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A multi-locus backbone tree for Pestalotiopsis, with a polyphasic

characterization of 14 new species
Article in Fungal Diversity · September 2012

DOI: 10.1007/s13225-012-0198-1
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Fungal Diversity (2012) 56:95–129
DOI 10.1007/s13225-012-0198-1
A multi-locus backbone tree for Pestalotiopsis,

with a polyphasic characterization of 14 new species
Sajeewa S. N. Maharachchikumbura & Liang-
Dong Guo & Lei Cai & Ekachai Chukeatirote &
Wen Ping Wu & Xiang Sun & Pedro W. Crous &
D. Jayarama Bhat & Eric H. C. McKenzie &
Ali H. Bahkali & Kevin D. Hyde
Received: 26 July 2012 / Accepted: 1 August 2012 / Published online: 1 September 2012
# Mushroom Research Foundation 2012
Abstract Pestalotiopsis is a taxonomically confused, path- β–tubulin and tef1 proved to be the better markers.
ogenic and chemically creative genus requiring a critical re- The other gene regions were less useful due to poor
examination using a multi-gene phylogeny based on ex-type success in PCR amplification and/or in their ability to
and ex-epitype cultures. In this study 40 isolates of Pesta- resolve species boundaries. As a single gene tef1 met
lotiopsis, comprised of 28 strains collected from living and the requirements for an ideal candidate and functions
dead plant material of various host plants from China were well for species delimitation due to its better species
studied by means of morphology and analysis of ITS, resolution and PCR success. Although β-tubulin showed
β–tubulin and tef1 gene sequence data. Based on mo- fairly good differences among species, a combination of
lecular and morphological data we describe 14 new ITS, β-tubulin and tef1 gene data gave the best resolu-
species (Pestalotiopsis asiatica, P. chinensis, P. chrysea, tion as compared to single gene analysis. This work
P. clavata, P. diversiseta, P. ellipsospora, P. inflexa, P. provides a backbone tree for 22 ex-type/epitypified spe-
intermedia, P. linearis, P. rosea, P. saprophyta, P. cies of Pestalotiopsis and can be used in future studies
umberspora, P. unicolor and P. verruculosa) and three of the genus.
species are epitypified (P. adusta, P. clavispora and P.
foedans). Of the 10 gene regions (ACT, β-tubulin,
CAL, GPDH, GS, ITS, LSU, RPB 1, SSU and tef1) Keywords β-tubulin . Epitype . ITS . Phylogeny .
utilized to resolve cryptic Pestalotiopsis species, ITS, Saprobe . tef1
S. S. N. Maharachchikumbura : E. Chukeatirote : K. D. Hyde P. W. Crous

Institute of Excellence in Fungal Research, CBS-KNAW Fungal Biodiversity Centre,
Mae Fah Luang University, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands
Chiang Rai 57100, Thailand
S. S. N. Maharachchikumbura : E. Chukeatirote : K. D. Hyde (*)

D. J. Bhat
Department of Botany, Goa University,
School of Science, Mae Fah Luang University,
Panaji, Goa 403 206, India
Chiang Rai 57100, Thailand
e-mail: kdhyde3@gmail.com
S. S. N. Maharachchikumbura : L.-D. Guo (*) : L. Cai : X. Sun E. H. C. McKenzie

State Key Laboratory of Mycology, Institute of Microbiology, Landcare Research,
Chinese Academy of Sciences, Private Bag,
Beijing 100101, People’s Republic of China 92170, Auckland, New Zealand
e-mail: guold@sun.im.ac.cn
W. P. Wu A. H. Bahkali
Novozymes China, College of Science, Botany and Microbiology Department,
14 Xin Xi Lu, Shangdi Zone, Haidian District, Beijing 100086, King Saud University,
People’s Republic of China P.O. Box: 2455, Riyadh 1145, Saudi Arabia
96 Fungal Diversity (2012) 56:95–129
Introduction The use of molecular data in resolving Pestalotiopsis

species has been reviewed by Hu et al. (2007), Tejesvi et
Pestalotiopsis, an appendage-bearing conidial asexual form al. (2007a), Liu et al. (2010) and Maharachchikumbura et al.
in the family Amphisphaeriaceae (Barr 1975, 1990; Kang et (2011). These studies have suggested that multi-locus phy-
al. 1998, 1999) is widely distributed throughout the tropical logenetic analysis is needed to resolve the cryptic species in
and temperate ecosystems (Bate-Smith and Metcalfe 1957). It the genus. Furthermore, species need to be epitypified so
is an important plant pathogenic genus (Yasuda et al. 2003; Das that we have sequence data pinned to names and thus can
et al. 2010; Maharachchikumbura et al. 2011) with more than confidently name species in future (Cai et al. 2011).
235 species, traditionally named according to their host associ- We have been studying the genus Pestalotiopsis and testing
ations (Guba 1961; Steyaert 1949; Venkatasubbaiah et al. 1991; the use of various genes to resolve species boundaries. In this
Kohlmeyer and Kohlmeyer 2001). Following the discovery of study, we report on 28 isolates sourced from plant material
taxol, the multimillion dollar anti-cancer drug, from P. micro- from China. All isolated species were first morphologically
spora (Speg.) G.C. Zhao & N. Li, an endophytic strain isolated characterised and then sequenced using ITS, β-tubulin and
from Taxus wallachiana (Strobel et al. 1996), the importance of tef1 genes. In order to select suitable gene regions for better
the genus has increased considerably (Strobel et al. 2002; Xu et species resolution, we analyzed nuclear ribosomal large sub-
al. 2010). Chemical exploration of endophytic Pestalotiopsis unit rDNA (LSU), nuclear ribosomal small subunit rDNA
species subsequently increased in an unprecedented way (Wei (SSU), partial actin (ACT), glutamine synthase (GS),
and Xu 2004; Tejesvi et al. 2007a, b). Species belonging to the glyceraldehyde-3-phosphate dehydrogenase (GPDH), RNA
genus Pestalotiopsis are thought to be a rich source for biopro- polymerase II (RPB1) and calmodulin (CAL) gene regions
specting when compared to other fungal genera (Aly et al. 2010; for several isolates of Pestalotiopsis. We compared the mor-
Xu et al. 2010). Xu et al. (2010) reviewed 130 different com- phological data versus the sequence data from single and
pounds isolated from species of Pestalotiopsis in the preceding combined genes to establish which characters satisfactorily
10 years. These included bioactive alkaloids, terpenoids, iso- resolve the species. As a result, we epitypified three species
coumarin derivatives, coumarins, chromones, quinones, semi- and describe 14 new saprobic Pestalotiopsis species. It is our
quinones, peptides, xanthones, xanthone derivatives, phenols, hope that this work will provide a backbone phylogenetic tree
phenolic acids, and lactones with a range of antifungal, antimi- for 22 type/epitypified species, which can be used in future
crobial, and antitumor activities. taxonomic work on the genus.
Due to their ability to switch life modes, many endophyt-
ic and pathogenic Pestalotiopsis species persist as saprobes
(Hyde et al. 2007; Zhou and Hyde 2001). Species of Pesta- Methods and materials
lotiopsis have been isolated as saprobes from dead leaves,
bark and twigs (Guba 1961). Several species have been Isolation and identification of pathogen
recovered from soil, polluted stream water, wood, paper,
fabrics and wool (Guba 1961). For example, P. bicolor (Ellis Dead plant tissues were collected from different sites in
& Everh.) A.R. Liu, T. Xu & L.D. Guo, P. funerea (Desm.) China. The samples were placed in separate plastic bags
Steyaert, P. monochaetioides (Doyer) Steyaert, P. montellica lined with tissue paper, sprayed with sterile water to create
(Sacc. & Voglino) Tak. Kobay., P. disseminata (Thüm.) humid conditions and incubated at room temperature. The
Steyaert, P. foedans (Sacc. & Ellis) Steyaert, P. versicolor fungi present on the samples were isolated by single spore
(Speg.) Steyaert and P. virgatula (Kleb.) Steyaert are common culture technique (Chomnunti et al. 2011). In short, a con-
saprobic species recorded either from decaying leaves or bark. idiomata was immersed in 300 μl of sterile distilled water on
However, there is less recent data on saprobic Pestalotiopsis a slide and left a few minutes so that the conidia were
species (Table 1). discharged. A conidial suspension was made, small drops
Table 1 Saprobic Pestalotiopsis species with their host/substrata
Species Host/ substrate References
Pestalotiopsis funerea Dead leaves of Rhododendron, Chamaecyparis, Dennis 1995; Ellis and Ellis 1997
Cupressus, Pinus, Juniperus
P. guepinii (Desm.) Steyaert Decaying leaves of Dracaena loureiri Thongkantha et al. 2008
P. palmarum (Cooke) Steyaert Dead culms of Schoenoplectus triqueter Wu et al. 1982
P. sydowiana (Bres.) B. Sutton Dead leaves of Calluna vulgaris, Erica, Dennis 1995; Ellis and Ellis 1997
Rhododendron ponticum, R. hybridum, Prunus laurocerasus
P. theae (Sawada) Steyaert Seeds of Diospyros crassiflora Douanla-Meli and Langer 2009
Fungal Diversity (2012) 56:95–129 97
were placed on water agar (WA) in Petri dishes and kept at (White et al. 1990), partial β-tubulin gene region was
room temperature for 8–12 h for conidia to germinate; single amplified with primer pairs BT2A (5′-GGTAAC
germinating conidia were transferred to potato dextrose agar C A A AT C G G T G C T G C T T T C - 3 ′ ) a n d B T 2 B ( 5 ′
(PDA) plates. The plates were incubated at 25 °C for 7 to ACCCTCAGTGTAGTGACCCTTGGC-3′) (Glass &
10 days. Colonies grown on PDA were transferred to PDA Donaldson 1995; O’Donnell & Cigelnik 1997) and tef1
slants, and stored at 4 °C for further study. Sporulation was was amplified using the primer pairs EF1-526 F (5′-
induced by placing sterilized carnation leaves on the surface GTCGTYGTYATY GGHCAYGT-3′) and EF1-1567R
of PDA with growing mycelia. The morphology of fungal (5′-ACHGTRCCRATACCACCRATCTT-3′) (Rehner
colonies was recorded following the method of Hu et al. 2001). In addition to above three gene regions selected
(2007). Fungal mycelia and spores were observed under a LSU, SSU, Actin, GS, GPDH, RPB1and CAL regions
light microscope and photographed. All microscopic measure- were amplified using primer pair/s listed in Table 2.
ments were done with Tarosoft image framework (v. 0.9.0.7) PCR was performed with the 25 μl reaction system con-
and 30 conidial measurements were taken for each isolate. taining 19.5 μl of double distilled water, 2.5 μl of 10× Taq
Isolates were deposited in Novozymes, Beijing and were also buffer with MgCl2, 0.5 μl of dNTP (10 mM each), 0.5 μl of
transferred to MFLUCC from Novozymes by Material Trans- each primer (10 μM), 0.25 μl Taq DNA polymerase (5 U
fer Agreement and cannot be distributed to a third party. μl−1), 1.0 μl of DNA template. The thermal cycling program
All other cultures dealt with in this study were obtained was as follows: For ITS an initial denaturing step of 95 °C
from China General Microbiological Culture Collection for 3 min, followed by 35 amplification cycles of 95 °C for
(CGMCC) and The International Collection of Micro- 30 s, 52 °C for 45 s and 72 °C for 90 s, and a final extension
organisms from Plants (ICMP). step of 72 °C for 10 min. For β-tubulin PCR conditions
were an initial step of 3 min at 95 °C, 35 cycles of 1 min at
DNA extraction 94 °C , 50 s at 55 °C , and 1 min at 72 °C, followed by
10 min at 72 °C. For tef1, an initial step of 5 min at 94 °C,
Total genomic DNA was extracted from fresh cultures using 10 cycles of 30 s at 94 °C, 55 s at 63 °C or 66 °C (decreasing
a modified protocol of Guo et al. (2000). Fresh fungal 1 °C per cycle), 90 s at 72 °C, plus 36 cycles of 30 s at
mycelia (500 mg) was scraped from the margin of a PDA 94 °C, 55 s at 53 °C or 56 °C, 90 s at 72 °C, followed by
plate incubated at 25 °C for 7 to 10 days and transferred into 7 min at 72 °C. The LSU, SSU, Actin, GS, GPDH, RPB
a 1.5 ml centrifuge tube with 100 μl of preheated (60 °C) 2X 1and CAL regions were tested under different optimal condi-
CTAB extraction buffer (2 % (w/v) CTAB, 100 mM Tris- tions (not shown). The PCR products were verified by staining
HCl, 1.4 M NaCl, 20 mM EDTA, pH 8.0), and 200 mg with Goldview (Guangzhou Geneshun Biotech, China) on 1 %
sterilized quartz sand. Mycelia were ground using a glass agarose electrophoresis gels.
pestle for 5 min and an extra 500 μl 2X CTAB preheated
(60 °C) was added and incubated in a 65 °C water bath for Phylogenetic analysis
30 min with occasional shaking. 500 μl of phenol:chloro-
form (1:1) was added to each tube and shaken thoroughly to DNAStar and SeqMan were used to obtain consensus
form an emulsion. The mixture was spun at 11,900g for sequences from sequences generated from forward and re-
15 min at 25 °C in a microcentrifuge and the supernatant verse primers. Single locus dataset and combination of
phase decanted into a fresh 1.5 ml tube. Supernatant con- multi-locus dataset of three gene regions were aligned using
taining DNA was re-extracted with phenol: chloroform (1:1) CLUSTALX (v. 1.83) (Thompson et al. 1997). The sequen-
at 4 °C until no interface was visible. 50 μl of 5 M KOAc ces were further aligned using default settings of MAFFTv6
was added into the supernatant followed by 400 μl of iso- (Katoh and Toh 2008; mafft.cbrc.jp/alignment/server/) and
propanol and inverted gently to mix. The genomic DNA was manually adjusted using BioEdit (Hall 1999) to allow max-
precipitated at 9,200g for 2 min at 4 °C in a microcentrifuge. imum alignment and minimum gaps. A maximum parsimo-
The DNA pellet was washed with 70 % ethanol twice and ny analysis (MP) was performed using PAUP (Phylogenetic
dried using SpeedVac® (AES 1010; Savant, Holbrook, NY, Analysis Using Parsimony) v. 4.0b10 (Swofford 2002).
USA) until dry. The DNA pellet was then resuspended in Ambiguously aligned regions were excluded and gaps were
100 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA). treated as missing data. Trees were inferred using the heu-
ristic search option with TBR branch swapping and 1,000
PCR amplification random sequence additions. Maxtrees were set up to 5,000,
branches of zero length were collapsed and all multiple
The ITS and 5.8 S region of rDNA fragment was amplified parsimonious trees were saved. Tree length [TL], consisten-
using primer pairs ITS5 (5′-GGAAGTAAAAGTCGTAA cy index [CI], retention index [RI], rescaled consistency
CAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) index [RC], homoplasy index [HI], and log likelihood
Table 2 Primers used

