Colonies
growing on PDA, reaching a diameter of 4 cm after ve days at 25 °C, euse, velvety,
with entire to slightly undulate edge.
Material examined. C, Sichuan Province, Jiangyou City, shuanghe County,
31°54'10"N, 104°55'57"E, elevation 656 m, on dead branches of Camellia oleifera,
10th June 2021, W.L Li, 288 (HUEST 22.0073), living culture UESTCC 22.0072.
Notes. e morphology of the taxa isolated from decaying woody oil plants is
similar to Diplodia seriata. In the multi-gene phylogenetic analysis, our new collection
clustered with the ex-type strain of Di. seriata (CBS 112555) with strong bootstrap
support. Diplodia seriata has been isolated from a wide range of hosts (121 species) and
has a worldwide distribution (reported in 46 countries) (Batista et al. 2021). is is the
rst report of Di. seriata isolated from Camellia oleifera.
Botryosphaerialean fungi associated with woody oil plants in China 93
Figure 11. Diplodia seriata (HUEST 22.0073, new host record) a–c appearance of ascomata on natu-
ral substrate d vertical section of ascomata e section of peridium f–i asci j–n ascospores. Scale bars:
100 µm (d); 10 µm (e, j–n); 20 µm (f–i).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
94
Dothiorella camelliae L.W. Li & Jian K. Liu, sp. nov.
MycoBank No: 847167
Fig. 12
Etymology. e epithet ‘‘camelliae’’ refers to the host genus Camellia, on which the
holotype was collected.
Holotype. HKAS 125892.
Description. Saprobic on decaying branches of Camellia oleifera. Sexual morph:
Ascomata 199–222 × 237–269 µm (x
– = 210.5 × 253 µm, n = 10), submerged in the
substrate, partly erumpent at maturity, solitary or gregarious, dark brown to black,
subglobose, multilocular or unilocular. Ostiole 17–37 µm diam., central. Peridium
35–43 µm wide, thick-walled, outer layers composed of 1–2 layers dark brown cells
of textura angularis, becoming hyaline towards the inner region. Pseudoparaphyses
3–4 µm wide, hyaline, frequently aseptate. Asci 80–96 × 22–25 µm (x
– = 88 × 23.5 µm,
n = 30), stipitate, clavate, thick-walled, bitunicate, (6–)8-spored, irregularly biseriate.
Ascospores 21–25 × 9.5–12 µm (x
– = 23 × 10.5 µm, n = 30), L/W ratio = 2, oblong,
ovate to sub-clavate, (0–)1-septate, slightly constricted at the septum, hyaline to dark
brown, moderately thick-walled, straight or inequilateral, basal cell tapering towards
the acute end. Asexual morph: Not observed.
Culture characteristics. Ascospores germinate on PDA within 12 h. Colonies
growing on PDA, reaching a diameter of 4 cm after ve days at 25 °C, euse, velvety,
with entire to slightly undulate edge. Surface initially white and later turning dark
olivaceous from the surrounding of the colony and dark gray in reverse.
Materials examined. C, Sichuan Province, Leshan City, Wutongqiao Dis-
trict, 29°22'28"N, 103°45'49"E, elevation 383 m, on dead branches of Camellia oleif-
era (eaceae), 23th July 2021, Z.P Liu, 351 (HKAS 125892, holotype), ex-type living
culture UESTCC 22.0081 = CGMCC 3.24158; ibid., 347 (HUEST 22.0081), living
culture UESTCC 22.0080; ibid., Shizhong District, 29°42'13"N, 103°52'25"E, eleva-
tion 356 m, on dead branches of Paeonia suruticosa, 23th July 2021, W.L Li, A240
(HUEST 22.0080), living culture UESTCC 22.0079; ibid., A234 (HUEST 22.0079),
living culture UESTCC 22.0078. Additional sequences: LSU: OQ164834 (CGMCC
3.24158), OQ164835 (UESTCC 22.0079), OQ164836 (UESTCC 22.0078).
Notes. Four strains isolated from Vernicia fordii and Camellia oleifera occupy a
basal position in the Dothiorella phylogenetic tree by forming a wellsupported sub-
clade sister to Do. zanthoxyli (ML/BI 97%/1, Fig. 4). e BLASTn searches of the ITS
sequence of Dothiorella zanthoxyli resulted in 97% matches with Neofusicoccum viti-
fusiforme BRIP64010, the tef1 showed 91.23% matches with Do. symphoricarposicola
BL158, and the tub2 BLASTn results indicated 96.53% similarity with Do. uruguay-
ensis CBS 124908 and Do. viticola B116-3. Dothiorella camelliae can be distinguished
from Do. zanthoxyli in the size of ascomata, ascus and L/W ratio of ascospores (Table 3).
