Phytotaxa 474 (3): 218–234
https://www.mapress.com/j/pt/
Copyright © 2020 Magnolia Press
ISSN 1179-3155 (print edition)
Article
PHYTOTAXA
ISSN 1179-3163 (online edition)
https://doi.org/10.11646/phytotaxa.474.3.2
Pseudocercospora dypsidis sp. nov. (Mycosphaerellaceae) on Dypsis lutescens leaves
in Thailand
YI-JYUN CHEN1,2,4, RUVISHIKA S. JAYAWARDENA1,2,5, CHITRABHANU S. BHUNJUN1,2,6, DULANJALEE L.
HARISHCHANDRA1,2,3,7 & KEVIN D. HYDE1,2,8*
1
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, People’s Republic of China
4
yui2134000@gmail.com; https://orcid.org/0000-0001-9954-0207
5
ruvi.jaya@yahoo.com; https://orcid.org/0000-0001-7702-4885
6
avnishbhunjun@gmail.com; https://orcid.org/0000-0001-8098-3390
7
dulanjalee.harishchandra@gmail.com; https://orcid.org/0000-0003-1538-4951
8
kdhyde3@gmail.com; https://orcid.org/0000-0002-2191-0762
* Corresponding author email: kdhyde3@gmail.com
2
Abstract
A cercosporoid fungus associated with leaf lesions of Dypsis lutenscens (Arecaceae) was observed in Chiang Rai, Thailand.
This study describes the new species Pseudocercospora dypsidis based on morphological characteristics and phylogenetic
analyses from three gene regions, ITS, TEF-1α, and ACT. Single gene analyses are generally insufficient for identification
of Pseudocercospora species, or for segregation of other genera in the Pseudocercospora complex. A list is provided of all
Pseudocercospora species known from Thailand and the need to confirm their identifications by a polyphasic approach is
stressed.
Key words: 1 new species, multigene analyses, Mycosphaerella, palm, Pseudocercospora spp. in Thailand
Introduction
Palms are one of the most important ornamental tree groups with approximately 3,000 species (Benítez & Soto 2010).
Areca palm (Dypsis lutescens), a member of Arecaceae, has a worldwide distribution and is used for landscaping and
decoration. Dypsis lutescens prefers warm and humid sub-tropical to tropical climates, conditions that are favorable
for many diseases (Basu & Mondol 2012). As ornamental palms have a high aesthetic value, foliar diseases may cause
economic loss (Elliott et al. 2004). In July 2019, numerous plants were observed with widespread leaf lesions in Mae
Fah Luang University, Chiang Rai Province, Thailand; these lesions were shown to be caused by a Pseudocercospora
sp.
Pseudocercospora typified by P. vitis was introduced by Spegazzini (1910). Pseudocercospora are asexual fungi
which belong to Mycosphaerecellaceae (Capnodiales, Dothideomycetes), closely related to mycosphaerella-like
sexual morphs (Braun et al. 2013, 2014, 2015, Crous et al. 2013a, Silva et al. 2016). Species of Pseudocercospora are
identified mainly from tropical and sub-tropical regions (Wanasinghe et al. 2018). They cause leaf spots, blight, and
necrotic lesions on flowers and fruits of various cultivated and native plants (Chupp 1954, Agrios 2005, Crous & Braun
2003). The typical characteristic of leaf spots caused by these species are distinct chlorotic margins that demarcate
the lesions (Crous et al. 2013a). These fungi typically have scolecosporous conidia and dematiaceous conidiophores
with unthickened and not darkened conidiogenous loci and hila (Crous & Braun 2003, Braun et al. 2013). The present
study aims to identify the causal agent associated with the foliar infection on Dypsis lutescens observed in Chiang Rai,
Thailand using morphological and molecular methods.
218
Accepted by Eric McKenzie: 24 Nov. 2020; published: 3 Dec. 2020
Materials and methods
Sample collection and examination of specimens
Leaves of Dypsis lutescens with necrotic lesions were collected in 2019 at Mae Fah Luang University, Chiang Rai,
Thailand. The specimens were placed in paper bags and taken to the laboratory. Leaf lesions were examined using
a stereo microscope and photographed by a Carl Zeiss GmbH (AxioCam ERc5s) stereo microscope. A compound
microscope (Nikon ECLIPSE 80i) connected with EOS 600D digital camera (Canon, Japan) was used to observe and
photograph the morphology.
The fungal strain was isolated in pure culture by single spore isolation on potato dextrose agar (PDA) as described
by Chomnunti et al. (2014) and incubated at 25 °C. After 18 hours, germinated spores were transferred to a fresh
PDA plate. A living ex-type culture was deposited in Mae Fah Luang University Culture Collection (MFLUCC). The
dried leaf specimen was deposited at Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand. Index
Fungorum number (Index Fungorum 2020) and Faces of Fungi number (Jayasiri et al. 2015) were registered.
DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh fungal mycelia using the modified CTAB method described by Guo et al.
(2000). The internal transcribed spacers (ITS), large subunit rRNA (LSU), translation elongation factor 1-alpha (TEF1α) and α-actin (ACT) regions were amplified with the primers ITS4/ITS5 (White et al. 1990), LROR/LR5 (Vilgalys &
Hester 1990), EF1-728F/EF2 (O’Donnell et al. 1998) and ACT512F/ACT783R (Carbone & Kohn 1999), respectively.
