A multi-locus backbone tree for Pestalotiopsis,
with a polyphasic characterization of14new 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:26July2012/Accepted:1August2012/Published online:1September2012
#Mushroom Rearch Foundation2012
Abstract Pestalotiopsis is a taxonomically confud,path-ogenic and chemically creative genus requiring a critical re-examination using a multi-gene phylogeny bad on ex-type and ex-epitype cultures.In this study40isolates of Pesta-lotiopsis,comprid of28strains collected from living and dead plant material of various host plants from China were studied by means of morphology and analysis of I
TS,β–tubulin and tef1gene quence data.Bad on mo-lecular and morphological data we describe14new species(Pestalotiopsis asiatica,P.chinensis,P.chrya, P.clavata,P.diversita,P.ellipsospora,P.inflexa,P. intermedia,P.a,P.saprophyta,P. umberspora,P.unicolor and P.verruculosa)and three species are epitypified(P.adusta,P.clavispora and P. foedans).Of the10gene regions(ACT,β-tubulin, CAL,GPDH,GS,ITS,LSU,RPB1,SSU and tef1) utilized to resolve cryptic Pestalotiopsis species,ITS,β–tubulin and tef1proved to be the better markers. The other gene regions were less uful due to poor success in PCR amplification and/or in their ability to resolve species boundaries.As a single gene tef1met the requirements for an ideal candidate and functions well for species delimitation due to its better species resolution and PCR success.Althoughβ-tubulin showed fairly good differences among species,a combination of ITS,β-tubulin and tef1gene data gave the best resolu-tion as compared to single gene analysis.This work provides a backbone tree for22ex-type/epitypified spe-cies of Pestalotiopsis and can be ud in future studies of the genus.
Keywordsβ-tubulin.Epitype.ITS.Phylogeny. f1
S.S.N.Maharachchikumbura
:E.Chukeatirote:K.D.Hyde Institute of Excellence in Fungal Rearch,
Mae Fah Luang University,
Chiang Rai57100,Thailand
S.S.N.Maharachchikumbura
:E.Chukeatirote:K.D.Hyde(*) School of Science,Mae Fah Luang University,
Chiang Rai57100,Thailand
e-mail:
S.S.N.Maharachchikumbura
:L.-D.Guo(*):L.Cai:X.Sun State Key Laboratory of Mycology,Institute of Microbiology, Chine Academy of Sciences,
Beijing100101,People’s Republic of China
e-mail:guold@sun.im.ac
W.P.Wu
Novozymes China,
14Xin Xi Lu,Shangdi Zone,Haidian District,Beijing100086, People’s Republic of China P.W.Crous
CBS-KNAW Fungal Biodiversity Centre,
P.O.Box85167,3508AD,Utrecht,The Netherlands
D.J.Bhat
Department of Botany,Goa University,
Panaji,Goa403206,India
E.H.C.McKenzie
你在哪里
Landcare Rearch,
Private Bag,
92170,Auckland,New Zealand
A.H.Bahkali
萎蕤College of Science,Botany and Microbiology Department, King Saud University,
P.O.Box:2455,Riyadh1145,Saudi Arabia
Fungal Diversity(2012)56:95–129 DOI10.1007/s13225-012-0198-1
Introduction
Pestalotiopsis,an appendage-bearing conidial axual form in the family Amphisphaeriaceae(Barr1975,1990;Kang et al.1998,1999)is widely distributed throughout the tropical and temperate ecosystems(Bate-Smith and Metcalfe1957).It is an important plant pathogenic genus(Yasuda et al.2003;Das et al.2010;Maharachchikumbura et al.2011)with more than 235species,traditionally named according to their host associ-ations(Guba1961;Steyaert1949;Venkatasubbaiah et al.1991; Kohlmeyer and Kohlmeyer2001).Following the discovery of taxol,the multimillion dollar anti-cancer drug,from P.micro-spora(Speg.)G.C.Zhao&N.Li,an endophytic strain isolated from Taxus wallachiana(Strobel et al.1996),the importance of the genus has incread considerably(Strobel et al.2002;Xu et al.2010).C
hemical exploration of endophytic Pestalotiopsis species subquently incread in an unprecedented way(Wei and Xu2004;Tejesvi et al.2007a,b).Species belonging to the genus Pestalotiopsis are thought to be a rich source for biopro-spectingwhencomparedtoother fungalgenera(Alyetal.2010; Xu et al.2010).Xu et al.(2010)reviewed130different com-pounds isolated from species of Pestalotiopsis in the preceding 10years.The included bioactive alkaloids,terpenoids,iso-coumarin derivatives,coumarins,chromones,quinones,mi-quinones,peptides,xanthones,xanthone derivatives,phenols, phenolic acids,and lactones with a range of antifungal,antimi-crobial,and antitumor activities.
