Virulence spectrum, molecular characterisation and fungicide sensitivity of the South African Rhynchosporium secalis population

Robbertse, Barbara (2000-12)

Thesis (PhDAgric)--University of Stellenbosch, 2000.

Thesis

ENGLISH ABSTRACT: Barley leaf scald, caused by Rhynchosporium secalis, is the most important disease of barley (Hordeum vulgare) in the Western Cape province of South Africa. The disease was first reported from South Africa in 1937. The present study is the first attempt to characterise the South African R. secalis population. Topics such as pathogenesisrelated proteins, virulence spectra, variability of pathotypes, sources of variation, host resistance, breeding strategies, molecular characterisation and fungicide sensitivity are summarised in Part 1 of this dissertation. In succeeding Parts the focus is on the characteristics of the local R. secalis population regarding virulence spectrum, DNA polymorphisms, in vitro as well as in vivo fungicide sensitivity. These aspects are treated as separate entities, leading to some duplication which is unavoidable. In Part 2 the virulence spectra of 50 R. secalis isolates from a population in the. Western Cape province were determined. Twenty-one races were detected using 17 differential barley cultivars. The two most prevalent races, namely races 4 and 7 had three and four virulence genes respectively. Both race 4 and 7 were virulent on the most susceptible cultivars, namely West China, Steudelli, C.I.8618 and C.I.2226. Considering the resistance genes reported for cultivars Atlas 46, Turk, and C.I.3515 which showed no susceptible cultivar-pathogen interaction, it would appear that the Rh- Rh3-Rh4 complex is primarily involved in conferring resistance to the local R. secalis isolates. A total of 20 races (47 isolates) characterised in Part 2 were selected for further characterisation by means of DNA fingerprinting. In Part 3 an anonymous multilocus DNA probe was used to characterise the genotypic structure of these isolates by means of RFLP analysis. No correlation between any particular fingerprint pattern, race, district, field or lesion was found. The two most prevalent races, 4 and 7, did not share the same genotypes, even when isolated from the same field or lesion. The genotypic diversity of the isolates studied was 46.5% of the theoretical maximum diversity. The high level of genotypic variation observed in the South African R. secalis population resembled the genotypic diversity observed in other cereal pathogens with known sexual structures. Although no teleomorph has yet been observed, these data suggest that sexual recombination may operate within the local population of R. secalis. In South Africa barley scald is primarily controlled by means of fungicides. The continued use of fungicides on cereal crops results in the build-up of fungicide resistance in the population, which could lower the efficacy of these compounds. These aspects were investigated in Part 4, where isolates (collected during 1993 to 1995) were evaluated in vitro for sensitivity to triadimenol, tebuconazole, flusilazole and propiconazole. The sensitivity fluctuated but in 1995 isolates were significantly less sensitive towards triadimenol than in the previous two years. In a second experiment, isolates collected from two fields with a 5-6 year-history of triadimenol seed treatments and tebuconazole applications, were evaluated for their fungicide sensitivity. A significant positive correlation was observed between tebuconazole and triadimenol sensitivity among,R. secalis populations from these fields. However, such a correlation was not found within the R. secalis population collected during 1993-1995 where shorter crop rotation patterns and a range of fungicides was applied. In a third experiment, the fungicide sensitivity of local R. secalis isolates was evaluated towards two new triazole fungicides, namely bromuconazole and triticonazole. Correlation coefficients observed between these new triazoles and those previously applied in South Africa were not significantly positive. The lack of significant cross-resistance has important practical implications regarding the management of fungicide resistance. In Part 5, isolates with different minimum inhibitory concentration (MIC) towards tebuconazole in vitro (1, 3 and 10 ug/ml) were compared in vivo. The aim of this study was to determine how MIC values would influence virulence (leaf area affected) and sporulation. Results indicated that all isolates were equally fit to induce lesions and sporulate in the absence of tebuconazole. Thus no fitness cost was associated with the degree of tebuconazole sensitivity in the present study. All R. secalis isolates were able to induce lesions on tebuconazole treated leaves, but differed significantly with respect to the percentage leaf area affected. Isolates, least sensitive (MIC = 10 ug/rnl) towards tebuconazole were more adapted on tebuconazole treated leaves, being able to repeatedly cause larger lesions than sensitive R. secalis isolates (MIC = 1 ug/rnl), Sporulation was not significantly different between isolates on lesions of untreated or tebuconazole treated leaves. Larger leaf areas affected and adequate sporulation suggest that a less sensitive population would result in more disease in tebuconazole treated fields. In conclusion, this study revealed the variability associated with the South African R. secalis population regarding virulence spectrum and genotypic structure. The data in this study suggest that it is likely that the local population will easily adapt to newly introduced, single gene resistance. For more durable resistance, higher levels of quantitative resistance should be introduced. This type of resistance is, however, more difficult to identify and incorporate than single gene resistance. Consequently, barley scald control will remain dependent on the efficacy of fungicide applications. Furthermore, the lack of cross-resistance and low frequency of resistant isolates indicates a low risk for the development of fungicide resistance in the local R. secalis population. Other factors such as current crop rotation practices and the range of fungicides being ~pplied also contribute to this low risk level. However, the status of these factors can change over time. The in vivo tebuconazole sensitivity study has indicated that a resistant field population of R. secalis may be able to build-up. It is, therefore, necessary to monitor the fungicide sensitivity of R. secalis isolates at timely intervals with view to successful barley cultivation in the future.

