The production of cereal crops is essential to secure a future that feeds the continuously growing population. Rust fungi reduce host fitness by feeding on their living tissue and interfere with the global production of crops. Cereal rusts, like Puccinia hordei (the causal agent of barley leaf rust) and Puccinia triticina (the causal agent of wheat leaf rust), have a narrow host range and colonize only one particular species. The most durable type of resistance, non-host resistance (NHR), is the immunity of an entire plant species to all strains of a pathogen species. Exploring the genetics of NHR has proven to be challenging because most interspecific hybrids are infertile. Previously, barley Rphq2 and Rph22, which encode orthologous lectin receptor-like kinases (LecRKs), were transformed into an experimental barley line, Golden SusPtrit, and showed resistance against adapted and non-adapted leaf rust species. We used these transgenic barley lines in the current project to explore the effect of the LecRKs on four wheat leaf rust (P. triticina) isolates. We used the settling tower method to inoculate four isolates of P. triticina on Rphq2 and Rph22 transgenic families. We found that most transgenic families showed an increase in resistance compared to the non-transgenic control 750-E1. By measuring the infection frequency of the infections, we identified that P. triticina isolates 93012 and 95012 had opposite virulence effects on two barley families, Rphq2-E5 and Rph22-E2A. Although the expression levels of Rphq2 and Rph22 followed an induction trend, we did not find significant differences between the isolates. We conclude that resistance mediated by Rphq2 and Rph22 against P. triticina isolates does not involve an isolate-specific component. Thus, we propose investigating differences between rust species to further explore the molecular aspect of non-host resistance.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Chemical control of diseases is the most used practice in wheat. The increase in mineral nutrition with foliar fertilizers has been a promising alternative for the plant resistance against leaf diseases. However, foliar fertilizers have been applied in association with fungicides and can change the performance of the fungicide on diseases control. This study aimed to evaluate the applicability of foliar fertilizer in combination with azoxystrobin + cyproconazole fungicide in wheat, based on biochemical, physiological , nutritional and yield parameters and determine the interference caused by the fertilizer on the evolution of leaf diseases in wheat. Isolated application rates of fertilizer and application in combination with the fungicide were performed on field and in the greenhouse works. The application of fertilizer increased the plant growth, green leaves and enhanced pigments levels (Chl a, Chl b and carotenoids). When the fungicide was applied with fertilizer, it reduced the stresses effect generated by fungicide application; it increased parameters of chlorophyll fluorescence, Fv / Fm and ETR. The levels of N, P and K in the leaves increased after fertilizer application. The fertilizer mixed with fungicide did not reduce the fungicide uptake. The diseases control was better when fertilizer was mixed with fungicide. The fertilizer applied alone had no effect on the diseases. Yield parameters were increased due to application of fungicide and foliar fertilizer. / O controle químico de doenças com o uso de fungicidas é umas das práticas mais empregadas na cultura do trigo em função da eficácia de controle. Incrementos na adubação mineral com fertilizantes foliares tem sido uma alternativa promissora em busca de maior resistência as doenças. Entretanto, fertilizantes foliares são comumente aplicados associados a fungicidas e podem interferir no desempenho de controle do produto. Este trabalho teve por objetivo avaliar a aplicabilidade do fertilizante foliar em mistura com o fungicida azoxistrobina + ciproconazol na cultura do trigo, com base em parâmetros bioquímicos, fisiológicos, nutricionais e produtivos e determinar a interferência causada pelo fertilizante sobre a evolução de doenças foliares na cultura. A partir da aplicação isolada de doses do fertilizante e da aplicação em associação com o fungicida foram realizados trabalhos a campo e em casa de vegetação. A aplicação do fertilizante refletiu em maior crescimento das plantas, manutenção de folhas verdes e maiores teores de pigmentos (Chl a, Chl b e carotenóides). Quando aplicado junto ao fungicida, o fertilizante teve efeito mitigatório dos estresses gerados pela aplicação do fungicida, com reflexos positivos em parâmetros da fluorescência da clorofila a, Fv/Fm e ETR. Houve aumento dos teores de N, P e K nas folhas em função do fertilizante foliar. Não houve redução da absorção do ingrediente ativo azoxistrobina + ciproconazol em mistura com o fertilizante. Houve melhor resposta de controle das doenças em função da mistura do fertilizante com o fungicida. O fertilizante isolado não teve nenhum efeito sobre as doenças. Parâmetros produtivos foram incrementados em função da aplicação do fungicida e do fertilizante foliar.