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Due to their ability to switch life modes,many endophyt-ic and pathogenic Pestalotiopsis species persist as saprobes (Hyde et al.2007;Zhou and Hyde2001).Species of Pesta-lotiopsis have been isolated as saprobes from dead leaves, bark and twigs(Guba1961).Several species have been recovered from soil,polluted stream water,wood,paper, fabrics and wool(Guba1961).For example,P.bicolor(Ellis &Everh.)A.R.Liu,T.Xu&L.D.Guo,P.funerea(Desm.) haetioides(Doyer)llica (Sacc.&Voglino)Tak.Kobay.,P.disminata(Thüm.) Steyaert,P.foedans(Sacc.&Ellis)Steyaert,P.versicolor (Speg.)Steyaert and P.virgatula(Kleb.)Steyaert
are common saprobic species recorded either from decaying leaves or bark. However,there is less recent data on saprobic Pestalotiopsis species(Table1).
The u of molecular data in resolving Pestalotiopsis species has been reviewed by Hu et al.(2007),Tejesvi et al.(2007a),Liu et al.(2010)and Maharachchikumbura et al. (2011).The studies have suggested that multi-locus phy-logenetic analysis is needed to resolve the cryptic species in the genus.Furthermore,species need to be epitypified so that we have quence data pinned to names and thus can confidently name species in future(Cai et al.2011).
We have been studying the genus Pestalotiopsis and testing the u of various genes to resolve species boundaries.In this study,we report on28isolates sourced from plant material from China.All isolated species were first morphologically characterid and then quenced using ITS,β-tubulin and tef1genes.In order to lect suitable gene regions for better species resolution,we analyzed nuclear ribosomal large sub-unit rDNA(LSU),nuclear ribosomal small subunit rDNA (SSU),partial actin(ACT),glutamine syntha(GS), glyceraldehyde-3-phosphate dehydrogena(GPDH),RNA polymera II(RPB1)and calmodulin(CAL)gene regions for veral isolates of Pestalotiopsis.We compared the mor-phological data versus the quence data from single and combined genes to establish which characters satisfactorily resolve the species.As a result,we epitypified three species
and describe14new saprobic Pestalotiopsis species.It is our hope that this work will provide a backbone phylogenetic tree for22type/epitypified species,which can be ud in future taxonomic work on the genus.
Methods and materials
Isolation and identification of pathogen
Dead plant tissues were collected from different sites in China.The samples were placed in parate plastic bags lined with tissue paper,sprayed with sterile water to create humid conditions and incubated at room temperature.The fungi prent on the samples were isolated by single spore culture technique(Chomnunti et al.2011).In short,a con-idiomata was immerd in300μl of sterile distilled water on a slide and left a few minutes so that the conidia were discharged.A conidial suspension was made,small drops
Table1Saprobic Pestalotiopsis species with their host/substrata
Species Host/substrate References
Pestalotiopsis funerea Dead leaves of Rhododendron,Chamaecyparis,
Cupressus,Pinus,Juniperus
Dennis1995;Ellis and Ellis1997 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,
Rhododendron ponticum,R.hybridum,Prunus laurocerasus
Dennis1995;Ellis and Ellis1997 P.theae(Sawada)Steyaert Seeds of Diospyros crassiflora Douanla-Meli and Langer2009
were placed on water agar(WA)in Petri dishes and kept at room temperature for8–12h for conidia to germinate;single germinating conidia were transferred to potato dextro agar (PDA)plates.The plates were incubated at25°C for7to 10days.Colonies grown on PDA were transferred to PDA slants,and stored at4°C for further study.Sporulation was induced by placing sterilized carnation leaves on the surface of PDA with growing mycelia.The morphology of fungal colonies was recorded following the method of Hu et al. (2007).Fungal mycelia and spores were obrved under a light micr
oscope and photographed.All microscopic measure-ments were done with Tarosoft image framework(v.0.9.0.7) and30conidial measurements were taken for each isolate. Isolates were deposited in Novozymes,Beijing and were also transferred to MFLUCC from Novozymes by Material Trans-fer Agreement and cannot be distributed to a third party. All other cultures dealt with in this study were obtained from China General Microbiological Culture Collection (CGMCC)and The International Collection of Micro-organisms from Plants(ICMP).