in this study to test Region Primer/s
different genes
LSU LR OR /5(Rehner and Samuels 1994; Moriya et al. 2005)
SSU NS 1/4 (White et al. 1990)
ACT ACT 512 F/783R (Carbone and Kohn 1999)
GS GS F1/R1 (Stephenson et al. 1997; Guerber et al. 2003)
GPDH GDF1/GPD2LM (Myllys et al. 2002; Guerber et al. 2003)
RPB1 RPB1 Af/Ac/Cr (http://www.clarku.edu/faculty/dhibbett/Protocols_Folder/Primers/Primers.pdf)
CAL CL 1/2; CAL 228 F/737R (Carbone and Kohn 1999; O’Donnell et al. 2000)
[-ln L] (HKY model) were calculated for trees generated comprised species having versicolorous median conidial cells
under different optimality criteria. The robustness of the most and Clade C species with dark concolorous median conidial
parsimonious trees was evaluated by 1,000 bootstrap replica- cells, with knobbed apical appendages. The species within each
tions resulting from maximum parsimony analysis, each with group were not well resolved at the terminal clades. Specifical-
10 replicates of random stemwise addition of taxa (Felsenstein ly, all taxa in Clade B did not separate into distinct species but
1985). The Kishino–Hasegawa tests (Kishino & Hasegawa clustered in two subclades. Species resolution was higher in
1989) were performed to determine whether the trees inferred Clade A, although a few species are not well resolved at the
under different optimality criteria were significantly different. terminal ends. Thus, ITS had lower inter-specific variation and,
Trees were viewed in Treeview (Page 1996). therefore, further gene sequences are needed to determine ge-
netic variation within each biological species.
Results Sequence analysis of β-tubulin gene data

from Pestalotiopsis strains
Phylogenetic trees were constructed using individual and
combined ITS, β-tubulin and tef1 sequences for our 40 The aligned dataset for β-tubulin sequences comprised 37
isolates of Pestalotiopsis with a Seiridium species as the taxa and 487 characters (including gaps). Parsimony analy-
outgroup taxon and other sequences downloaded from sis indicated that 285 characters were constant, 48 variable
GenBank (Table 3). We tested 10 genes in PCR amplifica- characters parsimony-uninformative and 154 characters
tion, alignment and the species delimitation in Pestalotiop- parsimony-informative. The parsimony analysis of the data
sis (Tables 4 and 5) and found that β-tubulin and tef1 were matrix resulted in two equally parsimonious trees and the
the optimal genes, while ITS is included as it is the accepted first tree (TL0410 steps, CI00.702, RI00.912, HI00.298
barcode for fungi (Schoch et al. 2012). We used the avail- and RC00.640) was shown here (Fig. 2).
able type ITS sequences from other studies (Pestalotiopsis Analysis of the β-tubulin gene sequences resulted in a phylo-
pallidotheae, P. hainanensis, P. jesteri and P. kunmingensis), gram (Fig. 2) in which the Pestalotiopsis species separated into
for comparison. three major clades, A, B and C with high bootstrap support.
Clade A comprised twelve well-resolved species. It was not
Sequence analysis of ITS from Pestalotiopsis strains possible to obtain PCR products from P. chinensis (MFLUCC
12-0273), P. intermedia (MFLUCC 12-0260), P. linearis
ITS sequences from the types (Pestalotiopsis pallidotheae, P. (MFLUCC 12-0272) and P. verruculosa (MFLUCC 12-0274)
hainanensis, P. jesteri and P. kunmingensis) for Pestalotiopsis using primer pair BT2A and BT2B. Although most of the
were analysed with our isolates used in this study. The align- species were well-resolved in the β-tubulin tree, the success
ment comprised 45 taxa and 527 characters (including gaps) rate of PCR has been low for this gene. Therefore, further
(Fig. 1). Parsimony analysis indicates that 398 characters were molecular loci were needed to resolve the species in this genus.
constant, 41 variable characters parsimony-uninformative and
88 characters are parsimony-informative. The parsimony anal- Sequence analysis of tef1 gene data from of Pestalotiopsis
ysis of the data matrix resulted in two equally parsimonious strains
trees and the first tree (TL0243, CI00.683, RI00.910, HI0
0.317, RC00.622) is shown here (Fig. 1). The aligned dataset for tef1 sequence data comprised 39 taxa
In the ITS phylogram, the Pestalotiopsis strains separated and 1,005 characters (including gaps). Among these, 723
into three major clades, named A, B and C with high bootstrap characters were constant, 87 variable characters parsimony-
support (Fig. 1). Clade A comprised species having pale brown uninformative and 195 characters parsimony-informative.
or olivaceous concolorous median conidial cells. Clade B The parsimony analysis resulted in six equally parsimonious
Table 3 Isolates used in this study

a
Taxon Isolates GenBank Accession Number
ITS β-tubulin tef1
P. adusta (Ellis & Everh.) Steyaert ICMP6088 JX399006 JX399037 JX399070

P. adusta MFLUCC10-146 JX399007 JX399038 JX399071
P. asiatica Maharachchikumbura & K.D. Hyde MFLUCC 12-0286/ NN047638 JX398983 JX399018 JX399049
P. camelliae Y. M. Zhang, Maharachchikumbura & K.D. Hyde MFLUCC12-0277 JX399010 JX399041 JX399074
P. camelliae MFLUCC 12-0278 JX399011 JX399042 JX399075
P. chinensis Maharachchikumbura & K.D. Hyde MFLUCC 12-0273/ NN047218 JX398995 - -
P. chrysea Maharachchikumbura & K.D. Hyde MFLUCC 12-0261/ NN042855 JX398985 JX399020 JX399051
P. chrysea MFLUCC 12-0262/ NN047037 JX398986 JX399021 JX399052
P. clavata Maharachchikumbura & K.D. Hyde MFLUCC 12-0268/ NN047134 JX398990 JX399025 JX399056
P. clavata MFLUCC 12-0269/ NN047005 JX398991 JX399026 JX399057
P. clavispora MFLUCC 12-0280/ NN043011 JX398978 JX399013 JX399044
P. clavispora MFLUCC 12-0281/ NN043133 JX398979 JX399014 JX399045
P. diversiseta Maharachchikumbura & K.D. Hyde MFLUCC 12-0287/ NN047261 JX399009 JX399040 JX399073
P. ellipsospora Maharachchikumbura & K.D. Hyde MFLUCC 12-0283 JX399016 JX399016 JX399047
P. ellipsospora MFLUCC 12-0284 JX399015 JX399015 JX399046
P. foedans (Sacc. & Ellis) Steyaert CGMCC 3.9178 JX398989 JX399024 JX399055
P. foedans CGMCC 3.9123 JX398987 JX399022 JX399053
P. foedans CGMCC 3.9202 JX398988 JX399023 JX399054
P. furcata Maharachchikumbura & K.D. Hyde MFLUCC 12-0054 JQ683724 JQ683708 JQ683740
P. hainanensis - GQ869902 - -
P. inflexa Maharachchikumbura & K.D. Hyde MFLUCC 12-0270/ NN047098 JX399008 JX399039 JX399072
P. intermedia Maharachchikumbura & K.D. Hyde MFLUCC 12-0259/ NN047642 JX398993 JX399028 JX399059
P. intermedia MFLUCC 12-0260/ NN047073 JX398997 JX399019 JX399062
P. jesteri Strobel, J.Yi Li, E.J. Ford & W.M. Hess - AF377282 - -
P. jesteri MFLUCC 12-0279/ NN042849 JX399012 JX399043 JX399076
P. kunmingensis J.G. Wei & T. Xu - AY373376 - -
P. linearis Maharachchikumbura & K.D. Hyde MFLUCC 12-0271/NN047190 JX398992 JX399027 JX399058
P. linearis MFLUCC 12-0272/ NN047141 JX398994 JX399060
P. pallidotheae Kyoto Watan. & Yas. Ono - AB482220 - -
P. rosea Maharachchikumbura & K.D. Hyde MFLUCC12-0258/ NN047135 JX399005 JX399036 JX399069
P. samarangensis Maharachchikumbura & K.D. Hyde MFLUCC 12-0233 JQ968609 JQ968610 JQ968611
P. saprophyta Maharachchikumbura & K.D. Hyde MFLUCC 12-0282/ NN047136 JX398982 JX399017 JX399048
P. theae MFLUCC12-0055 JQ683727 JQ683711 JQ683743
P. theae SC011 JQ683726 JQ683710 JQ683742
P. trachicarpicola Y. M. Zhang & K.D. Hyde MFLUCC 12-0263/ NN047072 JX399000 JX399031 JX399064
P. trachicarpicola MFLUCC 12-0264/ NN047196 JX399004 JX399035 JX399068
P. trachicarpicola OP068 JQ845947 JQ845945 JQ845946
P. umberspora Maharachchikumbura & K.D. Hyde MFLUCC 12-0285/ NN042986 JX398984 JX399019 JX399050
P. unicolor Maharachchikumbura & K.D. Hyde MFLUCC 12-0275/ NN047308 JX398998 JX399029 JX399063
P. unicolor MFLUCC 12-0276/ NN046974 JX398999 JX399030 -
P. verruculosa Maharachchikumbura & K.D. Hyde MFLUCC 12-0274/ NN047309 JX398996 - JX399061
Seiridium sp. SD096 JQ683725 JQ683709 JQ683741
a
Acronyms: NN Novozymes
Table 4 Comparison of gene

regions used in our study (this Region ITS β-tub tef1 Combined
excludes the ex-type ITS of P.
hainanensis, P. jesteri, PCR success/sequencing success 100 % 90 % 95 % -
P. kunmingensis and Characters in aligned dataset 546 487 1005 2038
P. pallidotheae)
Parsimony-informative characters 78 (14.3 %) 154 (31.6 %) 195 (13 %) 427 (21 %)
Number of bootstrap support >50 % 16 24 28 34
trees and the first tree (TL0606 steps, CI00.670, RI00.896, informative characters. ITS sequence data has relatively poor
HI00.330 and RC00.600) is shown here (Fig. 3). species resolution for the genus Pestalotiopsis, even though it
In the tef1 phylogram (Fig. 3), the Pestalotiopsis strains is now standardized as the universal DNA barcode marker for
separated into three major Clades, A, B and C with high the fungi (Schoch et al. 2012). Therefore, ITS can be used as
bootstrap support. In comparison to ITS and β-tubulin, the rough identification guide for some species in Pestalotiopsis.
tef1 gene clearly separated all species used in this study at β-tubulin and tef1 successfully resolved most of the strains
the species level, with high bootstrap support. The branch analyzed in this study to species within Pestalotiopsis, al-
lengths of neighboring clades are longest in the tef1 gene though tef1 had a higher PCR success rate when compared
region and thus signifies speciation in Pestalotiopsis. It was to β-tubulin. Thus, due to its better species resolution and
not successful in the amplification of species P. chinensis PCR success rate, we suggest that tef1 is an additional barcode
(MFLUCC 12-0273) and P. unicolor (MFLUCC 12-0276). for Pestalotiopsis species.
Although 235 species have been described in the genus,
Combined sequence analysis of ITS, β-tubulin and tef1 only those few with sequence data are included in this study.
gene data from Pestalotiopsis strains The new species described below are based on molecular data
and distinct morphological characteristics. At the terminal ends
The aligned data matrix for combined ITS, β-tubulin and tef1 of the clades, most species can be differentiated from closely
sequences consisted of 41 taxa and 2,047 characters (includ- related species in the β-tubulin and tef1 and combined ITS, β-
ing gaps). Parsimony analysis indicated that 1,450 characters tubulin and tef1 phylograms. All designated epitypes have
were constant, 170 variable characters parsimony- spore characters fitting those of the holotype and are supported
uninformative and 427 characters parsimony-informative. as distinct based on molecular data.
The parsimony analysis of the data matrix resulted in a single
parsimonious tree (TL01,193 steps, CI00.685, RI00.907, Taxonomy
HI00.315, RC00.621) (Fig. 4).
In the analysis of the combined dataset from ITS, β-tubulin Pestalotiopsis adusta (Ellis & Everh.) Steyaert, Trans. Br.
and tef1 genes, all species separated into three major clades A, mycol. Soc. 36: 82 (1953)
B and C with high bootstrap support. Combined sequence Basionym: Pestalotia adusta Ellis & Everh., J. Mycol. 4
analysis successfully resolved most of the Pestalotiopsis spe- (6): 51 (1888)
cies used in this study with high bootstrap supports. The MycoBank: MB302600
bootstrap support value of terminal and internal node has been Description from holotype (Fig. 5a–h)
increased as compared to the single gene phylogenetic trees. Conidiomata 80–150 μm diam., acervulus, subepidermal in
In this study we attempted to obtain sequence data from 10 origin, with basal stroma, with lateral wall 2–4 cells thick
genes. In contrast to the other genes, ITS, β-tubulin and tef1 comprising hyaline to pale brown cells of textura angularis.
were relatively easy to amplify, sequence and align. β-tubulin Conidiophores indistinct. Conidiogenous cells discrete, simple,
and tef1 also contained considerably more phylogenetic short, filiform. Conidia 16–20×5–7 μm (x ¼ 18:7 6:2 μm),
Table 5 Comparison of gene

regions tested but not used Region Primer/s PCR success (%) Sequence success (%) Species resolution
in the final phylogenetic study
LSU LROR /5 100 100 Very low
SSU NS 1/4 100 100 Very low
Actin ACT 512 F/783R 95 100 Low
GS GS F1/R1 0 - -
GPDH GDF1/GPD2LM 95 100 Low
RPB 1 RPB1 Af/Ac/Cr 60 50 High
CAL CL 1/2; CAL 228 F/737R 70 90 High
P. trachicarpicola MFLUCC 12-0263

