Dissertations / Theses on the topic 'Barley net blotch'

Pyrenophora teres f. teres is a fungal pathogen that causes barley net form net blotch. To evaluate the genetics of resistance in barley, a RIL population was developed using resistant barley lines CI5791 and Tifang and tested against a global collection of nine P. teres f. teres isolates. QTL analysis indicated that CI5791 resistance mapped to chromosome 6H and was effective against all isolates. Additionally, CI5791 harbored resistance on chromosome 3H effective against two Japanese isolates. Tifang also had resistance that mapped to 3H and was effective against four of the isolates. To evaluate the genetics of virulence in P. teres f. teres, a fungal population was developed and evaluated against ten barley lines. 19 unique QTL were identified on 12 different linkage groups. 1 or 2 major loci were identified for a few of the barley lines whereas for most lines, virulence was contributed by several loci.

The occurrence of spot type net blotch (Pyrenophora teres f. maculata) and net type net blotch (P. teres f. teres) as well as the occurrence of net blotch mating types has been investigated in Lithuania, Latvia and Estonia. Genetic diversity of barley net blotch isolates from various locations in Lithuania was investigated using ISSR and AFLP markers. The net blotch resistance of 150 spring barley varieties was investigated under artificial and natural infection conditions in the field.
Panaudojant molekulinius žymeklius ištirta tinkliškosios dryžligės patogeno Pyrenophora teres populiacijos genetinė įvairovė, nustatyti P. teres formų ir lytinio dauginimosi tipų sutinkamumas Lietuvoje, Latvijoje ir Estijoje. Lietuvos sąlygomis įvertintas Vakarų Europos ekotipo 150 vasarinių miežių veislių ir linijų jaurumas tinkliškajai dryžligei esant skirtingai pradinei infekcijai.

Net blotch is one of the most devastating diseases of barley (Hordeum vulgare L.) and occurs in two distinct forms, net-type net blotch (NTNB) and spot-type net blotch (STNB), caused by the fungal pathogens Pyrenophora teres f. sp. teres Smedeg. and P. teres f. sp. maculata Smedeg., respectively. Several sources of resistance have been previously reported, however, few barley cultivars with high levels of resistance have been developed from these sources. Efficient utilization of available resistance sources is dependent upon successful characterization of genes governing resistance in each resistant parent. Five net blotch resistant parents and one susceptible parent were crossed to identify novel resistance genes, postulate gene number and mode of inheritance, and conduct linkage mapping of novel genes for net blotch resistance. Results indicate that the highly resistant spring barley lines CIho 2291 and CIho 5098, and the winter barley cultivar Nomini each have single dominant genes for NTNB resistance. Resistance to NTNB in CIho 5098 is controlled by the same dominant gene conferring resistance in Nomini. Resistance to NTNB in CIho 2291 is controlled by one dominant gene which putatively is the same gene conferring resistance in ND B112, but differs from the resistance genes carried by the other parents in this study. An F2 population of 238 individuals derived from a cross between Nomini and the susceptible parent â Hectorâ , and an F2 population of 193 individuals derived from a cross between CIho 2291 and Hector were used to map the genes governing NTNB resistance in Nomini and CIho 2291. The dominant gene governing resistance in Nomini, temporarily designated Rpt-Nomini, was mapped to a 9.2 cM region near the centromere of barley chromosome 6H between the flanking microsatellite markers Bmag0344a (r2=0.70) and Bmag0103a (r2=0.90), which were 6.8 cM and 2.4 cM away from Rpt-Nomini, respectively. The dominant gene governing resistance in CIho 2291, temporarily designated Rpt-CIho2291, was mapped to the distal region of barley chromosome 6H between the flanking microsatellite markers Bmag0173 (r2=0.65) and Bmag0500 (r2=0.26), which were 9.9 cM and 24.4 cM from Rpt-CIho2291, respectively. Previous studies have reported genes governing net blotch resistance in this region; however, allelism tests have not been conducted to determine the relationship between these genes. Identification of the chromosomal location of Rpt-Nomini and Rpt-CIho2291 will facilitate future efforts in pyramiding multiple independent genes for net blotch resistance.
Ph. D.