DNA extraction
Total genomic DNA was extracted from fresh cultures using a modified protocol of Guo et al.(2000).Fresh fungal mycelia(500mg)was scraped from the margin of a PDA plate incubated at25°C for7to10days and transferred into a1.5ml centrifuge tube with100μl of preheated(60°C)2X CTAB extraction buffer(2%(w/v)CTAB,100mM Tris-HCl,1.4M NaCl,20mM EDTA,pH8.0),and200mg sterilized quartz sand.Mycelia were ground using a glass pestle for5min and an extra500μl2X CTAB preheated (60°C)was added and incubated in a65°C water bath for 30min with occasional shaking.500μl of phenol:chloro-form(1:1)was added to each tube and shaken thoroughly to form an emulsion.The mixture was spun at11,900g for 15min at25°C in a microcentrifuge and the supernatant pha decanted into a fresh1.5ml tube.Supernatant con-taining DNA was re-extracted w
ith phenol:chloroform(1:1) at4°C until no interface was visible.50μl of5M KOAc was added into the supernatant followed by400μl of iso-propanol and inverted gently to mix.The genomic DNA was precipitated at9,200g for2min at4°C in a microcentrifuge. The DNA pellet was washed with70%ethanol twice and dried using SpeedVac®(AES1010;Savant,Holbrook,NY, USA)until dry.The DNA pellet was then resuspended in 100μl TE buffer(10mM Tris-HCl,1mM EDTA).
PCR amplification
The ITS and5.8S region of rDNA fragment was amplified using primer pairs ITS5(5′-GGAAGTAAAAGTCGTAA CAAGG-3′)and ITS4(5′-TCCTCCGCTTA TTGA TA TGC-3′)(White et al.1990),partialβ-tubulin gene region was amplified with primer pairs BT2A(5′-GGTAAC CAAATCGGTGCTGCTTTC-3′)and BT2B(5′ACCCTCAGTGTAGTGACCCTTGGC-3′)(Glass& Donaldson1995;O’Donnell&Cigelnik1997)and tef1 was amplified using the primer pairs EF1-526F(5′-GTCGTYGTYATY GGHCAYGT-3′)and EF1-1567R (5′-ACHGTRCCRATACCACCRATCTT-3′)(Rehner 2001).In addition to above three gene regions lected LSU,SSU,Actin,GS,GPDH,RPB1and CAL regions were amplified using primer pair/s listed in Table2.
PCR was performed with the25μl reaction system con-taining19.5μl of double distilled water,2.5μl of
10×Taq buffer with MgCl2,0.5μl of dNTP(10mM each),0.5μl of each primer(10μM),0.25μl Taq DNA polymera(5U μl−1),1.0μl of DNA template.The thermal cycling program was as follows:For ITS an initial denaturing step of95°C for3min,followed by35amplification cycles of95°C for 30s,52°C for45s and72°C for90s,and a final extension step of72°C for10min.Forβ-tubulin PCR conditions were an initial step of3min at95°C,35cycles of1min at 94°C,50s at55°C,and1min at72°C,followed by 10min at72°C.For tef1,an initial step of5min at94°C, 10cycles of30s at94°C,55s at63°C or66°C(decreasing 1°C per cycle),90s at72°C,plus36cycles of30s at 94°C,55s at53°C or56°C,90s at72°C,followed by 7min at72°C.The LSU,SSU,Actin,GS,GPDH,RPB 1and CAL regions were tested under different optimal condi-tions(not shown).The PCR products were verified by staining with Goldview(Guangzhou Geneshun Biotech,China)on1% agaro electrophoresis gels.