P. trachicarpicola OP068
77 P. trachicarpicola MFLUCC 12-0264
67 P. clavata MFLUCC 12-0268
P. clavata MFLUCC 12-0269
66
P. rosea MFLUCC 12-0258
P. inflexa MFLUCC 12-0270
85 P. adusta ICMP 6088
Clade A, pale brown or
P. aducta MFLUCC 10-0146
olivaceous concolourous
85 P. linearis MFLUCC 12-0271 median conidial cells
P. linearis MFLUCC 12-0272
P. intermedia MFLUCC 12-0259
60
84 P. chinensis MFLUCC 12-0273
P. verruculosa MFLUCC 12-0274
67
P. unicolor MFLUCC 12-0276
P. Intermedia MFLUCC 12-0260
P. hainanensis GQ869902
81 P. camelliae MFLUCC 12-0277
73
P. furcata MFLUCC 12-0054
P. pallidotheae AB482220
92
P. kunmingensis AY373376
P. diversiseta MFLUCC 12-0287
70 P. jesteri MFLUCC 12-0279
P. jesteri AF377282
P. clavispora MFLUCC 12-0280
P. foedans CGMCC 3.9202
73
100 P. saprophyta MFLUCC 12-0282 Clade B, versicolorous
P. ellipsospora MFLUCC 12-0283 median conidial cells
P. ellipsospora MFLUCC 12-0284
100 P. samarangensis MFLUCC 12-0233
P. chrysea MFLUCC 12-0262
69 P. chrysea MFLUCC 12-0261
P. umberspora MFLUCC 12-0285
P. asiatica MFLUCC 12-0286
100 P. theae SC011
Clade C, dark concolorous median conidial
P. theae MFLUCC 12-0055 cells with knobbed apical appendages
Seiridium sp. SD096
1 changes
Fig. 1 Maximum parsimony phylogram generated from ITS dataset. Data were analyzed with random addition sequences, unweighted parsimony
and treating gaps as missing data. A Seiridium sp. was used as outgroup. Ex-type and ex-epitype sequences are in bold
fusiform to ellipsoid, straight to slightly curved, 4-septate, with (x ¼ 10 μm), arising from the apex of the apical cell; filiform
short basal cell, obtuse, hyaline, thin-walled and verruculose, basal appendage.
2.7–3.8 μm long (x ¼ 3:2 μm); with three median cells, dolii- Description from epitype (Fig. 6. a–g)
form to subcylindrical, concolorous, olivaceous, with septa and Conidiophores indistinct. Conidiogenous cells discrete,
periclinal walls darker than the rest of the cell, together 12.4– simple, short, filiform. Conidia 17–20×5.2–6.6 μm (x ¼ 19
13.8 μm long (x ¼ 13:2 μm) second cell from base 4.3–5.3 μm 6 μm), fusiform to ellipsoid, straight to slightly curved, 4-
(x ¼ 4:8 μm); third cell 4–4.7 μm (x ¼ 4:2 μm); fourth cell septate, basal cell short, obtuse, hyaline, thin-walled and ver-
3.8–4.4 μm (x ¼ 4 μm); apical cell hyaline, conic, 2.4–3.4 μm ruculose, 3–3.8 μm long ( x ¼ 3:3 μm ); with three median
long (x ¼ 3 μm); with two to three appendages, 7–15 μm long cells, doliiform to subcylindrical, concolorous, olivaceous,

100 P. trachicarpicola OP068

Clade A, pale brown or 89 P. trachicarpicola MFLUCC 12-0266

olivaceous concolourous P. trachicarpicola MFLUCC 12-0267
median conidial cells

100 P. linearis MFLUCC 12-0271
92 P. intermedia MFLUCC 12-0259

70 63
58

99
76
72
P. jesteri MFLUCC 12-0279
58
90 P. ellipsospora MFLUCC 12-0283
100 62
P. samarangensis MFLUCC 12-0233
54
73 Clade B, versicolorous
100
87
63 P. foedans CGMCC 3.9202

P. saprophyta MFLUCC 12-0282
100 P. theae SC011 Clade C, dark concolorous

median conidial cells with
P. theae MFLUCC 12-0055
knobbed apical appendages
Seiridium sp. SD096
10 changes
Fig. 2 The maximum parsimony phylogram generated from β-tubulin dataset. Data were analyzed with random addition sequence, unweighted
parsimony and treating gaps as missing data. A Seiridium sp. was used as the outgroup. Ex-type and ex-epitype sequences are in bold
septa and periclinal walls darker than the rest of the cell, Colonies on PDA attaining 7 cm diam. after 7 days at
together 12.5–14.2 μm long (x ¼ 13:6 μm) second cell from 25 °C, with undulate edge, whitish, with dense, aerial my-
base 4.4–5.5 μm (x ¼ 4:9 μm); third cell 4.3–5 μm (x ¼ 4:5 celium on surface; fruiting bodies black, gregarious; reverse
μm); fourth cell 4–4.8 μm (x ¼ 4:3 μm); apical cell hyaline, of the colony yellowish.
conic, 2.7–3.7 μm long ( x ¼ 3:2 μm ); with two to three Habitat/Distribution: Inhabiting leaves of Prunus cerasus,
appendages 6–14 ( x ¼ 10 μm ) μm long, arising from the USA, refrigerator door PVC gasket, Fiji and Syzygium sp.,
apex of the apical cell; filiform basal appendage. Thailand.

65 P. Intermedia MFLUCC 12-0260
99
86 P. unicolor MFLUCC 12-0275

Clade A, pale brown or
olivaceous concolourous
median conidial cells P. clavata MFLUCC 12-0269
P. verruculosa MFLUCC 12-0274



51 93

100
62
96

77
100
P. samarangensis MFLUCC 12-0233
78
P. saprophyta MFLUCC 12-0282
Clade B, versicolorous
100
93 P. clavispora MFLUCC 12-0281

96
94
100 P. theae SC011

Clade C, dark concolorous
P. theae MFLUCC 12-0055 median conidial cells with
Seiridium sp. SD096 knobbed apical appendages
10 changes
Fig. 3 Maximum parsimony phylogram generated from tef1 dataset. Data were analyzed with random addition sequence, unweighted parsimony
and treating gaps as missing data. A Seiridium sp. is used as outgroup. Ex-type and ex-epitype sequences are in bold
Material examined: USA, Newfield, New Jersey, on Notes: Pestalotiopsis adusta was described from cultivated
leaves of Prunus cerasus L., cultivated plum, 20 July 1887 plum in New Jersey (Steyaert 1949) and recently phenolic
(NY 00937391, holotype); FIJI, on refrigerator door PVC compounds isolated from one putative isolate of P. adusta
gasket, 1 June 1978, E.H.C. McKenzie (MFLU12-0425, showed antimicrobial activity against Fusarium culmorum, Gib-
epitype designated here; ex-type living culture ICMP berella zeae and Verticillium aiboatrum (Li et al. 2008). Pesta-
60880PDDCC 6088). lotiopsis adusta is characterized by its small conidia (16–20×5–
Additional culture examined: Thailand, Chiang Rai, on 7 μm) and two to three relatively short apical appendages (7–
leaves of Syzygium sp., 06 Febuary 2010, S.S.N. Mahara- 15 μm) (Fig. 5 e–g). According to Guba (1961), P. adusta
chchikumbura SS008 (MFLUCC 10-0146). occurs on various hosts and has a cosmopolitan distribution.

51 P. Intermedia MFLUCC 12-0260
76
53 P. unicolor MFLUCC 12-0275
88
Clade A, pale brown or P. unicolor MFLUCC 12-0276
olivaceous concolourous 86 P. chinensis MFLUCC 12-0273
90
median conidial cells P. verruculosa MFLUCC 12-0274
99
85 P. aducta MFLUCC 10-0146
100 100 P. camelliae MFLUCC 12-0277

