Tesi sul tema "Barley leaf scald disease"

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

In the context of increasing food insecurity, new integrated and more sustainable crop protection methods need to be developed. The phyllosphere, i.e. the leaf habitat, hosts a considerable number of microorganisms. However, only a limited number of these are pathogenic and the roles of the vast majority still remain unknown. Managing the leaf-associated microbial communities is emerging as a potential integrated crop protection strategy. This thesis reports the characterisation of the phyllosphere of barley, an economically important crop in Scotland, with the purpose of developing tools to manipulate it. Field experiments were carried out to determine the composition of the culturable bacterial phyllosphere. The leaf-associated populations were demonstrated to be dominated by bacteria belonging to the Pseudomonas genus. Two bacterial isolates, Pseudomonas syringae and Pectobacterium atrosepticum, hindered the growth of Rhynchosporium commune, the causal agent of the leaf scald, but promoted the development of powdery mildew symptoms, caused by Blumeria graminis f.sp. hordei. However, using a molecular fingerprinting technique, namely T-RFLP, the global community was shown to be significantly richer and more diverse than indicated by the culture-based methods, thus increasing the complexity of interactions taking place in the phyllosphere. Various factors were found to affect the structure of the phyllobacteria significantly. Under controlled conditions, a root-associated symbiont, Piriformospora indica, was shown to increase the plant fitness and shift the abundance of the most common bacteria. In the field, both agro-chemical treatments tested, conventional fungicide and an elicitor mixture, increased the bacterial diversity, but fungicide application resulted in a higher yield and better protection against diseases. Finally, the plant genotype also affected the phyllosphere structure. Mutations in the leaf epicuticular waxes led to significant changes in the bacterial diversity and differentially affected disease in the field. A negative correlation between bacterial diversity and scald infection was also observed.

Dissertation (PhD(Agric)) -- University of Stellenbosch, 2001.
ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most important diseases of this cereal in the south Western Cape Province of South Africa. This fungus exists as two different types (forms), namely a nettype and a spot-type that are distinguished by differential symptom expression on barley leaves. Based on this specific plant pathological difference a series of studies of agricultural importance were executed to investigate the effects of sexual recombination between these two types. In addition, studies were done to determine the difference between local net- and spot-type populations with regards to population structure and fungicide sensitivity. This dissertation therefore, consists of a collection of separate publications and as a result a certain degree of redundancy has been unavoidable. Recombination is one of the most important evolutionary forces involved with sexual reproduction. In plant-fungal agricultural ecosystems this may result in pathogenic fungal populations adapting more rapidly to control programs such as fungicide applications. The first section of the review in part 1 of this dissertation covers different aspects of sexual reproduction in ascomycetes, specifically focussing on mating-type genes, vegetative incompatibility and recombination. The major part of the review is then dedicated to various plant pathological aspects of P.teres, specifically addressing the differences between the two types, and in various cases highlighting the significance of sexual recombination within and between the net- and spot-type. Using morphological criteria for identification purposes there have been many conflicting reports concerning the identity of leaf spot isolates in the Western Cape Province of South Africa. In part 2, the correct identity was eventually achieved employing mating studies and molecular markers .: This was accomplished after single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on the differential cultivars susceptible to the net-blotch and leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. Fifteen of the net-spot hybrid progeny (F1) produced from the mating study in Part 2 were screened in Part 3 to assess their viability and genetic stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and