Dothiorella camelliae resembles the sexual morph of Do. sarmentorum in producing im-
mersed to sub-immersed ascomata, clavate asci and ovate to sub-clavate, hyaline to
Botryosphaerialean fungi associated with woody oil plants in China 95
Figure 12. Dothiorella camelliae (HKAS 125892, holotype) a, b appearance of ascomata on natural sub-
strate c vertical section of ascoma d section of peridium e–g asci h–m ascospores n germinated ascospore.
Scale bars: 50 µm (c); 10 µm (d, h–n); 20 µm (e–g).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
96
brown conidia with (0–)1-septate. However, Do. sarmentorum morphologically can be
distinguished from Do. camelliae in having larger ascomata (350–400 µm vs. 237–
269 µm), thicker peridium (50–75 µm vs. 35–43 µm), and longer asci (140–210 µm vs.
80–96 µm) (Table 3). Phylogenetically, these two species reside in two distinct clades.
Dothiorella sarmentorum (Fr.) A.J.L. Phillips, J. Luque & A. Alves, Mycologia 97:
522. (2005).
MycoBank No: 501403
Fig. 13
Sphaeria sarmentorum Fr., K. svenska Vetensk-Acad. Handl. 39: 107. 1818. Basionym.
≡ Diplodia sarmentorum (Fr.) Fr., Summ. veg. Scand. (Stockholm) 2: 417. 1849.
= Diplodia pruni Fuckel, Jahrb. Nassauischen Vereins Naturk., 23–24: 169. 1870 [1869].
= Botryosphaeria sarmentorum A.J.L. Phillips, J. Luque & A. Alves, Mycologia 97:
522. 2005.
Description. Saprobic on decaying branches of Pistacia chinensis. Sexual morph: Not
observed. Asexual morph: Conidiomata 278–338 × 240–280 µm (x
– = 308 × 260 µm,
n = 10), immersed, erumpent, forming split-like opening on the host, gregarious, glo-
bose to subglobose, dark brown to black, unilocular or multilocular, papillate, ostio-
late. Ostiole 52–57 µm diam., conical or circular, centrically located. Peridium 28.5–
44 µm, comprising 5–8 layers of thickwalled, dark brown to hyaline cells arranged in
a textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous
cells 2.5–3.5 × 6–9 µm (x
– = 3 × 7.5 µm, n = 20), holoblastic, discrete, cylindrical,
hyaline, smooth, indeterminate, proliferating at the same level giving rise to periclinal
thickenings, or rarely proliferating percurrently to form one or two close, indistinct an-
nellations. Conidia 21.5–24 × 9–10 µm (x
– = 22.5 × 9.5 µm, n = 30), L/W ratio = 2.4,
ellipsoid to obovoid, with rounded ends, initially hyaline and aseptate becoming pig-
mented brown and 1-septate often while still attached to conidiogenous cell, brown
walled, slightly constricted at the septum.
Culture characteristics. Conidia germinate on PDA within 12 h. Colonies grow-
ing on PDA, reaching a diameter of 4 cm after three days at 25 °C, euse, velvety, with
Table 3. A morphological comparison of the sexual morph of three Dothiorella species.
Taxa Ascomata (μm) Asci (μm) Peridium (μm) Ascospores
Size(μm) Color L/W ratio
Dothiorella camelliae 199–222 × 237–269 80–96 × 22–25 35–43 21–25 × 9.5–12 Hyaline to
dark brown
2
Dothiorella sarmentorum 350–400 140–210 × 17–24 50–75 24.5–25.5 × 11.5–12.5 Dark brown 2.4
Dothiorella zanthoxyli 258–280 × 170–174 63.5–77 × 20–24.5 35–40 22.5–25 × 9.5–11 Hyaline to
dark brown
2.6
Botryosphaerialean fungi associated with woody oil plants in China 97
Figure 13. Dothiorella sarmentorum (HUEST 22.0077, new host record) a, b appearance of conidiomata
on natural substrate c vertical section of conidioma d–g conidiogenous cells and developing conidia
h–lbrown conidia. Scale bars: 50 µm (c); 10 µm (d–l).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
98
entire to slightly undulate edge. Surface initially white and later turning dark oliva-
ceous from the surrounding of the colony and dark gray in reverse.
