Identification and characterization of Pestalotiopsislike fungi related to grapevine diseases in China

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Identification and characterization of Pestalotiopsis- like fungi related to grapevine diseases in China

Ruvishika S. JAYAWARDENA

^a,b,c

, Wei ZHANG

^a

, Mei LIU

^a

, Sajeewa S. N. MAHARACHCHIKUMBURA

^b,c

, Ying ZHOU

^a

, JinBao HUANG

^a

, Somrudee NILTHONG

^b,c

, ZhongYue WANG

^d

, XingHong LI

^a,

*, JiYe YAN

^a,

*, Kevin D. HYDE

^b,c

aInstitute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, People’s Republic of China

bInstitute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand

cSchool of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

dInstitute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China

a r t i c l e i n f o

Article history:

Received 29 September 2014 Received in revised form 30 October 2014

Accepted 9 November 2014 Corresponding Editors:

JiYe Yan, XingHong Li Keywords:

b-tubulin Fruit rot Neopestalotiopsis tef1

Trunk diseases

a b s t r a c t

Pestalotiopsis-like fungi are an important plant pathogenic genus causing postharvest fruit rot and trunk diseases in grapevine in many countries.Pestalotiopsis-like fungi diseases were studied in vineyards in nine provinces across China. Multi-gene (ITS,b-tubulin and tef1) analysis coupled with morphology showed that aNeopestalotiopsissp. andPestalotiopsis trachicarpicolaare associated in causing grapevine fruit rot and trunk diseases in China.Pes- talotiopsis trachicarpicola is reported as the causative agent of grapevine diseases in the world for the first time.Neopestalotiopsissp. caused significantly longer lesions than the other taxon present. This study represents the first attempt to identify and characterize thePestalotiopsis-like fungi causing grapevine diseases in China using both morphological and molecular approaches.

Introduction

Grape (Vitis viniferaL. familyVitaceae) 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, bac- terial 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),sa- jeewa83@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).

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / f u n b i o

http://dx.doi.org/10.1016/j.funbio.2014.11.001

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(Urbez-Torres et al.2009).Pestalotiopsis, Neopestalotiopsisand Pseudopestalotiopsis(Pestalotiopsis-like fungi) belong to the fam- ilyAmphisphaeriaceae(Barr 1975, 1990; Kanget al.1998, 1999;

Jeewonet al.2003; Tejesviet al.2007; Maharachchikumbura et al.2011, 2014; Hydeet al.2014) and its species are commonly present in tropical and subtropical ecosystems (Tejesviet al.

2007; Maharachchikumburaet al.2011).Pestalotiopsis-like fungi consists of members that are difficult to identify at the species level (Jeewonet al.2003; Maharachchikumburaet al.2012). It is an important plant pathogenic group (Yasudaet al.2003; Das et al.2010; Maharachchikumburaet al.2011, 2012, 2013, 2014;

Suwannarachet al.2013; Hydeet 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. Se- verely infected fruits become rotten and separate completely from the pedicel (Xuet al.1999; Denget al.2013). Shoot damage is the main symptom of the grapevine trunk infected byPesta- lotiopsis-like fungi. When the disease is severe, it results in the bleaching of canes and formation of fruiting bodies and some- times the surface of the shoots and canes appears split (Sergeevaet 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 (Gubaet al. 1961; Ryuet al. 1999; Xu et al.1999; Sergeevaet al.2005;Urbez-Torres et al.2009, 2012;

Denget al.2013).Pestalotiopsis menezesiana(Bres. & Torr.) Bis- sett. and Pestalotiopsis uvicola (Spegazzini) Bissett. were the first two species to be reported from Japan as causal agents of postharvest disease of grapes (Xuet al.1999).Pestalotiopsis uvicolahas been reported from variousVitissp. includingVitis viniferaandVitis indusain Australia, Brazil, Europe, Italy, Japan and United States (Guba 1961; Sergeeva et al. 2005;Urbez- Torreset 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 thatPestalotiopsis-like fungi occurred not only on leaves, but also on canes, wood, berries and flowers (Castillo-Pando et al.2001; Sergeevaet al.2001;Urbez-Torres et al.2009, 2012;

Denget al.2013).Urbez-Torreset al.(2009, 2012)have shown the association ofPestalotiopsis-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-Torreset 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 aPestalotiopsissp.