AFRIKAANSE OPSOMMING: Blaarvlek op gars (Hordeum vulgare), veroorsaak deur Rhynchosporium secalis, is die belangrikste siekte van gars in die Wes-Kaap provinsie van Suid-Afrika. Die voorkoms van R. secalis op gars is in Suid-Afrika vir die eertse keer in 1937 gerapporteer. Hierdie studie is die eerste poging tot karakterisering van die plaaslike R. secalis-populasie. Aspekte soos proteïene betrokke by patogenese, virulensiespektra, variabiliteit van patotipes, bronne van variasie, gasheerweerstand, teeltprogramme, molekulêre karakterisering en swamdodersensitiwiteit word in Deel I van die tesis opgesom. In die daaropvolgende gedeelte is die fokus op die karakterisering van die R. secalis-populasie en behels DNA karakterisering, virulensiespektrum, en swamdodersensitiwiteit in vitro asook in vivo. .. In Deel 2 is die virulensiespektra van 50 R. secalis isolate van 'n populasie in die. Wes-Kaap geëvalueer teenoor 17 differensiëel weerstandbiedende gars kultivars en hieruit is 21 rasse geïdentifiseer. Die twee mees algemene rasse (rasse 4 en 7), met onderskeidelik drie en vier virulensie gene, het virulent vertoon teenoor die mees vatbare kultivars soos West China, Steudelli, C.I.8618 en C.I.2226. Geen vatbare kultivar-patogeen interaksies is met kultivars Atlas 46, Turk en C.I.3515, wat al drie die Rh-Rh3-Rh4 kompleks dra, gevind nie. Dit wil dus voorkom asof hierdie genekompleks effektiewe gasheerweerstand teen die plaaslike R. secalis isolate kan bied. 'n Totaal van 20 rasse (47 isolate), gekarakteriseer in Deel 2, is geselekteer vir verdere karakterisering met behulp van DNA bandpatrone. In Deel 3 is 'n anonieme multilokus DNA peiler gebruik om deur middel van RFLP analise die genotipiese struktuur van hierdie R. secalis-isolate te bepaal. Geen assosiasie is gevind tussen DNA bandpatroon en ras, distrik, garsland of letsel nie. Die twee rasse (4 en 7) wat mees algemeen voorkom, het nie dieselfde bandpatroon vertoon nie, ook nie dié afkomstig vanuit dieselfde garsland of letsel nie. Die genotipiese diversiteit van isolate was 46.5% van die teoretiese maksimum diversiteit. Die hoë vlak van variasie waargeneem in die R. secalis populasie is soortgelyk aan variasie waargeneem in ander graanpatogene wat oor 'n geslagtelike stadium in die lewenssiklus beskik. Alhoewel geen geslagtelike stadium tot dusver geidentifiseer is nie, dui die vlak van variasie daarop dat geslagtelike rekombinasie moontlik wel plaasvind binne die plaaslike R. secalis populasie. In Suid-Afrika word blaarvlek op gars primêr deur swamdoders beheer. Die toenemende gebruik van swamdoders op graangewasse veroorsaak moontlik 'n opbou van swamdoderweerstand in die populasie. Dit kan die effektiwiteit van swamdoders verlaag. Hierdie veronderstelling is in Deel 4 ondersoek, waar die sensitiwiteit van isolate in vitro teenoor triadimenol, tebukonasool, flusilasool en propikonasool geëvalueer is. Die triasooi sensitiwiteit van R. secalis isolate wat gedurende die 1993- 1995 seisoen versamel is het gewissel en slegs vir triadimenol was daar 'n tendens na meer weerstandbiedenheid. 'n Swamdoder-evaluasie is in 'n aparte eksperiment op isolate gedoen wat versamel is vanaf twee garslande met 'n 5-6 jaar geskiedenis van triadimenol saadbehandelings en tebukonasool bespuitings. 'n Betekenisvolle positiewe korrelasie is waaJ~geneem tussen tebukonasool en triadimenol sensitiwiteit in R. secalis isolate afkomstig vanaf hierdie twee garslande. 