Spot form net blotch (SFNB) caused by Pyrenophora teres f. maculata is a major foliar disease of barley (Hordeum vulgare L.) worldwide. SFNB epidemics have recently been observed in major barley producing countries, suggesting that the local barley cultivars are not resistant and that virulence of the pathogen populations may have changed. Here we attempt to identify sources of resistance effective against diverse isolates of the SFNB pathogen. A total of 2062 world barley core collection accessions were phenotyped using isolates of this pathogen collected in the United States (FGO), Australia (SG1), New Zealand (NZKF2), and Denmark (DEN 2.6). Isolate-specific susceptibility was identified in several of the barley accessions tested, indicating variability in both pathogen virulence and host resistance/susceptibility. Collectively, only 15 barley accessions were resistant across all isolates tested. Future research will involve the characterization of host resistance, pathogen virulence, and the host-pathogen interaction associated with SFNB of barley.

The necrotrophic fungal pathogen Pyrenophora teres f. teres causes the foliar disease net form net blotch (NFNB) on barley (Hordeum vulgare). To investigate the genetics of virulence in the barley- P. teres f. teres pathosystem, we used 118 progeny derived from a cross between the isolates 15A and 6A. The barley lines, chosen based on their different reactions to 15A and 6A, were evaluated for NFNB caused by the 15A ? 6A progeny. Genetic maps generated with SSR and AFLP markers in the fungal population were scanned for quantitative trait loci (QTL) associated with virulence in P. teres f. teres. Two QTL were identified in Rika, two in Kombar and PI356715 had a single unique QTL. Therefore, a total of five virulence loci were identified in this pathogen population based on inoculation on three different barley lines.

Pyrenophora teres f. teres is the causal agent of net form net blotch of barley. P. teres f. teres is prevalent globally across all barley growing regions and globally is the most devastating foliar disease of barley. Though economically important, the molecular mechanism whereby P. teres f. teres causes disease is poorly understood and investigations into these mechanisms have been hindered by a lack of genomic resources. To set a genomic foundation for P. teres f. teres the reference isolate 0-1 was sequenced and assembled using PacBio single molecule real-time (SMRT) sequencing and scaffolded into 12 chromosomes to provide the first finished genome of P. teres f. teres. High confidence gene models were generated for the reference genome of isolate 0-1 using a combination of pure culture and in planta RNA sequencing. An additional four P. teres f. teres isolates were sequenced and assembled to the same quality as the reference isolate 0-1 and used in a comparative genomic study. Comparisons of the five P. teres f. teres isolates showed a two-speed genome architecture with the genome being partitioned into core and accessory genomic compartments. Accessory genomic compartments clustered in sub-telomeric regions of the P. teres f. teres genome with a majority of previously identified quantitative trait loci (QTL) associated with avirulence/virulence being spanned by these accessory regions. Using these genomic resources, with a bi-parental mapping population and a natural population for QTL analysis and genome wide association study (GWAS), respectively, we identified a candidate gene for the previously mapped AvrHar. QTL analysis identified a locus extending off the end of P. teres f. teres chromosome 5 and GWAS analysis identified significant associations with a gene encoding a small secreted protein. The candidate AvrHar gene was validated using CRISPR-Cas9-RNP gene disruption in parental isolates 15A and 0-1. Disruption of AvrHar in isolate 15A did not result in a phenotypic change while disruption of the 0-1 allele resulted in a complete loss of pathogenicity. This is the first identification of an effector from P. teres f. teres validated using CRISPR-Cas9-RNP gene editing.
North Dakota Barley Council