Polymerase chain reactions (PCR) were conducted in a total volume of 25 μl using PCR mixtures containing 16.2 μl
of ddH2O, 1 μl of each primer, 3.0 μl of dNTPs (TaKaRa, China), 2.5 μl of 10x Ex-Taq buffer (TaKaRa, China), 1 μl
of genomic DNA, and 0.3 μl of TaKaRa Ex-Taq DNA polymerase (TaKaRa, China). A BIORAD C1000 TouchTM
Thermal Cycler was used to perform PCR amplification (Applied Biosystems, Foster City, CA, USA). The thermal
cycling programs were accomplished by an initial denaturation for 3 min at 95 °C, followed by 34 cycles of denaturation
for 30 s at 95 °C, 30 s of annealing, elongation for 1 min at 72 °C, and a final extension for 10 min at 72 °C. The
annealing temperatures for ITS, LSU, TEF-1α, and ACT were 58 °C, 55 °C, 52 °C and 61 °C, respectively. The PCR
products were checked on 1% agarose gel stained with ethidium bromide under UV light. Sequences of PCR products
were obtained from the Beijing Biomed Gene Technology Co., China. Sequence quality was checked and sequences
derived from this study are listed in (Table 1).
Phylogenetic analyses
Resulting sequences were manually trimmed and subjected to a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.
cgi). Closest matched sequences were downloaded from GenBank and taxa from different clades of Pseudocercospora
were also included in this study (Silva et al. 2016, Wang et al. 2019). Sequence alignments were performed individually
by MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/), using default setting (Katoh et al. 2019). Alignments were
trimmed by trimAL with gappyout model (Capella-Gutierrez et al. 2009) and manually edited by BioEdit v.7.0.5.2
(Hall 1999).
Phylogenetic analyses were generated by Bayesian inference (BI), maximum likelihood (ML), and maximum
parsimony (MP) analysis with MrBayes on XSEDE v.3.2.6 (Miller et al. 2010), RAxML-HPC2 on XSEDE (8.2.8)
(Stamatakis 2014), and PAUP on XSEDE (Swofford 2002) in the CIPRES Science Gateway, respectively. An initial
BI analysis was performed with an individual alignment of LSU data to determine generic rank and a concatenated
alignment for the remaining three genes (ITS, TEF-1α and ACT) was run using BI, ML, and MP. Bayesian inference
analysis was conducted by GTR +G +I substitution model for 50,000,000 Markov chain Monte Carlo (MCMC)
generations, and trees were saved every 1,000th generations. The first 25% of generated trees were discarded as
the burn-in value. Posterior probabilities (PP) were estimated from the remaining trees. All trees were visualized
in FigTree v1.4.0 (Rambaut 2012). The ML analysis including 1,000 bootstrap replicates was performed using the
GTR+G+I substitution model. Maximum parsimony analysis was estimated with heuristic searches of tree-bisection
reconnection (TBR) algorithm and 1,000 random stepwise addition. Maximum trees were set to 1,000 and branches
of zero length were collapsed. Parsimony scores including homoplasy index (HI), rescaled consistency index (RC),
retention index (RI), consistency index (CI), and tree length (TL) were calculated for trees generated under different
optimal criteria.
PSEUDOCERCOSPORA DYPSIDIS
Phytotaxa 474 (3) © 2020 Magnolia Press • 219
220 • Phytotaxa 474 (3) © 2020 Magnolia Press
TABLE 1. Collection details and GenBank accession numbers of isolates included in this study.
Species
Culture accession numbers1
Host
Family
Origin
New Zealand
GenBank accession numbers2
LSU
ITS
TEF-1α
GU214410
–
–
Cladosporium herbarum
CBS 723.79
Allium ampeloprasum
Amaryllidaceae
ACT
–
Neopseudocercospora
terminaliae
Pallidocercospora acaciigena
CPC 22685 (ex-type)
Terminalia sp.
Combretaceae
Zambia
KF777228
–
–
–
CBS 112515 (ex-type)
Acacia mangium
Fabaceae
Venezuela
GQ852599
–
–
–
Pallidocercospora crystallina
CBS 116158
Eucalyptus bicostata
Myrtaceae
South Africa
DQ204747
–
–
–
Pallidocercospora heimii
CBS 110682
Eucalyptus sp.
Myrtaceae
Madagascar
DQ204751
–
–
–
Pallidocercospora holualoana
CBS 129063
Hedychium coronarium
Zingiberaceae
USA
JF770467
–
–
–
Paracercospora egenula
CPC 12537
Solanum melongena
Solanaceae
South Korea
GU253738
–
–
–
Passalora eucalypti
CBS 111318; CPC 1457
(ex-type)
CPC 19812
Eucalyptus saligna
Myrtaceae
Brazil
GU253860
GU269845
GU384558
GU320548
Colophospermum
mopane
Acer albopurpurascens
Fabaceae
South Africa
NG_042683
–
–
–
Aceraceae
Taiwan
GU253699
GU269650
GU384368
GU320358
Phaeocercospora
colophospermi
Pseudocercospora acericola
CBS 122279
Aeschynomene falcata
Fabaceae
Brazil
KT290173
KT290146
KT290200
KT313501
P. angolensis
CPC 25227; COAD 1972
(ex-type)
CBS 112933; CPC 4118
Citrus sp.
Rutaceae
Zimbabwe
GU214470
GU269836
GU384548
JQ325010
P. angolensis
CBS 149.53 (ex-type)
Citrus sinensis
Rutaceae
Angola
JQ324941
JQ324975
JQ324988
JQ325011
P. assamensis
CBS 122467; X988
Musa cv.
Musaceae
India
GU253705
GU269656
GU384374
GU320364
P. assamensis
CBS 122467 (ex-type)
Musa cv.
Musaceae
India
JX901882
EU514281
JX901673
JX902129
P. atromarginalis
CBS 114640
Solanum sp.
Solanaceae
New Zealand
GU253706
GU269658
GU384376
GU320365
P. atromarginalis
CPC 25230; COAD 1975
Solanum americanum
Solanaceae
Brazil
KT290176
KT290149
KT290203
KT313504
P. basitruncata
CBS 114664; CPC 1202
(ex-type)
CGMCC 3.19020
Eucalyptus grandis
Myrtaceae
Colombia
GU253710
GU269662
DQ211675
DQ147622
Euonymus japonicus
Celastraceae
China
–
MH255813
MH255819
MH392526
Bixa orellana
Bixaceae
Brazil
KT290180
KT290153
KT290207
KT313508
P. boehmeriigena
CPC 25244; COAD 1563
(ex-epitype)
CPC 25243; COAD 1562
Bohemia nivea
Urticaceae
Brazil
KT290179
KT290152
KT290206
KT313507
P. breonadiae
CBS 143489T = CPC 30153 Breonadia microcephala
Rubiaceae
South Africa
MH107959
NR_158961
MH108026
MH107985
P. aeschynomenicola
P. beijingensis
P. bixae
CHEN ET AL.