Clipper (both differentially susceptible to spot-type isolates) produced intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a healthy set of seedlings produced similar intermediate symptoms. RAPDs conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny revealed profiles similar to those obtained for F1 isolates. RAPD molecular data, therefore, indicated that hybrid progeny of this net x spot mating were genetically stable after having been subjected to two repetitive inoculation and reisolation cycles. Phylogenetic analysis of DNA sequences of the internal transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA gene and the 5' end partial histone-3 gene confirmed the genetic stability of the hybrid progeny. These results also indicated that the hybrid progeny produced consistent symptoms throughout the series of experiments, and maintained their virulence to the differential cultivars screened. Both types of P. teres are prevalent in the south Western Cape Province of South Africa, found on susceptible cultivars often grown within close proximity of each other. In Part 4, a net- and spot-type population were characterised in terms of their population structure using RAPD markers. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. A total of 65 loci were produced of which 54 were polymorphic. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spottype populations. A coefficient of genetic differentiation (Gs) of 0.0149 was obtained between sites within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis done on the two populations together with six progeny from the mating between a netand spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the nettype population also contained a unique spot-type RAPD fragment. This suggested that sexual recombination may be taking place between isolates of the net- and spot-type under field conditions. Fungicide application is the most important method used in the control of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values) of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol demethylation inhibiting fungicides were determined, based on the inhibitory effect on radial mycelial growth. The fungicides evaluated were triadimenol, bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and spot-type isolates revealed strong resistance to triadimenol while flusilazole was shown to be the strongest inhibitor of fungal growth. Spot-type isolates showed a higher resistance than net-type isolates to all five fungicides screened. The ICsD values indicated significant differences between four of the fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The ICsD values between propiconazole and bromuconazole were not significant. This study suggested that spot-type isolates showed a higher degree of resistance to commercially used fungicides than net-type isolates. The overall conclusion of this study is that the spot-type of P. teres is the pathogen associated with leaf spots of barley in the south western Cape province of South Africa and not P. japonica as earlier reported. Together with the net-type, both types exist as genetically variable populations in this barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to one another in which either net- or spot-type susceptible cultivars are being cultivated may lead to sexual progeny being produced. This in turn may lead to an increased rate at which fungal populations may become resistant to commercially used fungicides. It is furthermore suggested that an alternative fungicide seed treatment is used instead of triadimenol due to high resistance of P. teres to this fungicide.
AFRIKAANSE OPSOMMING: Netvlek op gars is een van die belangrikste siektes van hierdie graansoort in die suidelike deel van die Westelike Kaapprovinsie. Dié siekte word veroorsaak deur die swam Pyrenophora teres. Hierdie swam kom voor as twee verskillende tipes, naamlik 'n net-tipe en 'n kol-tipe wat onderskei word op grand van die voorkoms van hulle simptome op garsblare. Hierdie planpatologiese verskil in ag genome, is 'n reeks studies van landboukundige waarde uitgevoer om die effek van geslagtelike rekombinasie tussen die twee tipes te ondersoek. Daarbenewens is ook studies uitgevoer om om die verskil te bepaal tussen plaaslike net- en koltipe populasies ten opsigte van populasiestruktuur en fungisiedsensitiwiteit. Hierdie verhandeling bestaan dus uit 'n versameling afsonderlike publikasies en as gevolg daarvan is daar onvermydelik'n mate van oorvleueling. Rekombinasie is een van die belangrikste