Materials examined. C, Sichuan Province, Chengdu City, Pidu District,
30°19'57"N, 103°59'47"E, elevation 442 m, on dead branches of Pistacia chinensis,
19th March 2021, W.L Li, 072 (HUEST 22.0077), living culture UESTCC 22.0076;
ibid., Guangyuan City, Qingchuan County, 32°40'38"N, 105°28'57"E, elevation
638 m, 20th April 2021, W.L Li, A189 (HUEST 22.0078), living culture UESTCC
22.0077. Additional sequences: LSU: OQ164837 (UESTCC 22.0076), OQ164838
(UESTCC 22.0077).
Notes. Dothiorella sarmentorum was introduced by Phillips et al (2005) with
both asexual and sexual morphs. Recently, nine Dothiorella species (Do. californica,
Do. iberica, Do. italica, Do. guttulata, Do. omnivora, Do. parva, Do. sempervirentis,
Do. symphoricarpicola, Do. vidmadera) were synonymized under Do. Sarmentorum
by Zhang et al. (2021) based on phylogenetic analyses. Two isolates obtained in the
present study clustered with the group of Do. sarmentorum taxa in the phylogenetic
analyses (Fig. 4).
Dothiorella zanthoxyli L.W. Li & Jian K. Liu, sp. nov.
MycoBank No: 847168
Fig. 14
Etymology. e epithet ‘‘zanthoxyli’’ refers to the host genus Zanthoxylum, on which
the holotype was collected.
Holotype. HKAS 125893.
Description. Saprobic on decaying branches of Zanthoxylum bungeanum. Sexual
morph: Ascomata 258–280 × 170–174 µm (x
– = 269 × 172 µm, n = 10), submerged in
the substrate, partly erumpent at maturity, solitary or gregarious, dark brown to black,
subglobose, unilocular. Ostiole 42–44 µm diam., central. Peridium 35–40 µm wide,
thick-walled, outer layers composed of 3–5 layers dark brown cells of textura angu-
laris, becoming hyaline towards the inner region. Pseudoparaphyses 3–4.5 µm wide,
hyaline, frequently aseptate. Asci 63.5–77 × 20–24.5 µm (x
– = 70 × 22.5 µm, n = 30),
short stipe, clavate, thick-walled, bitunicate, 8-spored, irregularly biseriate. Ascospores
22.5–25 × 9.5–11 µm (x
– = 24 × 10 µm, n = 30), L/W ratio = 2.6, oblong, ovate to
sub-clavate, (0–)1-septate, slightly constricted at the septum, hyaline to dark brown,
moderately thick-walled, straight or inequilateral, basal cell tapering towards the acute
end. Asexual morph: Not observed.
Culture characteristics. Ascospores germinate on PDA within 12 h. Colonies
growing on PDA, reaching a diameter of 4 cm after ve days at 25 °C, euse, velvety,
with entire to slightly undulate edge. Surface initially white and later turning dark
olivaceous from the surrounding of the colony. Dark gray in reverse.
Botryosphaerialean fungi associated with woody oil plants in China 99
Figure 14. Dothiorella zanthoxyli (HKAS 125893, holotype) a, b appearance of ascomata on natural
substrate c vertical section of ascoma d ostiole e section of peridium f, k–n asci g–j brown ascospores.
Scale bars: 50 µm (c); 20 µm (d–f); 10 µm (g–n).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
100
Materials examined. C, Sichuan Province, Yanan City, Hanyuan County,
29°16'51"N, 102°37'48"E, elevation 1,689 m, on dead branches of Zanthoxylum
bungeanum (Rutaceae), 30th October 2021, W.L Li, 504 (HKAS 125893, holotype),
ex-type living culture UESTCC 22.0082 = CGMCC 3.24159; ibid., 506 (HUEST
22.0084), living culture UESTCC 22.0083; ibid., 507 (HUEST 22.0085), living cul-
ture UESTCC 22.0084. Additional sequences: LSU: OQ164839 (CGMCC 3.24159),
OQ164840 (UESTCC 22.0083), OQ164841 (UESTCC 22.0084).
Notes. ree strains of Dothiorella zanthoxyli isolated from Zanthoxylum bungeanum
correspond well with sexual morph of Dothiorella described by Phillips et al. (2013),
but morphologically dier from other species (Do. camelliae, Do. iberica and Do. sar-
mentorum) in the size of ascomata and asci (Table 3). A comparison of ITS and tef1
nucleotides shows that Do. zanthoxyli is signicantly dierent from its sister species,
Do. camelliae by 4/550 bp (0.72%) in ITS and 14/242 bp (5.8%) in tef1. In the phy-
logenetic analysis, these two species formed two distinct clades in Dothiorella (Fig. 4).
Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips,
Stud. Mycol. 55: 248. (2006).
MycoBank No: 500879
Fig. 15
Fusicoccum parvum Pennycook & Samuels, Mycotaxon 24: 455. 1985. Basionym.