92 P. furcata MFLUCC 12-0054
53 P. samarangensis MFLUCC 12-0233
100
99 P. clavispora MFLUCC 12-0281
73
98 Clade B, versicolorous
P. foedans CGMCC 3.9123 median conidial cells
100 P. saprophyta MFLUCC 12-0282
97
96 P. umberspora MFLUCC 12-0285
100 P. theae SC011
Clade C, dark concolorous, median
P. theae MFLUCC 12-0055 conidial cells with knobbed apical
Seiridium sp. SD096 appendages
10 changes
Fig. 4 Maximum parsimony phylogram generated from combination data. Seiridium spp. was used as the outgroup. Ex-type and ex-epitype
of ITS, β-tubulin and tef1 sequences. Data were analyzed with random sequences are in bold
addition sequence, unweighted parsimony and treating gaps as missing
Guba (1961) listed it from Acer platanoides in Point Pleasant, species in the genus. The epitype has identical conidiogenous
New Jersey; on stems of Barringtonia speciosa in Bermuda; cells and morphology, including three apical appendages and a
from circular spots on leaves of Bischofia javanica in Taiwan; spore size fitting that of the holotype. As we want to advance the
on leaves of Carpinus betulus in Italy; as causing fruit rot and understanding of this poorly defined species rich genus, the Fiji
grey leaf spot in Eriobotrya japonica in Japan; on leaves of collection is designated here as an epitype of P. adusta.
Homalomena philippinensis in the Philippines; and on spots and
dead areas of leaves of Pavonia multiflora in Brazil. Living Pestalotiopsis asiatica Maharachchikumbura & K.D. Hyde,
specimens from cultivated plum or from the USA would have sp. nov.
been desirable when epitypifying this taxon. The sample col- MycoBank: MB 800529
lected from Fiji, however, is characteristic of P. adusta, a distinct Figure 7 a–j.
Fig. 5 Pestalotiopsis adusta (holotype). a. Herbarium material – leaves of Prunus cerasus. b. Conidiomata, split irregularly. c–d. Section of
conidiomata. e. Conidiogenous cells f–h. Conidia with concolorous median cells. Scale Bars: e050 μm, f–h020 μm
Etymology: The specific epithet is based on the geograph- 4-septate; basal cell conical, hyaline, thin and verruculose,
ical region (Asia), in reference to where fungus was isolated. 3–5 μm long (x ¼ 4 μm); three median cells 13–15.5 μm
Conidiophores indistinct. Conidiogenous cells hyaline, long (x ¼ 14 μm), dark brown, verruculose, septa and peri-
simple, filiform, 3–12 μm long. Conidia 20–26×5–7 μm clinal walls darker than the rest of the cell, versicoloured,
(x ¼ 22:6 6:25 μm), fusiform, straight to slightly curved, second cell from base pale brown, 4–5.5 μm (x ¼ 4:5 μm);
Fig. 6 a. Pestalotiopsis adusta (epitype) b. Conidiogenous cells c–e. Conidia with concolorous median cells. f–g. Colony on PDA, f. from above,
g. from below. Scale Bars: a–e020 μm
Fig. 7 a. Pestalotiopsis asiatica (holotype). b–c. Conidiophores/ conidiogenous cells. d. e. Immature conidia. f–h. Mature conidia. i–j. Colony on
PDA, i. from above, j. from below. Scale Bars: a–h020 μm
third cell darker brown, 4–5 μm (x ¼ 4:8 μm); fourth cell mycelium on surface, fruiting bodies black, developing in con-
darker, 4–5 μm ( x ¼ 4:7 μm ); apical cell 3.5–5 μm long centric circles; reverse of culture yellow to pale orange.
(x ¼ 3:35 μm), hyaline, conical to cylindrical, comprising Habitat/Distribution: Endophyte in leaves of Taxus sp.,
2–4 appendages (mainly 3); apical appendages 20–30 μm long Kunming, Yunnan Province China.
(x ¼ 25:6 μm), tubular, arising from the apex of the apical cell; Material examined: CHINA, Yunnan Province, Kunming,
basal appendage, 4–8 μm long (x ¼ 5:65 μm), filiform. Kunming Botanical Garden, on living leaves of Taxus sp., 19
Colonies on PDA reaching 7 cm diam. after 6 days at March 2002, Wenping Wu KBG13-9 (HMAS047218, holo-
25 °C, with crenate edge, whitish, with aerial mycelium on type; MFLU12-0415, isotype; ex-type living culture
surface; fruiting bodies black, gregarious; reverse of culture NN0472180MFLUCC 12-0273).
whitish to pale yellow. Notes: The conidial size of Pestalotiopsis chinensis over-
Habitat/Distribution: Endophyte on unidentified tree, laps with P. funerea (Desm.) Steyaert (21–29×7–9.5 μm)
Yizhang County, Hunan Province China. (Steyaert 1949), P. macrochaeta (Speg.) J. Xiang Zhang &
Material examined: CHINA, Hunan Province, Yizhang T. Xu (22–31×8–10 μm) (Zhang et al. 2002), P. mayum-
County, Mangshan, isolated from living leaves of unidenti- bensis (Steyaert) Steyaert (22–28×6.5–8.5 μm) (Steyaert
fied tree, 12 April 2002, Wenping Wu HN51-1 1949) and P. osyridis (Thüm.) H.T. Sun & R.B. Cao (22–
(HMAS047638, holotype; MFLU12-0422, isotype; ex- 28×5–7 μm) (Guba 1961). However, P. chinensis can be
type living culture NN0476380MFLUCC 12-0286). distinguished from P. mayumbensis and P. osyridis by its
Notes: Pestalotiopsis asiatica is a distinct species in the relatively large conidial size and also by its long apical
versicolour group (Clade B) and clearly distinguishable from appendages (in P. mayumbensis 8–15 μm and in P. osyridis
P. chrysea and P. umberspora in the β-tubulin, tef1 and up to 14 μm). Pestalotiopsis chinensis (1–3 tubular apical
combined genes phylogram. Pestalotiopsis asiatica (20–26× appendages) can be differentiated by the number of apical
5–7 μm) is morphologically similar to P. pauciseta (Sacc.) appendages (3–6 apical appendages (mostly 4–5) in P. fune-
Y.X. Chen (conidia 20–24×4.5–5 μm) (Saccardo 1914) and P. rea and three apical appendages in P. macrochaeta). It was
gracilis (Kleb.) Steyaert (conidia 19–23×6–7 μm) (Saccardo not possible to obtain PCR products of P. chinensis for β-
1931). However, P. asiatica differs from P. pauciseta by its tubulin and tef1 and thus in phylogenetic tree it clusters with
wider conidia and from P. gracilis in having long apical P. verruculosa, which is morphologically distinct.
appendages (in P. gracilis 10–26 μm).
Pestalotiopsis chrysea Maharachchikumbura & K.D. Hyde,
Pestalotiopsis chinensis Maharachchikumbura & K.D. sp. nov.
Hyde, sp. nov. MycoBank: MB 800533
MycoBank: MB 800522 Figure 9 a–g.
Figure 8 a–h. Etymology: The specific refers to the golden yellow
Etymology: The specific epithet is referring to China, the colour of the colony (Latin- chryseus) of this species.
country from where the taxon was isolated. Conidiophores indistinct. Conidiogenous cells discrete or
Conidiophores most often indistinct, septate, hyaline, integrated, lageniform, hyaline, smooth-walled. Conidia 20–
smooth, rarely branched. Conidiogenous cells discrete, ampul- 24×5.5–7 μm ( x ¼ 22:3 6:1 μm ), fusiform, straight to
liform to lageniform, smooth, thin-walled, hyaline or pale slightly curved, 4-septate; basal cell obconic to conic, hyaline,
brown, with 2–3 proliferations. Conidia fusoid to ellipsoid, thin and smooth-walled, 3–5 μm long (x ¼ 4:3 μm ); three
straight to slightly curved, 4–septate, 23–32 × 7–9 μm median cells 14–16 μm long (x ¼ 14:8 μm), dark brown to
( x ¼ 29 8:3 μm ), basal cell conic to obconic, hyaline or olivaceous, septa and periclinal walls darker than the rest of
slightly olivaceous, thin-walled and verruculose, 5–7 μm long the cell, versicoloured, verruculose, second cell from base pale
(x ¼ 5:7 μm), with three median cells, doliiform to cylindrical, brown, 4–5 μm ( x ¼ 4:6 μm ); third cell darker brown, 4–
constricted at the septa, concolorous, olivaceous, septa and 5 μm (x ¼ 4:6 μm); fourth cell darker, 4–5 μm (x ¼ 4:5 μm);
periclinal walls darker than the rest of the cell, wall rugose, apical cell 3.5–4.5 μm long ( x ¼ 4 μm ), hyaline, conic to
together 20–22 μm long (x ¼ 20:2 μm) second cell from base obconic; apical appendages 22–30 μm long (x ¼ 26:8 μm), 3,
6–7 μm (x ¼ 6:5 μm); third cell 7–7.5 μm (x ¼ 7:1 μm); fourth tubular, arising from the apex; basal appendage, 3 –6 μm long
cell 6–7.5 μm ( x ¼ 6:8 μm ); apical cell hyaline, conic to (x ¼ 4:4 μm), filiform.
subcylindrical, 3–6 μm long (x ¼ 4:3 μm); with 1–3 tubular Colonies on PDA reaching 7 cm diam. after 10 days at
apical appendages (mostly 3), arising from the apex of the apical 25 °C, edge irregular, yellowish to pale brown, aerial my-
cell, 25–30 μm long (x ¼ 28 μm), unequal; basal appendage celium on surface, fruiting bodies black, gregarious; reverse
present 7–11 μm (x ¼ 8:7 μm). of the colony orange to brown.
Colonies on PDA reaching 7 cm diam. after 13 days at 25 °C, Habitat/Distribution: Saprobe on dead plant material,
with edge crenate, whitish to pale yellow, with dense aerial Guangxi and Hunan provinces, China.
Fig. 8 Pestalotiopsis chinensis (holotype). b–c. Conidiophores/ conidiogenous cells. d–f. Conidia. g–h. Colony on PDA, g from above, h from
below. Scale Bars: a–f020 μm
Material examined: CHINA, Guangxi Province, Shangsi, basal cell conic to obconic with obtuse end, hyaline, thin-
Shiwandashan, Wangle, dead leaves of unidentified plant, 2 walled and verruculose, 4–5 μm long (x ¼ 4:6 μm), with three
January 1997, Wenping Wu WUFH1303a (HMAS042855, median cells, doliiform, concolorous, olivaceous to brown, septa
holotype; MFLU12-0411, isotype; ex-type living culture and periclinal walls darker than the rest of the cell; wall rugose,
NN0428550MFLUCC 12-0261). together 15–16 μm long (x ¼ 15:2 μm) second cell from base
Additional culture examined: CHINA, Hunan Province, 5–6 μm (x ¼ 5:2 μm); third cell 4–5 μm (x ¼ 4:8 μm); fourth
Yizhang County, Mangshanon dead plant material, 12 cell 5–5.5 μm ( x ¼ 5:2 μm ); apical cell hyaline, conic to
April 2002, Wenping Wu HN27-10 (NN047037 0 cylindrical 3–5 μm long ( x ¼ 3:75 μm ), with 2– 3 tubular
MFLUCC 12-0262). apical appendages (mostly 3) arising from the apex of the apical
Notes: Pestalotiopsis chrysea is a morphologically dis- cell, 20–25 μm long ( x ¼ 23 μm ); basal appendage mostly
tinct species in the genus with its yellowish colony; its con- present, 7–9 μm long (x ¼ 7:8 μm).
idiogenous cells and conidia are also slightly yellowish. It can Colonies on PDA reaching 7 cm diam. after 8 days at
clearly be differentiated from its phylogenetically related sib- 25 °C, with edge entire, whitish to pale brown, with dense,
ling species, P. umberspora (19–29×6–8 μm) in having rela- aerial mycelium on the surface, with black fruiting bodies;
tively narrow conidia (20–24×5.5–7 μm) and also in tef1 and reverse of culture pale brown to brown.
combined genes phylogenetic trees (Figs. 3 and 4). Habitat/Distribution: Endophyte in living leaves of Buxus
sp. and Euonymus sp., Hunan and Yunnan provinces, China.
Pestalotiopsis clavata Maharachchikumbura & K.D. Hyde, Material examined: CHINA, Yunnan Province, Kunming,
sp. nov. Kunming Botanical Garden, living leaf of Buxus sp., 19
MycoBank: MB 800524 March 2002, Wenping Wu KBG26-5 (HMAS047134, holo-
Figure 10 a–f. type; MFLU12-0412, isotype; ex-type living culture
Etymology: In Latin, clavatus refers to the clavate conidia. NN0471340MFLUCC 12-0268).
Conidiophores most often indistinct. Conidiogenous cells Additional culture examined: CHINA, Hunan Province,
discrete ampulliform to lageniform, smooth, thin-walled, hy- Yizhang County, Mangshan, living leaf of Euonymus sp., 12
aline, short. Conidia fusoid to ellipsoid, straight to slightly April 2002, Wenping Wu HN49-6 (NN0470050MFLUCC
curved, 4-septate, 20–27×6.5–8 μm ( x ¼ 22:6 7:3 μm ), 12-0269)
Fig. 9 a. Pestalotiopsis chrysea (holotype). b. Conidiophores/ conidiogenous cells. c–e. Conidia. f–g. Colony on PDA, f. from above, g. from
below. Scale Bars: a–e020 μm
Notes: Pestalotiopsis clavata is a distinct species recog- Conidia 18–26×6.5–8.5 μm (x ¼ 21 7:5 μm), fusiform,
nized based on its morphology and phylogeny. It has similar 4-septate, straight or slightly curved and clavate-fusiform;
sized conidia to P. heterocornis (Guba) Y.X. Chen (18–26× basal cell long and conic, hyaline, thin and verruculose, 4–
6.5–8 μm) (Guba 1961). However, these species are distinct in 5 μm long ( x ¼ 4:2 μm ); with three median cells 13.7–
the length and number of their apical appendages. P. clavata has 15.3 μm long ( x ¼ 14:7 μm ), dark brown to olivaceous,
conidia with 2–3 apical appendages (mostly 3) which are 20– septa and periclinal walls darker than the rest of the cell,
25 μm long, while in P. heterocornis the apical appendages are versicolored, verruculose, second cell from base pale brown,
unequal in length being 9–21 μm long (Guba 1961). Pestalo- 4.3–5.3 μm ( x ¼ 4:8 μm ); third cell darker brown, 5.5–
tiopsis carveri (Guba) P.L. Zhu, Q.X. Ge & T. Xu (20–26×6– 6.4 μm (x ¼ 5:8 μm); fourth cell darker, 4.5–5.8 μm (x ¼ 5
7 μm) (Guba 1961) also has a somewhat similar conidial μm); apical cell 3.3–4.2 μm long (x ¼ 3:7 μm), short, broad
morphology with P. clavata, but they differ in the length and conic, hyaline, subcylindric; with apical appendages 19–
number of their apical appendages. In P. carveri the two apical 30 μm long (x ¼ 24:5 μm), tubular, 2–3 (rarely 2), arising
appendages are unequal in length being 12–26 μm long. from the apex of the apical cell; with basal appendage
present, filiform.
Pestalotiopsis clavispora (G.F. Atk.) Steyaert, Bull. Jard. bot. Description from epitype (Fig. 12 a–g)
État Brux. 19: 335 (1949) Conidiophores indistinct. Conidiogenous cells hyaline,
Basionym: Pestalotia clavispora G.F. Atk., Bulletin of simple, short or relatively long, filiform, 4–10 um long. Co-
Cornell University 3: 37 (1897) nidia 20–24×6–8 μm (x ¼ 22 7:2 μm), fusiform, straight
MycoBank: MB289191 to slightly curved, 4-septate, clavate-fusiform when mature;
Description from holotype (Fig. 11 a–h) basal cell conical, hyaline, thin and verruculose, 3–5 μm long
Conidiomata 150–250 μm in diam., black, numerous, (x ¼ 3:8 μm); three median cells 13–15 μm long (x ¼ 13:9
scattered, rupturing the epidermis and dehiscing irregularly. μm), dark brown to olivaceous, verruculose-walled, septa and
Fig. 10 a. Pestalotiopsis
clavata (holotype). b.
Conidiophores/ conidiogenous
cells. c. d. Conidia. e–f.
Colony on PDA, e. from
above, f. from below. Scale
Bars: a–g020 μm
periclinal walls darker than the rest of the cell, versicoloured, 4–5 μm (x ¼ 4:5 μm); apical cell 3–5 μm long (x ¼ 4:3 μm),
second cell from base pale brown, 4–5 μm (x ¼ 4:5 μm); third hyaline, subcylindric; with apical appendages 22–32 μm long
cell darker brown, 4–5 μm (x ¼ 4:6 μm); fourth cell darker, (x ¼ 26:5 μm), tubular, 2–3 (rarely 2), arising from apex of
clavispora (holotype). b. Fallen
leaves of Quercus sp.. c-d.
Conidiomata, split
irregularly. e. Section of
conidiomata. f–h. Conidia
with versicolorous median
cells. Scale Bars: e050 μm,
f–h015 μm
Fig. 12 a. Pestalotiopsis clavispora (epitype). b. Conidiophores/ conidiogenous cells. c–d. Conidia. e. Mature conidia f–g. Colony on PDA, f.
from above, g. from below. Scale Bars: a–e020 μm
the apical cell; with basal appendage, 3–5.5 μm (x ¼ 4 μm), leaves of Bruchellia bubalina in South Africa (Guba 1961).
filiform. Thus, P. clavispora appears to have a wide host range and
Colonies on PDA reaching 7 cm diam. after 7 days at 25°C, distribution. Since no ex-type culture is available for this species,
edge undulate, whitish, aerial mycelium on surface, fruiting an epitype with a living culture is designated from a sample
bodies black, concentric; reverse of culture pale luteous. collected in Guangxi Province, China. We would prefer to
Habitat/Distribution: Known to inhabit in Quercus rubra choose an epitype from USA and the original host however, in
in USA and Magnolia sp. in China. order to expedite the understanding of this poorly resolved
Material examined: USA, Auburn, Alabama, on fallen genus, we designate an epitype which has conidial characters
leaves of Quercus rubra L., 10 March 1891, F. Atkinson (length, width and length of apical appendages) fitting that of the
(CUP-A-032389, holotype); CHINA, Guangxi Province, holotype. The present material is a good match for P. clavispora.
Shiwandashan, on dead leaves of Magnolia sp., 28 Dec
1997, Wenping Wu WUFH1486c (HMAS043133 0 Pestalotiopsis diversiseta Maharachchikumbura & K.D.
MFLU12-0418, epitype designated here; ex-type living cul- Hyde, sp. nov.
ture NN0431330MFLUCC 12-0281). MycoBank: MB 800526
Additional culture examined: CHINA, Yunnan Guangxi Figure 13 a–g.
Province, Shiwandashan, on dead leaves of Magnolia sp., 28 Etymology: The specific epithet is based on the diverse
December 1997, Wenping Wu (NN043011 0MFLUCC arrangement, Latin0diversisetae of the apical appendages.
12-0280) Conidia fusoid to ellipsoid, straight to slightly curved, 4–
Notes: Pestalotiopsis clavispora is known as a plant path- septate, 27–34×5.5–8 μm ( x ¼ 29:7 6:3 μm ), with basal
ogen but has been isolated as a common endophyte in recent cell obconic and obtuse at the base, hyaline, thin-walled and
studies (Keith et al. 2006; Espinoza et al. 2008; Liu et al. 2007; verruculose, 3–6 μm long (x ¼ 4:5 μm), with three median
Wei et al. 2007). The holotype of P. clavispora was recorded cells, doliiform, concolorous, olivaceous, septa and periclinal
from fallen leaves of Quercus rubra, in Auburn, Alabama, walls darker than the rest of the cell, wall rugose, together 17–
USA. In addition, P. clavispora has been recorded from leaves 21 μm long ( x ¼ 19 μm ) second cell from base 5–7 μm
of black oak, Quercus minima and on fruit husks and leaves of (x ¼ 5:8 μm ); third cell 6–8 μm (x ¼ 6:8 μm ); fourth cell
Aleurites fordii grows in different parts of USA and on living 6–7 μm (x ¼ 6:3 μm); apical cell hyaline, cylindrical 4–7 μm
long (x ¼ 6 μm); with 3–5 tubular appendages (rarely 2); some in P. leucopogonis (Nag Rag 1993). Although P. diversiseta
appendages branched, slightly swollen at the tip, arising from and P. perseae have knobbed apical appendages with similar
the apex of the apical cell and sometimes arising from the attachment to the apical cell, and length (22–30 vs 10–
different parts of the apical cell, 22–30 μm long (x ¼ 26 μm); 23 μm), the species can be distinguished by its concolorous
with basal appendage 5–9 μm long, rarely absent. median cells which in P. perseae are versicoloured with
Colonies on PDA reaching 7 cm diam. after 8 days at irregular longitudinal ridges (Guba 1961; Nag Rag 1993).
25 °C, edge fimbriate, whitish, with dense, aerial mycelium
on surface, with black fruiting bodies, gregarious; reverse of Pestalotiopsis ellipsospora Maharachchikumbura & K.D.
the culture white. Hyde, sp. nov.
Habitat/Distribution: Endophyte on living leaf of Rhodo- MycoBank: MB 800528
dendron sp., Yunnan Province, China. Figure 14 a–h.
Material examined: CHINA, Yunnan Province, Kunming, Etymology: The specific epithet is based on the ellipsoid
Kunming Botanical Garden, living leaves of Rhododendron shape, Latin0ellipsospora, of the conidia.
sp., 19 March 2002, Wenping Wu HN26-5 (HMAS047261, Conidia 19–25×5–6.5 μm (x ¼ 21:7 6 μm), fusiform,
holotype; MFLU12-0423, isotype; ex-type living culture straight to slightly curved, 4-septate; with basal cell conical
NN0472610MFLUCC 12-0287). Table 6 with obtuse end, hyaline, thin and smooth-walled, 4–5 μm
Notes: long (x ¼ 4:3 μm); with three median cells 13–15 μm long
Pestalotiopsis diversiseta is a morphologically distinct (x ¼ 14:1 μm), dark brown, septa and periclinal walls darker
species, also shown in its DNA phylogeny. However, it than the rest of the cell, versicoloured, second cell from base
has an overlapping conidial size with P. leucopogonis, P. pale brown, 4–5 μm (x ¼ 4:8 μm); third cell darker brown,
perseae and P. theae (Guba 1961; Nag Rag 1993). Pestalo- 4–5 μm (x ¼ 4:7 μm); fourth cell darker, 4–5 μm (x ¼ 4:5
tiopsis diversiseta can be differentiated from all these spe- μm); apical cell 3–4 μm long (x ¼ 3:8 μm), hyaline, conical;
cies by its morphological distinction. P. diversiseta has 3–5 with apical appendages 5–12 μm long (x ¼ 8 μm), tubular,
apical appendages which differ from P. leucopogonis (7–11 1–3, arising from the apex of the apical cell; basal append-
apical appendages), P. perseae (2–4 apical appendages) and age small or absent, 3–4 μm long (x ¼ 3:4 μm), filiform.
P. theae (7–11 apical appendages) (Guba 1961; Nag Rag Colonies on PDA reaching 7 cm diam. after 6 days at
1993). Its apical appendages are also knobbed unlike those 25 °C, edge crenate, whitish, with aerial mycelium on the
Fig. 13 a. Pestalotiopis diversiseta (holotype). a–g. Conidia. h–i. Colony on PDA, h. from above, i. from below. Scale Bars: a–g020 μm
Table 6 Synopsis of Pestalotiopsis diversiseta and related species (Guba (1961))
Species P. diversiseta P. theae a P. leucopogonis b