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 (Zhanget al.2007). However, in many cases the specific iden- tity of thePestalotiopsisspecies causing disease of grapes has

not been given (Castillo-Pando et al. 2001; Sergeeva et al.

2001; Urbez-Torreset al.2009, 2012; Denget al.2013).

Although there have been many studies identifying the pathogens causing diseases of grapes in China (Penget al.

2013; Dissanayake et al.2014), no studies have been carried out to determinePestalotiopsis-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 25C. Cultures on the filter paper pieces were dried on sterilized filter paper and stored at 20C. The morphology of fungal colonies was recorded following the method used by Maharachchikumburaet al.(2012). Fungal mycelia and spores were observed and photographed using a Leica DM5500B mi- croscope. 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 (Urbez-Torres et al. 2009). In short, the pathogenicity tests were conducted on the shoots collected from matureVitis vi- niferacf. 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. Inocu- lations 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. In- oculated shoots were immediately placed in plastic con- tainers, 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.

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Fruit inoculation

An inoculation method was used for the pathogenicity test on grape fruits. Healthy grape fruits from Vitis viniferacf. 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 mm- diameter 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 contain- ing 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 Mini- tab, 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 Guoet 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 25C for 7e10 d. The ITS,b-tubulin andtef1 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 1000Thermal Cycler in a total volume of 25ml. The PCR mix- tures contained TaKaRa Ex-Taq DNA polymerase 0.3ml, 12.5ml of 2PCR buffer with 2.5ml of dNTPs, 1ml of each primer, 9.2ml of double-distilled water and 100e500 ng of DNA template.

The thermal cycling programme followed

Maharachchikumburaet al.(2012). The PCR products were verified by staining with Ethidium Bromide on 1.2 % agarose elec- trophoresis 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

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 (Thompsonet 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 genusPestalotiopsisandNeopestalotiopsis, 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). Ambigu- ously 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 swap- ping 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 calcu- lated 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 nu- cleotide substitution for each gene, as determined using MrModelTest (Nylander 2004). The GTRþIþG model was selected for ITS and the HKYþIþG model forb-tubulin andtef.

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 gen- eration. 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 inTable 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

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Table 1eStrains used in phylogenetic analyses and their GenBank accession numbers. Ex-type and ex-epitype strains are bolded.