= Botryosphaeria parva Pennycook & Samuels, Mycotaxon 24: 455. 1985.
Description. Saprobic on decaying branches of Idesia polycarpa. Sexual morph:
Ascomata 284–321 × 129–223 µm (x
– = 302.5 × 176 µm, n = 10), pseudothecial,
forming a botryose aggregation of up to 30, solitary or gregarious, stromatic, immersed,
partially erumpent when mature, dark brown to black, more or less circular, multi-
loculate, individual locules 143.5–161 µm diam, thick-walled. Peridium 59–78 µm
diam., composed of several layers of thick-walled, pale brown cells of textura angula-
ris. Ostiole 43.5–58 µm wide, circular, central, papillate. Asci 95–99 × 20–21.5 µm
(x
– = 97 × 20.5 µm, n = 30), (6–)8spored, bitunicate, ssitunicate, cylindrical to
clavate, apex rounded with an ocular chamber, sometimes short pedicellate. Ascospores
18.5–23 × 7–10.5 µm (x
– = 20.5 × 9 µm, n = 30), L/W ratio = 3, fusoid to ovoid, with
tapered ends and appearing spindle-shape, hyaline, aseptate, externally smooth, inter-
nally nely verruculose, biseriate in ascus. Asexual morph: Not observed.
Culture characteristics. Ascospores germinate on PDA within 12 h. Colonies
growing on PDA, reaching a diam., of 7 cm after ve days at 25 °C, euse, velvety,
with entire to slightly undulate edge. Surface initially white and later turning dark
olivaceous from the surrounding of the colony and dark gray in reverse.
Materials examined. C, Sichuan Province, Leshan City, Jingyan County,
29°30'27"N, 103°57'14"E, elevation 682 m, on dead branches of Idesia polycarpa, 23th
Botryosphaerialean fungi associated with woody oil plants in China 101
Figure 15. Neofusicoccum parvum (HUEST 22.0097, new host record) a, b appearance of ascomata on
natural substrate c, d vertical section of ascomata e ostiole f section of peridium g–j ascospores k , l im-
mature asci m, n mature asci. Scale bars: 100 µm (d); 25 µm (e , f ); 10 µm (g–j); 20 µm (k–n).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
102
July 2021, W.L Li, STZ 327 (HUEST 22.0095), living culture UESTCC 22.0094;
ibid., STZ 359 (HUEST 22.0094), living culture UESTCC 22.0093; ibid., Leshan
City, Shizhong Distinct, 29°42'13"N, 103°52'25"E, elevation 356 m, on dead branch-
es of Paeonia suruticosa, 23th July 2021, W.L Li, YMD 366 (HUEST 22.0096), living
culture UESTCC 22.0095; ibid., Guangyuan City, Qingchuan County, 32°40'38"N,
105°28'57"E, elevation 638 m, on dead branches of Vernicia fordii, 20th April 2021,
W.L. Li, YT 175 (HUEST 22.0097), living culture UESTCC 22.0096.
Notes. e morphology of our collections obtained from decaying woody oil
plants are similar to the original description of Neofusicoccum parvum (Crous et al.
2006). In the multi-gene phylogenetic analysis, these four isolates clustered together
(ML/BI 75%/0.99) with the ex-type of N. parvum. Neofusicoccum parvum has a wide
range of hosts and has a worldwide distribution (Phillips et al. 2013). is is the rst
report of N. parvum on Idesia polycarpa.
Sardiniella guizhouensis Y.Y. Chen & Jian K. Liu. Phytotaxa 508 (2): 190. (2021).
MycoBank No: 558352
Fig. 16
Description. Saprobic on decaying branches of Pistacia chinensis. Sexual morph: Not
observed. Asexual morph: Conidiomata 223–232 × 150–176 µm (x
– = 227.5 × 163 µm,
n = 10), dark brown to black, globose, submerged in the substrate, partially erumpent
at maturity, ostiolate. Ostiole 28.5–45 µm diam., circular, central. Peridium 21–30 µm
thick, composed of dark brown thick-walled cells of textura angularis, becoming
thin-walled and hyaline towards the inner region. Conidiophores reduced to conid-
iogenous cells. Conidiogenous cells 6–9.5 × 3.5–5 µm (x
– = 7.5 × 4 µm, n = 20), hya-
line, short obpyriform to subcylindrical, holoblastic, indeterminate. Conidia 20.5–
24 × 11.5–14 µm (x
– = 22 × 13 µm, n = 30), L/W ratio = 1.6, ellipsoid to ovoid with
both ends rounded, hyaline, aseptate, externally smooth, internally nely verruculose.
