Reporter gene transformation of grapevine pathogens Phaeomoniella chlamydospora and Phomopsis viticola, and biocontrol agent Trichoderma harzianum

Mclean, Tamaryn (2007-03)

Thesis (MScAgric)--University of Stellenbosch, 2007.

Thesis

ENGLISH ABSTRACT: Trunk diseases cause major economical losses in the grape growing industry due to decline and premature dieback, resulting in major reductions in quantity and quality in yield. Trunk diseases are caused by a complex of pathogens including basidiomycetes, species of Botryosphaeriaceae (including Botryosphaeria and aggregate genera, Lasiodiplodia and Neofusicoccum), Eutypa lata, Phaeoacremonium spp., Phaeomoniella chlamydospora and Phomopsis viticola. Limited knowledge is available on interactions between the grapevine host and these pathogens. Currently, there are no sustainable control measures available for management of trunk disease pathogens. Grapevine pruning wounds serve as the main entry portals for trunk disease pathogens, and therefore require prolonged protection. The biological grapevine pruning wound protectant, Trichoderma harzianum, have demonstrated the ability to colonise pruning wound sites, thereby providing a sustainable living barrier against trunk disease pathogen invasion. Although T. harzianum has the potential for controlling trunk disease pathogens, limited knowledge is available on its host colonisation ability and methods of antagonism. Knowledge on the interactions of trunk disease pathogens with the host, as well as with T. harzianum within grapevine host tissue will greatly aid the development of sustainable control strategies. However, host interaction studies have been hampered due to difficulty in observing and studying target fungi within the grapevine host, where several endophytic fungi are also present. The transformation of fungi with reporter genes, which convey detectable phenotypes such as green and red fluorescence to fungi, will greatly aid these studies. Therefore, the first aim of this study was to stably transform South African Pa. chlamydospora, P. viticola (two isolates) and T. harzianum isolates with the green (GFP) or red (DsRed-Express) reporter genes using a polyethylene glycol/calcium chloride transformation method and the selectable marker gene hygromycin phosphotransferase (hph). The second aim of the study was to determine whether the transformation process has altered biological characteristics [colony colour, spore size, germination percentage, growth rate at different temperatures, virulence (Pa. chlamydospora and P. viticola) and host colonisation (T. harzianum)] of the transformants. Furthermore, in addition to characterisation of these transformants and wild type isolates, a Pa. chlamydospora GFP transformant (pCT74-P7) and its wild type isolate from New Zealand were also characterised. Phaeomoniella chlamydospora (STE-U 7584) was stably transformed with the GFP or DsRed-Express protein reporter genes, yielding brightly fluorescing transformants. The presence of the trans genes was also confirmed through polymerase chain reaction amplifications. Characterisation of the colony colour, spore size, germination percentage and growth rate at different temperatures of two of the stable, highly fluorescent GFP (PcG 1 and PcG 10) and DsRed-Express (PcRl and PcR2) transformants as well the Pa. chlamydospora GFP transformant (pCT74-P7), revealed no differences between the transformants and their respective wild type isolates. The only exception was the significant lower germination percentage of transformant PcG 10, and the different colony colour of transformants PcG 1 and PcRl, when compared with the wild type isolate. Characterisation of the virulence of three of the reporter gene transformants (PcGl, PcRl and pCT74-P7) showed that they did not differ from their respective wild type isolates . . Two P. viticola isolates (STE-U 6048 and STE-U 6049) were stably transformed with the GFP and DsRed-Express reporter genes. However, brightly fluorescing transformants could not be obtained, limiting the usefulness of these isolates in host colonisation studies. All the GFP transformants exhibited low to intermediate levels of fluorescence in mycelia as well as in conidia over a 6-week growth period. Similarly, all the DsRed-Express transformants also showed low to intermediate levels of fluorescence in the mycelia of 1- to 3-week-old cultures, whereas no fluorescence was observed in 4- to 6-week-old cultures. Contrarily, the conidia of DsRed-Express transformants showed very bright fluorescence in 3- to 4-week-old cultures. The presence of the hph, GFP and DsRed-Express genes was shown through polymerase chain reaction amplifications. Biological characterisation of a subset of the isolates and their respective wild type isolates showed that they were similar to their respective wild type isolates. Trichoderma harzianum strain T77 was stably transformed with the GFP or DsRedExpress reporter genes. However, transformation of T. harzianum strain T77 was difficult, since most transformants proved to be unstable, and did not retain the selectable marker gene (hygromycin phosphotransferase) or the reporter genes. Only four stably transformed isolates, two expressing GFP (TGl and TG2) and two expressing DsRed-Express (TRI and TR2) were obtained. Characterisation of the growth rate, morphology, conidial size and germination percentage of these transformants showed that transformants TRI and TG 1 were altered in their growth rate and/or germination percentage. Characterisation of the host colonisation ability of transformants TRI and TG I on grapevine cuttings of cvs. Chenin blanc and Merlot, showed that both transformants were able to colonise grapevine cuttings in a similar manner than the wild type isolate, and that they retained their fluorescent phenotype following isolation from the host. All isolates, including the wild type, showed a significantly higher colonisation rate on Merlot than on Chenin blanc.