Net blotch is an economically important foliar disease of barley that occurs in two distinct forms: Spot Form Net blotch (SFNB) and Net Form Net Blotch (NFNB) caused by the necrotrophic fungal pathogens Pyrenophora teres f. maculata (Ptm) and Pyrenophora teres f. teres (Ptt), respectively. The recent emergence and the identification of both Ptm and Ptt isolates virulent on popular ND malting barley varieties have warranted the identification of new resistance sources. Association mapping was conducted on 2,062 diverse barley accessions phenotyped at the seedling stage with four diverse P. teres f. maculata isolates and genotyped with the 9k Illumina barley iSelect chip. A total of 138 significant marker-trait associations (MTA; ?log10P value > 3.0) corresponding to 27 resistance loci were identified of which 21 loci were novel and six corresponded to previously characterized SFNB resistance QTL. Further, two higly resistant lines PI67381 and PI84314 were crossed with the two susceptible cultivars Tradition and Pinnacle grown in the Upper Midwestern US to develop three bi-parental recombinant inbred line (RIL) mapping populations of Tradition x PI67381, Pinnacle x PI67381 and Pinnacle x PI84314. These RIL populations were phenotyped with six diverse Ptm isolates and genotyped using PCR-GBS. MapDisto and Qgene were used to analyze the data and a total of twelve QTL were identified on chromosome 2H, 3H, 4H, 6H and 7H, of which nine were previously reported and the remaining three are considered novel. These resistances and the markers delimiting the QTL are being utilized to develop prebreeding lines by introgressing SFNB resistance into the cultivars Pinnacle and Tradition utilizing marker assisted selection. The barley line CI5791 exhibits a high level of resistance to diverse Ptt isolates collected from around the world. A forward genetics approach and an exome capture-mediated mapping-by-sequencing identified a candidate HvWRKY6 transcription factor gene required for NFNB resistance on chr 3H. We hypothesize that the HvWRKY6 gene function as a component of a conserved basal defense mechanism, which regulates the expression of other defense response genes that restrict lesion growth. The resistance/susceptibility loci identified in this study will facilitate the development of net blotch resistant cultivars.
USDA-NIFA-AFRI grant #2011-68002-30029 (T-CAP)

Pyrenophora teres f. maculata causes spot form net blotch (SFNB) on barley and was recently documented in North Dakota. The impact of SFNB on barley, the genetic diversity of the pathogen, and virulence structure are unknown for the state. Yield and quality loss in North Dakota due to SFNB was investigated over eleven year-sites, and simple linear regression of percent yield loss on adjusted percent disease using year-site means of treatments predicted a 0.77% increase in yield loss for every 1% increase in disease. When virulence of isolates of P. teres f. maculata collected from geographically diverse regions in the northern United States was evaluated on differential barley genotypes, few isolates were identical in terms of virulence patterns, and the virulence profile of a population from Idaho differed from other populations. To understand population structure and genetic diversity, SNPs of 140 isolates were generated using genotyping-by-sequencing for analysis of population genetics and structure. Evidence for sexual recombination in each population includes the ratio of mating-type idiomorphs that do not significantly differ from a 1:1 ratio; low index of association values for most populations; and high variation within and low variation among populations. Association mapping detected forty-five significant marker-trait associations of SNPs associated with virulence or avirulence across 19 P. teres f. maculata scaffolds using 82 isolates of P. teres f. maculata from diverse areas in the northern United States. The most significant marker, 01700_198, was found on P. teres f. maculata-scaffold 8 when the population was challenged with four different barley lines. This research demonstrates that SFNB causes significant yield loss; that high diversity exists in the pathogen, with respect to virulence and population genetics; and that association mapping can be used to identify virulence/avirulence marker-trait associations to fill gaps in our understanding of host-parasite genetic interactions in this pathosystem.
American Malting Barley Association
Triticeae CAP
North Dakota Barley Commission

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.

Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), exists in two forms; P. teres f. teres and P. teres f. maculata. Both forms induce a combination of brown necrotic spots and extensive chlorosis in susceptible barley cultivars. Although a number of low molecular weight compounds (LMWCs) have been previously isolated from P. teres culture filtrates, they only induced certain components of symptoms. Fungal metabolites were extracted from culture filtrates of both forms of the pathogen and separated into low (<3kDa) and high molecular weight compounds (HMWCs, >10 kDa) with each fraction inducing a component of the net blotch symptoms in a barley leaf toxicity assay. Inactivation of both LMWCs (<1kDa) and HMWCs resulted in loss of activity confirming their potential role in symptom development. Low molecular weight compounds induced chlorosis and water soaking but not the brown necrotic spots or lesions usually seen during the infection of barley by P. teres. The high molecular weight compounds (>10 kDa) induced the brown necrotic spots or lesions with no chlorosis evident. Further characterisation of LMWCs showed that they are not host specific while HMWCs exhibited host specificity. LMWCs were purified and further analysed using high voltage paper electrophoresis, staining and mass spectrometry. Electrophoretic properties and staining of the LMWCs with ninhydrin indicated that both forms of P. teres produced similar LMWCs in the conditions grown. Each form produced eight ninhydrin-positive compounds with the samerelative mobilities. Each individual compound was shown to induce chlorosis in excised barley leaves. All compounds except the one isolated in this study appear to be derivatives of or are the previously described compounds; N-(2-amino-2carboxyethyl) aspartic acid (Toxin A), aspergillomarasmine A, anhydroaspergillomarasmine A and aspergillomarasmine B. The exception is a bioactive UV absorbing LMWC which appears to be a reductive conjugation of the α-keto acid of phenylalanine with Toxin A. The HMWCs (>10kDa) were proteinaceous since they were identifiable using Coomassie staining. Additionally, the loss of activity that occurred with incubation at 40, 60, and 80 °C for 30 and 60 min followed a pattern fairly typical for protein denaturation. Further, treatment with protease decreased their phytotoxicity in proportion to the amount of enzyme used. Enzyme and heat treatment of proteins extracted from each form showed that proteins of P. teres f. teres are more resistant to heat and enzyme treatment compared with those of P. teres f. maculata. This suggests the protein(s) involved in symptom induction by P. teres f. teres and P. teres f. maculata are different which contributes to the difference in the symptom expression during the interaction between the pathogens and barley. Proteinaceous metabolites extracted from P. teres f. teres and P. teres f. maculata ranged from 10 to 100 kDa. Fractions purified using gel filtration had biological activity when they contained eight proteins when extracted from P. teres f. maculata (90, 80, 75, 55, 48, 35, 14 and 12 kDa) and six proteins when extracted from P. teres f. teres (90, 80, 55, 48, 14 and 12 kDa). Additionally, intercellular washing fluids (IWF) extracted from barley plants inoculated with both forms of P. teres, contained proteins of the same size as those in the biologically active fractions extracted from culture filtrates of P. teres f. maculata (80, 14 and 12 kDa) and P. teres f. teres (80, 48 and 14 kDa). Automated MS/MS sequencing of the biologically active proteins showed no resemblance to the sequences or conserved domain information available in public databases and as a consequence, these proteins were considered as novel proteins for P. teres. However, exact short matches with fragments resulting from the 80, 48 and 14 kDa proteins, showed considerable homology with ATP-binding cassette (ABC) transporters and their components, cellulases, serine proteinases as well as some hypothetical proteins isolated from different fungal species. Reaction of six plant species including one susceptible barley cultivar (Sloop) and one resistant line (CI9214) to P. teres showed that partially purified proteins induce the symptoms selectively in barley cultivars where the proteinaceous metabolites only induced brown necrotic spot/lesions in barley with a greater response seen on the susceptible cultivar Sloop when compared to the resistant line CI9214. No symptoms were seen on other plant species employed in this study suggesting that the proteinaceous metabolites isolated in this study are host specific phytotoxins. This research has allowed the first isolation of proteinaceous host-specific toxins from P. teres as well as the identification of a UV-sensitive LMWC phytotoxin not previously described. Proteinaceous toxins induced brown necrotic spots/lesions specific to the host while the LMWCs induced chlorosis in a number of different plant species. This contributes significantly to the body of knowledge defining how symptoms are caused during the pathogenicity process in the interaction between P. teres and barley.