Culture characteristics. Conidia germinate on PDA within 12 h. Colonies grow-
ing on PDA, reaching a diameter of 7 cm after ve days at 25 °C, euse, velvety, with
entire to slightly undulate edge. Surface initially white and later turning dark oliva-
ceous from the surrounding of the colony and dark gray in reverse.
Material examined. C, Sichuan Province, Chengdu City, Pidu District,
29°16'50.70"N, 102°37'47.53"E, elevation 442 m, on dead branches of Pistacia chin-
ensis, 19th March 2021, W.L Li, 047 (HUEST 22.0101), living culture UESTCC
22.0100; ibid., 070 (HUEST 22.0102), living culture UESTCC 22.0101; ibid.,
071 (HUEST 22.0100), living culture UESTCC 22.0099; ibid., 150 (HUEST
22.0098), living culture UESTCC 22.0097; ibid., 151 (HUEST 22.0099), living
culture UESTCC 22.0098; ibid., A39 (HUEST 22.0103), living culture UESTCC
22.0102; ibid., A40 (HUEST 22.0104), living culture UESTCC 22.0103. Additional
Botryosphaerialean fungi associated with woody oil plants in China 103
sequences: LSU: OQ164842 (UESTCC 22.0100), OQ164843 (UESTCC 22.0101),
OQ164844 (UESTCC 22.0099), OQ164845 (UESTCC 22.0097), OQ164846
(UESTCC 22.0098), OQ164847 (UESTCC 22.0102).
Notes. Seven isolates of our collection are morphologically similar to the original
description of Sardiniella guizhouensis (Chen et al. 2021). e multi-gene phylogenetic
Figure 16. Sardiniella guizhouensis (HUEST 22.0100, new host record) a–c appearance of conidiomata
on natural substrate d vertical section of conidioma e section of peridium f ostiole g–j conidiogenous cells
and developing conidia k–n conidia. Scale bars: 40 µm (d); 20 µm (e, f ); 10 µm (g–n).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
104
analysis showed that the newly obtained isolates clustered together with ex-type of
Sa. guizhouensis (CGMCC 3.19222) and this is the rst report of Sa. guizhouensis from
Pistacia chinensis.
Sphaeropsis citrigena (A.J.L. Phillips, P.R. Johnst. & Pennycook) A.J.L. Phillips &
A. Alves. Stud. Mycol. 76, 157. (2013).
MycoBank No: 805463
Fig. 17
Description. Saprobic on decaying branches of Camellia oleifera. Sexual morph:
Ascomata 219–252 × 216–241 µm (x
– = 235.5 × 228.5 µm, n = 10), brown to black,
solitary or aggregated, immersed, becoming erumpent, ostiolate. Ostiole 71–92 µm,
central, relatively broad. Peridium 37.5–45 µm diam., composed of several layers of
dark brown cells of textura angularis. Pseudoparaphyses 1.5–2 µm wide, hyaline,
smooth, septate. Asci 93.5–107 × 28.5–33 µm (x
– = 100 × 30.5 µm, n = 30), bituni-
cate, 8-spored, stipitate, thick-walled, with well-developed apical chamber. Ascospores
29–35 × 13–15 µm (x
– = 32 × 14 µm, n = 30), L/W ratio = 2.3, yellowish brown to
dark brown, ellipsoid to ovoid with both ends rounded, with an apiculus at either end,
aseptate, externally smooth, internally nely verruculose, widest in middle to upper
third. Asexual morph: Not observed.
Culture characteristics. Ascospores germinate on PDA within 12 h. Colonies
growing on PDA, reaching a diam. of 7 cm after ve days at 25 °C, euse, velvety,
with entire to slightly undulate edge. Surface initially white and later turning dark
olivaceous from the surrounding of the colony and dark gray in reverse.
Materials examined. C, Sichuan Province, Chengdu City, Pidu District,
31°54'10"N, 104°55'57"E, 656 m, on dead branches of Camellia oleifera, 10th June
2021, W.L Li, 285 (HUEST 22.0107), living culture UESTCC 22.0106; ibid., on
dead branches of Acer truncatum, 30°19'57"N, 103°59'47"E, elevation 442 m, 19th
March 2021, W.L Li, A33 (HUEST 22.0106), living culture UESTCC 22.0105.
Additional sequence: LSU: OQ164848 (UESTCC 22.0105).