Phylogenetic analysis
DNAStar and SeqMan were ud to obtain connsus quences from quences generated from forward and re-ver primers.Single locus datat and combination of multi-locus datat of three gene regions were aligned using CLUSTALX(v.1.83)(Thompson et al.1997).The quen-ces were further aligned using default ttings of MAFFTv6 (Katoh and Toh2008;mafft.cbrc.jp/alignment/rver/)and manually adjusted using BioEdit(Hall1999)to allow max-
imum alignment and minimum gaps.A maximum parsimo-ny analysis(MP)was performed using PAUP(Phylogenetic Analysis Using Parsimony)v.4.0b10(Swofford2002). Ambiguously aligned regions were excluded and gaps were treated as missing data.Trees were inferred using the heu-ristic arch option with TBR branch swapping and1,000 random quence additions.Maxtrees were t up to5,000, branches of zero length were collapd and all multiple parsimonious trees were saved.Tree length[TL],consisten-cy index[CI],retention index[RI],rescaled consistency index[RC],homoplasy index[HI],and log likelihood
[-ln L](HKY model)were calculated for trees generated under different optimality criteria.The robustness of the most parsimonious trees was evaluated by1,000bootstrap replica-tions resulting from maximum parsimony analysis,each with 10replicates of random stemwi addition of taxa(Felnstein 1985).The Kishino–Hagawa tests(Kishino&Hagawa 1989)were performed to determine whether the trees inferred under different optimality criteria were significantly different. Trees were viewed in Treeview(Page1996).
Results
Phylogenetic trees were constructed using individual and combined ITS,β-tubulin and tef1quences
大专考公务员for our40 isolates of Pestalotiopsis with a Seiridium species as the outgroup taxon and other quences downloaded from GenBank(Table3).We tested10genes in PCR amplifica-tion,alignment and the species delimitation in Pestalotiop-sis(Tables4and5)and found thatβ-tubulin and tef1were the optimal genes,while ITS is included as it is the accepted barcode for fungi(Schoch et al.2012).We ud the avail-able type ITS quences from other studies(Pestalotiopsis pallidotheae,P.hainanensis,P.jesteri and P.kunmingensis), for comparison.
Sequence analysis of ITS from Pestalotiopsis strains
ITS quences from the types(Pestalotiopsis pallidotheae,P. hainanensis,P.jesteri and P.kunmingensis)for Pestalotiopsis were analyd with our isolates ud in this study.The align-ment comprid45taxa and527characters(including gaps) (Fig.1).Parsimony analysis indicates that398characters were constant,41variable characters parsimony-uninformative and 88characters are parsimony-informative.The parsimony anal-ysis of the data matrix resulted in two equally parsimonious trees and the first tree(TL0243,CI00.683,RI00.910,HI0 0.317,RC00.622)is shown here(Fig.1).
不知道说什么In the ITS phylogram,the Pestalotiopsis strains parated into three major clades,named A,B and C
with high bootstrap support(Fig.1).Clade A comprid species having pale brown or olivaceous concolorous median conidial cells.Clade B comprid species having versicolorous median conidial cells and Clade C species with dark concolorous median conidial cells,with knobbed apical appendages.The species within each group were not well resolved at the terminal clades.Specifical-ly,all taxa in Clade B did not parate into distinct species but clustered in two subclades.Species resolution was higher in Clade A,although a few species are not well resolved at the terminal ends.Thus,ITS had lower inter-specific variation and, therefore,further gene quences are needed to determine ge-netic variation within each biological species.
Sequence analysis ofβ-tubulin gene data
from Pestalotiopsis strains
The aligned datat forβ-tubulin quences comprid37 taxa and487characters(including gaps).Parsimony analy-sis indicated that285characters were constant,48variable characters parsimony-uninformative and154characters parsimony-informative.The parsimony analysis of the data matrix resulted in two equally parsimonious trees and the first tree(TL0410steps,CI00.702,RI00.912,HI00.298 and RC00.640)was shown here(Fig.2).
Analysisoftheβ-tubulingenequencesresultedinaphylo-gram(Fig.2)in which the Pestalotiopsis species parated into three major clades,A,B and C with high bootstrap support. Clade A comprid twelve well-resolved species.It was not possible to obtain PCR products from P.chinensis(MFLUCC 12-0273),P.intermedia(MFLUCC12-0260),P.linearis (MFLUCC12-0272)and P.verruculosa(MFLUCC12-0274) using primer pair BT2A and BT2B.Although most of the species were well-resolved in theβ-tubulin tree,the success rate of PCR has been low for this gene.Therefore,further molecular loci were needed to resolve the species in this genus.
