Auswahl der wissenschaftlichen Literatur zum Thema „Rhynchosporium secalis“

A method of spray inoculation with the scald (Rhynchosporium secalis) incitant was highly reliable in differentiating susceptible and resistant barley plants grown at high plant density in a controlled environment. Segregating populations can be efficiently screened under precise conditions in the limited space available in controlled environment chambers.Key words: Hordeum vulgare, Rhynchosporium secalis, scald, screening, resistance, high plant density

A worlwide list of 75 host plants of Rhynchosporium secalis (Oud.) Davis is given, as well as a list of the grasses that are not host, and of those that are secondary hosts. The aim is to help to achieve agronomic control of this barley parasite.

A polymerase chain reaction (PCR)-based diagnostic assay was developed to detect Rhynchosporium secalis, the barley scald fungus, in barley seed. Species-specific primers were designed based on sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers 1 and 2 of R. secalis. The sequenced regions showed 100% homology between the two R. secalis isolates and 93% homology between R. secalis and R. orthosporum. Five sets of synthesized oligonucleotide primers were tested for their specificity using 29 isolates of R. secalis of diverse geographic origins and from different barley cultivars. In addition, DNA extracts from 22 species of microbes either taxonomically related to or from the same niche as R. secalis were tested as negative controls. Among five sets of primers, a primer set, RS8 and RS9, was selected for use in detecting R. secalis because it amplified a 264-bp fragment from the DNA of all R. secalis isolates but not the DNA from other species used for validation of the specificity of this primer set. This primer set was also used to detect R. secalis in barley seed and successfully amplified the predicted size of the DNA fragment in the infected material. PCR detection of as little as 1 to 10 pg of R. secalis DNA was possible. The method described here requires 1 day for completion, compared to 10 days required for the cultural method.

Fifty-five isolates of <i>Rhynchosporium secalis</i> from <i>Hordeum vulgare</i> and 34 isolates from Secale cereale were compared for growth on different nutrient media, effect of temperature on growth and morphology of colonies. The pathogenicity of the isolates was assessed on 10 rye varieties, 10 triticale varieties and the susceptible barley variety Gambrinus. The triticale varieties differed in the number of rye chromosomes in the genome. Isozymes of <i>R. secalis</i> isolated from infected leaves of barley and rye were compared. The RAPD-PCR method was used for comparison of isolates on DNA-markers. The analysis indicated two specialised forms of the fungus; each of them able to develop only on its original host.

Restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of Australian field populations of the barley scald pathogen Rhynchosporium secalis. Fungal isolates were collected by hierarchical sampling from five naturally infected barley fields in different geographic locations during a single growing season. Genetic variation was high in Australian R. secalis populations. Among the 265 fungal isolates analyzed, 214 distinct genotypes were identified. Average genotype diversity within a field population was 65% of its theoretical maximum. Nei's average gene diversity across seven RFLP loci was 0.54. The majority (76%) of gene diversity was distributed within sampling site areas measuring ≈1 m2; 19% of gene diversity was distributed among sampling sites within fields; and 5% of gene diversity was distributed among fields. Fungal populations from different locations differed significantly both in allele frequencies and genotype diversities. The degree of genetic differentiation was significantly correlated with geographic distance between populations. Our results suggest that the R. secalis population in Western Australia has a different genetic structure than populations in Victoria and South Australia.

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