f u n g a l b i o l o g y x x x ( 2 0 1 4 ) 1 e1 4
journal homepage: www.elsevier.com/locate/funbio
Identification and characterization of Pestalotiopsislike fungi related to grapevine diseases in China
Ruvishika S. JAYAWARDENAa,b,c, Wei ZHANGa, Mei LIUa,
Sajeewa S. N. MAHARACHCHIKUMBURAb,c, Ying ZHOUa,
JinBao HUANGa, Somrudee NILTHONGb,c, ZhongYue WANGd,
XingHong LIa,*, JiYe YANa,*, Kevin D. HYDEb,c
a
Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097,
People’s Republic of China
b
Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
c
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
d
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China
article info
abstract
Article history:
Pestalotiopsis-like fungi are an important plant pathogenic genus causing postharvest fruit
Received 29 September 2014
rot and trunk diseases in grapevine in many countries. Pestalotiopsis-like fungi diseases
Received in revised form
were studied in vineyards in nine provinces across China. Multi-gene (ITS, b-tubulin and
30 October 2014
tef1) analysis coupled with morphology showed that a Neopestalotiopsis sp. and Pestalotiopsis
Accepted 9 November 2014
trachicarpicola are associated in causing grapevine fruit rot and trunk diseases in China. Pes-
Corresponding Editors:
talotiopsis trachicarpicola is reported as the causative agent of grapevine diseases in the
JiYe Yan, XingHong Li
world for the first time. Neopestalotiopsis sp. caused significantly longer lesions than the
other taxon present. This study represents the first attempt to identify and characterize
Keywords:
the Pestalotiopsis-like fungi causing grapevine diseases in China using both morphological
b-tubulin
and molecular approaches.
Fruit rot
ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Neopestalotiopsis
tef1
Trunk diseases
Introduction
Grape (Vitis vinifera L. family Vitaceae) is one of the most important economical crops cultivated worldwide mainly for wine
production and fruit consumption. Grapes have been cultivated in China for more than 2000 y and at present it is the
5th most important fruit produced in China (FAO 1999). There
has been a rapid increase in areas that grow grapes across
China. As the cultivation area has increased, many fungal, bacterial and viral diseases have become major problems for grape
cultivation (Urbez-Torres
et al. 2009). These diseases reduce
crop yields, vine growth and increase annual production costs
* Corresponding authors. Tel.: þ86 10 51503434; fax: þ86 10 51503899.
E-mail addresses: ruvi.jaya@yahoo.com (R. S. Jayawardena), zhwei1125@163.com (W. Zhang), liumeidmw@163.com (M. Liu), sajeewa83@yahoo.com (S. S. N. Maharachchikumbura), zhouying16_2013@163.com (Y. Zhou), jbhuang898@sina.com (JinBao Huang), somrudee@mfu.ac.th (S. Nilthong), wangzhy0301@sian.com (ZhongYue Wang), lixinghong1962@163.com (XingHong Li), jiyeyan@gmail.com
(JiYe Yan), kdhyde3@gmail.com (K. D. Hyde).
http://dx.doi.org/10.1016/j.funbio.2014.11.001
1878-6146/ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
2
et al. 2009). Pestalotiopsis, Neopestalotiopsis and
(Urbez-Torres
Pseudopestalotiopsis (Pestalotiopsis-like fungi) belong to the family Amphisphaeriaceae (Barr 1975, 1990; Kang et al. 1998, 1999;
Jeewon et al. 2003; Tejesvi et al. 2007; Maharachchikumbura
et al. 2011, 2014; Hyde et al. 2014) and its species are commonly
present in tropical and subtropical ecosystems (Tejesvi et al.
2007; Maharachchikumbura et al. 2011). Pestalotiopsis-like fungi
consists of members that are difficult to identify at the species
level (Jeewon et al. 2003; Maharachchikumbura et al. 2012). It is
an important plant pathogenic group (Yasuda et al. 2003; Das
et al. 2010; Maharachchikumbura et al. 2011, 2012, 2013, 2014;
Suwannarach et al. 2013; Hyde et al. 2014) and has been reported
as a pathogenic taxon causing postharvest fruit rot and trunk
diseases, including grapevine dieback in different parts of the
world. In the field, initial symptoms of fruit rot disease are
mostly observed at the splits between the pedicel and the berry
and at the wounds of the fruits. Later, the skin of the fruit will
turn reddish brown/brown, and form water-soaked lesions
covered by whitish mycelium with black conidial masses. Severely infected fruits become rotten and separate completely
from the pedicel (Xu et al. 1999; Deng et al. 2013). Shoot damage
is the main symptom of the grapevine trunk infected by Pestalotiopsis-like fungi. When the disease is severe, it results in the
bleaching of canes and formation of fruiting bodies and sometimes the surface of the shoots and canes appears split
(Sergeeva et al. 2005; Urbez-Torres
et al. 2009, 2012).
Pestalotiopsis-like fungi have been reported as pathogens of
grape cultivars causing grapevine dieback in Australia and
USA (Arkansas, Missouri and Texas) and causing fruit rot in
Italy, Japan and Korea (Guba et al. 1961; Ryu et al. 1999; Xu
et al. 1999; Sergeeva et al. 2005; Urbez-Torres
et al. 2009, 2012;
Deng et al. 2013). Pestalotiopsis menezesiana (Bres. & Torr.) Bissett. and Pestalotiopsis uvicola (Spegazzini) Bissett. were the
first two species to be reported from Japan as causal agents
of postharvest disease of grapes (Xu et al. 1999). Pestalotiopsis
uvicola has been reported from various Vitis sp. including Vitis
vinifera and Vitis indusa in Australia, Brazil, Europe, Italy, Japan
and United States (Guba 1961; Sergeeva et al. 2005; UrbezTorres et al. 2009, 2012). Pestalotiopsis menezesiana has been
recorded from India causing severe defoliation of grapevines
and rot of berries (Mundkur & Thirumalachar 1946; Mishra
et al. 1974). Recent studies in Australia and America showed
that Pestalotiopsis-like fungi occurred not only on leaves, but
also on canes, wood, berries and flowers (Castillo-Pando
et al. 2001; Sergeeva et al. 2001; Urbez-Torres
et al. 2009, 2012;
Deng et al. 2013). Urbez-Torres et al. (2009, 2012) have shown
the association of Pestalotiopsis-like fungi with grapevine dieback, particularly with wedge-shaped cankers and their association with dark streaking of the wood, with light-brown
discolouration and central necrosis. Pestalotiopsis-like fungi
was one of the most prevalent fungi isolated from the cankers
of grapevines in Arkansas and Missouri (Urbez-Torres et al.