...continued on the next page
PSEUDOCERCOSPORA DYPSIDIS
TABLE 1. (Continued)
Species
Culture accession numbers1
Host
Family
Origin
South Korea
GenBank accession numbers2
LSU
ITS
TEF-1α
GU253718
GU269670
GU384387
P. cercidis-chinensis
Cercis chinensis
Fabaceae
Chamaecrista sp.
P. chengtuensis
CBS 132109; CPC 14481
(ex-epitype)
CPC 25228; COAD 1973
(ex-epitype)
CBS 131924; CPC 10696
ACT
GU320376
Fabaceae
Brazil
KT290174
KT290147
KT290201
KT313502
Lycium chinense
Solanaceae
South Korea
JQ324942
GU269673
GU384390
GU320379
P. contraria
CBS 132108; CPC 14714
Dioscorea quinqueloba
Dioscoreaceae
South Korea
JQ324945
GU269677
GU384394
GU320385
P. cordiana
Cordia goeldiana
Boraginaceae
Brazil
GU214472
GU269681
GU384398
GU320387
P. corylopsidis
CBS 114685; CPC 2552
(ex-type)
MUCC 874
Hamamelis japonica
Hamamelidaceae
Japan
GU253757
GU269721
GU384437
GU320425
P. corylopsidis
MUCC 908 (ex-epitype)
Corylopsis spicata
Hamamelidaceae
Japan
GU253727
GU269684
GU384401
GU320390
P. cotoneastri
MUCC 876
Cotoneaster salicifolius
Rosaceae
Japan
GU253728
GU269685
GU384402
GU320391
P. crousii
CBS 119487
Eucalyptus sp.
Myrtaceae
New Zealand
GU253729
GU269686
GU384403
GU320392
P. cruenta
CBS 132021; CPC 10846
Vigna sp.
Fabaceae
Trinidad
GU214673
GU269688
GU384404
JQ325012
P. diplusodonii
CPC 25179; COAD 1476
(ex-type)
MFLUCC 20-0117 (extype)
MUCC 925
Diplusodon sp.
Lythraceae
Brazil
KT290162
KT290135
KT290189
KT313490
Dypsis lutescens
Arecaceae
Thailand
MT767884
MT767837
MT772098
MT772099
Elaeocarpus sp.
Elaeocarpaceae
Japan
GU253740
GU269701
GU384417
GU320405
Emmotum nitens
Icacinaceae
Brazil
KT290163
KT290136
KT290190
KT313491
P. euonymi-japonici
CPC 25187; COAD 1491
(ex-type)
CGMCC 3.18576
Euonymus japonicus
Celastraceae
China
–
MH255812
MH255818
MH392525
P. euphorbiacearum
CPC 25222; COAD 1537
Dalechampia sp.
Euphorbiaceae
Brazil
KT290172
KT290145
KT290199
KT313503
P. eustomatis
CBS 110822
Eustroma grandiflorum
Gentianaceae
Argentina
GU253744
GU269705
GU384421
GU320409
P. exilis
Chamaecrista orbiculata
Fabaceae
Brazil
KT290166
KT290139
KT290193
KT313494
P. fukuokaensis
CPC 25193; COAD 1501
(ex-epitype)
CBS 132111; CPC 14689
Styrax japonicus
Styracaceae
South Korea
GU253750
GU269713
GU384429
GU320417
P. fukuokaensis
MUCC 887 (ex-epitype)
Styrax japonicus
Styracaceae
Japan
GU253751
GU269714
GU384430
GU320418
P. fuligena
CBS 132017; CPC 12296
Lycopersicon sp.
Solanaceae
Thailand
JQ324953
GU269711
GU384427
GU320415
P. chamaecristae
P. dypsidis
Phytotaxa 474 (3) © 2020 Magnolia Press • 221
P. elaeocarpi
P. emmotunicola
...continued on the next page
222 • Phytotaxa 474 (3) © 2020 Magnolia Press
TABLE 1. (Continued)
Species
Culture accession numbers1
Host
Family
Origin
South Korea
GenBank accession numbers2
LSU
ITS
TEF-1α
GU253752
GU269715
GU384431
P. glauca
CBS 131884; CPC 10062
Albizzia julibrissin
Fabaceae
ACT
GU320419
P. guianensis
MUCC 855
Lantana camara
Verbenaceae
Japan
GU253755
GU269719
GU384435
GU320423
P. guianensis
MUCC 879
Lantana camara
Verbenaceae
Japan
GU253756
GU269720
GU384436
GU320424
P. hakeae
CBS 112226; CPC 3145
Grevillea sp.
Proteaceae
Australia
GU253805
GU269784
GU384495
JQ325017
P. hakeae
CBS 144520; CPC 32100
Hakea sp.
Proteaceae
Australia
MK442553
MK442617
MK442708
MK442642
P. latens
MUCC 763
Lespedeza wilfordii
Fabaceae
Japan
GU253763
GU269732
GU384445
GU320434
P. longispora
CBS 122470 (ex-type)
Musa sp.