evolusionêre kragte betrokke by geslagtelike voortplanting. In plant-swam landboukundige ekostelsels kan dit veroorsaak dat patogene swampopulasies vinniger aanpas by beheerpragramme soos fungisiedtoediening. Die eerste gedeelte in deel 1 van hierdie verhandeling dek die verskillende aspekte van geslagtelike voortplanting van ascomycetes, met spesifieke verwysing na paringstipe gene, vegetatiewe onverenigbaarheid en rekombinasie. Die grootste gedeelte van die oorsig word gewyaan verskeie plantpatologiese aspekte van P. teres,en wys veralop die verskille tussen die twee tipes. In verskeie gevalle word die betekenis van geslagsrekombinasie binne en tussen die net- en koltipe uitgelig. Deur morfologiese kenmerke vir identifikasiedoeleindes te gebruik, is daar baie teenstrydige verslae rakende die identifikasie van blaarvlekisolate in die Westlike Kaapprovinsie van Suid-Afrika. In deel 2 is die korrekte identifikasie eventueel verkry deur gebruik te maak van paringstudies en molekulêre merkers. Dit is bereik nadat enkel ascospore verkry is uit pseudothecia gevorm na in vitro paring plaasgevind het tussen 'n bevestigde P. teres netvlek isolaat uit Denemarke en 'n verteenwoordigende Pyrenophora blaarvlekisolaat van Suid- Afrika. Deur gebruik te maak van versterkte fragmentlengte polimorfisme [AFLP] en RAPD merkers, is rekombinasie gedemonstreer in die nasate wat DNA bandpatrone gehad het wat verskil het van dié van die "ouer" isolate. Patogenisiteitstoetse het ook bevestig dat rekombinasie tydens paring plaasgevind het. Inokulasies is uitgevoer op die verskillende cultivars wat vatbaar is vir die netvlek en blaarvlek vorme. Die twee ouers het tipiese netvlek of blaarvlek simptome veroorsaak, terwyl die nasate hoekige vlekke veroorsaak het op elke cultivar. Toetse vir fungisiedsensitiwiteit deur gebruik van die ergosterol biosintese inhibeerders het gewys dat a.g.v. rekombinasie sekere nasate verhoogde weerstand teen hierdie fungisiedes het. As gevolg van paring en daaropvolgende rekombinasie tussen 'n netvlek isolaat van P. teres en 'n verteenwoordigende blaarvlek isolaat is afgelei dat laasgenoemde P. teres f. maculata is. Vyftien van die netvlek hibried nakomelinge (F1) verkry van die paringstudie in deel 2 is ondersoek in deel 3 om hul lewensvatbaarheid en genetiese stabiliteit te bepaal. Hibried nasate (F1) geïnokuleer op garssaailinge bestaande uit die volgende cultivars: Stirling (soms vatbaar vir net-tipe isolate) , B87/14 en Clipper (albei soms vatbaar vir kol-tipe isolate) het intermediêre simptome op al die cultivars veroorsaak. Akseniese kulture (F1-1) geïsoleer uit blaarletsels gevolg deur herhaalde inokulasie en isolasie (F1-2) op 'n gesonde stel saailinge het dieselfde intermediêre simptome veroorsaak. RAPDs uitgevoer met twee 10-mer inleiers op al die isolate van F1-1 en F1-2 nasate het profiele opgelewer soortgelyk aan dié wat vir F1 isolate verkry is. RAPD molekulêre data het dus gewys dat die hibried nasate van hierdie net x kol paring geneties stabiel was nadat dit onderwerp is aan twee inokulasie en reïsolasie siklusse. Genetiese stabiliteit van die hibried nageslag is bevestig deur filogenetiese analise van die DNA volgorde van die interne getranskribeerde spasieerders (ITS1 en ITS2) reg langs die 5.8S nukluêre ribosomale RNA geen en die 5' end gedeeltelike histoon-3 geen. Hierdie resultate het ook gewys dat die hibried nasate konstante simptome getoon het tydens die hele reeks eksperimente en hulle virulensie behou het vir die kultivars wat getoets is. Beide tipes van P. teres kom algemeen voor in die suidelike deel van die Westelike Kaapprovinsie en word gevind op vatbare cultivars wat dikwels naby mekaar groei. In deel 4 is 'n net- en kol-tipe populasie gekarakteriseer in terme van hulle populasiestruktuur deur gebruik van RAPD merkers. Monsters is versamel van geïnfekteerde garsblare van twee aparte kwadrante in elke saailand. Die twee kwadrante is geplaas in die hoeke van die saailand, diagonaal tot mekaar. 'n Totaal van 65 lokusse is gevorm, waarvan 54 polimorfies was. Die algehele genetiese verskeidenheid bepaal vir alle lokusse, het gelei tot gemiddelde indekse van 0.063 en 0.082 soos gevind vir die net- en kol-tipe populasies. 