2012), while it was the second most common genus isolated
from grapevine cankers in Texas. It was isolated from the
woody stems, providing the first report of a Pestalotiopsis sp.
as a canker pathogen on grapevines (Urbez-Torres et al.
2009). Pestalotiopsis guepini (Desm.) Steyaert was reported to
cause disease of grape canes in Yunnan Province of China
(Zhang et al. 2007). However, in many cases the specific identity of the Pestalotiopsis species causing disease of grapes has
R. S. Jayawardena et al.
not been given (Castillo-Pando et al. 2001; Sergeeva et al.
2001; Urbez-Torres et al. 2009, 2012; Deng et al. 2013).
Although there have been many studies identifying the
pathogens causing diseases of grapes in China (Peng et al.
2013; Dissanayake et al. 2014), no studies have been carried
out to determine Pestalotiopsis-like fungi diseases. Therefore,
the aim of the current paper is to identify and characterize
the Pestalotiopsis-like fungi species occurring on grapes in
China using both morphological as well as molecular data.
Materials and methods
Isolation and identification
Isolates were collected from different provinces (Anhui,
Guangxi, Hubei, Hunan, Shandong, Shanxi, Sichuan, Yunan
and Zhejiang) of China from 2011 to 2013. Diseased grapevine
samples were collected and placed in separate plastic bags
with sterilized tissues dipped in distilled water to maintain
humid conditions. Samples were surface-sterilized with 70 %
ethanol for 1 min and then rinsed three times in sterilized water. The isolation of Pestalotiopsis-like fungi followed the
methods used by Maharachchikumbura et al. (2012). The
pure isolates were cultured on Potato Dextrose Agar (PDA)
plates with sterilized filter paper pieces and incubated for
7e10 d at 25 C. Cultures on the filter paper pieces were dried
on sterilized filter paper and stored at 20 C. The morphology
of fungal colonies was recorded following the method used by
Maharachchikumbura et al. (2012). Fungal mycelia and spores
were observed and photographed using a Leica DM5500B microscope. Forty conidial measurements were taken for each
isolate. All microscopic measurements were recorded with
a Nikon, NIS-Elements F3.0.
Pathogenicity test
Detached shoot inoculation
Pathogenicity tests were conducted by an inoculation method
et al. 2009). In short, the pathogenicity tests
(Urbez-Torres
were conducted on the shoots collected from mature Vitis vinifera cf. Summer Black grapevines. Shoots were cut in a uniform length and all leaves, lateral branches and tendrils were
removed. First, shoots were surface-sterilized in sodium hypochlorite (1 % NaOCl) for 3e5 min and rinsed three times
with distilled water. After air drying, ten canes were inoculated with fungal isolates selected from each species. Shoots
were wounded in the middle using a 4 mm cork borer. Inoculations were conducted by placing a 1-week-old 4 mm agar
plug from the edge of an actively growing culture. Wounds
were then wrapped with parafilm. Ten shoots were inoculated
with 4 mm non-colonized PDA plugs for negative controls. Inoculated shoots were immediately placed in plastic containers, arranged in a completely randomized design with
distilled water to maintain the humid environment (70e80 %
relative humidity) and incubated at room temperature
(25 C) under artificial light (12/12 h light-and-dark cycles).
Mean lesion length of the inoculated shoots was measured
from 5 to 10 d.
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Characterization of Pestalotiopsis-like fungi
Fruit inoculation
An inoculation method was used for the pathogenicity test on
grape fruits. Healthy grape fruits from Vitis vinifera cf. Red
Globe that were uniform in size and lacking visible disease
symptoms on the outside were washed with tap water and
then disinfected in sodium hypochlorite (1 % NaOCl) for
5e7 min. Disinfected fruits were washed three times with distilled and sterilized water and then dried with sterilized filter
paper. Superficial wounds in the epidermis were carried out
with a sterile scalpel. For inoculation with the isolate, 4 mmdiameter disks of PDA were removed from the edge of an actively growing culture and placed mycelium-side down on
the wound. Fruits inoculated with agar plugs of sterile PDA
were used as a negative control. Ten fruits from each plant
were inoculated with one fungal isolate selected from each
species of the phylogenetic tree. Fruits were kept individually
in a 12 cm diam. petri dish with a swab of cotton wool containing distilled water to maintain humidity (70e80 % relative humidity) and incubated at room temperature (25 C). Lesion
diameters were measured 7 d after inoculation.
The method of non-wound inoculation involved placing
mycelium plugs on shoots and fruits without wounding. To
fulfil Koch’s postulates, diseased tissues were placed on
PDA. Pestalotiopsis-like fungi were re-isolated and the fungal
identification was verified based on colony and conidial characters. All inoculated fruits and shoots were sterilized and
autoclaved before disposing.
Data analysis
Data from the pathogenicity tests were analysed using Minitab, V.15.1.1.0 (Minitab release 15.1.1.0, Minitab Inc., Boston,
MA, USA). One-way analysis of variance (ANOVA) was performed to assess the differences in the extent of vascular discolouration of shoots and the lesions on fruits induced by the
fungi tested. Treatment means were compared using Turkeys’
test at the 5 % significance level.