Musaceae
Malaysia
GU253764
GU269734
GU384447
GU320436
P. lonicericola
MUCC 889 (ex-neotype)
Caprifoliaceae
Japan
GU253766
GU269736
JQ324999
GU320438
P. luzardii
CPC 2556
Lonicera
gracilipes var. glabra
Hancornia speciosa
Apocynaceae
Brazil
GU214477
GU269738
GU384450
GU320440
P. luzardii
Apocynaceae
Brazil
KT290167
KT290140
KT290194
KT313495
Lythraceae
South Korea
GU253771
GU269742
GU384454
GU320444
P. lythri
CPC 25196; COAD 1505 (ex- Harcornia speciosa
epitype)
CBS 132115; CPC 14588
Lythrum salicaria
(ex-epitype)
MUCC 865
Lythrum salicaria
Lythraceae
Japan
GU253772
GU269743
GU384455
GU320445
P. macrospora
CBS 114696; CPC 2553
Bertholletia excelsa
Lecythidaceae
Brazil
GU214478
GU269745
GU384457
GU320447
P. mali
MUCC 886
Malus sieboldii
Rosaceae
Japan
GU253773
GU269744
GU384456
GU320446
P. manihotii
Manihot sp.
Euphorbiaceae
Brazil
KT290171
KT290144
KT290198
KT313499
P. musae
CPC 25219; COAD 1534
(ex-type)
CBS 116634
Musa sp.
Musaceae
Cuba
GU253775
GU269747
GU384459
GU320449
P. nephrolepidis
CBS 119121
Nephrolepis auriculata
Oleandraceae
Taiwan
GU253779
GU269751
GU384462
GU320453
P. nogalesii
CBS 115022
Chamaecytisus proliferus
Fabaceae
New Zealand
JQ324960
GU269752
GU384463
GU320454
P. norchiensis
CBS 114641
Rubus sp.
Rosaceae
New Zealand
–
GU269772
GU384484
GU320475
P. norchiensis
Eucalyptus sp.
Myrtaceae
Italy
GU253780
GU269753
GU384464
GU320455
P. oenotherae
CBS 120738; CPC 13049
(ex-type)
CBS 131885; CPC 10290
Oenothera odorata
Onagraceae
South Korea
JQ324961
GU269856
GU384567
GU320559
P. oenotherae
CBS 131920; CPC 10630
Oenothera odorata
Onagraceae
South Korea
GU253781
GU269755
GU384466
GU320457
P. pallida
CBS 131889; CPC 10776
Campsis grandiflora
Bignoniaceae
South Korea
GU214680
GU269758
GU384469
GU320459
P. paraguayensis
CBS 111286; CPC 1459
Eucalyptus nitens
Myrtaceae
Brazil
GU214479
DQ267602
DQ211680
DQ147606
P. lythri
CHEN ET AL.
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PSEUDOCERCOSPORA DYPSIDIS
TABLE 1. (Continued)
Species
Culture accession numbers1
Host
Family
Origin
Brazil
GenBank accession numbers2
LSU
ITS
KT290159
KT290132
P. perae
Pera glabrata
Euphorbiaceae
P. pini-densiflorae
CPC 25171, COAD 1465
(ex-type)
MUCC 534
TEF-1α
KT290186
ACT
KT313487
Pinus thunbergii
Pinaceae
Japan
GU253785
GU269760
GU384471
GU320461
P. piperis
FBR 151
Piper aduncum
Piperaceae
Brazil
JX875063
JX875062
JX896123
P. planaltinensis
CPC 25189; COAD 1495
(ex-type)
CPC 25191; COAD 1498
(ex-epitype)
CPC 26081; COAD 1548
Chamaecrista sp.
Fabaceae
Brazil
KT290164
KT290137
KT290191
KT313492
Himatanthus obovatus
Apocynaceae
Brazil
KT290165
KT290138
KT290192
KT313493
Mikania hirsutissima
Asteraceae
Brazil
KT290178
KT290151
KT290205
KT313506
Pothomorphe umbellata
Piperaceae
Brazil
KT290158
KT290131
KT290185
KT313486
P. pouzolziae
CPC 25166; COAD 1450
(ex-type)
CBS 122280
Gonostegia hirta
Urticaceae
Taiwan
GU253786
GU269761
GU384472
GU320462
P. prunicola
CBS 132107; CPC 14511
Prunus xyedoensis
Rosaceae
South Korea
GU253723
GU269676
GU384393
GU320382
P. pseudomyrticola
CBS 145554
Myrtus sp.
Myrtaceae
Italy
MK876446
MK876405
MK876499
MK876461
P. purpurea
CBS 114163; CPC 1664
Persea americana
Lauraceae
Mexico
GU253804
GU269783
GU384494
GU320486
P. pyracanthae
MUCC 892
Pyracantha angustifolia
Rosaceae
Japan
GU253792
GU269767
GU384479
GU320470
P. pyracanthigena
CBS:131589; CPC 10808
(ex-type)
CBS 131590; CPC 12500
(ex-type)
CBS 282.66
Pyracantha angustifolia
Rosaceae
South Korea
–
GU269766
GU384478
GU320469
Rhamnella frangulioides
Rhamnaceae
South Korea
GU253813
GU269795
GU384505
GU320496
Rhapis flabellifornis
Arecaceae
Japan
GU253793
GU269770
GU384482
GU320473
Richardia brasiliensis
Rubiaceae
Brazil
KT290181
KT290154
KT290208
KT313509
Palicourea rigida
Rubiaceae
Brazil
KT290161
KT290134
KT290188
KT313489
P. rubi
CPC 25248; COAD 1568
(ex-epitype)
CPC 25175; COAD 1472
(ex-epitype)
MUCC 875
Rubus allegheniensis
Rosaceae
Japan
GU253795
GU269773
GU384485
GU320476
P. sawadae
CBS 115024
Psidium guajava
Myrtaceae
New Zealand
JQ324967
GU269775
–
GU320478
P. sennae-multijugae
Senna multijuga
Fabaceae
Brazil
KT290169
KT290142
KT290196
KT313497
P. serpocaulonicola
CPC 25206; COAD 1519
(ex-type)
CPC 25077
Serpocaulon triseriale
Polypodiaceae
Brazil
KT037566
NR_147291
KT037485
KT037607
P. solanipseudocapsicicola
CPC 25229; COAD 1974
(ex-type)
Solanum pseudocapsicum
Solanaceae
Brazil
KT290175
KT290148
KT290202
KT313503
P. plumeriifolii
P. plunkettii
P. pothomorphes
Phytotaxa 474 (3) © 2020 Magnolia Press • 223
P. rhamnellae
P. rhapisicola
P. richardsoniicola
P. rigidae
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224 • Phytotaxa 474 (3) © 2020 Magnolia Press
TABLE 1. (Continued)
Species
Host
Family
Origin
GenBank accession numbers2
LSU
ITS
TEF-1α
ACT
P. sordida
MUCC 913
Campsis radicans
Bignoniaceae
Japan
GU253798
GU269777
GU384488
GU320480
P. stephanandrae
MUCC 914 (ex-epitype)
Stephanandra incisa
Rosaceae
Japan
GU253831
GU269814
GU384526
GU320516
P. struthanthi
Struthanthus flexicaulis
Loranthaceae
Brazil
KT290168
KT290141
KT290195
KT313496
P. styracina
CPC 25199; COAD 1512
(ex-epitype)
COAD 2369
Styrax sp.
