Dissertationen zum Thema „Rhynchosporium secalis“

Rhynchosporium secalis is a major fungal pathogen of barley. Fungicides, including triazoles and QoIs, play an important part in R. secalis control programmes, but can select for resistance. Reduced triazole sensitivity had been reported in R. secalis, but the mechanism was not known. QoI resistance had not been reported in R. secalis until 2008, when the G 143A substitution in cytochrome b was reported in two R. secalis isolates from France. A high-throughput fungicide sensitivity assay was developed for R. secalis, and isolates were screened for Quinone outside Inhibitor (QoI) and triazole sensitivity. QoI sensitivity was reduced by over lOO-fold in the isolates with G143A. This was not found in any UK isolates, but smaller sensitivity shifts were detected. These sensitivity shifts were mostly reversed by alternative oxidase (AOX) inhibitors, and there is preliminary evidence of AOXupregulation following exposure to azoxystrobin. Shifts in triazole sensitivity were not correlated with point mutations or constitutive over- expression of the target site encoding gene, CYP 51, or reversed by putative efflux inhibitors. However, a second CYP51 paralogue, CYP51A, was sequenced from less-sensitive isolates but absent from sensitive isolates. CYP51A was upregulated more than CYP51B following exposure to tebuconazole. Pyrosequencing analysis of the Hoosfield archive showed that levels of CYP 51 A were low until 1998, then rapidly increased. Phylogenetic analysis suggests a CYP 51 gene duplication event basal to the filamentous ascomycetes, followed by multiple losses of CYP51A. Therefore it appears that CYP51A was almost lost from the R. secalis population, but re-emerged due to selection by triazoles. This project has identified the mechanism responsible for an initial shift in R. secalis sensitivity that compromised the effectiveness of some older triazoles, and reported further variation in sensitivity to newer triazoles that currently provide control in the field. Furthermore, the G143A cytochrome b substitution can confer QoI resistance in R. secalis. Therefore resistance management is important for sustainable R. secalis control.

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.

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.

De nouvelles stratégies de lutte contre la rhynchosporiose de l'orge sont proposées et certaines caractéristiques épidémiologiques de l'agent pathogène Rhynchosporium secalis (Oud. ) Davis, notamment le rôle des semences, précisées en révélant directement la présence du champignon sur ces dernières, ou indirectement lors d'essais au champ. Une première méthode d'analyse, "classique", comprend une désinfection préalable des grains d'orge (éthanol, hypochlorite de sodium et solution antibactérienne et antifongique), avant leur dépôt sur un milieu électif ("orge") et sélectif (terramycine + iprodione + procymidone + 2,4 D). Son taux de réussite est toutefois relativement faible. La deuxième, par immunofluorescence, permet l'analyse de routine des lots de grains. Une plus grande spécificité des immunsérums est obtenue lors de protocoles d'immunisation complexes faisant intervenir des formes antigéniques différentes (spores, mycélium). Les essais de traitements chimiques des semences et de pulvérisations de produits antifongiques sur le sol permettent d'estimer l'importance relative des types d'inoculum "semence" et "sol". Ils démontrent également l'intérêt de telles interventions en tant que techniques culturales. Les mélanges de variétés enfin peuvent aussi constituer une stratégie limitant l'extension de la maladie
New cultural practices against barley scald disease were proposed and some epidemiological characteristics of the fungal pathogen Rhynchosporium secalis (Oud. ) Davis, especially importance of seeds, assessed both directly showing its presence and indirectly by field trials. The first analysis method, a "classical" one, associated barley seeds disinfection with ethanol, sodium hypochlorite and bactericidal and fungicidal solution, before placing them on the elective ("barley") and selective medium (terramycine + iprodione + procymidone + 2,4 D). But the experiments were not adequatly successful. The second method, immunofluorescence, allowed routine analysis of seeds samples. Moreover, a higher specificity of immunsera was obtained by complex immunization procedures including different antigenic forms. The chemical treatments of seeds and sprayings of fungicides on the soil that were tested allowed estimation of the comparative importance of "seeds" and "soil" inoculum. They also demonstrated that such applications can be useful as cultural techniques. Lastly, varietal mixtures can be used as a strategy to limit disease spreading in the crop