Molecular phylogeny
DNA extraction, PCR amplification, and DNA sequencing
Total genomic DNA was extracted by the modified protocol of
Guo et al. (2000). Total genomic DNA was extracted from fresh
mycelium (500 mg), scraped from the margin of a colony on
a PDA plate incubated at 25 C for 7e10 d. The ITS, b-tubulin
and tef1 genes were amplified using primer pairs ITS5/ITS4
(White et al. 1990), BT2A/BT2B (Glass & Donaldson 1995;
O’Donnell & Cigelnik 1997) and 728F/1567R or 728F/EF2
(O’Donnell & Cigelnik 1997; Carbone & Kohn 1999; Rehner
2001) respectively. The PCR were performed in a BIORAD
1000 Thermal Cycler in a total volume of 25 ml. The PCR mixtures contained TaKaRa Ex-Taq DNA polymerase 0.3 ml, 12.5 ml
of 2 PCR buffer with 2.5 ml of dNTPs, 1 ml of each primer, 9.2 ml
of double-distilled water and 100e500 ng of DNA template.
The
thermal
cycling
programme
followed
Maharachchikumbura et al. (2012). The PCR products were verified by staining with Ethidium Bromide on 1.2 % agarose electrophoresis gels and purified according to the manufacturer’s
instructions of a Qiagen purification kit (Qiagen, USA). DNA
sequencing of the genes were conducted by Sunbiotech
3
Company, Beijing, China. The DNA sequences of ITS, b-tubulin
and tef1 regions generated in this study were submitted to
GenBank.
Phylogenetic analysis
DNAStar V.5.1 and SeqMan V.5.00 were used to obtain consensus sequences from sequences generated from forward and
reverse primers. Combined dataset of three gene regions
were aligned using Clustal X1.81 (Thompson et al. 1997). The
sequences were further aligned using default settings of
MAFFT v.7 (Katoh & Toh 2008; http://mafft.cbrc.jp/alignment/server/) and manually adjusted using BioEdit V.7.0.9.0
(Hall 1999) where necessary. Two separate phylogenetic trees
were constructed for genus Pestalotiopsis and Neopestalotiopsis,
Pseudopestalotiopsis genera respectively, based on the initial
blast results obtained from NCBI blast tool (http://
www.ncbi.nlm.nih.gov/BLAST/Blast.cgi). A maximum parsimony analysis (MP) was performed using PAUP (Phylogenetic
Analysis Using Parsimony) v. 4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded and gaps were treated as
missing data. Trees were inferred using the heuristic search
option with Tree Bisection Reconnection (TBR) branch swapping and 1000 random sequence additions. Maxtrees were
set up to 5000, branches of zero length were collapsed and
all multiple parsimonious trees were saved. Tree Length
(TL), Consistency Index (CI), Retention Index (RI), Rescaled
Consistency index (RC), and Homoplasy index (HI) were calculated for trees generated under different optimality criteria.
The robustness of the most parsimonious trees was evaluated
by 1000 bootstrap replications resulting from maximum parsimony analysis (Hillis & Bull 1993). The Kishino-Hasegawa
tests (Kishino & Hasegawa 1989) were performed in order to
determine whether the trees inferred under different optimality criteria, were significantly different.
In addition, Bayesian inference (BI) was used to construct
the phylogenies using Mr. Bayer’s v. 3.1.2 (Ronquist et al.
2003). Suitable models were first selected using models of nucleotide substitution for each gene, as determined using
MrModelTest (Nylander 2004). The GTRþIþG model was selected for ITS and the HKYþIþG model for b-tubulin and tef.
The above mentioned models were incorporated into the
analysis. Six simultaneous Markov chains were run for
1 000 000 generations and trees were sampled every 100th generation. The first 2000 trees, representing the burn-in phase of
the analyses, were discarded and the remaining 8000 trees
used for calculating posterior probabilities (PP) in the majority
rule consensus tree. Phylogenetic trees were viewed using
Treeview (Page 1996). The alignments and trees are deposited
in TreeBASE under accession numbers S16573 and S16594 respectively. The fungal strains that were used for this study are
listed in Table 1.
Results
Isolation of fungi
Pestalotiopsis-like fungi were isolated from wedge-shaped cankers as well as canes showing bleaching symptoms (Fig 1).
Species of Pestalotiopsis-like fungi were also isolated from
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
4
R. S. Jayawardena et al.
Table 1 e Strains used in phylogenetic analyses and their GenBank accession numbers. Ex-type and ex-epitype strains are
bolded.
Species
Neopestalotiopsis aotearoa
Neopestalotiopsis asiatica
Neopestalotiopsis australis
Neopestalotiopsis clavispora
Neopestalotiopsis clavispora
Neopestalotiopsis clavispora
Neopestalotiopsis chrysea
Neopestalotiopsis chrysea
Neopestalotiopsis cubana
Neopestalotiopsis ellipsospora
Neopestalotiopsis ellipsospora
Neopestalotiopsis eucalypticola
Neopestalotiopsis foedans
Neopestalotiopsis foedans
Neopestalotiopsis foedans
Neopestalotiopsis honoluluana
Neopestalotiopsis magna
Neopestalotiopsis mesopotamicum
Neopestalotiopsis mesopotamicum
Neopestalotiopsis mesopotamicum
Neopestalotiopsis natalensis
Neopestalotiopsis piceana
Neopestalotiopsis piceana
Neopestalotiopsis piceana
Neopestalotiopsis protearum
Neopestalotiopsis rosa
Neopestalotiopsis samarangensis
Neopestalotiopsis saprophyta
Neopestalotiopsis steyaertii
Neopestalotiopsis surinamensis
Neopestalotiopsis umbrinospora
Neopestalotiopsis zimbabwana
Neopestalotiopsis sp.
Neopestalotiopsis sp.