Styracaceae
Brazil
MH480643
MH397664
MH480642
MH480641
P. subsessilis
CBS 136.94
–
–
Cuba
GU253832
GU269815
GU384527
GU320517
P. subtorulosa
CBS 117230
Melicope sp.
Rutaceae
Taiwan
GU253833
GU269816
GU384528
GU320518
P. tecomicola
CPC 25260; COAD 1585
Tecoma stans
Bignoniaceae
Brazil
KT290183
KT290156
KT290209
KT313511
P. trinidadensis
CPC 26082; COAD 1756
Croton urucurana
Euphorbiacea
Brazil
KT290184
KT290157
KT290210
P. udagawana
CBS 131931; CPC 10799
Hovenia dulcis
Rhamnaceae
South Korea
–
GU269824
GU384537
GU320527
P. variicolor
MUCC 746
Paeoniaceae
Japan
GU253843
GU269826
GU384538
GU320530
P. vassobiae
CPC 25251; COAD 1572
(ex-type)
CBS 125998; CPC 15249
(ex-epitype)
MUCC 899
Paeonia lactiflora var.
trichocarpa
Vassobia breviflora
Solanaceae
Brazil
KT290182
KT290155
–
KT313510
Viburnum davidii
Caprifoliaceae
Netherlands
GU253827
GU269809
GU384520
GU320512
Weigela coraeensis
Caprifoliaceae
Japan
GU253847
GU269831
GU384543
GU320535
Wulffia stenoglossa
Asteraceae
Brazil
KT290177
KT290150
KT290204
KT313505
Xylopia aromatica
Annonaceae
Brazil
KT290160
KT290133
KT290187
KT313488
P. zelkovae
CPC 25232; COAD 1976
(ex-type)
CPC 25173; COAD 1469
(ex-type)
CBS 132118; CPC 14717
Zelkova serrata
Ulmaceae
South Korea
GU253850
GU269834
GU384546
JQ325028
P. zelkovae
MUCC 872
Zelkova serrata
Ulmaceae
Japan
GU253851
GU269835
GU384547
GU320537
P. viburnigena
P. weigelae
P. wulffiae
P. xylopiae
1
Culture accession numbers1
CHEN ET AL.
CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; COAD, CGMCC China General Microbiological Culture
Collection Center; MFLUCC: Mae Fah Luang University Culture Collection; MUCC: Culture Collection, Laboratory of Plant Pathology, Mie University, Tsu, Mie Prefecture, Japan.
2
LSU: partial 28S nrRNA gene; ITS: internal transcribed spacer regions 1 & 2 including 5.8S nrRNA gene; TEF-1α: partial translation elongation factor 1-alpha gene; ACT: partial actin
gene.
The newly generated taxon is highlighted in green and bold. Type strains (ex-type, ex-epitype, and ex-neotype) are in bold.
Results
Phylogenetic analyses
Large subunit (LSU) phylogeny showed that our pseudocercospora-like isolate clustered in Pseudocercospora sensu
lato and for further clarification multi-gene phylogenetic analyses was performed.
The final aligned LSU dataset comprised 111 ingroup taxa with 1,282 characters and Cladosporium herbarum
(CBS 723.79) as the outgroup taxon. Bayesian analysis resulted in a total of 2,111 trees. The first 25% of generated
trees were discarded as the burn-in. The consensus trees and posterior probabilities were estimated from the remaining
1,584 trees (Fig. 1).
The final concatenated alignment (ITS, TEF-1α, ACT) comprised 102 strains with 971 characters (ITS 1–471,
TEF-1α 475–780, ACT 784–971) including gaps. Passalora eucalypti (CBS 111318) served as the outgroup based on
its position as sister taxon to Pseudocercospora (Crous et al. 2006). Bayesian inference generated 9,501 trees when the
average standard deviation of split frequencies reached 0.01 (stop value). The consensus trees and posterior probabilities
were calculated from the remaining 7,126 trees after discarding the first 25% of generated trees for burn-in. The best
scoring RAxML tree with the final optimization likelihood value of -10477.381841 (α = 0.300136, TL = 2.802134,
substitution rates AC = 1.350659, AG = 3.246614, AT = 1.424190, CG = 0.784644, CT = 4.575591, GT = 1.0000 and
estimated base frequencies A = 0.220122, C = 0.280648, G = 0.249246, T = 0.249984). The MP analysis resulted in one
most parsimonious tree including 519 constant characters, 115 parsimony-uninformative variable characters, and 337
parsimony-informative characters (TL = 1,803, CI = 0.433, RI = 0.798, RC = 0.345, HI = 0.567). Maximum likelihood
and MP analyses produced mostly identical tree topologies with the result of Bayesian analyses. Bootstrap support
values of the ML and MP trees were incorporated into the tree that resulted from BI analyses (Fig. 2).
