Genetic diversity in Plasmopara viticola in South Africa

Abstract

ENGLISH ABSTRACT: Downy mildew, caused by the obligate pathogen Plasmopara viticola, is a very destructive grapevine disease. The asexual phase (sporangia) of the pathogen has long been viewed as the life cycle stage that is most important in causing expansion of epidemics. Contrarily, the role of the sexual phase (oospores) has primarily been viewed as only providing the initial primary inoculum of the epidemic at the start of the season. However, population genetic studies in Europe have challenged these long standing epidemiological views. Downy mildew is mainly controlled through the application of fungicides, since no commercially acceptable resistant cultivars are available. The use of reliable high throughput in vitro resistance screening methods is very important for identifying new sources of resistance, as well as for mapping of quantitative trait loci (QTLs) involved in downy mildew resistance. Resistance screenings also require the use of effective longterm pathogen storage methods, since it allows the continual use of the same well characterised P. viticola isolates in different resistance screenings over seasons. The first main aim of this study was to investigate the population genetic structure of P. viticola populations in South Africa in two vineyards. The second aim was to determine whether an in vitro leaf disk method is a reliable and reproducible resistance screening method for determining downy mildew resistance of grapevine seedlings. The third aim was to evaluate long-term storage techniques for P. viticola isolates. The population genetic structure of P. viticola was investigated m two consecutive grape growmg seasons in an organically managed and a conventional fungicide sprayed vineyard. The study showed that population differentiation between the two vineyards was low (0.004 and 0.016) in both growing seasons, suggesting one metapopulation. New genotypes (12% to 74%) contributed to the epidemic throughout the growing seasons in both years and vineyards. The epidemic in both years and vineyards were dominated by one or two genotypes, which contributed between 14% and 67% through asexual reproduction to the epidemic. The remaining genotypes showed low levels of asexual reproduction, with most genotypes never being able to reproduce asexually. Ten genotypes were able to survive asexually from one season to the next. Moreover, the predominant genotype in the organically grown vineyard during 2004/05 survived asexually to the next season, where it also dominated the epidemic. Evaluation of an in vitro leaf disk method showed that the method was a reliable and reproducible method for screening the downy mildew resistance of the progeny of a Regent x Red Globe cross. Spearman correlation analyses revealed a moderate to high (0.64 to 0.82) correlation between three screening trails that were conducted over two growing seasons. However, the percentage seedlings that belonged to the different OIV 452 rating classes differed between the third (2005/06) and the first two (2004/05) resistance screening trials. This difference was statistically supported by one-way analysis of variance of rank means of these screenings, as well as Chi-square test of the screening x rating scale contingency table. This discrepancy indicates the importance of the inclusion of tolerant and sensitive reference seedlings, as well as the parents of the cross in each screening trial. Evaluation of different long-term storage methods for P. viticola showed that the pathogen was best stored as lesions. Successful storage methods included the storage of whole leaves with sporulating lesions in sealed Petri dishes, or the storage of small leaf lesion fragments within 2 ml centrifuge tubes at -20 and -80°C. Viability testing of these storage methods after a period of 6 (leaves within Petri plates) and 1 7 months (lesions within centrifuge tubes) showed that the pathogen remained viable for these periods, although the viability of sporangia were reduced.