Neopestalotiopsis sp.
Neopestalotiopsis sp.
Neopestalotiopsis sp.
Neopestalotiopsis sp.
Neopestalotiopsis sp.
Neopestalotiopsis sp. (JZB340002)
Neopestalotiopsis sp. (JZB340003)
Neopestalotiopsis sp. (JZB340004)
Neopestalotiopsis sp. (JZB340005)
Neopestalotiopsis sp. (JZB340009)
Neopestalotiopsis sp. (JZB340011)
Neopestalotiopsis sp. (JZB340012)
Neopestalotiopsis sp. (JZB340013)
Neopestalotiopsis sp. (JZB340014)
Neopestalotiopsis sp. (JZB340006)
Neopestalotiopsis sp. (JZB340015)
Neopestalotiopsis sp. (JZB340010)
Neopestalotiopsis sp. (JZB340008)
Neopestalotiopsis sp. (JZB340007)
Neopestalotiopsis sp. (JZB340017)
Pestalotiopsis adusta
Pestalotiopsis anacardiacearum
Pestalotiopsis arceuthobii
Pestalotiopsis arenga
Pestalotiopsis australis
Pestalotiopsis australis
Pestalotiopsis australis
Pestalotiopsis australis
Isolate
CBS 367.54
MFLUCC 12-0286
CBS 114159
MFLUCC 12-0280
MFLUCC 12-0281
ICMP 20405
MFLUCC 12-0261
MFLUCC 12-0262
CBS 600.96
MFLUCC 12-0283
MFLUCC 12-0284
CBS 264.37
CGMCC 3.9123
CGMCC 3.9178
CGMCC 3.9123
CBS 114495
MFLUCC 12-652
CBS 336.86
CBS 299.74
CBS 464.69
CBS 138.41
CBS 394.48
CBS 254.32
CBS 225.30
CBS 114178
CBS 101057
MFLUCC 12-0233
MFLUCC 12-0282
IMI 192475
CBS 450.74
MFLUCC 12-0285
CBS 111495
CBS 110.20
CBS 177.25
CBS 274.29
CBS 322.76
CBS 664.94
CBS 360.61
CBS 266.80
ICMP 20406
ICMP 20407
ICMP 20408
ICMP 20409
ICMP 20410
ICMP 20411
ICMP 20412
ICMP 20413
ICMP 20414
ICMP 20415
ICMP 20416
ICMP 20417
ICMP 20418
ICMP 20419
ICMP 20421
ICMP 6088
IFRDCC 2397
CBS 434.65
CBS 331.92
CBS 114193
CBS 111503
CBS 119350
CBS 114474
GenBank Accession numbers
ITS
b-tubulin
tef1
KM199369
JX398983
KM199348
JX398978
JX398979
KJ623224
JX398985
JX398986
KM199347
JX399016
JX399015
KM199376
JX398987
JX398989
JX398987
KM199364
KF582795
KM199362
KM199361
KM199353
KM199377
KM199368
KM199372
KM199371
JN712498
KM199359
JQ968609
KM199345
KF582796
KM199351
JX398984
JX556231
KM199342
KM199370
KM199375
KM199366
KM199354
KM199346
KM199352
KJ623216
KJ623217
KJ623218
KJ623221
KJ623220
KJ623219
KJ623214
KJ623225
KJ623213
KJ623222
KJ623211
KJ623212
KJ623215
KJ623223
KJ623210
JX399006
KC247154
KM199341
KM199340
KM199332
KM199331
KM199333
KM199334
KM199454
JX399018
KM199432
JX399013
JX399014
KJ623206
JX399020
JX399021
KM199438
JX399016
JX399015
KM199431
JX399022
JX399024
JX399022
KM199457
KF582793
KM199441
KM199435
KM199436
KM199466
KM199453
KM199452
KM199451
KM199463
KM199429
JQ968610
KM199433
KF582794
KM199465
JX399019
KM199456
KM199442
KM199445
KM199448
KM199446
KM199449
KM199440
e
KJ994532
KJ623196
KJ623197
KJ623198
KJ623200
KJ623199
KJ623207
KJ623195
KJ623194
KJ623204
KJ623201
KJ623202
KJ623203
KJ623205
KJ623192
JX399037
KC247155
KM199427
KM199426
KM199383
KM199382
KM199384
KM199385
KM199526
JX399049
KM199537
JX399044
JX399045
KJ623238
JX399051
JX399052
KM199521
JX399047
JX399046
KM199551
JX399053
JX399055
JX399053
KM199548
KF582791
KM199555
KM199541
e
KM199552
KM199527
KM199529
KM199535
KM199542
KM199523
JQ968611
KM199538
KF582792
KM199518
JX399050
KM199545
KM199540
KM199533
KM199534
KM199536
KM199525
KM199522
KM199532
KJ623236
KJ623234
KJ623240
KJ623239
KJ623226
KJ623227
KJ623231
KJ623228
KJ623237
KJ623233
KJ623230
KJ623235
KJ623232
KJ623229
KJ632073
JX399070
KC247156
KM199516
KM199515
KM199475
KM199557
KM199476
KM199477
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Characterization of Pestalotiopsis-like fungi
5
Table 1 e (continued )
Species
Pestalotiopsis autralasiae
Pestalotiopsis autralasiae
Pestalotiopsis biciliata
Pestalotiopsis biciliata
Pestalotiopsis biciliata
Pestalotiopsis brassicae
Pestalotiopsis camelliae
Pestalotiopsis camelliae
Pestalotiopsis chamaeropis
Pestalotiopsis chamaeropis
Pestalotiopsis chamaeropis
Pestalotiopsis chamaeropis
Pestalotiopsis clavata
Pestalotiopsis colombiensis
Pestalotiopsis diploclisiae
Pestalotiopsis diploclisiae
Pestalotiopsis diploclisiae
Pestalotiopsis diversiseta
Pestalotiopsis ericacearum
Pestalotiopsis furcata
Pestalotiopsis gaultheria
Pestalotiopsis grevillea
Pestalotiopsis hawaiiensis
Pestalotiopsis hollandica
Pestalotiopsis humus
Pestalotiopsis humus
Pestalotiopsis inflexa
Pestalotiopsis intermedia