Doctor of Philosophy / Department of Plant Pathology / John P. Fellers / Harold Trick / In Kansas, wheat (Triticum aestivum L.) is severely affected by the biotrophic fungus Puccinia triticina (leaf rust). Although resistant varieties have been developed, the fungus tends to overcome new sources resistance very quickly. Plants have evolved a single gene (R genes) defense network that can recognize specific pathogen effectors (Avr), in a gene-for-gene manor. In rusts, effectors are secreted proteins responsible for inducing the uptake of nutrients and inhibit host defense responses. Identification of secreted proteins during the infection may help to understand the mode of infection of P. triticina. Little is known about molecular interactions in the pathosystem wheat-leaf rust and no Avr genes from cereal rusts have been cloned. In order to understand pathogenicity in leaf rust and generate new alternatives for disease control, the goal of this research is identify P. triticina secreted proteins from a collection of expressed genes during the infection, and to characterize putative Avr function for three candidates. From 432 EST’s derived from haustoria and infected plants, fifteen secreted proteins were identified and 10 were selected as potential avirulence candidates. Pt3 and Pt 51 are two P. triticina (Pt) candidates expressed specifically in the haustoria and encode small cysteine-rich secreted proteins. Eight candidates are expressed at early stages of infection, during spore germination and 6 days after inoculation. They are small-secreted proteins. None are repetitive elements or have nuclear localization signals. They also do not share a conserved motif with known filamentous fungus Avr proteins. Five candidates are novel proteins, two have similarity with predicted proteins, one is homologous with Hesp-379-like protein, one is homologous with superoxide dismutase, and one has a cell glucanase predicted function. Pt3, Pt12 and Pt27 were tested by transient expression experiments using co-bombardment with GUS into leaf rust resistant isogenic lines. Reduction in the expression of reporter gene GUS co-expressed with Pt27 indicates a potential avirulence factor for Lr26 in wheat.

Disease resistance is critical in soft red winter wheat (Triticum aestivum L.) cultivars. Leaf rust caused by Puccinia triticina Eriks and stripe rust caused by Puccinia striiformis Westend. f.sp. tritici Eriks. are destructive pathogens of wheat. From 2014 to 2015 phenotypic data was collected at diverse locations for resistance to leaf rust (North Carolina, Texas, and Virginia) and stripe rust (Arkansas, North Carolina, Georgia, Texas, and Virginia) in a Pioneer ‘25R47’ /‘Jamestown’ (P47/JT) population composed of 186 F5:9 recombinant inbred lines (RILs). Analysis of the P47/JT population identified two quantitative trait loci (QTL) for leaf rust resistance on chromosome 5B and two QTL for stripe rust resistance on chromosomes 3B and 6A. Phenotypic variation (%) explained by the putative leaf rust resistance QTL of Jamestown on 5B was as high as 22.1%. Variation explained by the putative stripe rust resistance QTL of Jamestown on 3B and 6A was as high as 11.1 and 14.3%, respectively. Jamestown is postulated to contain gene Lr18. Seedlings of 186 F5:9 recombinant inbred lines from the P47/JT population and 200 F2 seedlings from eight other crosses including Jamestown and/or the Lr18 host differential line RL6009 (Thatcher*6/Africa 43) were screened with P. triticina race TNRJJ. Genetic analysis of the populations was conducted to validate the presence of Lr18 in Jamestown. Results of linkage analysis identified SNP maker IWB41960 linked within 5 cM of gene Lr18 in all three populations. From 2016 to 2017 phenotypic data was collected at diverse locations for resistance to leaf rust (Illinois, North Carolina, and Virginia) in a ‘2013412’ (PI 667644) / VA10W-21 (PI 676295) population (412/21) composed of 157 doubled haploid (DH) lines. The 412/21 DH lines were genotyped via genotyping by sequence (GBS). Analysis of the 412/21 population identified one quantitative trait loci (QTL) region associated with adult plant resistance to leaf rust on chromosome 1B. Phenotypic variation (%) explained by the putative leaf rust resistance QTL of 2013412 on 1B was as high as 40.1%. Kompetitive allele-specific (KASP) markers KASP_S1B_8414614 and KASP_S1B_8566239 were developed as markers for use in marker assisted selection. / Ph. D. / Disease resistance to leaf rust and stripe rust is important when growing soft red winter wheat. Genetic resistance can have a benefit to cost ratio of up to 27:1, considerably better than that of fungicide treatments. From 2013 to 2017 disease data was collected across multiple locations spanning the eastern United States (Arkansas, Georgia, Illinois, North Carolina, Texas, and Virginia). DNA molecular markers were used to identify specific chromosome regions containing genes associated with leaf and stripe rust resistance. DNA markers associated with genes conferring resistance to leaf rust resistance were identified in three chromosome regions, and genes in two regions were associated with stripe rust resistance. These genes and molecular markers associated with them can be used by scientists to further enhance resistance in wheat cultivars. Another study was conducted to determine if Lr18, a gene for leaf rust resistance that has a large effect, is present in the Virginia Tech soft red winter wheat breeding material. This gene (Lr18) is known to have been introduced from an ancestral species highly related to wheat. Wheat seedlings derived from crosses between lines postulated to carry Lr18 with susceptible lines were tested for resistance to a specific strain of leaf rust lacking virulence to Lr18. Genetic analysis of the ratio of resistant versus susceptible seedlings and association between DNA molecular markers and resistant seedlings were conducted to validate the presence of gene Lr18. A molecular marker linked tightly to gene Lr18 was identified in the study. This gene was found to be widely distributed in soft red winter wheat breeding materials and the molecular marker associated with gene Lr18 will be useful for scientists to further improve resistance in wheat cultivars.