The purpose of this study was to identify pathogenicity genes in the fungal pathogen of cultivated barley, Rhynchosporium secalis. Pathogenicity genes are described as genes that are critical for the successful invasion and colonisation of the host plant but not necessary for life cycle completion in culture. To identify genes a pool of insertion mutants was generated. Insertional mutants were generated by two methods, restriction enzyme-mediated integration (REMI) and Agrobacterium tumefaciens-mediated transformation (ATMT). A detailed REMI study showed circular pAN7-1 vector produced higher transformation efficiencies than linear vector at all enzyme levels tested. Fungal strain 5, in combination with 20 units of the restriction enzyme BamHI produced the highest observed transformation efficiency with approximately 40% of these mutants producing simple, single integrations based on interpreted Southern data. The addition of BamHI increased transformation efficiency at all enzyme levels tested with the exception of the highest enzyme concentration: 200 units of enzyme/transformation reaction. In comparison to REMI, the ATMT protocol proved more efficient than REMI and the binary vector backbone pPZP200 produced >50% simple single copy integrations, interpreted from Southern data. This study is the first ATMT protocol for R. secalis and was successfully adapted from other fungal species. In total, 534 BamHI and HindIII REMI mutants of R. secalis fungal strain UK7 (83) and strain 5 (453) were screened on the universally susceptible barley cultivar Sloop yielding 10 non-pathogenic mutants, eight from strain 5 and two from UK7, respectively. During screening experiments strain 5 mutants failed to produce enough spores for a spore suspension to be prepared and inoculated. Strain 5 loses the ability to sporulate after four generations, or successive subculture steps. The inability to sporulate was not correlated to an observable, macroscopic loss in fungal biomass. Starvation experiments utilising carbon and nitrogen sources did not alter sporulation in the sporulating strain 5 sample or reverse the loss of sporulation. However, an overall trend was observed in the sporulation of strain UK7 where sporulation decreased with increasing nitrogen and increased with increasing carbon. Genomic sequence flanking the integration site was isolated and analysed from six of the ten non-pathogenic mutants. Four putative genes were identified with integrations located in their putative promoter sequences. Sequence similarity searches showed three of these putative genes had similarities to amino acid permeases, cytochrome p450 and rhomboid-like genes. The two putative genes with similarities to amino acid permease and cytochrome p450 genes were selected for targeted gene disruption studies using homologous recombination (HR). ATMT was used as the delivery system for the HR construct in an attempt to generate a disruption mutant and prove gene function. Over 200 mutants transformed with the two knock out vectors were screened. However, gene disruption experiments failed and could not be repeated due to a lack of resources and time. In conclusion, this study has demonstrated that the REMI transformation technique is feasible for gene disruption studies in R. secalis. Furthermore, ATMT is a viable alternative transformation method that, for future studies, would be the preferable technique.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

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

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

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.
Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Specific information towards each fungicidal active ingredient and its effects on all local organisms, whether they’re pathogens, non-target organisms or crops is still largely lacking. In this study, the potential effects of azoxystrobin, tebuconazole and a commercial formulation with these two active ingredients combined, known as Custodia 320 SC, were tested on two fungal species, Pyrenophora teres and Rhynchosporium secalis, both well known worldwide pathogens responsible for worldwide losses in important crops such as barley and rye. The results indicated significant resistance to both active ingredients by P. teres, with tebuconazole-treated cultures showing the lowest inhibition in the growth response. R. secalis showed also low and poorly variable growth inhibition rates for both fungicides, never reaching to 50% inhibition in comparison to the respective control. These results were obtained considering exposure concentration ranges within the limits of water solubility for both compounds and including concentrations that correspond to typical application rates used in the field to treat affected crops. Nevertheless, bacterial contamination in early trials allowed to observe that under these conditions the efficacy of the fungicides is superior (fungal growth inhibition rate over 50%) until a certain level of exposure, from which onwards the efficacy is again limited; this effect can be explained by a reduction of the fungi resistance capacity in competitive scenarios. The efficacy of the combined active ingredients was lower than equivalent treatments in trials where they were applied singly, suggesting the possibility of antagonistic interactions between azoxystrobin and tebuconazole. Additionally, the comparison of effects promoted by the combination of the active substances with their equivalent combinations applied via commercial formulation demonstrated that the formulants other than the active substances used in the commercial formulation do not promote their efficacy when the target organisms are P. teres or R. secalis.
Informação específica sobre cada ingrediente ativo fungicida, e os seus efeitos combinados em todos os organismos potencialmente afetados, quer sejam patogénicos, organismos não-alvo ou colheitas é ainda limitada. Neste estudo, os efeitos potenciais da azoxystrobina, do tebuconazole e de uma formulação comercial com a combinação destes dois ingredientes ativos, Custodia 320 SC, foram testados em duas espécies fúngicas, Pyrenophora teres e Rhynchosporium secalis, ambos fungos patogénicos conhecidos mundialmente por grandes perdas em colheitas agrícolas importantes como o centeio e a cevada. Os resultados indicaram resistência significativa a ambos os ingredientes ativos por P. teres, com as culturas tratadas com tebuconazole a demonstrarem as mais baixas taxas de inibição do crescimento relativamente ao respetivo controlo. R. secalis mostrou também taxas baixas e pouco variáveis de inibição de crescimento em resposta à exposição a cada um dos fungicidas, nunca chegando aos 50% de inibição em relação ao respetivo controlo. Estes resultados foram obtidos para gamas de concentrações de exposição dentro dos limites de solubilidade em água dos compostos e que incluíram concentrações que refletem as taxas de aplicação utilizadas para tratamento de culturas. Não obstante, a contaminação bacteriana em ensaios iniciais permitiu observar que, nestas condições, a eficácia dos fungicidas é superior (inibição de crescimento dos fungos alvo de mais de 50%) até um determinado nível de exposição, a partir do qual a eficácia se torna de novo muito limitada; este efeito será explicável pela redução da capacidade de resistência dos fungos aos fungicidas em cenários competitivos. A eficácia da combinação dos ingredientes ativos foi inferior à dos tratamentos equivalentes em que os mesmos foram aplicados individualmente, sugerindo a possibilidade de ocorrência de interações antagonísticas entre a azoxystrobina e o tebuconazole. Adicionalmente, a comparação dos efeitos de combinações de ingredientes ativos com as combinações equivalentes aplicadas através da formulação comercial demonstra que os restantes formulantes não promovem a eficácia dos ingredientes ativos quando as espécies alvo são P. teres ou R. secalis.
Mestrado em Microbiologia

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