'n Soortgelyke korrelasie is egter nie gevind in die populasie wat gedurende die 1993-1995 seisoene versamel IS me. Laasgenoemde kan moontlik toegeskryf word aan korter wisselboupatrone en die toediening van 'n verskeidenheid van swamdoders. In 'n derde eksperiment is die sensitiwiteit van plaaslike R. secalis isolate teenoor twee nuwe triasole, naamlik bromukonasool en tritikonasool getoets. Die korrelasie waargeneem tussen die twee nuwe triasole en triasooi swamdoders reeds voorheen in gebruik in die Wes-Kaap was me betekenisvol positief me. Die gebrek aan betekenisvolle kruisweerstandbiedendheid het belangrike praktiese implikasies vir die bestuur van swamdoder -weerstandbiedendheid. In Deel 5 is isolate met wisselende minimum inhiberende konsentrasies (MIKs) teenoor tebukonasool in vitro (1, 3 en 10 ug/ml) en in vivo vergelyk. Die doel van hierdie studie was om te bepaal hoe wisselende MIK-waardes virulensie (blaaroppervlakte geïnfekteer) en sporulasie sal beïnvloed. Resultate dui daarop dat alle R. secalis isolate in hierdie studie ewe fiks was om, in die afwesigheid van tebukonasool, letsels te induseer en te sporuleer. Die bevinding is dat die verlies in fiksheid nie geassosieer is met die mate van tebukonasool weerstand nie. Alle R. secalis isolate het die vermoë gehad om letsels op tebukonasool-behandelde blare te veroorsaak maar het betekenisvol verskil ten opsigte van die blaaroppervlakte geaffekteer. Isolate wat minder sensitief (MIK = 10 ug/rnl) teenoor tebukonasool in vitro is, het meer aangepastheid op tebukonasool-behandelde blare getoon. Gevolglik het hierdie isolate herhaaldelik meer letsels veroorsaak as sensitiewe isolate (MIK = 1 ug/ml), Sporulasie het nie betekenisvol verskil tussen isolate vanaf letsels op ondehandelde of tebukonsoolbehandelde blare nie. Hierdie resultate dui egter daarop dat 'n minder sensitiewe populasie tot meer siektevoorkoms in tebukonasool-bespuite lande kan lei. Die studie het die veranderlike karakter van die Suid-Afrikaanse R. secalispopulasie aangaande virulensiespektrum en genotipiese struktuur blootgelê. Dit is dus baie moontlik dat die R. secalis-populasie maklik sal aanpas by teelmateriaal met nuwe enkelgeen-weerstand. Vir volgehoue gasheerweerstand is dit egter nodig dat hoër vlakke van kwantitatiewe weerstand ingeteel moet word. In die praktyk is hierdie tipe weerstand egter baie moeiliker om te identifiseer en by nuwe teelmateriaal in te sluit as in die geval van enkelgeen-weerstand, Dit bring mee dat blaarvlekbeheer afhanklik bly van swamdodertoedienings as beheermaatreël. Die resultate van hierdie studie dui daarop dat daar tans 'n lae risiko vir die ontwikkeling van swamdoderweerstand in die plaaslike populasie is, as gevolg van die afwesigheid van kruisweerstandbiedendheid en die lae voorkoms van weerstandbiediende isolate. Ander faktore soos die wisselboustelsels wat toegepas word en die verskeidenheid van swamdoders toegedien dra ook daartoe by. Ten spyte hiervan kan die status van hierdie faktore egter oor tyd verander. Die in vivo tebukonasool studie het daarop gedui dat 'n weerstandbiedende veldpopulasie van R. secalis die potensiaal het om te vermeerder. Gevolglik is die tydige monitering van swamdodersenisitiwiteit van R. secalis isolate noodsaaklik om 'n volhoubare garsproduksie te verseker.

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