Taxonomy
Pseudocercospora dypsidis Y.J. Chen, Jayaward. & K.D. Hyde, sp. nov. (Fig. 3)
Index Fungorum number: IF557830; Faces of Fungi number: FoF 09219
Type:—THAILAND. Chiang Rai: Thasud, Mueang, Mae Fah Luang University, on leaf of Dypsis lutescens, 31 July 2019, Y.J. Chen,
holotype MFLU 20-0502; ex-type culture MFLUCC 20-0117.
Associated with leaf blight of Dypsis lutescens Wendel. Symptoms form yellowish brown streaks from the leaf tip,
becoming grey in the center with brown border, surrounded by a chlorotic yellow zone, amphigenous, coalescing
to form large, brown necrotic areas. Sexual morph: not observed. Asexual morph on host. Sporodochia 64−117 ×
63−111 ( = 88 × 88 μm, n=20), erumpent, pale olivaceous brown. Conidiophores 10−30.5 × 1.8−3.9 μm ( = 17.6 ×
2.7 μm, n=30), grouped in dense fascicles, unbranched, subcylindrical, straight to curved, 1−2-septate, hyaline to pale
olivaceous. Conidiogenous cells 8−15.7 × 1.1−3.3 μm ( = 11.3 × 2 μm, n=25), unbranched, terminal, proliferating
sympodially and percurrently, smooth, unthickened wall, pale brown. Conidiogenous loci inconspicuous, unthickened
and not darkened. Conidia 21−67 × 1.3−2.9 μm ( = 39 × 2 μm, n=50), solitary, holoblastic, straight or slightly curved,
smooth, narrowly obclavate, apex sub-obtuse, base rounded to long obconic-truncate, 1−5-septate, unthickened wall,
subhyaline to pale olivaceous brown. Hilum unthickened, not darkened.
Culture characteristics:—Colony on PDA reaching a diameter of 80 mm after 7 days at 25 °C, not sporulating.
Edge circular to irregular, umbonate, margin entire to undulate, pale grey at center, greenish grey to dark grey towards
the margin from above, dark grey to black from below.
Etymology:—Refers to the host, Dypsis lutescens.
Habitat/Distribution:—Known to inhabit Dypsis lutescens, Chiang Rai Province, Thailand.
GenBank accession numbers:—LSU: MT767884; ITS: MT767837; TEF-1α: MT772098; ACT: MT772099.
Notes:—Pseudocercospora dypsidis formed a monophyletic lineage with high statistical support (BYPP: 1.0,
ML: 93%, MP: 97%; Fig. 2) sister to P. hakeae isolated from leaves of Hakea sp. and Grevillea sp. in Australia
(Crous et al. 2019a). Pseudocercospora dypsidis can be distinguished from P. hakeae by its narrower conidia (P.
hakae: 30–50 × 4–5 μm) with unthickened wall and narrower and smaller conidiophores (P. hakae: 30–70 × 6–8 μm).
Pseudocercospora dypsidis differs from its sister clade taxa, P. musae (Chupp 1954) in having narrower and longer
conidiophores (P. musae: 5–25 × 2–2.3 μm) with narrower and shorter conidia (P. musae: 10–80 × 2−4 μm) and from
P. longispora (Arzanlou et al. 2008) in having shorter conidia (P. longispora: 82–120 × 2.5−4 μm). To our knowledge
P. dypsidis is the first record of Pseudocercospora species on the host Dypsis.
PSEUDOCERCOSPORA DYPSIDIS
Phytotaxa 474 (3) © 2020 Magnolia Press • 225
FIGURE 1. Phylogram (50% majority rule) of 1,584 trees generated from a Bayesian analysis of the LSU sequence
alignment. The tree was rooted with Cladosporium herbarum (CBS 723.79). The scale bar represents the expected
number of nucleotide substitutions per site. The newly generated taxon is highlighted in red and bold. Ex-type (exepitype and ex-neotype) strains are in bold.
226 • Phytotaxa 474 (3) © 2020 Magnolia Press
CHEN ET AL.
FIGURE 2. Phylogram of Pseudocercospora species and the outgroup taxon Passalora eucalypti (CBS 111318)
generated from Bayesian analysis based on ITS−TEF-1α−ACT sequence data. Support values were calculated via BI
(≥0.90 PP), and ML, MP (≥50%) indicated above or below the nodes or with a black arrow, respectively. The scale bar
represents the expected number of nucleotide substitutions per site. The newly generated taxon is highlighted in red
and bold. Ex-type (ex-epitype and ex-neotype) strains are in bold.
PSEUDOCERCOSPORA DYPSIDIS
Phytotaxa 474 (3) © 2020 Magnolia Press • 227
FIGURE 3. Pseudocercospora dypsidis. a. Host b, c. Leaf blight on host. d, e. Stromata on host substrate. f.
Conidiogenous cells and conidiophores. g. Conidia. h. Germinated conidium. i. Colony on PDA after 2 months. Scale
bars c = 3 mm, d, e = 200 μm, f–h = 20 μm.
Discussion
Pseudocercospora is a large genus comprising about 1,500 morphological species in Species Fungorum, although
less than 300 species have molecular data (Hongsanan et al. 2020). The genus is polyphyletic (Crous et al. 2013a).
There are few informative morphologies to distinguish taxa within the Pseudocercospora complex (Bakhshi et al.
2014). For example, Neopseudocercospora, Pallidocercospora, Paracercospora and Phaeocercospora belong to
Pseudocercospora complex (Bakhshi et al. 2014) and are morphologically similar to Pseudocercospora, but they can
be distinguished based on phylogenetic analyses (Crous et al. 2013a, b, Hyde et al. 2013, Hongsanan et al. 2020).
Thus, it is important to use sequence data at generic rank to illustrate our pseudocercospora-like isolate which clustered
in Pseudocercospora s. lat (Fig. 1).