'n Koëffisiënt van genetiese differensiasie (Gs ) van 0.0149 is gevind tussen gebiede tussen populasies, terwyl 'n koëffisiënt (GT) van 0.63 gevind is tussen die twee populasies. Genotipiese variasie het 13 duidelike multilokus genotipes (haplotipes) getoon in die net-tipe populasie, terwyl daar twaalf was in die kol-tipe populasie. UPGMA groeperingsanalises wat gedoen is op die twee populasies tesame met ses nasate van die paring van 'n net- en koltipe isolaat het tot gevolg gehad dat drie hoof groepe gevorm is, een vir elke populasie en een vir die nasate. Een isolaat wat versamel is, van die net-tipe populasie het 'n unieke kol-tipe RAPD fragment bevat. Dit wys daarop dat geslagtelike rekombinasie in veldomstandighede mag voorkom tussen isolate van die net- en kol-tipe. Fungisiedtoediening is die belangrikste metode wat gebruik word om netvlek in Suid-Afrika te beheer. In deel 5 is die fungisiedsensitiwteit (Ieso waardes) van 89 enkelkonidiale isolate (46 net-tipe en 43 kol-tipe) van P. teres teen sterol demetielasie inhiberende fungisiedes bepaal, op die basis van die onderdrukkende effek op die radiale groei van die miselium. Die volgende fungisiedes is geëvalueer: triadimenol, bromuconazole, flusilazole, propiconazole en tebuconazole. Beide net- en kol-tipe isolate het 'n sterk weerstand teen triadimenol openbaar, terwyl flusilazole gevind is as die sterkste onderdrukker van swamgroei. Kol-tipe isolate het 'n hoër weerstand as die net-tipe isolate teen al vyf fungisiedes wat getoets is, gehad. Die lesowaardes het aangedui dat daar beduidende verskille tussen vier van die fungisiedes IS (triadimenol, tebuconazole, flusilazole en propiconazole). Die leso waardes tussen propiconazole en bromuconazole was nie beduidend nie. Die gevolgtrekking van hierdie studie is dus dat die kol-tipe isolate 'n hoër graad van weerstand teen kommersiëel gebruikte fungisiedes as die net-tipe isolate gehad het. Die algehele gevolgtrekking van hierdie studie is dat die kol-tipe van P. teres, die patogeen is wat geassosieer word met blaarvlekke op gars in die suidwestelike Kaapprovinsie van Suid-Afrika, en nie P. japonica soos voorheen gerapporteer nie. Tesame met die net-tipe, kom altwee tipes voor as geneties veranderlike populasies in hierdie gars verbouingstreek. Paring tussen die twee tipes lei tot geslagtelike nasate wat geneties stabiel is. Dit impliseer dat aangrensende garsvelde waarop net- óf kol-tipe vatbare kultivars verbou word, mag lei tot die produksie van geslagtelike nasate. Dit kan weer lei tot 'n verhoogde tempo waarteen swampopulasies weerstandbiedend teenoor kommersiële fungisiedes raak. Daar word verder ook voorgestel dat alternatiewe fungisied saadbehandelings gebruik word in plaas van triadimenol as gevolg van verhoogde weerstand van P. teres teenoor laasgenoemde.

Scald disease (Rhynchosporium secalis) is one of the major plant diseases causing considerable yield loss in barley (Hordeum vulgare) plantations in Turkey. To develop, scald resistant barley varieties, C.R.I.F.C. of Turkey has a large accumulated collection of barley seed sources in hand, but these samples are difficult to be followed and used in the breeding programs due to lack of genetic studies on them. Thus, the objective of this study was to characterize and fingerprint of eighty barley seed sources, and assess the magnitude and pattern of genetic diversity that could be used to have more efficient scald disease resistant breeding programs in the future. Forty scald disease resistant and 40 scald sensitive Turkish barley seed sources were screened using 6 simple sequence repeats (SSR) primers. Each of barley seed source were represented with four seeds, assuming they are genetically uniform since barley is a self-pollinated crop. Estimated genetic parameters indicated that scald disease resistant and sensitive barley seed sources still maintain large amount of genetic diversity. For example, expected heterozygosity was 0.62±
0.01 and 0.64±
0.01 for resistant and sensitive Turkish barley seed sources, respectively. Thirty-nine percent of total genetic variation was between populations for resistant and 46% for sensitive group, while 61% of total variation was within populations for resistant group and 54% for sensitive group. When overall Turkish barley seed sources were considered, genetic distances between scald sensitive seed source S18 and resistant R1 as well as between sensitive S28 and resistant R1 were large. Scald resistant and sensitive barley seed sources were generally located in different clusters in dendrogram. The presence of R25, R39 and S16 barley seed sources with high genetic diversity parameters among studied seed sources, suggests that this diversity could be important drive in future barley breeding program in Turkey. However, further study is needed to illustrate genetic divergence of Turkish barley seed sources with use of more molecular markers.