P. perseae b
Conidia size (μm) 27–34×5.5–8 22–32×5–8 27–32×7.5–9.5 24–36×7–8

Median cells Concolorous, olivaceous Concolorous, dark brown Concolorous, brown Versicolorous
to olivaceous
Apical appendages: 3–5 (sometimes branched) 2–4 (not branched) 7–11(not branched) 2–4(not branched)
Apical appendage length (μm) 22–30, unequal 25–50 12–19 10–23
Appendage tip Knobbed Knobbed Not knobbed Knobbed
Position of appendage on apical cell Top to middle Apex only 3 rows (top, middle Top row and
and bottom) subapical row
a
Guba (1961)
b
Nag Rag (1993)
surface, with black, gregarious fruiting bodies; reverse of the apical cell; basal appendage present (rarely absent), fili-
the culture white. form 3–5 μm (x ¼ 4 μm).
Habitat/Distribution: Saprobe on dead plant material in Description from epitype (Fig. 16 a–h)
Yunnan Province, China and Chiang Rai Province Thailand. Conidiophores indistinct, arising in the concavity of acer-
Material examined: CHINA, Yunnan Province, on dead vuli. Conidiogenous cells discrete, simple, short, filiform, 2–4
plant materials, Guo Liang-Dong Guo986 (MFLU12-0420; um. Conidia 19.2–23.4×5.5–7 μm (x ¼ 20:6 6:7 μm), fu-
holotype; ex-type living culture MFLUCC 12-0283). siform to ellipsoid, straight to slightly curved, 4-septate; basal
Additional culture examined: THAILAND, Chiang Rai, Tool cell conic, hyaline, thin and smooth-walled, 3.2–5 μm long
Kwan, Huay Mesak waterfall, on dead plant material, 12 Janu- (x ¼ 4:4 μm), three median cells hyaline, versicoloured, verru-
ary 2010, S.S.N Maharachchikumbura (MFLUCC 12-0280) culose, 12.7–15.3 μm long (x ¼ 13:7 μm); second cell from
Notes: Pestalotiopsis ellipsospora (conidia 19–25×5– base pale brown to olivaceous, 4.1–5.2 μm (x ¼ 4:8 μm); third
6.5 μm) can be morphologically distinguished from its cell darker brown to olivaceous, 4.7–5.3 μm (x ¼ 5 μm); fourth
phylogenetically closely related species, P. samarangensis cell darker, 4.9–5.7 μm (x ¼ 5:3 μm); apical cell 4–5 μm long
(conidia 18–21×6.5–7.5 μm) (Maharachchikumbura et al. ( x ¼ 4:3 μm ), hyaline, cylindric to subcylindric; apical cell
2012b). Pestalotiopsis samarangensis has three long apical hyaline, subcylindric to conic 3.3–4.4 μm (x ¼ 3:7 μm); apical
appendages (12–18 μm long) whereas in P. ellipsospora the appendages 8–15 μm long ( x ¼ 12:6 μm ), 2–3 (mostly 3),
1–3 appendages are shorter (5–12 μm). arising from the apex of the apical cell; basal appendage present
(rarely absent), filiform, 3–6 μm long (x ¼ 4:3 μm).
Pestalotiopsis foedans (Sacc. & Ellis) Steyaert, Bull. Jard. Colonies on PDA reaching 7 cm diam. after 6 days at
bot. État Brux. 14: 329 (1949) Basionym: Pestalotia foe- 25°C, edge undulate, whitish, aerial mycelium on surface,
dans Sacc. & Ellis, Michelia 2(no. 8): 575 (1882) with black fruiting bodies, gregarious; reverse of culture
MycoBank: MB289196 whitish (rarely pale luteous).
Description from holotype (Fig. 15 a–h) Habitat/Distribution: Saprobe on bark of Thuja occidenta-
Conidiomata acervuli, with basal stroma and lateral wall lis, USA and mangrove leaves, Calliandra haematocephala
1–3 cells thick; the wall cells pale brown, textura angularis, and Neodypsis decaryi, China.
200– 400×150– 300 μm. Conidiophores reduced to con- Material examined: USA, Newfield, New Jersey, on decay-
idiogenous cells arising in the concavity of acervuli. Coni- ing bark of white cedar, Thuja occidentalis L., October 1880,
diogenous cells discrete, simple, short, filiform. Conidia 19– Ellis and Harkness (BPI 0405695, holotype); CHINA, Xing-
24×5.7–6.9 μm (x ¼ 20:7 6:4 μm), fusiform to ellipsoid, long, Hainan, on mangrove leaves, April 2005, A.R. Liu L443
straight to slightly curved, 4-septate; basal cell conic, hyaline, (MFLU 12-0424, epitype designated here; extype living
thin and smooth-walled, 3.2–4.5 μm long (x ¼ 4 μm); three culture-CGMCC 3.9123).
median cells 12.5–14.6 μm long (x ¼ 14 μm), hyaline, versi- Additional culture examined: CHINA, Xinglong, Hainan,
coloured, verruculose; second cell from base pale brown to on leaves of Calliandra haematocephala, May 2004, A.R. Liu
olivaceous, 4.3–5.7 μm (x ¼ 4:9 μm); third cell darker brown L101 (CGMCC 3.9202); CHINA, Xinglong, Hainan, on
to olivaceous, 4.7–6 μm (x ¼ 5 μm); fourth cell darker, 4.5– leaves of Neodypsis decaryi, May 2004, A.R. Liu L96
5 μm (x ¼ 4:7 μm); apical cell 4–5 μm long (x ¼ 4:3 μm), (CGMCC 3.9178).
hyaline, cylindric to subcylindric; apical appendages 6–18 μm Notes: The holotype of P. foedans was recorded from decay-
long (x ¼ 13:3 μm), 2–3 (mostly 3), arising from the apex of ing bark of white cedar, in New Jersey, USA. In addition,
Fig. 14 a. Pestalotiopsis ellipsospora (holotype). a–f. Conidia. g–h. Colony on PDA, g. from above, h. from below. Scale Bars: a–g020 μm
P. foedans was recorded from Cupressus thyoides in New Jersey, Etymology: From the Latin, inflexus in reference to the
USA; on Cryptomeria japonica in Philadelphia and Japan; curved nature of the conidia.
leaves and twigs of C. japonica in Princeton and on needles of Conidiophores most often reduced to conidiogenous
Pinus mugo in Pennington (Guba 1961). Thus, P. foedans cells, simple, hyaline, smooth-walled. Conidiogenous cells
appears to have a wide host range and distribution. Recently, discrete, ampulliform to lageniform, smooth, thin-walled,
P. foedans was discovered as a source of bioactive metabolites hyaline or pale olivaceous. Conidia fusoid to ellipsoid,
of high economic importance (Ding et al. 2008). Since no ex- straight to slightly curved, 4-septate, 24–31 × 6–9 μm
type culture is available for this species, an epitype with a living (x ¼ 27 7:6 μm), basal cell conic to obconic, hyaline or
culture is designated from a sample collected in Hainan Prov- slightly olivaceous, thin-walled and verruculose, 5–7 μm
ince, China. We would prefer to choose an epitype from USA long (x ¼ 5:7 μm), with 3 median cells, doliiform to cylin-
and the original host however, in order to expedite the under- drical, with thick verruculose walls, constricted at the septa,
standing of this poorly resolved genus we choose to be prag- concolorous, olivaceous, with septa and periclinal walls
matic and designated an epitype which has conidial characters darker than the rest of the cell, wall rugose, together 15–
(length, width and length of apical appendages) similar to that of 19 μm long (x ¼ 17:1 μm) second cell from base 5–7 μm
the holotype and hence this is a good match for P. foedans. (x ¼ 5:7 μm); third cell 5–7 μm (x ¼ 5:8 μm); fourth cell
4.5–6 μm (x ¼ 5:3 μm); apical cell hyaline, subcylindrical
Pestalotiopis inflexa Maharachchikumbura & K.D. Hyde, to cylindrical 4–5 μm long (x ¼ 4:6 μm); 2–5 tubular apical
sp. nov. appendages (mostly 3–4), often arising from the apex of the
MycoBank: MB 800530 apical cell or rarely arising from just below the apex of
Figure 17 a–i. apical cell, 20–30 μm long ( x ¼ 24 μm ), unequal, rarely
foedans (holotype). b. on
decaying bark of white cedar
Thuja occidentalis c.
Conidiomata, split irregularly.
d. Section of conidiomata.
e. Conidiogenous cells
f–h. Conidia with versicolorous
median cells. Scale Bars:
d050 μm, e–h020 μm
branched; basal appendage present, relatively long 9–15 μm of the apical cell. In P. inflexa, the appendages usually arise
(x ¼ 12 μm). from the tip of the apical cell and rarely from the middle.
Colonies on PDA reaching 7 cm diam. after 18 days at
25 °C, edge undulate, whitish, with dense, aerial mycelium Pestalotiopsis intermedia Maharachchikumbura & K.D.
on surface, with black, gregarious fruiting bodies; reverse of Hyde, sp. nov.
the culture yellowish. MycoBank: MB 800532
Habitat/Distribution: Endophyte in living leaves of un- Figure 18 a–h.
identified plant, Hunan Province, China. Etymology: From Latin, intermediate pertaining to the
Material examined: CHINA, Hunan Province, Yizhang intermediate size of the conidia.
County, Mangshan, living leaf of unidentified tree, 12 April Conidiophores indistinct. Conidiogenous cells discrete, sim-
2002, Wenping Wu HN14-2 (HMAS047098, holotype; ple, filiform, smooth, thin-walled, hyaline, and short. Conidia
MFLU12-0413, isotype; ex-type living culture NN0470980 fusoid to ellipsoid, straight to slightly curved, 4-septate, 24–28×
MFLUCC 12-0270). 5.5–6.5 μm (x ¼ 25:7 6 μm), basal cell conic to obconic with
Notes: Pestalotiopsis inflexa can be differentiated from obtuse end, hyaline, thin- and verruculose, 4–5 μm long (x ¼
its close relatives in the β-tubulin, tef1 and combined phylo- 4:8 μm), with three median cells, doliiform, concolorous, oli-
gram in Clade A. The characteristic morphology of P. vaceous to brown, septa and periclinal walls darker than the rest
inflexa is due to its divergent, 2 to 5 apical appendages, of the cell, wall rugose, together 15–19 μm long (x ¼ 17 μm)
sometimes arising from the middle of apical cell and by a second cell from base 5–6 μm (x ¼ 5:7 μm); third cell 5–6 μm
relatively long basal appendage (9–15 μm). Morphological- (x ¼ 5:7 μm); fourth cell 5–6.5 μm (x ¼ 5:2 μm); apical cell
ly similar species to P. inflexa in conidial size is P. thujicola hyaline, conic to cylindrical 4–5 μm long (x ¼ 4:5 μm); with 2–
(J.L. Maas) Y. Suto & Tak. Kobay (25–31×6.5–10 μm) 3 tubular apical appendages (rarely 4), arising from the apex of
(Maas 1971). However, P. thujicola can be differentiated the apical cell, 10–28 μm long (x ¼ 18:5 μm), unequal; basal
by its 3–6 apical appendages radiating from different parts appendage present 6–10 μm (x ¼ 7:5 μm), rarely absent.
Fig. 16 a. Pestalotiopsis foedans (epitype). b–c. Conidiogenous cells d–f. Conidia with versicolorous median cells. g–h. Colony on PDA, g. from
above, h. from below. Scale Bars: a–f020 μm
Colonies on PDA reaching 7 cm diam. after 6 days at Pestalotiopsis jesteri Strobel, J.Yi Li, E.J. Ford & W.M.
25 °C, edge entire, whitish, with dense, aerial mycelium on Hess, in Strobel, Li, Ford, Worapong, Baird & Hess, Myco-
surface, fruiting bodies black; reverse of culture whitish to taxon 76: 260 (2000)
pale yellow. MycoBank: MB466231
Habitat/Distribution: Saprobe/endophyte on unidentified Figure 19 a–j.
trees, Hubei and Yunnan provinces, China. Conidiophores indistinct. Conidiogenous cells lageni-
Material examined: CHINA, Hubei Province, Sheng- form to subcylindrical, colourless, smooth, proliferation
nongjia, on dead leave of unidentified tree, 24 March once or twice. Conidia fusoid to ellipsoid, straight to slight-
2003, Wenping Wu WUFH7033 (HMAS047642, holotype; ly curved, 4-septate, 23–29×5.5–7 μm (x ¼ 25 6:1 μm),
MFLU12-0410, isotype; ex-type living culture NN0476420 basal cell obconic, colorless, thin- and verruculose, 5–6 μm
MFLUCC 12-0259). long (x ¼ 5:3 μm), with three median cells, subcylindrical,
Additional culture examined: CHINA, Yunnan Hunan with thick verruculose walls, constricted at the septa, con-
Province, Yizhang County, Mangshan, on living leaf of colorous, pale brown, together 14–16.5 μm long (x ¼ 15:2
unidentified plant, 12 April 2002, Wenping Wu HN28-16 μm) second cell from base 4.5–6.5 μm (x ¼ 5:8 μm); third
(NN0470730MFLUCC 12-0260). cell 4–6 μm (x ¼ 5:2 μm); fourth cell 5–6 μm (x ¼ 5:2 μm);
Notes: The morphologically similar species to P. inter- apical cell colorless, obconic, acute at the apex, 4–5 μm
media (24–28×5.5–6.5 μm) in conidial size are P. lespede- long (x ¼ 4:3 μm); with 4 tubular appendages, 14–20 μm
zae (Syd.) Bilgrami (20–25 × 7–9 μm) (Guba 1961), P. long, one arising from the apex and rest arising from just
osyridis (Thüm.) H.T. Sun & R.B. Cao (22–28×5–7 μm) above the septum separating upper median and apical cell;
and P. cocculi (Guba) G.C. Zhao & N. Li (22–29×5.5– basal appendage present, 4–5 μm long (x ¼ 4:5 μm).
7 μm) (Guba 1961). Pestalotiopsis intermedia can be dif- Colonies on PDA reaching 7 cm diam. after 7 days at
ferentiated from P. lespedezae by its long and thin conidia; and 25 °C, edge lobate, whitish yellow, with dense, aerial my-