Notes. e phylogenetic tree shows that two isolates of Sphaeropsis from our col-
lection clustered together with the ex-type strain of Sp. citrigena (ICMP 16812) with
high bootstrap support (ML/BI 100%/1). Sphaeropsis citrigena was rst described as
Phaeobotryosphaeria citrigena by Phillips et al. (2008), later transferred to Sphaeropsis
based on morphological and phylogenetic analyses (Phillips et al. 2013). e new
collection (UESTCC 22.0105) isolated from Camellia oleifera resembles Sp. citrigena
isolated from Citrus sinensis (Phillips et al. 2013) in the shape of asci and ascospores,
though their asci are somewhat smaller than those of Sp. citrigena (93.5–107 × 28.5–
33 µm vs. 180–230 × 35–43 µm). In addition, there are no base pair dierences in
ITS and tef1 sequences of these two strains. We, thus, identify the new collection as
Sp. citrigena and this is the rst record of Sp. citrigena from Camellia oleifera.
Botryosphaerialean fungi associated with woody oil plants in China 105
Figure 17. Sphaeropsis citrigena (HUEST 22.0107, new host record) a, b appearance of ascomata on
natural substrate c vertical section of ascomata d section of peridium e–h mature asci i–m dark brown
ascospores. Scale bars: 100 µm (c); 20 µm (d–h); 10 µm (i–m).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
106
Sphaeropsis guizhouensis Y.Y. Chen, A. J. Dissanayake & Jian K. Liu., J. Fungi 7,
893. (2021).
MycoBank No: 558475
Fig. 18
Description. Saprobic on decayed branched of Camellia oleifera. Sexual morph:
Ascostromata 166–198 × 146.5–175 µm (x
– = 182 × 160.5 µm, n = 20), initially
immersed under host epidermis, becoming semiimmersed to erumpent, solitary or
gregarious, uniloculate, black, globose to subglobose, membraneous, ostiolate. Ostiole
75–80 µm wide, central, papillate, pale brown, relatively broad, periphysate. Peridium
23–27 µm wide, comprising 3–5 layers of relatively thickwalled, dark brown to black
walled cells arranged in a textura angularis. Pseudoparaphyses 2–2.5 µm diam., hy-
phaelike, numerous, embedded in a gelatinous matrix. Asci 87.5–135 × 28.5–35 µm
(x
– = 111 × 32 µm, n = 20), 8spored, bitunicate, ssitunicate, cylindrical to clavate,
sometimes short pedicellate, mostly long pedicellate, apex rounded with an ocular
chamber. Ascospores 28.5–33 × 13–15 µm (x
– = 30.5 × 14 µm, n = 20), overlapping un-
iseriate to biseriate, ellipsoidal to obovoid, pale brown to dark brown, septate, slightly
wide at the center, minutely guttulate, smoothwalled. Asexual morph: Not observed.
Culture characteristics. Ascopores germinate on PDA within 12 h. Colonies
growing on PDA, reaching a diam. of 7 cm after ve days at 25 °C, euse, velvety,
with entire to slightly undulate edge. Surface initially white and later turning dark
olivaceous from the surrounding of the colony and dark gray in reverse.
Material examined. C, Sichuan Province, Chengdu City, Pidu District, on
dead branches of Pistacia chinensis, 30°19'57"N, 103°59'47"E, elevation 442 m, 24th
March 2021, W.L Li, 290 (HUEST 22.0105), living culture UESTCC 22.0104.
Notes. Sphaeropsis guizhouensis was introduced by Dissanayake et al. (2021) and
isolated from an unknown host. One isolate obtained in the present study clustered
with the ex-type isolate of Sp. guizhouensis (CGMCC 3.20352) in the phylogenetic
analyses of combined ITS and tef1 sequence data with high bootstrap support. A com-
parison of ITS and tef1 shows that there are no base pair dierences between the iso-
lates of UESTCC 22.0104 and CGMCC 3.20352. e new collection is morphologi-
cally similar to Sp. guizhouensis, with immersed to erumpent, black ascostromata and
biseriate, aseptate, ellipsoid to obovoid, thickwalled conidia. In addition, ascospores
become brown and septate when aged. Considering similar morphology and strong
molecular evidence, we identify UESTCC 22.0104 as Sp. guizhouensis and this is the
rst record of Sp. guizhouensis on Camellia oleifera.
Diversity of Botryosphaerialean fungi collected in this study
Based on the phylogenetic and morphological analyses, 50 Botryosphaeriales iso-
lates collected from the ve regions (Chengdu, Guangyuan, Leshan, Mianyang and
Yaan City) in Sichuan Province were identied as 16 species. Of these, Botryosphaeria
dothidea was the most prevalent species (20%), followed by Sphaeropsis guizhouensis
Botryosphaerialean fungi associated with woody oil plants in China 107
(14%) and Diplodia mutila (12%) (Fig. 19a). Aplosporella ginkgonis, Barriopsis tectonae
and Sphaeropsis guizhouensis were identied only once. ere are 14 isolates (28%)
isolated from Pistacia chinensis, including Di. acerigena, Di. mutila, Di. pistaciicola,
Figure 18. Sphaeropsis guizhouensis (HUEST 22.0105, new host record) a, b appearance of ascomata on
natural substrate c vertical section of ascoma d section of peridium e–h mature asci i–l brown ascospores.