In the past two decades a new barley threat has emerged as the disease Ramularia leaf spot (RLS) became more prevalent in temperate regions worldwide. This disease, first identified in the late 19th century, is caused by the filamentous fungus Ramularia collo-cygni (Rcc) and can cause substantial yield losses as well as reduce grain quality. RLS typically occurs late in the growing season and characteristic disease symptoms are usually seen after the crop has flowered. Expression of RLS lesions is thought to be associated with the action of fungal secondary metabolism products. The one group of secondary metabolites (SMs) characterised to date from Rcc, the anthraquinone toxins rubellins, are known to cause necrosis to plant tissues in a non-host specific manner. Therefore, it appears that fungal secondary metabolism might be a key component in understanding the interaction between Rcc and its host. In this study, more than 23 core genes involved in the biosynthesis of SMs belonging to the polyketide and non-ribosomal peptide pathways were identified in the genome of Rcc. Putative clusters containing genes with a predicted function relating to secondary metabolism were identified by in silico genome walking in the genetic loci adjacent to Rcc SM core genes. Two gene clusters containing no SM core gene were also identified. Five of the putative SM clusters exhibited similarity to the known fungal SM biochemical pathways involved in gliotoxin, monodictyphenone, ferricrocin, betaenone and chaetoglobosins biosynthesis. Several gene clusters exhibited similarity to SM clusters from fungal species where the SM pathway is uncharacterised. Changes in transcript abundance of selected SM core genes during RLS development in artificially inoculated barley seedlings were tested. Transcript levels were found to be the highest at an early stage of disease development, typically during the asymptomatic and early lesions formation stages and declined over time, suggesting that the associated SMs in Rcc, may not necessarily be involved in symptoms appearance. The in planta mode of action of the non-host specific photoactivated toxin rubellin D was studied in the model plant Arabidopsis thaliana. Rubellin-induced cell death appeared phenotypically reminiscent of programmed cell death (PCD). Full expression of rubellin D-induced cell death required the host salicylic acid (SA) pathway and the host proteasome supporting the PCD response to this fungal SM. However, a clear correlation between toxin sensitivity and disease susceptibility could not be found, suggesting a potential alternative role for rubellin in disease symptom development.

Proportional loss models commonly used in disease surveys are based on the assumption that per cent yield loss is the same in all crops, regardless of their yield potential. Estimates of regional crop loss may be inaccurate if the relationship between disease and yield loss is affected by crop yield potential. The importance of crop yield potential in disease: yield loss modelling was investigated and models for more accurate regional crop loss estimates were developed, taking crop yield potential into account. Two spring sown barley (cv. Triumph) experiments were conducted in 1987/88 and 1988/89 in Canterbury, New Zealand, to study the effect of crop yield potential on the relationship between disease and yield loss. Crop yield potentials of 323 to 806gDM/m² were generated in seven crops by varying nitrogen and water inputs, sowing date (mid-spring and early-summer) and season. Leaf rust (Puccinia hordei Otth) epidemics of different severity were generated by applying fungicides at different times, frequencies and rates to control the natural epidemics. Disease was measured as per cent disease severity (%DS), green leaf area, radiation interception and near-infrared radiation (NIR) reflectance from crop canopies. Yield was measured as total and grain dry weight. Epidemics were severe in the fully diseased plots from GS 34 and 46 to maturity in the late and early sown crops respectively. Disease reduced grain yield by 50 to 63% in 1987/88 and 24 to 38% in 1988/89 in the fully diseased plots. Disease: yield loss models were derived by regression analysis for each crop in 1987/88. Single point, multiple point and area under curve models were derived from %DS and GLAI variables, and proportional (%) and actual (gDM/m²) grain yield. The effect of yield potential was determined by comparing regression equation coefficients for each crop with crop yield potential. An area under green leaf area index curve (AUGLAIC): actual yield model was best suited to determining the effect of yield potential on yield loss. This model was selected because AUGLAIC summarised the effect of disease on plant growth over the season and actual yield represented the crop yield potential in the absence of disease and the response of actual yield to disease. Crop yield potential did not affect actual yield loss caused by leaf rust. Disease measured as AUGLAIC explained most of the variation in yield (R²adj=0.93) for all crops in both years. Assessment of GLAI is not suitable for estimation of regional crop loss because of the requirement for a rapid and low cost method. Reflectance of NIR from the crop canopy was investigated as an alternative to GLAI measurements. Reflectance was correlated significantly (P<0.001) with GLAI (r=0.66 to 0.89) and green area index (r=0.76 to 0.92). Reflectance measured at grain-filling (GS 85-87) explained most (R²adj=0.94) of the variation in yield for all crops in both years. The relationship between AUGLAIC and yield was validated with data from independent diseased and healthy barley crops. The AUGLAIC: yield model described the effects of disease on yield accurately but overestimated yield by 49 to 108% in the healthy crops. Models based on accumulated PAR (photosynthetically active radiation) intercepted by green leaves explained the observed deviations in yield of these crops from the AUGLAIC: yield model. Accumulated PAR models accounted for differences in incident radiation, canopy structure, radiation interception by green leaves, radiation use efficiency and harvest index which are important in determining dry matter production and grain yield. Accumulated PAR models described the effects of disease on crop growth which were not represented by GLAI alone. Variation in crop yield potential at the regional scale is important in disease: yield loss modelling and can be accounted for by using either separate equations for each yield potential crop or crop category, robust models, inclusion of a form function for yield potential or choice of disease and yield variables which integrate yield potential.