from P. osyridis and P. cocculi by its long apical appendages celium on surface, with black, gregarious fruiting bodies;
(P. osyridis usually has 3 apical appendages (rarely 2) mea- reverse of the colony whitish yellow.
suring up to 14 μm long and in P. cocculi there are three apical Habitat/Distribution: Endophyte on Fagraea bodenii, Papua
appendages (sometimes 2), up to 11–12 μm long). New Guinea and saprobic on dead plant material, China.
Fig. 17 a. Pestalotiopsis inflexa (holotype). b–c. Conidiophores/ conidiogenous cells. d–g. Conidia. h–i. Colony on PDA, h. from above, i. from
below. Scale Bars: a–g020 μm
Culture examined: CHINA, Yunnan Province, on proliferations, sometimes remain vegetative. Conidia fusiform,
dead plant material, Wenping Wu (NN042849 0 straight to slightly curved, 4-septate, 24–33 × 4.7–6 μm
MFLUCC 12-0279). ( x ¼ 29 5:5 μm ), basal cell conic to obconic, hyaline or
Notes: Pestalotiopsis jesteri has a similar morphology to slightly olivaceous, thin- and verruculose, 3.5–5.5 μm long
that of P. montellica (Sacc. & Voglino) Tak. Kobay (Guba (x ¼ 4:4 μm), with three median cells, doliiform to cylindrical,
1961) and may be a synonym. There are no sequence data with thick verruculose walls, constricted at the septa, concolo-
available for P. montellica in GenBank, while ITS sequence rous, olivaceous, septa and periclinal walls darker than the rest
data is available for the type of P. jesteri (Strobel et al. 2000). of the cell, together 17–21 μm long (x ¼ 19 μm) second cell
We therefore refrain from taking any action concerning syn- from base 5–6.2 μm (x ¼ 5:5 μm); third cell 6–7 μm (x ¼ 6:
onymy at this stage. Pestalotiopsis jesteri is, however, distinct 3 μm); fourth cell 6–8 μm (x ¼ 6:6 μm); apical cell hyaline,
from all other species in the genus both in morphology and cylindrical to subcylindrical 4–5 μm long (x ¼ 4:2 μm); with
phylogeny. The attachment and arrangement of apical appen- 2–3 tubular apical appendages (rarely 1), arising from
dages at the apical cell are noticeably distinct. the apex of the apical cell, 10–20 μm long ( x ¼ 15 μm ),
unequal in length; basal appendage present, rarely two, 4–
Pestalotiopis linearis Maharachchikumbura & K.D. Hyde, 7 μm long (x ¼ 5:7 μm).
sp. nov. Colonies on PDA reaching 7 cm diam. after 6 days at
MycoBank: MB 800531 25 °C, edge entire, whitish, with dense, aerial mycelium on
Figure 20 a–h. surface, with black, gregarious fruiting bodies; reverse of
Etymology: The specific epithet is based on the linear culture white.
shape of the conidia and in Latin, linear is linearis. Habitat/Distribution: Endophytes on living leaves of Tra-
Conidiophores often reduced to conidiogenous cells, some- chelospermum sp. and Tsuga sp., Yunnan Province, China.
times sparsely septate at the base and unbranched or branched, Material examined: CHINA, Yunnan Province, Kunm-
hyaline, smooth. Conidiogenous cells discrete ampulliform ing, Kunming Botanical Garden, on living leaves of Trache-
to lageniform, smooth, thin-walled, hyaline, with 1–2 lospermum sp., 19 March 2002, Wenping Wu KBG14-3
Fig. 18 a. Pestalotiopsis intermedia (holotype). b–c. Conidiophores/ conidiogenous cells. d–f. Conidia. g–h. Colony on PDA, g. from above, h.
from below. Scale Bars: a–f020 μm
(HMAS047190 holotype; MFLU12-0414, isotype; ex-type thin-walled, slightly red, rarely hyaline, with 2–3 proliferations.
living culture NN0471900MFLUCC 12-0271). Conidia fusoid to ellipsoid, straight to slightly curved, 4-
Additional culture examined: CHINA, Yunnan Province, septate, 17.5–21.8×5.7–7 μm (x ¼ 19:2 6:2 μm), basal cell
Kunming, Kunming Botanical Garden, on living leaves of obconic, hyaline, thin- and verruculose, 3.1–4 μm long (x ¼ 3
Tsuga sp., 19 March 2002, Wenping Wu KBG16-7 :6 μm ), with three median cells, doliiform to subcylindrical,
(NN0471410MFLUCC 12-0272). with thick verruculose walls, constricted at the septa, concolo-
Notes: Pestalotiopsis linearis is a distinct species both in rous, olivaceous with slightly red, septa and periclinal walls
conidial morphology and phylogeny. It can be easily differ- darker than the rest of the cell, wall rugose, together 11.8–
entiated from its phylogenetically related species P. inter- 13.8 μm long (x ¼ 12:9 μm) second cell from base 4–5.3 μm
media both in tef1 and combined trees (Figs. 3 and 4) and (x ¼ 4:5 μm); third cell 3.3–5.1 μm (x ¼ 4:3 μm); fourth cell
morphologically related species in the concolorous groups 4.2–5.4 μm (x ¼ 4:7 μm); apical cell hyaline, conic to cylin-
such as P. macrochaeta (Speg.) J. Xiang Zhang & T. Xu drical 2.6–4.2 μm long (x ¼ 3:3 μm); with 1–3 tubular apical
(22–31×8–10 μm) and P. caudata (Syd.) B. Sutton (22– appendages, some appendages branched, arising from the apex
31×8–10 μm) (Saccardo 1902). In P. linearis (24–33×4.7– of the apical cell, 14–22 μm long ( x ¼ 16:5 μm ); basal
6 μm) conidia are much thinner than these two species. appendage present 2–5.7 μm (x ¼ 4:1 μm), rarely absent.
Colonies on PDA reaching 7 cm diam. after 27 days at
Pestalotiopsis rosea Maharachchikumbura & K.D. Hyde, 25 °C, edge undulate, whitish or pale red, with dense, aerial
sp. nov. mycelium on surface, with black to reddish brown fruiting
MycoBank: MB 800521 bodies, gregarious; reverse of culture white or slightly red to red.
Figure 21 a–k. Habitat/Distribution: Endophyte on living leaves of
Etymology: The specific epithet is based on the Latin Pinus sp., Yunnan Province, China.
roseus in reference to the rose-colored, colony of this Material examined: CHINA, Yunnan Province, Kunming,
species Kunming Botanical Garden, on living leaves of Pinus sp., 19
Conidiophores septate, unbranched, up to 20 μm long, March 2002, Wenping Wu KBG25-3 (HMAS047135, holo-
often reduced to conidiogenous cells, smooth walled; Coni- type; MFLU12-0409, isotype; ex-type living culture
diogenous cells discrete, ampulliform to lageniform, smooth, NN0471350MFLUCC 12-0258).
Fig. 19 a. Pestalotiopsis jesteri (NN042849). b.c. Conidiophores/ conidiogenous cells. d–g. Conidia. i–j. Colony on PDA, i. from above, j. from
below. Scale Bars: a–g020 μm
Notes: Pestalotiopsis rosea is a distinct species in the periclinal walls darker than the rest of the cell, versicol-
genus. The reddish colony is unique to the species and this oured, verruculose, second cell from base pale brown to
can be found even in conidiogenous cells and in some olivaceous, 4.5–7 μm (x ¼ 5:3 μm); third cell darker brown
conidia. This species was quite similar to the type species to dark olivaceous, 4–5 μm (x ¼ 4:7 μm); fourth cell darker,
of the genus Pestalotiopsis; P. guepinii (Desm.) Steyaert 4–6 μm (x ¼ 5 μm); apical cell 4–5 μm long (x ¼ 4:3 μm),
(Guba 1961; Nag Rag 1993) isolated from Camellia japon- hyaline, cylindric to subcylindric; apical appendages 23–
ica. In P. guepini, conidia are 14–21×5.5–6.6 μm, with 1–3 35 μm long (x ¼ 27:3 μm), tubular, 2–4 (often 3), arising
apical appendages that are sometimes knobbed at their api- from the apex of the apical cell; basal appendage, 4–7 μm
ces. However, in P. rosea apical appendages are not (x ¼ 6 μm), filiform.
knobbed and according to Guba (1961) P. guepinii is re- Colonies on PDA reaching 7 cm diam. after 7 days at 25 °C,
stricted to Camellia species. edge crenate, off white, aerial mycelium on surface, fruiting
bodies black, gregarious; reverse of culture off white.
Pestalotiopsis saprophyta Maharachchikumbura & K.D. Habitat/Distribution: Saprobes on leaves of Magnolia
Hyde, sp. nov. sp., Yunnan Province, China.
MycoBank: MB 800525 Material examined: CHINA, Yunnan Province, Kunm-
Figure 22. a–h. ing, Kunming Botanical Garden, on leaves of Magnolia sp.,
Etymology: From the Latin saprophyta. 19 March 2002, Wenping Wu KBG29-2 (HMAS047136,
Conidiophores 0–1-septate, unbranched or irregularly holotype; MFLU12-0419, isotype; ex-type living culture
branched, colorless, smooth-walled. Conidiogenous cells NN0471360MFLUCC 12-0282).
discrete or integrated, lageniform, subcylindric to cylindric, Notes: Pestalotiopsis saprophyta is a distinct species in
hyaline. Conidia 22–30 × 5–6 μm ( x ¼ 24:9 5:7 μm ), the versicolour group (Clade B) with a higher conidial
fusiform, straight to slightly curved, 4-septate; basal cell length to width ratio compared with other species. In β-
conical to obtuse, hyaline, thin and smooth-walled, 4– tubulin and tef1 phylograms, it separates well with other
7 μm long (x ¼ 5 μm); three median cells 14–20 μm long species in the Clade B. P. saprophyta separates from its
( x ¼ 15:5 μm ), dark brown to olivaceous, septa and phylogenetic relative, P. foedans (19–25×5.5–7 μm) by
Fig. 20 a. Pestalotiopsis linearis (holotype). b–c. Conidiophores/ conidiogenous cells. d–f. Conidia. g. h. Colony on PDA, g. from above, h. from
below. Scale Bars: a–f020 μm
having larger conidia (22–30×5–6 μm) and longer apical to cylindrical, concolorous, olivaceous, verruculose, septa
appendages (23–35 μm in P. saprophyta and 6–18 um in P. and periclinal walls darker than the rest of the cell, together
foedans). Other morphologically related species are P. bata- 13–17 μm long (x ¼ 14 μm) second cell from base 4–6 μm
tas (Ellis & Everh.) G.C. Zhao & N. Li (23–28×7–8 μm) (x ¼ 4:7 μm); third cell 4–6 μm (x ¼ 4:5 μm); fourth cell 4–
(Zhao and Li 1995), P. matildae (Richatt) S.J. Lee & Crous 6 μm (x ¼ 4:4 μm); apical cell hyaline, conic to subcylin-
(22–32 × 6–8 μm) (Lee et al. 2006), and P. paeoniae drical 4–6 μm long (x ¼ 4:8 μm); with 2–3 tubular apical
(Servazzi) Steyaert (20–28×6–8 μm) (Guba 1961). Howev- appendages, arising from the apex of the apical cell, 9–
er, in P. saprophyta conidia are thinner and apical appen- 18 μm long (x ¼ 13:6 μm); with a basal appendage, rarely
dages are longer. two, 4–8 μm (x ¼ 6:3 μm) long.
Colonies fast growing on PDA, reaching 7 cm diam. after
Pestalotiopsis trachicarpicola Y. M. Zhang & K.D. Hyde in 6 days at 25 °C, edge fimbriate, yellowish white, dense,
Zhang, Maharachchikumbura, McKenzie and Hyde, Cryp- aerial mycelium on surface, fruiting bodies black; reverse of
togamie mycol (in press) (2012a) the culture yellowish white.
MycoBank: MB 564879 Habitat/Distribution Pathogen on Trachycarpus fortunei
Figure 23 a–f. and saprobes on dead plant materials in China.
Conidiophores indistinct. Conidiogenous cells discrete, Material examined: CHINA, Yunnan Province, Kunming,
simple, filiform, smooth, thin-walled, hyaline. Conidia Kunming Botanical Gardens, on leaf spots on living leaves of
fusoid to ellipsoid, broad-clavate, straight to slightly curved, Trachycarpus fortunei, March 2011, K.D. Hyde OP068
4-septate, 20–25×5.5–7.2 μm ( x ¼ 23:5 6:5 μm ), basal (IFRD 9026,holotype; ex-type living culture IFRDCC 2440).
cell conic to acute, hyaline, thin-walled and verruculose, 4– Additional culture examined: CHINA, Hunan Province,
6 μm long (x ¼ 4:9 μm), with three median cells, doliiform Yizhang County, Mangshan, on living leaf of unidentified
Fig. 21 a. Pestalotiopsis rosea (holotype). b–e. Conidiophores/ conidiogenous cells. f–i. Conidia. j. k. Colony on PDA, j from above, k from
below. Scale Bars: a–i020 μm
tree, 12 April 2002, Wenping Wu HN53-2 (NN0470720 Notes: Pestalotiopsis trachicarpicola is a recently de-
MFLUCC 12-0263); CHINA, Yunnan Province, Kunm- scribed species from Kunming, China, and it causes serious
ing, Kunming Botanical Garden, on living leaf of leaf blotch and defoliation in Trachycarpus fortunei (Zhang
Chrysophullum sp., 19 March 2002, Wenping Wu et al. 2012a). The isolates we obtained as endophytes are
KBG19-3 (NN047196 0MFLUCC 12-0264); CHINA, morphologically and phylogenetically consistent to the type
Hunan Province, Yizhang County, Mangshan, on living of P. trachicarpicola.
leaf of Schima sp., 12 April 2002, Wenping Wu HN40-
8 (NN046983 0MFLUCC 12-0265); CHINA, Hunan Pestalotiopsis umberspora Maharachchikumbura & K.D.