AFRIKAANSE OPSOMMING: Donsskimmel, veroorsaak deur die verpligte patogeen Plasmopara viticola, is 'n vernietigende siekte op wingerd. Tot op hede is die ongeslagtelike fase (sporangiums) van die patogeen beskou as die belangrikste fase in die lewenssiklus wat uitbreiding van epidemies veroorsaak. Hierteenoor is die rol van die geslagtelike fase (oospore) hoofsaaklik gesien as die fase wat verantwoordelik is vir die verskaffing van die primere inokulum van die epidemie, aan die begin van die seisoen. Populasie-genetika studies in Europa bevraagteken egter hierdie epidemiologiese sienswyses. Donsskimmel word hoofsaaklik deur die toedien van fungisiedes beheer, aangesien geen kommersieel aanvaarbare weerstandbiedende kultivars beskikbaar is nie. Die gebruik van betroubare hoe deurset in vitro weerstandstoetse is baie belangrik vir die identifikasie van nuwe bronne van weerstand, sowel as vir die kartering van kwantitatiewe eienskap gene, betrokke by weerstand teen donsskimmel. Om weerstandstoetse volhoubaar uit te voer, word 'n effektiewe opbergingsmetode benodig om die patogeen oor lang periodes op te berg. Sodoende word verseker dat dieselfde P. viticola isolate in veskillende weerstandstoetse, oor verskillende seisoene, gebruik kan word. Die eerste doelwit van hierdie studie was om die genetiese struktuur van P. viticola populasies in twee Suid-Afrikaanse wingerde te ondersoek. Die tweede doelwit was om te bepaal of 'n in vitro blaarskyf metode 'n akkurate en herhaalbare weerstandstoets vir die bepaling van donsskimmel weerstand van wingerdsaailinge is. Die derde doelwit was om langtermyn opbergingsmetodes vir P. viticola isolate te evalueer. Die populasie genetiese struktuur van P. viticola is in twee opeenvolgende groeiseisoene in 'n organies-verboude en konvensioneel fungisied-behandelde wingerd ondersoek. Die studie het getoon dat populasie differensiasie tussen die twee wingerde in beide seisoene laag (0.004 en 0.016) was, wat een meta-populasie aandui. Nuwe genotipes (12% tot 74%) het deur die seisoen tot die epidemie, in beide jare en wingerde, by gedra. In beide jare en wingerde, is die epidemie deur een of twee genotipes, wat tussen 14% en 67% deur ongeslagtelike voorplanting tot die epidemie bygedra het, gedomineer. Die oorblywende genotipes het lae vlakke van ongeslagtelike voorplanting getoon, met die meeste genotipes wat nooit in staat was om ongeslagtelik voor te plant nie. Tien genotipes was in staat om ongeslagtelik van een seisoen na die volgende te oorleef. Verder het die predominante genotipe gedurende 2004/05 in die organiesverboude wingerd ongeslagtelik na die volgende seisoen oorleef, waar dit ook die epidemie gedomineer het. Evaluasie van die in vitro blaarskyf metode het getoon dat dit 'n betroubare en herhaalbare metode is vir die toets vir donsskimmelweerstand van saailinge van 'n Regent x Red Globe kruising. Spearman korrelasie analise het 'n gemiddeld tot hoe (0.64 tot 0.82) korrelasie tussen drie weerstandstoetse getoon, wat oor twee groeiseisoene uitgevoer is. Die persentasie saailinge wat tot die verskillende OIV 452 klassifikasieklasse behoort het, het egter tussen die derde (2005/06) en eerste twee (2004/05) weerstandstoetse verskil. Hierdie verskil is statisties deur eenrigting analise van variansie van rang gemiddeldes van hierdie weerstandstoetse ondersteun, asook deur die Chi-kwadraat toets van die weerstand x groepering skaal gebeurlikheidstabel. Hierdie teenstrydigheid dui op die belang van die insluiting van bestande en vatbare verwysing saailinge, asook die ouers van die kruising, in elke weerstandstoets. Evaluasie van verskillende langtermyn opbergingsmetodes vir P. viticola het getoon dat die patogeen die beste as letsels gestoor word. Suksesvolle opbergingmetodes sluit die berging van heel blare met sporulerende letsels in geseelde Petri bakkies in, of die opberg van klein blaarletsel deeltjies in 2 ml sentrifugebuisies by -20 en -80°C. Lewensvatbaarheidstoetse van hierdie opbergingsmetodes het getoon dat die patogeen na 6 (blare in Petri bakkies) en 17 maande (letsels in sentrifugebuisies) nog steeds lewensvatbaar was, hoewel die lewensvatbaarheid van sporangiums afgeneem het.