Xanthomonas albilineans est la bactérie responsable de l'échaudure des feuilles, une maladie vasculaire et létale de la canne à sucre. Cette plante, domestiquée depuis plus de 5000 ans, est aujourd'hui cultivée sur près de 26 millions d'hectares. Depuis le début du 20ème siècle, les variétés naturelles de canne à sucre ont été remplacées par des variétés hybrides issues de croisements artificiels et présentant des caractères agronomiques plus intéressants. La transmission de X. albilineans est essentiellement liée au mode de multiplication par bouturage de la canne à sucre, les boutures produites à partir d'un plant infecté ayant de fortes chances d'être contaminées. L'utilisation d'outils de coupe favorise également la transmission de la bactérie à partir d'un plant infecté. Une transmission aérienne a toutefois été décrite pour au moins un groupe génétique de souches de X. albilineans appelé PFGE-B et caractérisé à l'aide de la technique d'électrophorèse en champ pulsé. Mon sujet de thèse visait, par une étude de génomique comparative et évolutive sur plusieurs souches de X. albilineans, à, d'une part, comprendre l'histoire de l'association de cette bactérie avec la canne à sucre, et, d'autre part, identifier des gènes candidats qui pourraient expliquer la pathogénie de X. albilineans et la capacité des souches du groupe PFGE-B à être transmises par voie aérienne.En comparant 12 souches de X. albilineans appartenant à neuf groupes PFGE différents, j’ai construit une phylogénie solide proposant l'existence de huit lignées regroupées en deux clades. Cette phylogénie a été confirmée en utilisant trois méthodes de phylogénomique différentes. De façon intéressante, cette phylogénie permet de proposer des hypothèses sur l'évolution de X. albilineans et sur l'introduction de cette bactérie chez la canne à sucre. L'analyse des gènes présents dans le génome de ces 12 souches appartenant à neuf groupes PFGE différents m'a également permis d'identifier un système RM (restriction/modification) qui n'est présent que chez les souches PFGE-B et qui pourrait expliquer pourquoi ces souches sont plus résistantes aux phages que celles appartenant aux autres groupes PFGE. En facilitant la survie des bactéries dans l'environnement, cette résistance aux phages pourrait jouer un rôle important dans la transmission aérienne.La comparaison de 11 souches de X. albilineans appartenant au groupe PFGE-B m'a permis de construire, à partir de 67 SNP (Single Nucleotide Polymorphism), une phylogénie qui indique que les Caraïbes, où la canne à sucre n'a été introduite qu'à la fin du 15ème siècle, pourraient être un centre de dispersion des souches PFGE-B. Cette étude a également mis en évidence un polymorphisme au niveau des spacers d'un locus CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats system). L'étude de ce polymorphisme sur 18 souches de X. albilineans non séquencées a permis de démontrer que les souches de Floride sont originaires des Caraïbes et de proposer des hypothèses concernant l'origine des souches PFGE-B d’autres régions du monde, même si le séquençage d'un plus grand nombre de souches sera nécessaire pour confirmer ces hypothèses.En analysant la séquence du génome de deux souches qui avaient été initialement classées dans l'espèce X. albilineans mais qui n'avaient pas été isolées à partir de tiges de canne à sucre, j’ai mis en évidence une nouvelle espèce que nous avons appelée 'Xanthomonas pseudalbilineans'. Enfin, la comparaison du génome fini et annoté de la souche PFGE-B GPE PC73 avec le génome d'autres espèces bactériennes phytopathogènes m'a permis de proposer des hypothèses pour expliquer la pathogénie de X. albilineans
Xanthomonas albilineans is the bacterium causing leaf scald, a vascular disease being lethal for sugarcane. This plant, domesticated for over 5,000 years, is nowadays grown across almost 26 million hectares. Since the beginning of 20th century, natural varieties of sugarcane have been replaced by hybrid varieties resulting from artificial crossings. The transmission of X. albilineans is mainly linked to the fact that sugarcane is propagated by taking cuttings, with the cuttings from an infected plant likely to be contaminated. The use of cutting instruments also facilitates transmission of the bacteria from an infected plant. An aerial transmission has nevertheless been described, at least for one genetic group of strains from X. albilineans, namely PFGE-B, characterized using the pulsed-field gel electrophoresis technique. My thesis topic consisted in performing a comparative and evolutive genomic study of several strains of X. albilineans in order, on one hand, to better understand the history of association of this bacterium with the sugarcane, and, on the other hand, to identify candidate genes which may explain the pathogenicity of X. albilineans as well as the ability of PFGE-B strains