It is recommended to use multi-loci phylogenetic analyses to resolve cryptic species in Pseudocercospora (Crous
et al. 2013a, Bakhshi et al. 2014). According to previous studies, the systematic relationships of Pseudocercospora
species were evident by the gene regions, ITS, TEF-1α and ACT (Crous et al. 2013a, Bakhshi et al. 2014, Silva et al.
2016, Nesamari et al. 2017). The ITS region alone is not enough to differentiate most taxa at species level based on BI,
ML and MP methods (data not shown). This is in accordance to previous studies by Crous et al. (2013a), Bakhshi et
al. (2014) and Silva et al. (2016). Pseudocercospora dypsidis could not be distinguished from other Pseudocercospora
species based solely on ACT phylogeny, but it is distinct in the TEF-1α (sister taxon to P. hakeae) phylogeny (data not
shown).
Thailand is a well-known biodiversity hotspot (Hyde et al. 2018). There are 138 records of Pseudocercospora
species listed in fungus/host database (Farr & Rossman 2020) (Table 2). Most of these species (94%) have been
identified solely on morphological characteristics and host association. Therefore, there is an urgent need to identify
and characterize species of Pseudocercospora based on a polyphasic approach.
228 • Phytotaxa 474 (3) © 2020 Magnolia Press
CHEN ET AL.
TABLE 2. Records of Pseudocercospora species in Thailand.
Species
Pseudocercospora
abelmoschi
P. atromarginalis
P. balsaminae
P. basiramifera
Host
Hibiscus sp.
Host family
Malvaceae
References
Meeboon et al. (2007)
Lycianthes biflora
Impatiens balsamina
Eucalyptus camaldulensis
Eucalyptus pellita
Solanaceae
Balsaminaceae
Myrtaceae
Myrtaceae
P. bauhiniae
P. bischofiae
P. bradburyae
P. buddleiae
P. carbonacea
Bauhinia racemosa
Bischofia javanica
Centrosema pubescens
Buddleja asiatica
Dioscorea bulbifera
Dioscorea glabra var. glabra
Terminalia tomentosa
Centrosema pubescens
Eucalyptus camaldulensis
Fabaceae
Phyllanthaceae
Fabaceae
Scrophulariaceae
Dioscoreaceae
Dioscoreaceae
Combretaceae
Fabaceae
Myrtaceae
Christella parasitica
Justicia gendarussa
Dioscorea alata
Crotalaria uncinella subsp.
elliptica
Crateva religiosa
Cuphea hyssopifolia
Cyclea fissicalyx
Cyclea peltata
Cyclea sp.
Dalbergia cultrata
Dalbergia stipulacea
Duabanga grandiflora
Dypsis lutescens
Chromolaena odorata
Chromolaena odorata
Ficus rumphii
Eucalyptus camaldulensis
Thelypteridaceae
Acanthaceae
Dioscoreaceae
Fabaceae
Phengsintham et al. (2013)
Phengsintham et al. (2010, 2013)
Crous (1998), Hunter et al. (2011)
Crous (1998), Crous et al. (2013b*, 2019b*), Hunter
et al. (2011), Quaedvlieg et al. (2014*), Guatimosim et
al. (2016*)
Nakashima et al. (2007)
Phengsintham et al. (2013)
Lenné (1990)
Nakashima et al. (2007)
Meeboon et al. (2007), Phengsintham et al. (2013)
Nakashima et al. (2007)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Cheewangkoon et al. (2008), Hunter et al. (2011),
Crous et al. (2013b*, 2019b*), Quaedvlieg et al.
(2014*)
Phengsintham et al. (2010, 2013), Braun et al. (2013*)
Phengsintham et al. (2013)
Meeboon et al. (2008)
Phengsintham et al. (2013)
P. catappae
P. centrosematicola
P. chiangmaiensis
P. christellae
P. consociata
P. contraria
P. cotizensis
P. cratevae
P. cupheae
P. cycleae
P. dalbergiae
P. duabangae
P. dypsidis
P. eupatorii-formosani
P. eupatorii-formosanii
P. fici
P. flavomarginata
P. fuligena
P. getoniae
P. glochidionis
P. griseola
P. heveae
P. holmskioldiae
P. houttuyniae
P. jahnii
P. jussiaeae
P. lygodii
P. lythracearum
P. malloticola
Lycopersicon esculentum
Lycopersicon esculentum
var. pyriforme
Lycopersicon sp.
Solanum lycopersicum
Solanum sp.
Getonia floribunda
Glochidion sphaerogynum
Phaseolus vulgaris
Hevea sp.
Holmskioldia sanguinea
Houttuynia cordata
Tabebuia chrysotricha
Ludwigia prostrata
Lygodium flexuosum
Lagerstroemia macrocarpa
Mallotus barbatus
Mallotus japonicus
PSEUDOCERCOSPORA DYPSIDIS
Capparaceae
Lythraceae
Menispermaceae
Menispermaceae
Menispermaceae
Fabaceae
Fabaceae
Fabaceae
Arecaceae
Asteraceae
Asteraceae
Moraceae
Myrtaceae
Solanaceae
Solanaceae
Solanaceae
Solanaceae
Solanaceae
Combretaceae
Phyllanthaceae
Fabaceae
Euphorbiaceae
Lamiaceae
Saururaceae
Bignoniaceae
Onagraceae
Lygodiaceae
Lythraceae
Euphorbiaceae
Euphorbiaceae
Phengsintham et al. (2013)
Meeboon et al. (2007)
Phengsintham et al. (2012)
Phengsintham et al. (2012, 2013)
Phengsintham et al. (2012)
Meeboon et al. (2007)
Nakashima et al. (2007)
Phengsintham et al. (2010, 2013)
Present study
Phengsintham et al. (2013)
Barreto & Evans (1994)
Meeboon et al. (2007)
Hunter et al. (2006, 2011), Crous et al. (2013a*,
2013b*, 2019b*), de Miranda et al. (2014*),
Quaedvlieg et al. (2014*), Videira et al. (2016*,
2017*)
Phengsintham et al. (2013)
Meeboon et al. (2008)
Osorio et al. (2015*), Guatimosim et al. (2016*)
Phengsintham et al. (2012)
Silva et al. (2016*)
Phengsintham et al. (2013)
Meeboon et al. (2007)
Meeboon et al. (2007)
Crous & Braun (2003)
Nakashima et al. (2007)
Nakashima et al. (2007)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Phengsintham et al. (2012, 2013)
Phengsintham et al. (2012)
...continued on the next page
Phytotaxa 474 (3) © 2020 Magnolia Press • 229
TABLE 2. (Continued)
Species
Host
Mallotus thorelii
P. melanolepidis
Mallotus pierrei
P. mombin
Spondias pinnata
P. mori
Morus alba
P. musae
Musa acuminata
Musa paradisiaca
P. nephrolepidigena
Nephrolepis biserrata
P. nymphaeacea
Nymphaea stellata
P. olacicola
Olax scandens
Olax sp.