AFRIKAANSE OPSOMMING: Stamsiektes veroorsaak groot ekonomiese verliese in die wingerdbedryf as gevolg van agteruitgang en terugsterwing van wingerde, wat 'n verlaging in kwaliteit en kwantiteit van oesopbrengste tot gevolg het. Stamsiektes word deur 'n kompleks van patogene veroorsaak, insluitend basidiomysete, Botryosphaeriaceae spesies (insluitende Botryosphaeria en die kompleks genera, Lasiodiplodia en Neofusicoccum), Eutypa lata, Phaeoacremonium spp., Phaeomoniella chlamydospora en Phomopsis viticola. Beperkte kennis is oor die interaksies tussen die wingerdgasheer en hierdie patogene beskikbaar, en daar is ook geen volhoubare beheermaatreels beskikbaar vir die bestuur van stamsiektepatogene nie. Stamsiektepatogene verkry hoofsaaklik toegang tot die wingerdgasheer via snoeiwonde, gevolglik is langdurige beskerming van die snoeiwonde nodig. Die biologiese beskermingsagent van wingerdsnoeiwonde, Trichoderma harzianum, is al bewys om snoeiwonde te koloniseer en daardeur 'n volhoubare, lewende versperring teen die indringing van stamsiektepatogene te verleen. Alhoewel T. harzianum die vermoe het om stamsiektepatogene te beheer, is beperkte inligting beskikbaar oor die vermoe van die swam om die gasheer te koloniseer, sowel as watter meganismes van antagonisme gebruik word. Kennis oor die interaksies van stamsiektepatogene met die gasheer, asook met T. harzianum binne-in die wingerdgasheerweefsel, sal grootskaals tot die ontwikkeling van volhoubare beheerstrategiee hydra. Gasheer-interaksiestudies is egter in die verlede deur struikelblokke in die waarneming en bestudering van teikenswamme binne-in die wingerdgasheer belemmer, veral in die teenwoordigheid van verskeie ander endofitiese swamme. Die transformasie van swamme met verklikkergene, wat rapporteerbare fenotipes soos groen en rooi fluoressensie na swamme oordra, sal grootskaals tot hierdie studies bydra. Die eerste doel van hierdie studie was dus om stabiele transformante van Suid-Afrikaanse Pa. chlamydospora, P. viticola (twee isolate) en T. harzianum isolate met die groen (GFP) of rooi (DsRed-Express) verklikkergene te verkry, deur van 'n polietileen glikol/kalsium chloried transformasie metode en die selektiewe merkergeen, hygromycin fosfotransferase (hph), gebruik te maak. Die tweede doel van die studie was om te bepaal of die transformasieproses die biologiese karakter-eienskappe [koloniekleur, spoorgrootte, ontkiemingspersentasie, groeitempo by verskillende temperature, virulensie (Pa. chlamydospora en P. viticola) en gasheerkolonisasie (T. harzianum)] van die transformante verander het. Bykomend tot die karakterisering van hierdie transformante en hul natuurlike isolate, is 'n Pa. chlamydospora GFP transformant (pCT74-P7) en sy wilde-tipe isolaat van Nieu-Seeland (Bradshaw et al., 2005) ook gekarakteriseer. Phaeomoniella chlamydospora (STE-U 7584) is stabiel met die GFP of DsRedExpress prote"ien verklikkergene getransformeer, wat helder fluoresserende transformante opgelewer het. Die teenwoordigheid van die transgene is ook met polimerase-kettingreaksie- amplifiserings