Sequence analysis of tef1gene data from of Pestalotiopsis strains
The aligned datat for tef1quence data comprid39taxa and1,005characters(including gaps).Among the,723 characters were constant,87variable characters parsimony-uninformative and195characters parsimony-informative. The parsimony analysis resulted in six equally parsimonious
Table2Primers ud in this study to test different genes Region Primer/s
LSU LR OR/5(Rehner and Samuels1994;Moriya et al.2005)
SSU NS1/4(White et al.1990)
ACT ACT512F/783R(Carbone and Kohn1999)
GS GS F1/R1(Stephenson et al.1997;Guerber et al.2003)
GPDH GDF1/GPD2LM(Myllys et al.2002;Guerber et al.2003)
RPB1RPB1Af/Ac/Cr(www.clarku.edu/faculty/dhibbett/Protocols_Folder/Primers/Primers.pdf) CAL CL1/2;CAL228F/737R(Carbone and Kohn1999;O’Donnell et al.2000)
Table3Isolates ud in this study百家讲坛汉武帝
Taxon Isolates a GenBank Accession Number
ITSβ-tubulin tef1
P.adusta(Ellis&Everh.)Steyaert ICMP6088JX399006JX399037JX399070 P.adusta MFLUCC10-146JX399007JX399038JX399071 P.asiatica Maharachchikumbura&K.D.Hyde MFLUCC12-0286/NN047638JX398983JX399018JX399049 P.camelliae Y.M.Zhang,Maharachchikumbura&K.D.Hyde MFLUCC12-0277JX399010JX399041JX399074 P.camelliae MFLUCC12-0278JX399011JX399042JX399075 P.chinensis Maharachchikumbura&K.D.Hyde MFLUCC12-0273/NN047218JX398995--
P.chrya Maharachchikumbura&K.D.Hyde MFLUCC12-0261/NN042855JX398985JX399020JX399051 P.chrya MFLUCC12-0262/NN047037JX398986JX399021JX399052 P.clavata Maharachchikumbura&K.D.Hyde MFLUCC12-0268/NN047134JX398990JX399025JX399056 P.clavata MFLUCC12-0269/NN047005JX398991JX399026JX399057 P.clavispora MFLUCC12-0280/NN043011JX398978JX399013JX399044 P.clavispora MFLUCC12-0281/NN043133JX398979JX399014JX399045 P.diversita Maharachchikumbura&K.D.Hyde MFLUCC12-0287/NN047261JX399009JX399040JX399073 P.ellipsospora Maharachchikumbura&K.D.Hyde MFLUCC12-0283JX399016JX399016JX399047 P.ellipsospora MFLUCC12-0284JX399015JX399015JX399046 P.foedans(Sacc.&Ellis)Steyaert CGMCC3.9178JX398989JX399024JX399055 P.foedans CGMCC3.9123JX398987JX399022JX399053 P.foedans CGMCC3.9202JX398988JX399023JX399054 P.furcata Maharachchikumbura&K.D.Hyde MFLUCC12-0054JQ683724JQ683708JQ683740 P.hainanensis-GQ869902--
P.inflexa Maharachchikumbura&K.D.Hyde MFLUCC12-0270/NN047098JX399008JX399039JX399072 P.intermedia Maharachchikumbura&K.D.Hyde MFLU
初二历史上册
CC12-0259/NN047642JX398993JX399028JX399059 P.intermedia MFLUCC12-0260/NN047073JX398997JX399019JX399062 P.jesteri Strobel,J.Yi Li,E.J.Ford&W.M.Hess-AF377282--
怎么戒烟最有效
P.jesteri MFLUCC12-0279/NN042849JX399012JX399043JX399076 P.kunmingensis J.G.Wei&T.Xu-AY373376--
P.linearis Maharachchikumbura&K.D.Hyde MFLUCC12-0271/NN047190JX398992JX399027JX399058 P.linearis MFLUCC12-0272/NN047141JX398994JX399060 P.pallidotheae Kyoto Watan.&Yas.Ono-AB482220--
achicarpicola MFLUCC12-0266/achicarpicola MFLUCC12-0267/achicarpicola OP068JQ845947JQ845945JQ845946 P.umberspora Maharachchikumbura&K.D.Hyde MFLUCC12-0285/NN042986JX398984JX399019JX399050 P.unicolor Maharachchikumbura&K.D.Hyde MFLUCC12-0275/NN047308JX398998JX399029JX399063 P.unicolor MFLUCC12-0276/NN046974JX398999JX399030-
P.verruculosa Maharachchikumbura&K.D.Hyde MFLUCC12-0274/NN047309JX398996-JX399061 Seiridium sp.SD096JQ683725JQ683709JQ683741
a Acronyms:NN Novozymes