Province, Yizhang County, Mangshan, on living leaf of Hyde, sp. nov.
Sympolocos sp., 12 April 2002, Wenping Wu HN38-6 MycoBank: MB 800534
(NN0469780MFLUCC 12-0266); CHINA, Hunan Province, Figure 24 a–g.
Yizhang County, Mangshan, on living leaf of unidentified tree, Etymology: The specific epithet is based on the Latin0
12 April 2002, Wenping Wu HN14-4 (NN0470990MFLUCC umber, in reference to the umber earth brown colour of the
12-0267). median cells of the conidia.
Fig. 22 a. Pestalotiopsis saprophyta (holotype). b–c. Conidiophores/ conidiogenous cells. d–e. Conidia. f–g. Colony on PDA, f. from above, g.
from below. Scale Bars: a– e020 μm
Conidiophores reduced to conidiogenous cells. Conidioge- MFLU12-0421, isotype; ex-type living culture
nous cells discrete or integrated, lageniform, hyaline, smooth NN0429860MFLUCC 12-0285).
walled, sometimes septate. Conidia 19–25×6–8 μm (x ¼ 21:3 Notes: Pestalotiopsis umberspora is a phylogenetically
6:5 μm), fusiform, straight to slightly curved, 4-septate; basal distinct species in the genus and separates well in tef1 and
cell obconic to conic, hyaline or pale brown, thin and verrucu- combined multi-locus tree with its phylogenetically related
lose, 3–4.5 μm long ( x ¼ 3:8 μm ); three median cells 12– species P. crysea. Its umber coloured and relatively wider
14 μm long (x ¼ 13:1 μm), umber brown to olivaceous, septa mature conidia are characteristic to the species.
and periclinal walls darker than the rest of the cell, versicoloured,
verruculose, second cell from base pale brown, 3–4.5 μm Pestalotiopsis unicolor Maharachchikumbura & K.D.
( x ¼ 3:9 μm ); third cell darker brown, 3.5–5 μm Hyde, sp. nov.
(x ¼ 4:3 μm); fourth cell darker, 3.5–4.5 μm (x ¼ 4:2 μm); MycoBank: MB 800523
apical cell 3–4.5 μm long ( x ¼ 3:9 μm ), hyaline, conic Figure 25 a–g.
to obconic; with apical appendages 22–35 μm long (x ¼ 27:7 Etymology: Specific epithet in reference to concolorous
μm), tubular, 1–3 (mainly 3), arising from the upper portion of median cells.
the apical cell; basal appendage, 5–7 μm (x ¼ 5:9 μm), filiform. Conidiophores indistinct. Conidiogenous cells discrete
Colonies on PDA reaching 7 cm diam. after 6 days at 25 °C, ampulliform to lageniform, smooth, thin-walled, hyaline,
edge entire, whitish, aerial mycelium on surface, fruiting bodies with 1–2 proliferations. Conidia fusoid to ellipsoid, straight
black, gregarious; reverse of culture pale yellow. to slightly curved, 4-septate, 20–24.5×4–6 μm (x ¼ 22 5
Habitat/Distribution: Saprobe on dead plant material, :1 μm ), basal cell conic to obconic, hyaline or slightly
Guangxi Province, China. olivaceous, thin- and verruculose, 4–5.5 μm long (x ¼ 4:9
Material examined: CHINA, Guangxi Province, Shiwan- μm), with three median cells, doliiform to cylindrical, with
dashan, on dead leaves of unidentified plant, 30 December thick verruculose walls, constricted at the septa, concolo-
1997, Wenping Wu WU1554j (HMAS042986, holotype; rous, olivaceous, septa and periclinal walls darker than the
Fig. 23 Pestalotiopsis
trachicarpicola a. Conidia in
culture. b. Conidiogenous cells.
c. d. Conidia. e–f.
Colony in culture, e. from
above, f. from below. Scale
Bars: a–d020 μm
rest of the cell, wall rugose, together 13–16 μm long (Sacc. & Berl.) W.P. Wu. (17–23×5–7 μm) (Guba 1961).
(x ¼ 14:7 μm) second cell from base 4–5 μm (x ¼ 4:8 μ However, 2–3 tubular apical appendages (11–20 μm long) of
m); third cell 4–5 μm (x ¼ 4:8 μm); fourth cell 4–6 μm P. unicolor are longer than in Pestalotia kawakamii (3 apical
(x ¼ 5 μm); apical cell hyaline, conic to subcylindrical appendages; 5–10 μm long). The conidial width of P. alger-
3–5 μm long ( x ¼ 4:2 μm ); with 2–3 tubular apical iensis is similar to P. unicolor but the length of conidia and
appendages, arising from the apex of the apical cell, 11– apical appendages are smaller in P. algeriensis and
20 μm long (x ¼ 17:5 μm), of unequal length; basal appen- length (up to 16 μm) is shorter than in P. unicolor.
dages present, rarely two, 4–10 μm (x ¼ 6:9 μm) long.
Habitat/Distribution: Endophyte on Rhododendron sp. Pestalotiopsis verruculosa Maharachchikumbura, & K.D.
and unidentified plant, Hunan Province, China. Hyde, sp. nov.
Material examined: CHINA, Hunan Province, Yizhang MycoBank: MB 800527
County, Mangshan, on living leaf of Rhododendron sp., 12 Figure 26 a–h.
April 2002, Wenping Wu HN42-1 (HMAS046974, holo- Etymology: The specific epithet is based on the Latin
type; MFLU12-0417, isotype; ex-type living culture verruculose in reference to the verrucose pattern in walls of
NN0469740MFLUCC 12-0276). three median cells.
Additional culture examined: CHINA, Hunan Province, Conidia ellipsoid, straight to slightly curved, 4-septate,
Yizhang County, Mangshan, on living leaf of unidentified 28–35×9–11 μm ( x ¼ 30:6 10:3 μm ), basal cell conic
tree, 12 April 2002, Wenping Wu HN51-1 (NN0473080 with obtuse end, hyaline, thin-walled and verruculose, 5–
MFLUCC 12-0275). 7 μm long (x ¼ 5:7 μm), with three median cells, doliiform
Notes: Pestalotiopsis unicolor is a distinct species in the to cylindrical, with thick verruculose walls, constricted at
genus from molecular and morphological characters. The mor- the septa, concolorous, olivaceous, septa and periclinal
phologically similar species in conidial size are P. kawakamii walls darker than the rest of the cell, wall rugose, together
Sawada (20–24×5–7 μm) (Guba 1961) and P. algeriensis 18–26 μm long ( x ¼ 21:6 μm ) second cell from base 6–
Fig. 24 a. Pestalotiopsis umberspora (holotype). b. Conidiophores/ conidiogenous cells. c–e. Conidia. f. g. Colony on PDA, f. from above, g.
from below. Scale Bars: a–e020 μm
9 μm (x ¼ 6:8 μm); third cell 6–9 μm (x ¼ 7:5 μm); fourth Notes: Pestalotiopsis verruculosa is a distinct species in
cell 6–9 μm (x ¼ 7:3 μm); apical cell hyaline, conic to sub- terms of morphology, and its molecular phylogeny. It has a
cylindrical 4–6 μm long (x ¼ 4:8 μm); with 2–6 tubular apical relatively large conidial size (28–35×9–11 μm) when com-
appendages (mostly 3–4), arising from the apex of the apical pared with morphologically similar species (P. funerea, 21–
cell (rarely 1 appendage arising from just above the septum 29×7–9.5 μm); (P. multiseta, 22–26×7.5–9.5 μm). It also
separating upper median and apical cell), 25–40 μm long has a long apical appendage (25–40 μm) when compared to
(x ¼ 34 μm); basal appendage present 8–12 μm (x ¼ 9 μm). P. funerea (5–20 μm), P. multiseta (9–16 μm) and P. macro-
Colonies on PDA reaching 7 cm diam. after 15 days at spora (15–22 μm) (Nag Rag 1993).
25 °C, edge undulate, whitish to pale yellow, with dense,
aerial mycelium on surface, with black, gregarious fruiting
bodies; reverse of culture yellow to pale orange. Discussion
Habitat/Distribution: Endophytes on living leaf of Rho-
dodendron sp., Yunnan Province, China. In this study, we include 40 sequenced isolates of Pestalo-
Material examined: CHINA, Yunnan Province, Kunming, tiopsis to provide a backbone tree for the genus Pestalotiop-
Kunming Botanical Garden, on living leaf of Rhododendron sis. Based on morphological and molecular data, we
sp., 19 March 2002, Wenping Wu KBG25-8 (HMAS047309, determined that the 40 sequenced isolates comprise 23 spe-
holotype; MFLU12-0416, isotype; ex-type culture cies of Pestalotiopsis, of which 14 are new. Three species
NN0473090MFLUCC 12-0274). Table 7 are epitypified, and six are recognized as existing species,
unicolor. b.
Conidiophores/conidiogenous
cells. c–e. Conidia. f–g.
Colony on PDA, f. from
above, g. from below. Scale
Bars: a–e020 μm
namely; P. camelliae (Zhang et al. 2012b), P. furcata ellipsospora, P. foedans, P. samarangensis and P. saprophyta,
(Maharachchikumbura et al. 2012a), P. jesteri (Strobel et Clade B diverges from a common ancestor. Within this Sub-
al. 2000), P. samarangensis (Maharachchikumbura et al. clade P. ellipsospora and P. foedans have pale brown, versi-
2012b), P. theae (Maharachchikumbura et al. 2012a) and colorous median conidial cells, while all other species belong-
P. trachicarpicola (Zhang et al. 2012a). There is a need to ing in this sub-clade have dark brown versicolorous median
deposit more sequences of epitypified species of Pestalotiop- conidial cells. Thus, we agree with Jeewon et al. (2003), Liu et
sis in GenBank and this communication provides data on three al. (2010) and Maharachchikumbura et al. (2011) in conclud-
genes for 14 new species, three epitypified species and six ing that the division of the “versicolor group” based on colour
species with type/epitype sequences from GenBank. intensities of the median conidial cell is not a taxonomically
In our studies, we analyzed sequence data from three gene good character. In addition to differences in median, conidial
regions, individually and in combination, to evaluate their cells (Clades A, B and C); the size variation in conidia, and
ability to resolve species limits of Pestalotiopsis. In all cases, presence or absence of basal appendages, can be used as
the genes resolved the species into three strongly supported additional taxonomic characters for defining Pestalotiopsis
Clades (A, B and C). These clades corresponded to three species. Apical appendage characters, such as branching pat-
conidial types: Clade A with pale brown or olivaceous con- tern, number, position of attachment to apical cell, and
colorous median conidial cells, Clade B with versicolorous knobbed or knotted nature are also useful as a species,
median conidial cells and Clade C with dark-coloured con- but not as a generic character (Crous et al. 2012). Differenti-
colorous median conidial cells and with knobbed apical appen- ation of species based on these characters is also sup-
dages. These Clades were also evident in the studies of Jeewon ported by molecular data and has been previously noted by
et al. (2003), Liu et al. (2010) and Maharachchikumbura et al. Jeewon et al. (2003), Hu et al. (2007), Liu et al. (2010) and
(2011). Steyaert (1949) and Guba (1961) had previously Maharachchikumbura et al. (2011).
grouped species with versicolorous conidia into two groups In our study, we tested 10 gene regions for suitability in
based on the intensity of colour of the median cells (umber resolving species in Pestalotiopsis, but narrowed this down
olivaceous and fuliginous olivaceous). Our findings, based on to three most applicable regions, which were tested individ-
combined genes analysis, did not support this arrangement. In ually and in combination, to evaluate the differences be-
the combined phylogenetic tree (Fig. 4) P. clavispora, P. tween species. The ITS is the universal barcode for fungi
Fig. 26 a. Pestalotiopsis verruculosa (holotype). a–f. Conidia. g–h. Colony on PDA, g. from above, h. from below. Scale Bars: a–f020 μm
(Schoch et al. 2012). It has a high sequence and PCR A gene-by-gene assessment of phylogenetic resolution
success rate with higher resolving power within some fungal yielded higher levels with protein genes as compared to
lineages (Bridge et al. 2005; Schoch et al. 2012). The ribosomal regions (Schoch et al. 2009). Cryptic taxa can
species of Pestalotiopsis sequenced with ITS in this study be better resolved using slow evolving protein coding genes
had a high PCR and sequence success rate. However, ITS (Liu et al. 1999; Liu and Hall 2004). Hu et al. (2007) and
could not fulfill the role as candidate gene for species discrim- Liu et al. (2010) used a β-tubulin fragment to study species
ination, as the data did not have high variation between relationships within Pestalotiopsis. This region has also
species. Thus, possible cryptic taxa could not be discriminated been shown to resolve species in other genera in groups
from one another. such as Discosia (Tanaka et al. 2011), Seimatosporium
Table 7 Synopsis of P. verruculosa and related species (Guba (1961))