Olax wightiana
Olax zeylanica
Ximenia sp.
P. oroxyli
Oroxylum indicum
P. paederiae
Paederia chinensis
Paederia foetida
Paederia scandens
Paederia tomentosa
P. panacis
Polyscias balfouriana
P. paraguayensis
Eucalyptus sp.
P. phyllitidis
Nephrolepis biserrata
Nephrolepis cordifolia
P. polysciatis
Polyscias balfouriana
Polyscias guilfoylei
Polyscias sp.
P. pteridophytophila
Cyclosorus parasiticus
P. puderi
Rosa sp.
P. puerariicola
Pueraria phaseoloides
P. punicae
Punica granatum
P. radermachericola
Radermachera ignea
P. rhinacanthi
Rhinacanthus nasutus
P. riachueli
Vitis sp.
P. riachueli var. horiana Vitis vinifera
P. rosae
Rosa canina
P. schizolobii
Eucalyptus camaldulensis
P. scopariicola
Scoparia dulcis
P. solani-melongenicola Solanum melongena
Pseudocercospora sp.
Musa acuminata
P. sphaerellae-eugeniae Syzygium cumini
P. stahlii
Passiflora foetida
P. stizolobii
Mucuna bracteata
Mucuna pruriens
P. subsessilis
Melia azedarach
P. tamarindi
Tamarindus indica
P. tecomae-heterophyllae Tecoma stans
P. thailandica
Acacia mangium
P. timorensis
P. trematicola
Eucalyptus camaldulensis
Operculina sp.
Trema orientale
Trema orientalis
Solanum sp.
P. trichophila var.
punctata
P. viticicola
Vitex quinata
P. vitis
Vitis sp.
P. wrightiae
Wrightia pubescens
* Indicates the species has sequence data.
230 • Phytotaxa 474 (3) © 2020 Magnolia Press
Host family
Euphorbiaceae
Euphorbiaceae
Anacardiaceae
Moraceae
Musaceae
Musaceae
Nephrolepidaceae
Nymphaeaceae
Olacaceae
Olacaceae
Olacaceae
Olacaceae
Olacaceae
Bignoniaceae
Rubiaceae
Rubiaceae
Rubiaceae
Rubiaceae
Rubiaceae
Myrtaceae
Nephrolepidaceae
Nephrolepidaceae
Araliaceae
Araliaceae
Araliaceae
Thelypteridaceae
Rosaceae
Fabaceae
Lythraceae
Bignoniaceae
Acanthaceae
Vitaceae
Vitaceae
Rosaceae
Myrtaceae
Plantaginaceae
Solanaceae
Musaceae
Myrtaceae
Passifloraceae
Fabaceae
Fabaceae
Meliaceae
Fabaceae
Bignoniaceae
Fabaceae
Myrtaceae
Convolvulaceae
Cannabaceae
Cannabaceae
Solanaceae
References
Phengsintham et al. (2012)
Meeboon et al. (2007)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Meeboon et al. (2008)
Phengsintham et al. (2013)
Braun et al. (2013)
Meeboon et al. (2008)
Phengsintham et al. (2012, 2013)
Phengsintham et al. (2012)
Phengsintham et al. (2012)
Phengsintham et al. (2012)
Phengsintham et al. (2012)
Meeboon et al. (2007); Phengsintham et al. (2013)
Phengsintham et al. (2012)
Phengsintham et al. (2012)
Phengsintham et al. (2012)
Phengsintham et al. (2012, 2013)
Phengsintham et al. (2013)
Meeboon et al. (2007); Phengsintham et al. (2013)
Nakashima et al. (2007)
Meeboon et al. (2007)
Phengsintham et al. (2012)
Phengsintham et al. (2012)
Phengsintham et al. (2012)
Kirschner & Liu (2014)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Phengsintham et al. (2011, 2013)
Phengsintham et al. (2010, 2013)
Meeboon et al. (2007)
Jayawardena et al. (2018b*)
Phengsintham et al. (2013)
Wanasinghe et al. (2018)
Crous et al. (2009a, b), Hunter et al. (2011)
Phengsintham et al. (2013)
Meeboon et al. (2007)
Lumyong et al. (2003)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Nakashima et al. (2007)
Phengsintham et al. (2013)
Meeboon et al. (2007)
Liu et al. (2015)
Nakashima et al. (2007)
Crous et al. (2004), Braun et al. (2014*), Liang et al.
(2016*)
Hunter et al. (2011)
Phengsintham et al. (2013)
Phengsintham et al. (2010)
Phengsintham et al. (2013)
Phengsintham et al. (2013)
Lamiaceae
Vitaceae
Apocynaceae
Nakashima et al. (2007)
Jayawardena et al. (2018a*, b*)
Phengsintham et al. (2013)
CHEN ET AL.
Acknowledgements
Y.J. Chen and R.S. Jayawadena would like to thank Mae Fah Luang University for financial support. K.D. Hyde
thanks the Thailand Research Fund for the grant RDG6130001MS Impact of climate change on fungal diversity and
biogeography in the Greater Mekong Subregion.
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