to be aerially transmitted. Comparing 12 strains of X. albilineans belonging to nine different PFGE groups, I built a robust phylogeny which proposes the existence of eight lineages clustered in two clades. This phylogeny has been confirmed by three different phylogenomic methods. Interestingly, this phylogeny allows to propose hypotheses about the evolution of X. albilineans and the introduction of this bacterium in sugarcane. The analysis of genes from the genomes of these 12 strains belonging to nine different PFGE groups also allowed me to identify a RM (Restriction/Modification) system which is present only in PFGE-B strains and which may explain why these strains are more resistant to phages that those belonging to other PFGE groups. By facilitating the survival of bacteria in the environment, this resistance to phages may play an important role in aerial transmission. The comparison of 11 strains of X. albilineans belonging to the PFGE-B group allowed me to build, based on 67 SNPs (Single Nucleotide Polymorphism), a phylogeny which indicates that the Caribbean area, where sugarcane was introduced not before the end of the 15th century, might be a center for dispersion of PFGE-B strains. This study also revealed a polymorphism of spacers of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats system) locus. The study of this polymorphism in 18 non-sequenced strains of X. albilineans allowed to demonstrate that strains from Florida originated in the Caribbean region, and to propose hypotheses regarding the origin of PFGE-B strains from other regions of the world, even if the sequencing of a higher number of strains will be required to confirm these hypotheses.Analyzing the sequence of the genome of two strains, initially thought to belong to the X. albilineans species but which weren’t isolated from sugarcane stalks, I revealed a new species that we called ‘Xanthomonas pseudalbilineans’. Finally, the comparison of the finished and annotated genome of the PFGE-B strain GPE PC73 with the genome of other phytopathogenic bacteria allowed me to propose hypotheses to explain X. albilineans’ pathogenicity

Bibliography: leaves 218-232.
232 leaves : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
This thesis investigates the resistance response of barley towards R. secalis by examining the in vitro interaction between various components of the apoplastic fluid and the fungus.
Thesis (Ph.D.)--Adelaide University, Dept. of Applied and Molecular Ecology, 2001

Bibliography: leaves 218-232.
232 leaves : ill. (some col.) ; 30 cm.
This thesis investigates the resistance response of barley towards R. secalis by examining the in vitro interaction between various components of the apoplastic fluid and the fungus.
Thesis (Ph.D.)--Adelaide University, Dept. of Applied and Molecular Ecology, 2001

The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375755
Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley.
Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Fall-sown barley will be increasingly important in the era of climate change due to higher yield potential and efficient use of water resources. Resistance/tolerance to biotic and abiotic stresses will be critical. Low temperature is an abiotic stress of great importance. Resistance to barley stripe rust (incited by Puccinia striifomis f. sp. hordei) and scald (incited by Rhynchosporium secalis) will be important in higher rainfall areas. Simultaneous gene discovery and breeding will accelerate the development of agronomically relevant germplasm. The role of FR-H1 and FR-H2 in low temperature tolerance (LTT) has been well documented. However the question still remains: is LTT due only to FR-H1 and FR-H2 or are there other, undiscovered, determinants of this critical trait? We developed two doubled haploid mapping populations using two lines from the University of Nebraska (NE) with superior cold tolerance and one line from Oregon State University (OR) with good malting quality and disease resistance: NB3437f/OR71 (facultative x facultative) and NB713/OR71 (winter x facultative). Both were genotyped with a custom 384 oligonucleotide pool assay (OPA). QTL analyses were performed for LTT, vernalization sensitivity (VS), and resistance to barley stripe rust and scald. Disease resistance QTL were identified with favorable alleles from both NE and OR germplasm. The role of VRN-H2 in VS was confirmed and a novel alternative winter allele at VRN-H3 was discovered in the Nebraska germplasm. FR-H2 was identified as a determinant of LTT and a new QTL, FR-H3, was discovered on chromosome 1H that accounted for up to 48% of the phenotypic variation in field survival at St. Paul, Minnesota, USA. The discovery of FR-H3 is a significant advancement in barley LTT genetics and will assist in developing the next generation of fall-sown varieties.
Graduation date: 2012