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Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), exists in two forms; P. teres f. teres and P. teres f. maculata. Both forms induce a combination of brown necrotic spots and extensive chlorosis in susceptible barley cultivars. Although a number of low molecular weight compounds (LMWCs) have been previously isolated from P. teres culture filtrates, they only induced certain components of symptoms. Fungal metabolites were extracted from culture filtrates of both forms of the pathogen and separated into low (<3kDa) and high molecular weight compounds (HMWCs, >10 kDa) with each fraction inducing a component of the net blotch symptoms in a barley leaf toxicity assay. Inactivation of both LMWCs (<1kDa) and HMWCs resulted in loss of activity confirming their potential role in symptom development. Low molecular weight compounds induced chlorosis and water soaking but not the brown necrotic spots or lesions usually seen during the infection of barley by P. teres. The high molecular weight compounds (>10 kDa) induced the brown necrotic spots or lesions with no chlorosis evident. Further characterisation of LMWCs showed that they are not host specific while HMWCs exhibited host specificity. LMWCs were purified and further analysed using high voltage paper electrophoresis, staining and mass spectrometry. Electrophoretic properties and staining of the LMWCs with ninhydrin indicated that both forms of P. teres produced similar LMWCs in the conditions grown. Each form produced eight ninhydrin-positive compounds with the samerelative mobilities. Each individual compound was shown to induce chlorosis in excised barley leaves. All compounds except the one isolated in this study appear to be derivatives of or are the previously described compounds; N-(2-amino-2carboxyethyl) aspartic acid (Toxin A), aspergillomarasmine A, anhydroaspergillomarasmine A and aspergillomarasmine B. The exception is a bioactive UV absorbing LMWC which appears to be a reductive conjugation of the α-keto acid of phenylalanine with Toxin A. The HMWCs (>10kDa) were proteinaceous since they were identifiable using Coomassie staining. Additionally, the loss of activity that occurred with incubation at 40, 60, and 80 °C for 30 and 60 min followed a pattern fairly typical for protein denaturation. Further, treatment with protease decreased their phytotoxicity in proportion to the amount of enzyme used. Enzyme and heat treatment of proteins extracted from each form showed that proteins of P. teres f. teres are more resistant to heat and enzyme treatment compared with those of P. teres f. maculata. This suggests the protein(s) involved in symptom induction by P. teres f. teres and P. teres f. maculata are different which contributes to the difference in the symptom expression during the interaction between the pathogens and barley. Proteinaceous metabolites extracted from P. teres f. teres and P. teres f. maculata ranged from 10 to 100 kDa. Fractions purified using gel filtration had biological activity when they contained eight proteins when extracted from P. teres f. maculata (90, 80, 75, 55, 48, 35, 14 and 12 kDa) and six proteins when extracted from P. teres f. teres (90, 80, 55, 48, 14 and 12 kDa). Additionally, intercellular washing fluids (IWF) extracted from barley plants inoculated with both forms of P. teres, contained proteins of the same size as those in the biologically active fractions extracted from culture filtrates of P. teres f. maculata (80, 14 and 12 kDa) and P. teres f. teres (80, 48 and 14 kDa). Automated MS/MS sequencing of the biologically active proteins showed no resemblance to the sequences or conserved domain information available in public databases and as a consequence, these proteins were considered as novel proteins for P. teres. However, exact short matches with fragments resulting from the 80, 48 and 14 kDa proteins, showed considerable homology with ATP-binding cassette (ABC) transporters and their components, cellulases, serine proteinases as well as some hypothetical proteins isolated from different fungal species. Reaction of six plant species including one susceptible barley cultivar (Sloop) and one resistant line (CI9214) to P. teres showed that partially purified proteins induce the symptoms selectively in barley cultivars where the proteinaceous metabolites only induced brown necrotic spot/lesions in barley with a greater response seen on the susceptible cultivar Sloop when compared to the resistant line CI9214. No symptoms were seen on other plant species employed in this study suggesting that the proteinaceous metabolites isolated in this study are host specific phytotoxins. This research has allowed the first isolation of proteinaceous host-specific toxins from P. teres as well as the identification of a UV-sensitive LMWC phytotoxin not previously described. Proteinaceous toxins induced brown necrotic spots/lesions specific to the host while the LMWCs induced chlorosis in a number of different plant species. This contributes significantly to the body of knowledge defining how symptoms are caused during the pathogenicity process in the interaction between P. teres and barley.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