Species Isolate GenBank Accession numbers

ITS b-tubulin tef1

Neopestalotiopsis aotearoa CBS 367.54 KM199369 KM199454 KM199526

Neopestalotiopsis asiatica MFLUCC 12-0286 JX398983 JX399018 JX399049

Neopestalotiopsis australis CBS 114159 KM199348 KM199432 KM199537

Neopestalotiopsis clavispora MFLUCC 12-0280 JX398978 JX399013 JX399044

Neopestalotiopsis clavispora MFLUCC 12-0281 JX398979 JX399014 JX399045

Neopestalotiopsis clavispora ICMP 20405 KJ623224 KJ623206 KJ623238

Neopestalotiopsis chrysea MFLUCC 12-0261 JX398985 JX399020 JX399051

Neopestalotiopsis chrysea MFLUCC 12-0262 JX398986 JX399021 JX399052

Neopestalotiopsis cubana CBS 600.96 KM199347 KM199438 KM199521

Neopestalotiopsis ellipsospora MFLUCC 12-0283 JX399016 JX399016 JX399047

Neopestalotiopsis ellipsospora MFLUCC 12-0284 JX399015 JX399015 JX399046

Neopestalotiopsis eucalypticola CBS 264.37 KM199376 KM199431 KM199551

Neopestalotiopsis foedans CGMCC 3.9123 JX398987 JX399022 JX399053

Neopestalotiopsis foedans CGMCC 3.9178 JX398989 JX399024 JX399055

Neopestalotiopsis foedans CGMCC 3.9123 JX398987 JX399022 JX399053

Neopestalotiopsis honoluluana CBS 114495 KM199364 KM199457 KM199548

Neopestalotiopsis magna MFLUCC 12-652 KF582795 KF582793 KF582791

Neopestalotiopsis mesopotamicum CBS 336.86 KM199362 KM199441 KM199555

Neopestalotiopsis mesopotamicum CBS 299.74 KM199361 KM199435 KM199541

Neopestalotiopsis mesopotamicum CBS 464.69 KM199353 KM199436 e

Neopestalotiopsis natalensis CBS 138.41 KM199377 KM199466 KM199552

Neopestalotiopsis piceana CBS 394.48 KM199368 KM199453 KM199527

Neopestalotiopsis piceana CBS 254.32 KM199372 KM199452 KM199529

Neopestalotiopsis piceana CBS 225.30 KM199371 KM199451 KM199535

Neopestalotiopsis protearum CBS 114178 JN712498 KM199463 KM199542

Neopestalotiopsis rosa CBS 101057 KM199359 KM199429 KM199523

Neopestalotiopsis samarangensis MFLUCC 12-0233 JQ968609 JQ968610 JQ968611

Neopestalotiopsis saprophyta MFLUCC 12-0282 KM199345 KM199433 KM199538

Neopestalotiopsis steyaertii IMI 192475 KF582796 KF582794 KF582792

Neopestalotiopsis surinamensis CBS 450.74 KM199351 KM199465 KM199518

Neopestalotiopsis umbrinospora MFLUCC 12-0285 JX398984 JX399019 JX399050

Neopestalotiopsis zimbabwana CBS 111495 JX556231 KM199456 KM199545

Neopestalotiopsissp. CBS 110.20 KM199342 KM199442 KM199540

Neopestalotiopsissp. CBS 266.80 KM199352 e KM199532

Neopestalotiopsissp. (JZB340002) ICMP 20406 KJ623216 KJ994532 KJ623236

Pestalotiopsis adusta ICMP 6088 JX399006 JX399037 JX399070

Pestalotiopsis anacardiacearum IFRDCC 2397 KC247154 KC247155 KC247156

Pestalotiopsis arceuthobii CBS 434.65 KM199341 KM199427 KM199516

Pestalotiopsis arenga CBS 331.92 KM199340 KM199426 KM199515

Pestalotiopsis australis CBS 114193 KM199332 KM199383 KM199475

Pestalotiopsis australis CBS 111503 KM199331 KM199382 KM199557

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Table 1e(continued)