Scale bars: 20 µm (c–h); 5 µm (i–l).
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
108
Dothiorella sarmentorum and Sardiniella guizhouensis. Ten isolates were from Camel-
lia oleifera (20%), including Bo. dothidea, Bo. fabicerciana, Di. mutila, Do. camelli-
ae, Sp. citrigena and Sp. guizhouensis. Nine isolates were from Olea europaea (18%),
Figure 19. Botryosphaeriales species composition a the proportion of each species to the total number of
isolates b the number of Botryosphaeriales fungi on each host and host distribution of species.
Botryosphaerialean fungi associated with woody oil plants in China 109
including Ba. tectonae, Bo. dothidea, Bo. fabicerciana, Di. mutila and Do. sarmentorum.
Relatively few strains were found on Idesia polycarpa, Paeonia suruticosa and Vernicia
fordii, as each host presents two species, respectively. As of nal conclusion, Bo. doth-
idea were isolated from ve hosts, Di. mutila were isolated from four hosts, N. parvum
were isolated from three hosts, Bo. fabicerciana, Di. acericola, Do. camelliae, Do. sar-
mentorum and Sp. citrigena were isolated from two hosts, but several fungal isolates
were only isolated from one host species, such as A. prunicola, Sa. guizhouensis and
Sp. guizhouensis (Fig. 19b).
Discussion
In this study, 48 Botryosphaeriaceae isolates and two Aplosporellaceae isolates were
obtained from woody oil plants in Sichuan Province, China, and they were identied
as 16 species based on morphological characters and multi-gene phylogenetic anal-
yses. ese species included Aplosporella prunicola, A. ginkgonis, Barriopsis tectonae,
Botryosphaeria dothidea, Bo. fabicerciana, Diplodia acerigena, Di. mutila, Di. pistacii-
cola, Di. seriata, Dothiorella camelliae, Do. sarmentorum, Do. zanthoxyli, Neofusicoccum
parvum, Sardiniella guizhouensis, Sphaeropsis citrigena and Sp. guizhouensis. Of these,
Di. acerigena, Di. pistaciicola, Do. camelliae and Do. zanthoxyli are introduced as novel
species. Descriptions, illustrations and notes were provided for 13 species, and only
sequences data were provided for the remaining three species viz. Barriopsis tectonae,
Botryosphaeria dothidea and Bo. fabicerciana due to low specimen quality.
According to previous studies, Barriopsis tectonae, Sardiniella guizhouensis,
Sphaeropsis citrigena and Sp. guizhouensis have limited geographical distribution. So far,
Barriopsis tectonae has been reported from China, ailand and South Africa (Doilom
et al. 2014; Dissanayake et al. 2021). Sardiniella guizhouensis and Sphaeropsis guizho-
uensis were only found in China while Sp. citrigena was isolated from China, Colombia
and New Zealand. It’s worth noting that most of the species obtained from this study
were also reported previously from Guizhou province (Dissanayake et al. 2021). Earlier
studies have shown that the distribution of Botryosphaeriaceae species is inuenced by
the climate condition (Úrbez-Torres et al. 2006; Pitt et al. 2010; Li et al. 2020; Vivas
et al. 2021). us, we speculate that the adjacent geographical location and similar cli-
matic conditions may be important reasons for the similarity of fungal species isolated
from the Sichuan and Guizhou provinces.
e remaining Botryosphaeriaceae species identied in this study are all well-
known and reported from various geographic regions. Botryosphaeria dothidea, Di. se-
riata and Ne. parvum are recognized to be globally distributed while Di. mutila and
Do. sarmentorum are founded only in the temperate and Mediterranean areas. In addi-
tion, these species have a broad host range. Batista et al. (2021) reported Neofusicoccum
parvum from 223 hosts, B. dothidea from 403 hosts and Di. seriata from 121 hosts.
Diplodia mutila and Di. seriata have previously been reported on Olea europaea in Uru-
guay (Hernández-Rodríguez et al. 2022). Botryosphaeria dothidea was recently isolated
from diseased Camellia oleifera in China (Hao et al. 2022). In this study, Bo. dothidea,
Wen-Li Li et al. / MycoKeys 97: 71–116 (2023)
110
Di. mutila and Ne. parvum occurred on most of the woody oil plants species we exam-
ined. However, some common genera e. g. Lasiodiplodia, Neodeightonia and Phaeobot-
ryon have never been collected from this group of hosts (Fig. 19). e absence of these
genera from there is likely a sampling eect.