Pestalotiopsis karstenii
Pestalotiopsis knightiae
Pestalotiopsis knightiae
Pestalotiopsis linearis
Pestalotiopsis malayana
Pestalotiopsis monocaheta
Pestalotiopsis monocaheta
Pestalotiopsis novaehollandiae
Pestalotiopsis proteacearum
Pestalotiopsis proteacearum
Pestalotiopsis proteacearum
Pestalotiopsis papuana
Pestalotiopsis papuana
Pestalotiopsis parva
Pestalotiopsis parva
Pestalotiopsis portugalica
Pestalotiopsis rhododendri
Pestalotiopsis rhodomyrtus
Pestalotiopsis rosea
Pestalotiopsis scorparia
Pestalotiopsis spathulata
Pestalotiopsis teleopa
Pestalotiopsis teleopa
Pestalotiopsis teleopa
Pestalotiopsis trachicarpicola
Pestalotiopsis trachicarpicola
Pestalotiopsis trachicarpicola
Pestalotiopsis trachicarpicola
Pestalotiopsis trachicarpicola
Pestalotiopsis trachicarpicola
Pestalotiopsis trachicarpicola
Pestalotiopsis trachicarpicola (JZB340016)
Pestalotiopsis unicolor
Pestalotiopsis verruculosa
Isolate
CBS 114141
CBS 114126
CBS 124463
CBS 790.68
CBS 236.38
CBS 170.26
MFLUCC 12-0277
MFLUCC 12-0278
CBS 113604
CBS 113607
CBS 186.71
CBS 237.38
MFLUCC 12-0268
CBS 118553
CBS 115587
CBS 115585
CBS 115449
MFLUCC 12-0287
IFRDCC 2439
MFLUCC 12-0054
IFRD 411-014
CBS 114127
CBS 114491
CBS 265.33
CBS 336.97
CBS 115450
MFLUCC 12-0270
MFLUCC 12-0259
IFRDCC OP13
CBS 114138
CBS 111963
MFLUCC 12-0271
CBS 102220
CBS 144.97
CBS 440.83
CBS 130973
CBS 111522
CBS 171.26
CBS 353.69
CBS 331.96
CBS 887.96
CBS 265.37
CBS 278.35
CBS 393.48
IFRDCC 2399
HGUP4230
MFLUCC12-0258
CBS 176.25
CBS 356.86
CBS 114137
CBS 114161
CBS 113606
MFLUCC 12-0263
MFLUCC 12-0264
MFLUCC 12-0265
MFLUCC 12-0266
MFLUCC 12-0267
IFRDCC 2403
OP068
ICMP 20420
MFLUCC 12-0275
MFLUCC 12-0274
GenBank Accession numbers
ITS
b-tubulin
tef1
KM199298
KM199297
KM199308
KM199305
KM199309
KM199379
JX399010
JX399011
KM199323
KM199325
KM199326
KM199324
JX398990
KM199307
KM199320
KM199315
KM199314
JX399009
KC537807
JQ683724
KC537805
KM199300
KM199339
KM199328
KM199317
KM199319
JX399008
JX398993
KC537806
KM199310
KM199311
JX398992
KM199306
KM199327
KM199329
KM199337
KM199294
KM199304
KM199299
KM199321
KM199318
KM199312
KM199313
KM199335
KC537804
KF412648
JX399005
KM199330
KM199338
KM199301
KM199296
KM199295
JX399000
JX399004
JX399003
JX399002
JX399001
KC537809
JQ845947
KJ623209
JX398998
JX398996
KM199410
KM199409
KM199399
KM199400
KM199401
e
JX399041
JX399042
KM199389
KM199390
KM199391
KM199392
JX399025
KM199421
KM199419
KM199417
KM199416
JX399040
KC537821
JQ683708
KC537819
KM199407
KM199428
KM199388
KM199420
KM199418
JX399039
JX399028
KC537820
KM199408
KM199406
JX399027
KM199411
KM199386
KM199387
KM199425
KM199394
KM199397
KM199398
KM199413
KM199415
KM199404
KM199405
KM199422
KC537818
KF412642
JX399036
KM199393
KM199423
KM199469
KM199403
KM199402
JX399031
JX399035
JX399034
JX399033
JX399032
KC537823
JQ845945
KJ623193
JX399029
e
KM199501
KM199499
KM199505
KM199507
KM199506
KM199558
JX399074
JX399075
KM199471
KM199472
KM199473
KM199474
JX399056
KM199488
KM199486
KM199483
KM199485
JX399073
KC537814
JQ683740
KC537812
KM199504
KM199514
KM199481
KM199484
KM199487
JX399072
JX399059
KC537813
KM199497
KM199495
JX399058
KM199482
KM199479
KM199480
KM199511
KM199493
KM199494
KM199496
KM199491
KM199492
KM199508
KM199509
KM199510
KC537811
KF412645
JX399069
KM199478
KM199513
KM199559
KM199500
KM199498
JX399064
JX399068
JX399067
JX399066
JX399065
KC537816
JQ845946
KJ623241
JX399063
JX399061
(continued on next page)
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grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
6
R. S. Jayawardena et al.
Table 1 e (continued )
Species
Pseudopestalotiopsis cocos
Pseudopestalotiopsis indica
Pesudopestalotiopsis theae
Pesudopestalotiopsis theae
Isolate
CBS 272.29
CBS 459.78
MFLUCC12-0055
SC011
fruits that had reddish brown, water-soaked lesions covered
with whitish mycelium with black conidial masses. Seventeen
isolates were obtained from the disease samples and deposited in ICMP culture collection. Isolates were obtained from
six grape varieties including three traditional Chinese varieties (Vitis vinifera cv. Guifei Meigui, V. vinifera cv. Brier grape
and V. vinifera cv. Wuhe Cuibao).