Pestalotiopsis autralasiae CBS 114141 KM199298 KM199410 KM199501

Pestalotiopsis autralasiae CBS 114126 KM199297 KM199409 KM199499

Pestalotiopsis biciliata CBS 124463 KM199308 KM199399 KM199505

Pestalotiopsis biciliata CBS 790.68 KM199305 KM199400 KM199507

Pestalotiopsis biciliata CBS 236.38 KM199309 KM199401 KM199506

Pestalotiopsis brassicae CBS 170.26 KM199379 e KM199558

Pestalotiopsis camelliae MFLUCC 12-0277 JX399010 JX399041 JX399074

Pestalotiopsis camelliae MFLUCC 12-0278 JX399011 JX399042 JX399075

Pestalotiopsis chamaeropis CBS 113604 KM199323 KM199389 KM199471

Pestalotiopsis chamaeropis CBS 113607 KM199325 KM199390 KM199472

Pestalotiopsis chamaeropis CBS 186.71 KM199326 KM199391 KM199473

Pestalotiopsis chamaeropis CBS 237.38 KM199324 KM199392 KM199474

Pestalotiopsis clavata MFLUCC 12-0268 JX398990 JX399025 JX399056

Pestalotiopsis colombiensis CBS 118553 KM199307 KM199421 KM199488

Pestalotiopsis diploclisiae CBS 115587 KM199320 KM199419 KM199486

Pestalotiopsis diploclisiae CBS 115585 KM199315 KM199417 KM199483

Pestalotiopsis diploclisiae CBS 115449 KM199314 KM199416 KM199485

Pestalotiopsis diversiseta MFLUCC 12-0287 JX399009 JX399040 JX399073

Pestalotiopsis ericacearum IFRDCC 2439 KC537807 KC537821 KC537814

Pestalotiopsis furcata MFLUCC 12-0054 JQ683724 JQ683708 JQ683740

Pestalotiopsis gaultheria IFRD 411-014 KC537805 KC537819 KC537812

Pestalotiopsis grevillea CBS 114127 KM199300 KM199407 KM199504

Pestalotiopsis hawaiiensis CBS 114491 KM199339 KM199428 KM199514

Pestalotiopsis hollandica CBS 265.33 KM199328 KM199388 KM199481

Pestalotiopsis humus CBS 336.97 KM199317 KM199420 KM199484

Pestalotiopsis humus CBS 115450 KM199319 KM199418 KM199487

Pestalotiopsis inflexa MFLUCC 12-0270 JX399008 JX399039 JX399072

Pestalotiopsis intermedia MFLUCC 12-0259 JX398993 JX399028 JX399059

Pestalotiopsis karstenii IFRDCC OP13 KC537806 KC537820 KC537813

Pestalotiopsis knightiae CBS 114138 KM199310 KM199408 KM199497

Pestalotiopsis knightiae CBS 111963 KM199311 KM199406 KM199495

Pestalotiopsis linearis MFLUCC 12-0271 JX398992 JX399027 JX399058

Pestalotiopsis malayana CBS 102220 KM199306 KM199411 KM199482

Pestalotiopsis monocaheta CBS 144.97 KM199327 KM199386 KM199479

Pestalotiopsis monocaheta CBS 440.83 KM199329 KM199387 KM199480

Pestalotiopsis novaehollandiae CBS 130973 KM199337 KM199425 KM199511

Pestalotiopsis proteacearum CBS 111522 KM199294 KM199394 KM199493

Pestalotiopsis proteacearum CBS 171.26 KM199304 KM199397 KM199494

Pestalotiopsis proteacearum CBS 353.69 KM199299 KM199398 KM199496

Pestalotiopsis papuana CBS 331.96 KM199321 KM199413 KM199491

Pestalotiopsis papuana CBS 887.96 KM199318 KM199415 KM199492

Pestalotiopsis parva CBS 265.37 KM199312 KM199404 KM199508

Pestalotiopsis parva CBS 278.35 KM199313 KM199405 KM199509

Pestalotiopsis portugalica CBS 393.48 KM199335 KM199422 KM199510

Pestalotiopsis rhododendri IFRDCC 2399 KC537804 KC537818 KC537811

Pestalotiopsis rhodomyrtus HGUP4230 KF412648 KF412642 KF412645

Pestalotiopsis rosea MFLUCC12-0258 JX399005 JX399036 JX399069

Pestalotiopsis scorparia CBS 176.25 KM199330 KM199393 KM199478

Pestalotiopsis spathulata CBS 356.86 KM199338 KM199423 KM199513

Pestalotiopsis teleopa CBS 114137 KM199301 KM199469 KM199559

Pestalotiopsis teleopa CBS 114161 KM199296 KM199403 KM199500

Pestalotiopsis teleopa CBS 113606 KM199295 KM199402 KM199498

Pestalotiopsis trachicarpicola MFLUCC 12-0263 JX399000 JX399031 JX399064

Pestalotiopsis trachicarpicola IFRDCC 2403 KC537809 KC537823 KC537816

Pestalotiopsis trachicarpicola OP068 JQ845947 JQ845945 JQ845946

Pestalotiopsis trachicarpicola(JZB340016) ICMP 20420 KJ623209 KJ623193 KJ623241

Pestalotiopsis unicolor MFLUCC 12-0275 JX398998 JX399029 JX399063

Pestalotiopsis verruculosa MFLUCC 12-0274 JX398996 e JX399061

(continued on next page)