a a a
Species P. verruculosa P. funerea P. multiseta P. macrospora
Conidia size (μm) 28–35×9–11 21–29×7–9.5 22–26×7.5–9.5 30–40×7–9

Median cells Olivaceous, concolorous Umber or olivaceous, Umber, Olivaceous, versicolour
concolorous concolorous
Apical appendages: 2–6 (mostly 3–4, rarely branched) 3–6 (unbranched) 3–5 (branched) 4–5 (arising separately or
pairs and often branched)
Length (μm) 25–40 5–20 9–16 15–22
Tip Not knobbed Not knobbed Not knobbed Not knobbed
a
Guba (1961)
(Tanaka et al. 2011) and Seiridium (Barnes et al. 2001). Tef1 In the present study multi-locus phylogeny for Pestalo-
is a widely used taxonomic marker and this has been suc- tiopsis species is presented and strives towards providing
cessfully utilized to investigate the kingdom-level phyloge- biological species concepts based on taxonomically impor-
ny of eukaryotes (Roger et al. 1999; Baldauf et al. 2000) and tant morphological characters. This backbone tree needs
species in fungal genera such as Diaporthe (Santos et al. expanding by re-examining type materials of some of the
2010; Udayanga et al. 2012), Fusarium (Geiser et al. 2004; important species described in Pestalotiopsis and using
O’Donnell et al. 2010) and Trichoderma and Hypocrea multi-locus analysis to establish epitypes. We have not
(Druzhinina et al. 2005). In the present study, β-tubulin epitypified some of the more commonly known Pestalotiop-
and tef1 gene regions proved to be favorable taxonomic sis names (e.g. P. disseminata, P. fici, P. longiseta, P. micro-
markers for Pestalotiopsis since they resolved the taxonom- spora, P. neglecta, P. pauciseta, P. photiniae and P. uvicola)
ic relationships of most species studied. Further, tef1 had due of lack of fresh collections and living material. Our new
better PCR amplification success rates (95 %) and was species, however, differ from putatively named examples of
found to be superior to β-tubulin (90 %). Tef1 is therefore the common species in GenBank (e.g. P. microspora, P.
a powerful tool to resolve lineages within Pestalotiopsis. neglecta) and thus we are confident that the species newly
Because of the better PCR and sequencing success rate and introduced in this paper are distinct.
fewer difficulties with alignment, editing and better resolu-
tion, the tef1 gene appears to be a very good molecular Acknowledgments We thank Thailand Research Fund (grant:
marker for phylogenetic investigation of Pestalotiopsis. BRG5280002); The National Research Council of Thailand (grant for
Combined sequence analysis of ITS, β-tubulin and tef1 Pestalotiopsis No: 55201020008); Mae Fah Luang University (grant
for Pestalotiopsis No: 55101020004); The National Natural Science
genes successfully resolved most of the Pestalotiopsis spe-
Foundation of China (grant: 30930005); the Knowledge Innovation
cies used in this study with high bootstrap support. Hu et al. Program of the Chinese Academy of Sciences (grant: KSCX2-YW-Z-
(2007) and Liu et al. (2010) have previously shown that a 0935); Mushroom Research Foundation, Chiang Mai, Thailand; State
combination of β-tubulin and ITS genes gave better species Key Laboratory of Mycology, Institute of Microbiology, Chinese
Academy of Sciences, Beijing, China and the King Saud University
resolution than a single gene and they suggested that at least
for supporting this research. We kindly acknowledge Novozymes,
two genes should be used to resolve species in Pestalotiopsis. CGMCC and ICMP for providing cultures and BPI, CUP and NY
Similar results have been shown in Fusarium (Summerell herbaria for providing type materials for this study.
et al. 2010); Calonectria (Lombard et al. 2010), Phyllosticta
(Wikee et al. 2011), and Colletotrichum (Phoulivong et al.
2010), however the genes best suited for each genus differed. References
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A multi-locus backbone tree for Pestalotiopsis, with a polyphasic

Article in Fungal Diversity · September 2012

Sajeewa Maharachchikumbura Liangdong Guo

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Lei Cai Ekachai Chukeatirote

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Phylogeny and taxonomy of Karst Fungi View project

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A multi-locus backbone tree for Pestalotiopsis,

S. S. N. Maharachchikumbura : E. Chukeatirote : K. D. Hyde P. W. Crous

S. S. N. Maharachchikumbura : E. Chukeatirote : K. D. Hyde (*)

S. S. N. Maharachchikumbura : L.-D. Guo (*) : L. Cai : X. Sun E. H. C. McKenzie

Introduction The use of molecular data in resolving Pestalotiopsis

Table 1 Saprobic Pestalotiopsis species with their host/substrata

Species Host/ substrate References

Table 2 Primers used

Results Sequence analysis of β-tubulin gene data

Table 3 Isolates used in this study

ITS β-tubulin tef1

P. adusta (Ellis & Everh.) Steyaert ICMP6088 JX399006 JX399037 JX399070

Table 4 Comparison of gene

Table 5 Comparison of gene

P. trachicarpicola MFLUCC 12-0263

P. trachicarpicola MFLUCC 12-0263

100 P. trachicarpicola OP068

Clade A, pale brown or 89 P. trachicarpicola MFLUCC 12-0266

100 P. adusta ICMP 6088

100 P. linearis MFLUCC 12-0271

92 P. intermedia MFLUCC 12-0259

94 P. camelliae MFLUCC 12-0277

63 P. foedans CGMCC 3.9202

100 P. theae SC011 Clade C, dark concolorous

61 P. intermedia MFLUCC 12-0259

86 P. unicolor MFLUCC 12-0275

P. inflexa MFLUCC 12-0270

53 P. trachicarpicola MFLUCC 12-0264

P. trachicarpicola MFLUCC 12-0267

100 P. adusta ICMP 6088

P. foedans CGMCC 3.9178

86 P. chrysea MFLUCC 12-0261

100 P. theae SC011

51 P. intermedia MFLUCC 12-0259

100 100 P. camelliae MFLUCC 12-0277

Table 6 Synopsis of Pestalotiopsis diversiseta and related species (Guba (1961))

Species P. diversiseta P. theae a P. leucopogonis b

Conidia size (μm) 27–34×5.5–8 22–32×5–8 27–32×7.5–9.5 24–36×7–8

Table 7 Synopsis of P. verruculosa and related species (Guba (1961))

Conidia size (μm) 28–35×9–11 21–29×7–9.5 22–26×7.5–9.5 30–40×7–9

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