Pathogenicity studies
To comply with Koch’s postulates, lesions resembling initial
symptoms were observed after 7 d on artificial inoculation of
shoots and 5 d on fruits. No symptoms were observed in the
control fruits and shoots. Two Pestalotiopsis-like species isolated from Chinese grapevines were pathogenic and reisolated (100 %) from the inoculated fruits and shoots. These
taxa were identical with the original isolates. For any given
isolate, disease scores were not significantly different between replicates (P < 0.05). In both fruits and shoots, isolates
belonging to Neopestalotiopsis sp. caused significantly longer
lesions (F ¼ 11.63, P > 0.05) than the other species of Pestalotiopsis recorded. Neopestalotiopsis strains ICMP 20415, ICMP 20416,
ICMP 20417, ICMP 20418 and ICMP 20419 showed longer lesions (mean lesion length ¼ 5.5 cm) than the other strains of
GenBank Accession numbers
ITS
b-tubulin
tef1
KM199378
KM199381
JQ683727
JQ683726
KM199467
KM199470
JQ683711
JQ683710
KM199553
KM199560
JQ683743
JQ683742
this species. Pestalotiopsis trachicarpicola was less virulent
with mean lesions of 2.4 cm. Therefore, Neopestalotiopsis sp.
was more virulent towards Red Globe and Summer Black varieties of grapes than the other species recorded in this study.
Detached shoot inoculation
Following artificial infection, circular, sunken, necrotic spots
appeared on detached shoots. After 4 d the area of discolouration of the skin increased and whitish mycelium developed on
the lesions. Raised masses of black conidia developed on the
surface of the lesions after 7 d. After 10 d the spots coalesced
and formed large irregular necrotic areas extending with lesions into the tissues of the shoots. Lesions extended upwards
and downwards from the point of infection (Fig 2). Neopestalotiopsis strains ICMP 20415, ICMP 20416, ICMP 20417, ICMP 20418
and ICMP 20419 caused significantly longer lesions in shoots
(F ¼ 1.27, P > 0.05), than other species and strains recorded
in this study.
Fruit inoculation
Following artificial inoculation small, circular, water-soaked,
sunken, brown spots appeared on the fruit skin. After 3 d
the area of discolouration of the skins increased and whitish
mycelium developed in the lesions. Raised masses of black
Fig 1 e Symptom on grapevine caused by Pestalotiopsis-like fungi. (A) Damaged grape shoot in the field (B) Bleached canes
with slitting, (C) Wedged-shaped canker, (D) Bleached cane with fruiting bodies.
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Characterization of Pestalotiopsis-like fungi
conidia developed on the surface of the lesions after 5 d. After
8 d the spots coalesced and formed large, irregular, rotting
areas, with lesions extending into the pulp of the fruits
(Fig 2). ICMP 20415, ICMP 20416, ICMP 20417, ICMP 20418 and
ICMP 20419 strains caused significant longer lesions (length:
F ¼ 7.33, P > 0.05; width: F ¼ 8.05, P > 0.05) than the other species and the strains recorded in this study.
7
Phylogenetic analysis
Phylogenetic trees were constructed using combined ITS,
b-tubulin and tef1 sequences for our 17 isolates of Pestalotiopsis-like
fungi
with
those
that
originated
from
Maharachchikumbura et al. (2012, 2013b, 2014), Song et al.
(2013, 2014), and Hyde et al. (2014). Two separate phylogenetic
Fig 2 e Symptom on grape fruits and shoots caused by Pestalotiopsis-like fungi. (A) Control, (B, C) Water soaked, necrotic
lesion on grape fruit after 3 d of infection, (D, E) White mycelium and conidial mass on lesion on grape fruit after 5 d of infection, (F) Grape fruit after 8 d of infection, (G) Control, (H) Necrotic lesions on shoot after 4 d of infection, (I) Grape shoot after
10 d of infection.
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
8
R. S. Jayawardena et al.
Fig 3 e Maximum Parsimonious tree obtained from a heuristic search of the combined ITS, b-tubulin, tef1 sequence alignment. Bootstrap support values above 50 % and Bayesian posterior probability values above 0.7 are shown above and below
the nodes. Neopestalotiopsis saprophyta (CBS 447.73) is used as outgroup. Isolates obtained in this study are shown in blue
colour. Ex-type and ex-epitype strains are bolded. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Conidiomata
Conidiogenous
cells
Neopestalotiopsis
sp. (Fig 5)
120e550 mm
diam, acervuli,
globose- oval,
black, scattered,
semi-immersed
on PDA black
conidia in
a slimy,
glistening mass
Pestalotiopsis
trachicarpicola
(Fig 6)
120e410 mm
Fusiform,
diam., acervuli,
hyaline, short,
globose, black,
thin-walled
semi-immersed
on PDA releasing
black in a black
conidia in
a slimy,
glistening mass
Fusiform,
hyaline, simple,
short
Conidia
4th Cell
Appendages
Basal cell
2nd Cell
3rd Cell
Apical cell
Basal
Apical
Conical,
hyaline, thin
and
verruculose
to smoothwalled,
1.9e6.2 mm
long
(x ¼ 4.1 mm,
n ¼ 25)
Pale brown to
olivaceous,
3.4e5.9 mm
(x ¼ 4.7 mm,
n ¼ 25)
Darker brown
to olivaceous,
4.1e6.5 mm
(x ¼ 5.5 mm,
n ¼ 25)
Darker brown,
3.8e6.5 mm
(x ¼ 5.2 mm,
n ¼ 25)
Cylindrical to
subcylindric,
hyaline, 2.3e5.8 mm
(x ¼ 4.5 mm, n ¼ 25)
Single basal
appendage
present,
filiform
3.4e7 mm
(x ¼ 5.2 mm,
n ¼ 60).