bevestig. Karakterisering van die koloniekleur, spoorgrootte, ontkiemingspersentasie en groeitempo by verskillende temperature van twee van die stabiele, helder fluoresserende GFP (PcGl en PcGlO) en DsRed-Express (PcRl en PcR2) transformante, asook die Pa. chlamydospora GFP transformant (pCT74-P7), het geen verskille tussen die transformante en hul onderskeie natuurlike isolate aangetoon nie. Die enigste uitsondering was transformant PcG 10 wat 'n betekenisvolle laer ontkiemingspersentasie getoon het, asook die verskil in koloniekleur van transformante PcGl en PcRl, in vergelyking met die wilde-tipe isolaat. Karakterisering van die virulensie van drie van die verklikkergeen transformante (PcGl, PcRl en pCT74-P7) het aangetoon dat hulle nie van die onderskeie natuurlike isolate verskil het nie. Twee P. viticola isolate (STE-U 6048 en STE-U 6049) is stabiel met die GFP en DsRed-Express verklikkergene getransformeer. Helder fluoresserende transformante kon egter nie verkry word nie, wat die gebruik van hierdie isolate in gasheerkolonisasiestudies beperk. Al die GFP transformante het lae tot intermediere vlakke van fluoressensie in die miselia, asook in die konidia, oor 'n 6-week groeiperiode getoon. Soortgelyk het al die DsRed-Express transformante ook lae tot intermediere vlakke van fluoressensie in die miselia van I-week-oue tot 3-week-oue kulture getoon, maar geen fluoressensie is in 4-week-oue tot 6-week-oue kulture waargeneem nie. Hierteenoor het die konidia van DsRed-Express transformante helder fluoressensie in 3-week-oue to 4- week-oue kulture getoon. Die teenwoordigheid van hph, GFP en DsRed-Express gene is deur die polimerase-ketting-reaksie-amplifisering bevestig. Biologiese karakterisering van geselekteerde isolate en hul onderskeie wilde-tipe isolate het getoon dat hulle nie van hul onderskeie wilde-tipe isolate verskil het nie. Trichoderma harzianum ras T77 is stabiel met die GFP of DsRed-Express verklikkergene getransformeer. Die transformasie van T harzianum ras T77 was egter moeilik aangesien meeste van die transformante onstabiel was, en hulle kon nie die selektiewe merkergeen (hygromycin fosfotransferase) of die verklikkergene behou het nie. Slegs vier stabiel getransformeerde isolate is verkry, twee wat GFP (TG I en TG2) uitdruk en twee wat DsRed-Express (TRI en TR2) uitdruk. Karakterisering van die groeitempo, morfologie, konidiagrootte en ontkiemingspersentasie van hierdie transformante het getoon dat transformante TRI en TG I se groeitempo en/of ontkiemingspersentasie verander is. Karakterisering van die gasheerkolonisasie-vermoe van transformante TRI en TG I op wingerdlote van cvs. Chenin blanc en Merlot, het getoon dat beide transformante die vermoe het om die wingerdlote op 'n soortgelyke manier as die wilde-tipe isolaat te koloniseer, en hulle het hulle fluoressensie fenotipe behou mi isolasie uit die gasheer. Al die isolate, insluitende die wilde-tipe isolaat, het 'n betekenisvolle hoer kolonisasie-persentasie op Merlot, in vergelyking met Chenin blanc, getoon.

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