Aplosporella (Aplosporellaceae) was introduced by Spegazzini (1880) with A. chlo-
rostroma as the genetic type. In a previous study, Aplosporella represents anamorph
lineage within the Botryosphaeriaceae. Slippers et al. (2013) later proposed the family
Aplosporellaceae to accommodate Aplosporella and Bagnisiella. Aplosporella species are
infrequently isolated in China. Aplosporella ginkgonis, isolated from Gansu Province,
was rst descripted by Du et al. (2017) while Aplosporella macropycnidia was reported in
Yunnan Province. Subsequently, Jiang et al. (2021) isolated a new collection of A. pru-
nicola. However, other species have not been recorded in China. Our study revealed
new host records of A. ginkgonis and A. prunicola. ough the phylogenetic analyses
indicated that A. yalgorensis and A. prunicola have a low genetic divergence (Taylor et
al. 2008, in this study), A. yalgorensis is still considered as a dierent species as it dif-
fers from other Aplosporella species (including A. prunicola) by its pitted conidial walls.
ough there are more than 1,000 Diplodia epithets listed in Index fungorum
(www. Index Fungorum. Accessed in November 2022), presently only 30 species are
accepted in this genus based on phylogenetic analyses (Slippers et al. 2017; Wu et al.
2021). Holomorphic species in Diplodia are Di. tsugae, Di. seriata, Di. mutila and
Di. sapinea. is study revealed two previously known Diplodia species, Di. mutila
and Di. seriata, and two new species, Di. acerigena and Di. pistaciicola. Among them,
Di. acerigena is a holomorphic species, as its sexual stage was observed on the dead
branches of Acer truncatum, and the asexual stage produced on culture (PDA). How-
ever, the sexual morph of Di. mutila and Di. pistaciicola, as well as the asexual morph
of Di. seriata have not been observed on woody oil plants.
Dothiorella was established by Saccardo with Do. pyrenophora as the type species (Sac-
cardo 1880). Recently, Dothiorella encountered a series of revisions as many species in
this genus have been reduced to synonymy, such as Do. americana, Do. eriobotryae and
Do. iberica (Dissanayake et al. 2021; Zhang et al. 2021). So far, 31 species are valid in
Dothiorella. Most of the species were reported as the asexual morph of Dothiorella and the
sexual stage is rarely founded on nature (Dissanayake et al. 2016). Phillips et al. (2013)
initiated a link of asexual-sexual morph for Do. sarmentorum, Do. iberica and Do. vidmad-
era. However, the latter two species were synonymized under Do. sarmentorum (Zhang et
al. 2021). In this study, two new species Do. camelliae and Do. zanthoxyli are introduced
based on their sexual morphs as well as strong molecular evidences. Besides, new collec-
tions of Do. sarmentorum is reported on Pistacia chinensis for the rst time.
Multiple molecular systematic studies, mainly of pathogenic fungi of woody plants
(Phillips et al. 2013; Slippers et al. 2013; Dissanayake et al. 2021; Zhang et al. 2021),
have generated a robust phylogeny for Botryosphaeriaceae. However, the classication
and identication of some species in this family remains a major challenge, due to the
reasons 1) With the increase of the number of Botryosphaeriaceae species, morpho-
logical feature of inter-genera and inter-species is vague, 2) Some species occurred as
asexual morph on nature and it is dicult to establish the link of asexual and sexual
Botryosphaerialean fungi associated with woody oil plants in China 111
morph, 3) In general, Botryosphaeriaceae species do not show an obvious host speciali-
zation, while some populations displayed a certain degree of host association. us,
the traditional host-based classication system made taxonomic position confusion of
some species. erefore, collection of more fresh specimens is very important for better
understanding the life cycle of Botryosphaeriaceae species, their host range (e. g. native
plants) and potential pathogenicity.
Acknowledgements
Wen-Li Li thanks Tian Zhang for her help with sample collections and Yan-Peng Chen
for the fungal diversity analysis.
is study was supported by the Joint Fund of the National Natural Science Foun-
dation of China and the Karst Science Research Center of Guizhou province (Grant
No. U1812401).
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Supplementary material 1
Taxa and GenBank accession numbers of sequences used in this study
Authors: Wen-Li Li, Rui-Ru Liang, Asha Dissanayake, Jian-Kui Liu
Data type: table (excel document)
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/mycokeys.97.103118.suppl1
Available via license: CC BY 4.0
Content may be subject to copyright.