Leaf rust and stripe rust of wheat (Triticum aestivum L.) are caused by the fungal pathogens Puccinia triticina, and Puccinia striiformis f.sp. tritici, respectively. In North America, the incorporation of adult-plant resistance (APR) genes into breeding lines has been an important strategy to achieve durable resistance to both diseases. Previously, the spring wheat cultivar AC Domain was reported to express an effective level of adult-plant resistance (APR) to leaf rust under field conditions. Early gene postulation work had suggested AC Domain might carry the APR gene Lr34 due to its phenotypic similarity to other Lr34 carrying lines. However, new gene specific markers have shown that AC Domain is not a carrier of Lr34. The objective of this research was to genetically localize the resistance in AC Domain, which is important because the cultivar has frequently been used as a parent in Canadian breeding programs, primarily for its value as a source of pre-harvest sprouting resistance. A mapping population of 185 doubled haploid (DH) lines derived from the cross ‘RL4452’ by ‘AC Domain’ was used for this study. RL4452 is a known carrier of Lr34. During 2011-2012, the DH population was evaluated in field leaf rust nurseries at Saskatoon, SK and Portage, MB and at a stripe rust nursery at Lethbridge, AB. Field results indicated that rust resistance in the mapping population was variable, with lines ranging from highly resistant, to highly susceptible. DH lines carrying Lr34 showed a high level of resistance to both diseases. Thus, the non-Lr34 carriers were genotyped using select SSR markers, and by an Illumina 9k Infinium iSelect SNP assay for subsequent quantitative trait loci (QTL) analysis. QTL analysis revealed that AC Domain donated a major resistance QTL located on chromosome 2BS, that mapped 46 cM proximal to markers linked to Lr16, and explained a significant portion of the leaf and stripe rust phenotypic variance in all test environments. In addition, this QTL was significantly associated with the expression leaf tip necrosis (LTN), reduction in area under the disease progress curve (AUDPC), and coefficient of infection (CI). In certain environments the interaction between the 2B QTL and Lr34 was additive resulting in a superior level of rust resistance. Indoor rust testing showed AC Domain was susceptible to both diseases at the seedling stage. Taken together these results suggest that the identified resistance in AC Domain is likely due to the presence of an APR gene, on chromosome 2BS.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Microsatellites were used to differentiate Leaf (Puccinia triticina Eriks.) and Yellow rust (Puccinia striiformis Westend. f. sp. tritici Eriks.) pathotypes. There was sufficient diversity in the Leaf rust microsatellite markers to differentiate the pathotypes and create a phylogenetic tree of Leaf rust. Three of the microsatellite markers were sufficient to differentiate all the Leaf rust pathotypes. Sufficient diversity in the Yellow rust microsatellite markers was also observed which made it possible to differentiate the pathotypes. Only three pathotypes were used so no phylogenetic inference was made. Two microsatellite markers were sufficient to differentiate all the yellow rust pathotypes. Microsatellite and Amplified Fragment Length Polymorphisms (AFLP) markers were used to differentiate Stem rust (Puccinia graminis f. sp. tritici Eriks. and Henn.) pathotypes, and the data was combined for phylogenetic analysis. AFLP bands unique to each Stem rust pathotype were converted to Sequence Characterised Amplified Region (SCAR) markers. A single specific SCAR marker was created for UVPgt52. A second SCAR marker amplified four of the eight pathotypes. None of the other SCAR markers were specific. A 270 basepair fragment of the ITS1 region of the rDNA gene of all the Puccinia spp. was also sequenced in order to develop pathotype specific primers that could be used in a Real Time-PCR to determine