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

[摘要] ENGLISH ABSTRACT: Trunk diseases cause major economical losses in the grape growing industry due todecline 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 ofBotryosphaeriaceae (including Botryosphaeria and aggregate genera, Lasiodiplodia andNeofusicoccum), Eutypa lata, Phaeoacremonium spp., Phaeomoniella chlamydospora andPhomopsis viticola. Limited knowledge is available on interactions between the grapevine hostand these pathogens. Currently, there are no sustainable control measures available formanagement 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, therebyproviding a sustainable living barrier against trunk disease pathogen invasion. Although T.harzianum has the potential for controlling trunk disease pathogens, limited knowledge isavailable 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 controlstrategies. However, host interaction studies have been hampered due to difficulty in observingand studying target fungi within the grapevine host, where several endophytic fungi are alsopresent. The transformation of fungi with reporter genes, which convey detectable phenotypessuch as green and red fluorescence to fungi, will greatly aid these studies. Therefore, the firstaim of this study was to stably transform South African Pa. chlamydospora, P. viticola (twoisolates) and T. harzianum isolates with the green (GFP) or red (DsRed-Express) reporter genesusing a polyethylene glycol/calcium chloride transformation method and the selectable markergene hygromycin phosphotransferase (hph). The second aim of the study was to determinewhether 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 werealso characterised.Phaeomoniella chlamydospora (STE-U 7584) was stably transformed with the GFP orDsRed-Express protein reporter genes, yielding brightly fluorescing transformants. Thepresence of the trans genes was also confirmed through polymerase chain reaction amplifications.Characterisation of the colony colour, spore size, germination percentage and growth rate atdifferent temperatures of two of the stable, highly fluorescent GFP (PcG 1 and PcG 10) andDsRed-Express (PcRl and PcR2) transformants as well the Pa. chlamydospora GFPtransformant (pCT74-P7), revealed no differences between the transformants and their respectivewild type isolates. The only exception was the significant lower germination percentage oftransformant PcG 10, and the different colony colour of transformants PcG 1 and PcRl, whencompared with the wild type isolate. Characterisation of the virulence of three of the reportergene transformants (PcGl, PcRl and pCT74-P7) showed that they did not differ from theirrespective wild type isolates .. Two P. viticola isolates (STE-U 6048 and STE-U 6049) were stably transformed with theGFP and DsRed-Express reporter genes. However, brightly fluorescing transformants could notbe obtained, limiting the usefulness of these isolates in host colonisation studies. All the GFPtransformants exhibited low to intermediate levels of fluorescence in mycelia as well as inconidia over a 6-week growth period. Similarly, all the DsRed-Express transformants alsoshowed 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 ofDsRed-Express transformants showed very bright fluorescence in 3- to 4-week-old cultures. Thepresence of the hph, GFP and DsRed-Express genes was shown through polymerase chainreaction amplifications. Biological characterisation of a subset of the isolates and theirrespective 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 DsRedExpressreporter genes. However, transformation of T. harzianum strain T77 was difficult, sincemost 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 germinationpercentage of these transformants showed that transformants TRI and TG 1 were altered in theirgrowth rate and/or germination percentage. Characterisation of the host colonisation ability oftransformants TRI and TG I on grapevine cuttings of cvs. Chenin blanc and Merlot, showed thatboth transformants were able to colonise grapevine cuttings in a similar manner than the wildtype 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 Merlotthan on Chenin blanc.

[发布机构] Stellenbosch University