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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 viniferacv. Guifei Meigui,V. viniferacv. Brier grape andV. viniferacv. 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. TwoPestalotiopsis-like species isolated from Chinese grapevines were pathogenic and re- isolated (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 toNeopestalotiopsissp. caused significantly longer lesions (F¼11.63,P>0.05) than the other species ofPestalotiop- sisrecorded.Neopestalotiopsisstrains ICMP 20415, ICMP 20416, ICMP 20417, ICMP 20418 and ICMP 20419 showed longer lesions (mean lesion length¼5.5 cm) than the other strains of

this species. Pestalotiopsis trachicarpicola was less virulent with mean lesions of 2.4 cm. Therefore,Neopestalotiopsissp.

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).Neopestalo- tiopsisstrains ICMP 20415, ICMP 20416, ICMP 20417, ICMP 20418 and ICMP 20419 caused significantly longer lesions in shoots (F¼1.27,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 Table 1e(continued)

Pseudopestalotiopsis cocos CBS 272.29 KM199378 KM199467 KM199553

Pseudopestalotiopsis indica CBS 459.78 KM199381 KM199470 KM199560

Pesudopestalotiopsis theae MFLUCC12-0055 JQ683727 JQ683711 JQ683743

Pesudopestalotiopsis theae SC011 JQ683726 JQ683710 JQ683742

Fig 1eSymptom on grapevine caused byPestalotiopsis-like fungi. (A) Damaged grape shoot in the field (B) Bleached canes with slitting, (C) Wedged-shaped canker, (D) Bleached cane with fruiting bodies.

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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:

F¼7.33,P>0.05; width:F¼8.05,P>0.05) than the other species and the strains recorded in this study.

Phylogenetic analysis

Phylogenetic trees were constructed using combined ITS, b-tubulin andtef1sequences for our 17 isolates ofPestalotiop- sis-like fungi with those that originated from Maharachchikumbura et al. (2012, 2013b, 2014), Song et al.

(2013, 2014), andHydeet al.(2014). Two separate phylogenetic

Fig 2eSymptom on grape fruits and shoots caused byPestalotiopsis-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.

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Fig 3eMaximum Parsimonious tree obtained from a heuristic search of the combined ITS,b-tubulin,tef1sequence 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.)

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Table 2eMorphological comparison ofPestalotiopsisspecies recorded in vineyards of China.

Species Conidiomata Conidiogenous cells

Conidia Appendages Culture

characteristics

Basal cell 2nd Cell 3rd Cell 4th Cell Apical cell Basal Apical

Neopestalotiopsis sp. (Fig 5)

120e550mm diam, acervuli, globose- oval, black, scattered, semi-immersed on PDA black conidia in a slimy, glistening mass

Fusiform, hyaline, simple, short

Conical, hyaline, thin and verruculose to smooth- walled, 1.9e6.2mm long (x¼4.1mm, n¼25)

Pale brown to olivaceous, 3.4e5.9mm (x¼4.7mm, n¼25)

Darker brown to olivaceous, 4.1e6.5mm (x¼5.5mm, n¼25)

Darker brown, 3.8e6.5mm (x¼5.2mm, n¼25)

Cylindrical to subcylindric, hyaline, 2.3e5.8mm (x¼4.5mm,n¼25)

Single basal appendage present, filiform 3.4e7mm (x¼5.2mm, n¼60).