relative levels of pathogen inoculum in a sample. Unfortunately insufficient diversity in the sequences of the ITS1 region of the rDNA gene did not allow unique primers to be designed for each pathotype making it impossible to proceed with the relative quantification using Real Time-PCR. Following marker development ninety one field isolates were collected from eleven sites in the Overberg and Swartland regions during 2008 and 2009. In the field isolates, four different Leaf rust pathotypes were identifiable. UVPgt13 and UVPgt10 were most prevalent. The most prevalent Stem rust pathotypes were UVPgt50, UVPgt52, UVPgt54 and UVPgt57. Only 6E16A- was identifiable in the Yellow rust isolates. There were no apparent patterns in the distribution of Leaf, Stem or Yellow rust. Leaf and Stem rust were widely distributed, while Yellow rust was confined to three sites in the central South Cape, the only sites where climatic conditions were favourable for its development during the sampling period. The low levels of diversity found in the rust population when compared to international populations are probably due to the relatively small population size, the lack of a host for sexual reproduction, the small sample size, the effective monoculture and the strong selective pressure created by artificial control methods. / AFRIKAANSE OPSOMMING: Mikrosatellietmerkers is gebruik om Blaar- (Puccinia triticina Eriks.) en Geelroes-( Puccinia striiformis Westend. f. sp. tritici Eriks.) patotipes te onderskei. Daar was genoeg diversiteit in die Blaarroesmerkers om verskillende patotipes te kon onderskei en om „n filogenetiese-boom te kon saamstel. Met drie van die mikrosatellietmerkers was dit moontlik om al die Blaarroespatotipes te kon onderskei. Daar was genoeg diversiteit in die Geelroesmerkers om al die patotipes te kon skei en met twee van die mikrosatellietmerkers kon al drie Geelroespatotipes van mekaar onderskei word. Mikrosatelliet- en ge-Amplifiseerde-Fragment-Lengte-Polimorfismes (AFLP) is gebruik om die Stamroes- (Puccinia graminis f. sp. tritici Eriks. and Henn.) patotipes te skei. AFLP-fragmente uniek aan „n spesifieke patotipe is omgeskakel na Volgorde-Spesifieke-ge-Amplifiseerde-Streek (SCAR) merkers. „n Spesifieke SCAR-merker is gemaak vir UVPgt52. „n Tweede SCAR-merker het vier van die patotipes geidentifiseer. Nie een van die ander SCAR-merkers was spesifiek t.o.v. „n spesifieke patotipe nie. Die volgorde van „n 270 basispaar fragment van die ITS1-streek van die rDNS-geen van al die Puccinia spp. is bepaal om patotipe spesifieke inleiers te kon ontwerp. Hierdie inleiers kan gebruik word om „n Intydse-Polimerase-Ketting-Reaksie (RT-PCR) te ontwerp om sodoende die relatiewe vlakke van die patogeen besmetting in „n monster te bepaal. Daar was nie genoeg diversiteit in die bepaalde volgordes om die spes1fieke inleiers te kon identifiseer nie en dus is RT-PCR laat vaar. Na die ontwikkeling van die merkers was een-en-negentig veldmonsters ingesamel afkomstig van elf lokaliteite in die Overberg en Swartland gedurende 2008 en 2009. Vier Blaarroespatotipes was uitkenbaar. Blaarroespatotipes UVPrt10 en UVPrt13 was die mees algemeenste. UVPgt50, UVPgt52, UVPgt54 en UVPgt57 was die mees algemene Stamroespatotipes. Net 6E16A- is geidentifiseer by die Geelroes-isolate. Daar was geen patroon in die verspreiding van Blaar-, Stam- of Geelroes patotipes. Blaar- en Stamroes was die wydste versprei, maar Geelroes het net by drie lokale in die sentrale Suid-Kaap voorgekom. Die lokaliteite is die enigste waar die weersomstandighede gunstig was vir Geelroes ontwikkeling gedurende die periode van monsterneming. Die lae vlakke van diversiteit wat in die roespopulasie gevind was is in teenstelling met internasionale populasies. Dit mag moontlik wees as gevolg van die relatief beperkte populasie grootte, die afwesigheid van „n gasheer vir seksuele voortplanting, die beperkte hoeveelheid monsters wat ingesamel is en die sterk selektiewe druk weens kunsmatige beheer.