Long, tubular,
11e53 mm
(x ¼ 31.8 mm,
n ¼ 60), 2e4
(mostly 3) arising
from the apex of
the apical cell
Conic to
acute,
hyaline, thin
and
verruculose,
2.8e6.8 mm
long
(x ¼ 4.4 mm,
n ¼ 25)
Concolorous,
2.9e7.4 mm
(x ¼ 4.7 mm,
n ¼ 25)
Concolorous,
3.4e6.8 mm
(x ¼ 5.3 mm,
n ¼ 25)
Concolorous,
3e5.8 mm
(x ¼ 4.7 mm,
n ¼ 25)
Conic to
subcylindrical,
hyaline, 2.7e6.4 mm
(x ¼ 4 mm, n ¼ 25),
Single basal
appendage
present,
filiform
2.2e8.3 mm
(x ¼ 4.2 mm,
n ¼ 60)
5e17 mm
(x ¼ 10 mm, n ¼ 60)
long, tubular, 2e4
(mostly 3) arising
from the apex of
the apical cell
Culture
characteristics
Colonies on PDA
reaching 8 cm diam.
after 7 days at 25 C,
edge undulate,
whitish, aerial
mycelium with black
fruiting bodies,
concentric,
gregarious, reverse
of culture white to
pale yellow
Colonies on PDA
reaching 6 cm diam.
after 7 d at 25 C,
edge fimbriate,
whitish, dense aerial
mycelium with black
fruiting bodies,
concentric, reverse
of culture pale
yellow
Characterization of Pestalotiopsis-like fungi
9
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Table 2 e Morphological comparison of Pestalotiopsis species recorded in vineyards of China.
Species
10
R. S. Jayawardena et al.
Fig 4 e Maximum Parsimonious tree obtained from a heuristic search of the combined ITS, b-tubulin, tef1 sequence alignment. Bootstrap support values above 50 % and Bayesian posterior probability values above 0.90 are shown above and below
the nodes. Pestalotiopsis trachicarpicola (OP068) is used as outgroup. Isolates obtained in this study are shown in blue colour.
Ex-type and ex-epitype strains are bolded. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Characterization of Pestalotiopsis-like fungi
11
Fig 5 e Neopestalotiopsis sp. (ICMP20417) (A, B) Colony on PDA, (A) from above, (B) from below, (C) Conidiomata, (DeH) Conidia
with versicolorous median cells, Scale bars [ deh 10 mm.
trees were constructed. Maximum-parsimony and Bayesian
inference produced nearly identical topologies (Bayesian trees
are not shown).
The combined gene alignment for Pestalotiopsis comprised
of 72 taxa and 1516 characters including gaps (ITS: 1e554, btubulin: 555e1010 and tef1: 1011e1516). Parsimony analysis indicated that 1060 characters were constant, 173 variable characters parsimony-uninformative and 291 characters
parsimony-informative. The parsimony analysis of the data
matrix yielded single parsimonious tree (Fig 3) (TL ¼ 1180,
CI ¼ 0.564, RI ¼ 0.808, RC ¼ 0.455, HI ¼ 0.436). Only one isolate
obtained in this study (ICMP 20420) was clustered together
with Pestalotiopsis trachicarpicola with a strong support.
The combined gene alignment for Neopestalotiopsis and
Pseudopestalotiopsis comprised of 59 taxa and 1140 characters
including gaps (ITS: 1e525, b-tubulin: 526e762 and tef1:
763e1140) of which 49 characters were excluded. Parsimony
analysis indicated that 853 characters were constant, 107 variable characters parsimony-uninformative and 131 characters
parsimony-informative. The parsimony analysis of the data
matrix yielded single parsimonious tree (Fig 4) (TL ¼ 360,
CI ¼ 0.794, RI ¼ 0.871, RC ¼ 0.692, HI ¼ 0.206). Sixteen isolates
obtained in this study clustered together with Neopestalotiopsis
species isolated from Vitis vinifera of India (CBS 266.80) with
a strong support.
Morphological characters
Morphological characters of the species identified are summarized including colony appearance, conidiogenous cells, conidia and colony characters (Table 2).
Discussion
Xu et al. (1999) isolated Pestalotiopsis from rotted grape berries
in Japan and found that Pestalotiopsis menezesiana and Pestalotiopsis uvicola initiating postharvest disease of grapes. Many
studies on grapes worldwide, mainly using morphological
characters have identified the above mentioned two species
as the most common species of Pestalotiopsis-like fungi found
on grapes (Guba 1961; Ryu et al. 1999; Sergeeva et al. 2005;
Urbez-Torres
et al. 2009, 2012), even though in this study these
two species were not recorded. Maharachchikumbura et al.
(2013a) referred P. menezesiana as P. cf. menezesiana, as the GenBank data on this species is very confusing and it is in urgent
need of study in order to clarify its phylogenetic position.
Zhang et al. (2007) isolated Pestalotiopsis guepinii from grape
canes in Yunnan Province, China. These three species lack
ex-type or ex-epitype strains and thus have not been included
in the phylogenetic analysis of this study. Pestalotiopsis uvicola
possess concolorous median cells which is similar to Pestalotiopsis trachicarpicola, but the conidiogenous cells of this species are ampuliform and the apical appendages often form
a closely aggregated crest, which cannot be observed in P. trachicarpicola. Pestalotiopsis menezesiana is characterized by conidiogenous cells with the presence of zero to two closely
spaced annular scars (Bissett 1982), which were not observed
in the species recorded in our study. Further studies on P. cf.
menezesiana, P. guepini and P. uvicola must be carried out in order to clarify their phylogenetic status within the Pestalotiopsis-like
fungi
as
well
as
to
epitypify
them.