Long, tubular, 11e53mm (x¼31.8mm, n¼60), 2e4 (mostly 3) arising from the apex of the apical cell

Colonies on PDA reaching 8 cm diam.

after 7 days at 25C, edge undulate, whitish, aerial mycelium with black fruiting bodies, concentric, gregarious, reverse of culture white to pale yellow Pestalotiopsis

trachicarpicola (Fig 6)

120e410mm diam., acervuli, globose, black, semi-immersed on PDA releasing black in a black conidia in a slimy, glistening mass

Fusiform, hyaline, short, thin-walled

Conic to acute, hyaline, thin and verruculose, 2.8e6.8mm long (x¼4.4mm, n¼25)

Concolorous, 2.9e7.4mm (x¼4.7mm, n¼25)

Concolorous, 3.4e6.8mm (x¼5.3mm, n¼25)

Concolorous, 3e5.8mm (x¼4.7mm, n¼25)

Conic to subcylindrical, hyaline, 2.7e6.4mm (x¼4mm,n¼25),

Single basal appendage present, filiform 2.2e8.3mm (x¼4.2mm, n¼60)

5e17mm (x¼10mm,n¼60) long, tubular, 2e4 (mostly 3) arising from the apex of the apical cell

Colonies on PDA reaching 6 cm diam.

after 7 d at 25C, edge fimbriate, whitish, dense aerial mycelium with black fruiting bodies, concentric, reverse of culture pale yellow

zationofPestalotiopsis-likefungi9

citethisarticleinpressas:JayawardenaRS,etal.,IdentificationandcharacterizationofPestalotiopsis-likefungirelatedtodiseasesinChina,FungalBiology(2014),http://dx.doi.org/10.1016/j.funbio.2014.11.001

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Fig 4eMaximum Parsimonious tree obtained from a heuristic search of the combined ITS,b-tubulin,tef1sequence 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.)

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trees were constructed. Maximum-parsimony and Bayesian inference produced nearly identical topologies (Bayesian trees are not shown).

The combined gene alignment forPestalotiopsiscomprised of 72 taxa and 1516 characters including gaps (ITS: 1e554,b- tubulin: 555e1010 andtef1: 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) (TL¼ 1180, CI¼0.564, RI¼0.808, RC¼0.455, HI¼0.436). Only one isolate obtained in this study (ICMP 20420) was clustered together withPestalotiopsis trachicarpicolawith a strong support.

The combined gene alignment for Neopestalotiopsis and Pseudopestalotiopsiscomprised 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, CI¼0.794, RI¼0.871, RC¼0.692, HI¼0.206). Sixteen isolates obtained in this study clustered together withNeopestalotiopsis species isolated fromVitis viniferaof India (CBS 266.80) with a strong support.

Morphological characters

Morphological characters of the species identified are summa- rized including colony appearance, conidiogenous cells, conidia and colony characters (Table 2).

Discussion

Xuet al.(1999)isolatedPestalotiopsisfrom rotted grape berries in Japan and found thatPestalotiopsis menezesianaandPestalo- tiopsis 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 ofPestalotiopsis-like fungi found on grapes (Guba 1961; Ryu et al.1999; Sergeevaet al.2005;

Urbez-Torres et al.2009,2012), even though in this study these two species were not recorded.Maharachchikumbura et al.

(2013a)referredP.menezesianaasP. cf.menezesiana, as the Gen- Bank 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)isolatedPestalotiopsis guepiniifrom 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 toPestalo- tiopsis trachicarpicola, but the conidiogenous cells of this species are ampuliform and the apical appendages often form a closely aggregated crest, which cannot be observed inP. tra- chicarpicola.Pestalotiopsis menezesianais 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 onP.cf.

menezesiana,P. guepiniandP. uvicolamust be carried out in order to clarify their phylogenetic status within thePestalotiop-

sis-like fungi as well as to epitypify them.

Maharachchikumburaet al.(2014)introduced two new genera Fig 5eNeopestalotiopsissp. (ICMP20417) (A, B) Colony on PDA, (A) from above, (B) from below, (C) Conidiomata, (DeH) Conidia with versicolorous median cells, Scale bars[deh 10mm.