Pyrenophora teres f. teres (Ptt) causes net form net blotch disease (NFNB), an important disease of barley in Australia and worldwide. This fungus uses proteinaceous toxins to cause necrosis and different isolates of Ptt differ in their ability to cause symptoms on different cultivars of barley. However, little is known about the roles of pathogen growth and individual toxins in symptom development. This project therefore aimed to determine whether there is a relationship between toxin production, fungal growth and virulence in NFNB. Conidial germination, extent of fungal growth and culture filtrate toxicity were compared for six South Australian Ptt isolates with different virulence on the barley cultivar ‘Sloop’. In addition, Ptt toxin production was optimised before identification and selection of virulence-related candidate proteins (VRCPs) for further characterisation. The biological activity of recombinant VRCPs on susceptible and resistant cultivars and VRCPs gene expression during the interaction of Sloop with each isolate were also compared. In general, the more virulent isolates had higher rates of conidial germination (both in vitro and in planta) and fungal development in planta, represented by longer hyphae and more appressoria, compared with less virulent isolates. Similarly, PttGAPDH and its transcript were more abundant during the interaction of barley with more virulent isolates. A proteomics approach was used to identify proteins unique to the more virulent isolate, proteins from bioactive fractions on either susceptible (Sloop) or resistant cultivars (CI9214 and Beecher) and proteins from the intercellular washing fluids (ICWFs) of infected barley. These analyses revealed that Ptt produced proteins between 37 and 150 kDa that have biological activity. Liquid Chromatography-Electrospray Ionisation Ion-Trap Mass Spectrometry (LC-eSI-IT MS), of individual biologically active proteins was used to identify peptides which matched to 17 proteins that belong to three groups of fungal proteins including virulence-related proteins; fungal growth and development proteins; and those with unknown function (hypothetical proteins). However, Ptt toxins were not detected in the ICWF protein profiles suggesting that Ptt toxins were either in trace amounts or might be internalised into the cell. The four VRCPs selected, were identified as hypothetical proteins with unknown function in the Ptt database. Further bioinformatic analysis characterised these VRCPs as an isochorismatase (PttCHFP1), an endo-1, 4-β- xylanase A (PttXyn11A), a glycophosphatidylinositol (GPI)-anchored common in fungal extracellular membrane (CFEM) domain-containing protein (PttGPICFEM) and an unknown proteinaceous secreted (but conserved) hypothetical protein (PttSP1). These VRCPs were heterologously expressed and characterised using gene expression studies. PttXyn11A had strong homology with the well characterised endoxylanases, TrXyn11A from Trichoderma reesei and BcXyn11A from Botrytis cinerea, known to contribute to virulence. A necrosis-inducing region on the surface of the enzyme was also identified in PttXyn11A, suggesting a potential role in necrosis induction. The culture filtrates for more virulent isolates had significantly greater xylanase activity than those from less virulent isolates. Even though heterologously expressed PttXyn11A was toxic to Escherichia coli, xylanase activity was detectable at very low levels and was not enough to cause symptoms in the bioassay. In addition, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and RT-quantitative PCR (RT-qPCR) analysis demonstrated that PttXyn11A was expressed more abundantly by the more virulent isolates compared with the other isolates in culture and during the plant-pathogen interaction. Together, these results suggest that PttXyn11A plays a role in virulence, either through its ability to degrade the plant cell wall to assist fungal growth or through its necrosisinducing ability. PttCHFP1 showed homology to an isochorismatase, an enzyme that has been proposed to have a role in plant defence via inhibition of salicylic acid production. PttSP1 showed homology to a membrane lipoprotein proposed to have a role in fungal development. Bioassay of recombinant PttCHFP1 and PttSP1 induced chlorosis symptoms in the susceptible barley cultivar (Sloop). The cysteine-rich CFEM domain identified in PttGPI-CFEM has been suggested to have an important role in hyphal attachment and fungal networking. However, E. coli was not able to express this gene probably due to its attachment to the plasma membrane and/or cell wall. Analysis of the gene expression profiles for PttCHFP1, PttGPI-CFEM and PttSP1 showed no significant differences between isolates in vitro and in planta suggesting that all isolates regulated the expression of these genes to the essential level possibly required for pathogenesis. This is the first study to identify the relationship between fungal growth and proteinaceous toxin production, characterise individual proteinaceous toxins in the mixture of Ptt culture filtrate and investigate the expression profiles of genes encoding VRCPs during the Ptt-barley interaction. This study therefore provides a better understanding of the Ptt-barley interaction by identifying the potential toxins which might lead to identify the toxin targets and ultimately support the breeding of resistant cultivars of barley.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013