Maharachchikumbura et al. (2014) introduced two new genera
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
12
R. S. Jayawardena et al.
Fig 6 e Pestalotiopsis trachicarpicola (ICMP20420) (A, B) Colony on PDA, (A) from above, (B) from below, (C) Conidiomata, (DeG)
Conidia with concolorous median cells, Scale bars [ deg [ 10 mm.
into Pestalotiopsis-like fungi: Neopestalotiopsis and Pseudopestalotiopsis. Neopestalotiopsis species are characterized by indistinct to reduced conidiophores and the two upper median
cells are darker than the lower median cells. Genus Pseudopestalotiopsis is characterized by dark concolorous median cells
with knobbed apical appendages. Pestalotiopsis-like fungal
species appear to have a wide host range (Guba 1961;
Maharachchikumbura et al. 2012) and most species were previously been named according to host association and only
a small number of morphological characters were available
to differentiate between species (Maharachchikumbura et al.
2012). Pestalotiopsis trachicarpicola has been recorded from Trachycarpus fortunei (Chinese windmill palm), Chrysophullum sp.
(Rare star Apple), Schima sp., and Symplocos sp. in China
(Maharachchikumbura et al. 2012; Zhang et al. 2012). Neopestalotiopsis sp. (CBS 266.80) had been recorded from V. vinifera in
India (Maharachchikumbura et al. 2014). The use of molecular
data in resolving Pestalotiopsis-like fungi reviewed by various
studies suggested that multi-locus phylogenetic analysis is
needed to resolve the cryptic species in this genus (Hu et al.
2007;
Liu
et
al.
2007;
Tejesvi
et
al.
2007a;
Maharachchikumbura et al. 2012, 2013c, 2014). Use of ITS sequences alone does not resolve Pestalotiopsis-like fungi well,
however, the combined gene tree (ITS, b-tubulin and tef1)
analysis
has
resolved
species
successfully
(Maharachchikumbura et al. 2012) and the present study
agreed with this. This approach has also been followed in
the important genera Bipolaris, Colletotrichum, Diaporthe and
Phyllosticta (Hyde et al. 2014; Udayanga et al. 2014; Yan et al.
2014).
The combined gene tree of this study consists of the strains
that have ex-types or ex-epitypes with the isolates used for
this study. There have been some researches carried out on
the incidence and role that Pestalotiopsis-like fungi play in
et al. 2009). Pestalotiopsis-like
grapevine disease (Urbez-Torres
fungi have been often isolated from the bleached canes of
grapevine with Phomopsis sp. and Botryosphaeria sp. (Sergeeva
et al. 2005).
To our knowledge this study represents the first attempt to
identify and characterize Pestalotiopsis-like fungi causing
grapevine diseases in China using both morphological and
molecular approaches. Genus Pestalotiopsis has commonly
been reported as a pathogen of grapevine causing die back
and postharvest fruit rot (Castillo-Pando et al. 2001; Sergeeva
et al. 2001; Urbez-Torres
et al. 2009; Deng et al. 2013). More recently, several new species have been introduced based on
morphological and molecular data. Currently there are more
than 70 ex-type or ex-epitype sequences for Pestalotiopsislike fungi (Hyde et al. 2014; Maharachchikumbura et al. 2014;
Nilsson et al. 2014). In their study Maharachchikumbura et al.
(2014) showed that CBS 266.80 is morphologically similar to
N. australis although phylogenetically and ecologically they
are distinct. Therefore, until more cultures and collections become available, they prefer to maintain this as Neopestalotiopsis sp. and we would like to follow that in this paper. Conidial
characters alone are insignificant criteria in distinguishing
Pestalotiopsis
species
(Jeewon
et
al.
2003;
Maharachchikumbura et al. 2011, 2013a, b). Many sequences
for genus Pestalotiopsis deposited in GenBank are unreliable.
Therefore, identification of Pestalotiopsis to species level is
presently difficult. There is a need for epitypification in genus
Pestalotiopsis (Maharachchikumbura et al. 2012, 2013d). Correct
species identification is essential in plant pathogenic genera
(Maharachchikumbura et al. 2013a; Rossman & Palm ndez 2008). In order to have effective measures to preHerna
vent the unwanted entry of diseases into a country, the plant
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Characterization of Pestalotiopsis-like fungi
pathologists should be able to name the Pestalotiopsis-like
fungi confidently (Maharachchikumbura et al. 2011).
Careful handling of grape berries in the field as well as during storage can prevent the fruit rot caused by Pestalotiopsislike fungi. Proper maintenance of the grape orchards during
pruning can help to prevent the canker disease caused by Pestalotiopsis-like fungi.
Conclusion
This study represents the first attempt to identify and characterize the Pestalotiopsis-like fungi causing diseases in grapevine: fruit rot and trunk diseases in China using both
morphological and molecular approaches. This is the first report of Neopestalotiopsis sp. and of Pestalotiopsis trachicarpicola
causing diseases in grapevine.
Acknowledgements
The research was funded by CARS-30, National Research
Council of Thailand (grant for Pestalotiopsis No: 55201020008)
and authors would like to thank the grape cultivators. We
are grateful to the Mushroom Research Foundation, Chiang
Rai, Thailand. KD Hyde thanks The Chinese Academy of Sciences, project number 2013T2S0030, for the award of Visiting
Professorship for Senior International Scientists at Kunming
Institute of Botany.
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Keith LM, Velasquez ME, Zee FT, 2006. Identification and characterization of Pestalotiopsis spp. causing scab disease of Guava,
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Theaceae and Taxaceaein southern China. Fungal Diversity 24:
55e74.
Please cite this article in press as: Jayawardena RS, et al., Identification and characterization of Pestalotiopsis-like fungi related to
grapevine diseases in China, Fungal Biology (2014), http://dx.doi.org/10.1016/j.funbio.2014.11.001
Ruvishika S Jayawardena