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intoPestalotiopsis-like fungi:NeopestalotiopsisandPseudopesta- lotiopsis.Neopestalotiopsisspecies are characterized by indis- tinct to reduced conidiophores and the two upper median cells are darker than the lower median cells. GenusPseudopes- talotiopsisis characterized by dark concolorous median cells with knobbed apical appendages. Pestalotiopsis-like fungal species appear to have a wide host range (Guba 1961;

Maharachchikumburaet al.2012) and most species were pre- viously been named according to host association and only a small number of morphological characters were available to differentiate between species (Maharachchikumburaet al.

2012).Pestalotiopsis trachicarpicolahas been recorded fromTra- chycarpus fortunei(Chinese windmill palm),Chrysophullumsp.

(Rare star Apple), Schima sp., and Symplocos sp. in China (Maharachchikumburaet al.2012; Zhanget al.2012).Neopesta- lotiopsissp. (CBS 266.80) had been recorded fromV. viniferain India (Maharachchikumburaet al.2014). The use of molecular data in resolvingPestalotiopsis-like fungi reviewed by various studies suggested that multi-locus phylogenetic analysis is needed to resolve the cryptic species in this genus (Huet al.

2007; Liu et al. 2007; Tejesvi et al. 2007a;

Maharachchikumburaet al.2012, 2013c, 2014). Use of ITS sequences alone does not resolvePestalotiopsis-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 generaBipolaris, Colletotrichum, Diaportheand Phyllosticta(Hydeet al.2014; Udayangaet al.2014; Yanet 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 grapevine disease (Urbez-Torres et al.2009).Pestalotiopsis-like fungi have been often isolated from the bleached canes of grapevine withPhomopsissp. andBotryosphaeriasp. (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-Pandoet al.2001; Sergeeva et al.2001;Urbez-Torres et al.2009; Denget al.2013). More re- cently, 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 Pestalotiopsis- like fungi (Hydeet al.2014; Maharachchikumburaet al.2014;

Nilssonet al.2014). In their studyMaharachchikumburaet 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 asNeopestalotiop- sissp. and we would like to follow that in this paper. Conidial characters alone are insignificant criteria in distinguishing

Pestalotiopsis species (Jeewon et al. 2003;

Maharachchikumburaet al.2011, 2013a, b). Many sequences for genusPestalotiopsisdeposited in GenBank are unreliable.

Therefore, identification of Pestalotiopsis to species level is presently difficult. There is a need for epitypification in genus Pestalotiopsis(Maharachchikumburaet al.2012, 2013d). Correct species identification is essential in plant pathogenic genera (Maharachchikumbura et al. 2013a; Rossman & Palm- Hernandez 2008). In order to have effective measures to prevent the unwanted entry of diseases into a country, the plant Fig 6ePestalotiopsis trachicarpicola(ICMP20420) (A, B) Colony on PDA, (A) from above, (B) from below, (C) Conidiomata, (DeG) Conidia with concolorous median cells, Scale bars[deg[10mm.

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pathologists should be able to name the Pestalotiopsis-like fungi confidently (Maharachchikumburaet al.2011).

Careful handling of grape berries in the field as well as during storage can prevent the fruit rot caused byPestalotiopsis- like fungi. Proper maintenance of the grape orchards during pruning can help to prevent the canker disease caused byPes- talotiopsis-like fungi.

Conclusion

This study represents the first attempt to identify and characterize thePestalotiopsis-like fungi causing diseases in grapevine: fruit rot and trunk diseases in China using both morphological and molecular approaches. This is the first report ofNeopestalotiopsissp. and ofPestalotiopsis trachicarpicola causing diseases in grapevine.

Acknowledgements

The research was funded by CARS-30, National Research Council of Thailand (grant forPestalotiopsisNo: 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 Sci- ences, project number 2013T2S0030, for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany.

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