Tesis sobre el tema "Bunt (Disease of wheat)"

2 pp.<br>The weather near heading is the overriding factor in disease development. Cultural practices may be partially effective in controlling Karnal bunt, but cannot eliminate the disease completely.

Thesis (M.S. in crop science)--Washington State University, December 2009.<br>Title from PDF title page (viewed on Jan. 12, 2010). "Department of Crop and Soil Sciences." Includes bibliographical references.

Originally Published: 2002; Revised<br>3 pp.<br>Environmental conditions between awn emergence and the end of flowering is the overriding factor in disease development. 2 The University of Arizona Cooperative Extension Cultural practices may be partially effective in controlling Karnal bunt but cannot eliminate the disease completely. Karnal bunt is most likely to be found in areas where lodging or water ponding have occurred.

Disease tolerance is defined as the ability to maintain grain yield in the presence of disease and could be a potential defence mechanism to be incorporated into breeding programmes. It is an attractive goal, as disease tolerance has the potential to be a broad-spectrum, durable defence mechanism while exerting little selection pressure on pathogen populations. Relatively little is known about how disease tolerance is conferred, but most of the hypotheses suggest resource capture and resource-use traits such as large green canopy area, increased light extinction coefficient and a high source to sink balance. Disease tolerance in current wheat genotypes is generally associated with low yield potential, and for disease tolerance to be incorporated into commercial breeding it is important to determine whether this link can be disassociated. In this study, an attempt was made to identify physiological traits conferring disease tolerance to Septoria tritici blotch (STB) in winter wheat. Wheat genotypes contrasting in disease tolerance were selected for in-depth phenotyping of selected physiological traits to determine their association with disease tolerance. A number of publications have attempted to link disease tolerance to physiological traits in wheat, based on their yield loss to disease symptom relationship. However, in this study it was proposed that variation in non-symptomatic disease could influence the appearance of disease tolerance which has not previously been investigated. The ratio of in-leaf pathogen biomass to visual disease symptoms was studied in both controlled-environment experiments and in field experiments to determine whether a high in-leaf pathogen biomass was associated with disease tolerance. Two field experiments were conducted during the field seasons 2011/12 and 2013/14 at Teagasc Oak Park, Carlow, Ireland and ADAS Rosemaund, Herefordshire, UK, respectively. A field experiment was also conducted in 2012/13 at Teagasc Oak Park, but due to dry conditions and little disease presence this field experiment was excluded from nearly all experimental analyses. In each experiment, there were two fungicide treatments, non-target disease control and full disease control. In order to increase genetic variability, 38 selected lines from a L14 x Rialto doubled-haploid (DH) mapping population developed by the International Maize and Wheat Improvement Centre (CIMMYT) were screened alongside 10 UK-adapted reference genotypes for contrasting disease tolerance in 2012. Tolerance was quantified as yield loss per unit of green lamina area index (GLAI) loss to disease. L14 is a CIMMYT spring wheat large-ear phenotype advanced line and Rialto is a UK winter wheat which has high radiation-use efficiency and stem soluble carbohydrate. The DH lines displayed an increased range of disease tolerance compared to the UK-adapted reference genotypes. Selected genotypes were subjected to in-depth phenotyping for an extended range of physiological traits in 2014 to identify traits associated with increased disease tolerance. The traits measured included pre- and post- anthesis radiation interception, light extinction coefficient at anthesis, pre- and post anthesis radiation-use efficiency and stem water soluble carbohydrate accumulation at ear emergence + 7 days. In general, there was a wide range of physiological traits displaying weak associations with disease tolerance. The main traits associated with disease tolerance were related to large and/or maintained source capacity in the presence of disease, such as increased GLAI at anthesis and increased post-anthesis light interception. There was also a general association with low grain yield in the absence of disease and decreased harvest index. Increased disease tolerance was associated with high source capacity and low sink capacity, and there was an association between a high source to sink balance, measured as increased Healthy Area Duration (HAD) per grain, and disease tolerance. The impact of genotype variation on the amount of non-symptomatic disease to visual disease expression was investigated in controlled-environment (CE) experiments. In-leaf Zymoseptoria tritici fungal biomass (pathogen load) was quantified by a Real Time qPCR assay targeting the β-tubulin gene (Accession no. AY547264) and compared to visual disease expression. In the first CE experiment, two wheat genotypes were exposed to increasing concentrations of Z. tritici inoculum. There were differences in rates of pathogen development and pathogen presence between inoculum concentrations in both visual disease symptoms and pathogen loads. In the following CE experiment, a wider range of genotypes exposed to a high inoculum level were shown to differ significantly in the relationship between visual disease symptoms and pathogen loads. In order to determine the impact of genotype variation on the visual disease symptoms to pathogen load ratio, flag leaves of genotypes screened for in-field disease tolerance in 2012 and 2014 were analysed. Large variations in the disease symptoms to pathogen load ratio were identified, which has not previously been shown in wheat experiments. An attempt was made to relate the visual symptoms – pathogen load ratio to non-lesion green area loss as a measure of a potential metabolic cost of increased pathogen pressure, but no such relationship was found. An increased pathogen load per unit visual symptoms did not account for larger yield losses than predicted for a given disease level and there was no direct relationship between symptom expression - pathogen load ratios and disease tolerance. The consistency of high/low displays of disease tolerance calculated by different disease measures was investigated using three different ways of measuring disease; HAD, area under disease progress curve (AUDPC) and pathogen DNA quantified by qPCR. In general, the two measures of pathogen presence (AUDPC and pathogen load) tended to quantify disease tolerance similarly, while the HAD-based tolerance contrasted. There were also differences in which traits were associated with disease tolerance for the different methods of calculating tolerance; the calculations based on AUDPC and pathogen DNA tended to associate a decreased source capacity to disease tolerance while the HAD-based tolerance indicated an association with increased source capacity. All methods, however, indicated that a low yield potential was associated with disease tolerance. In conclusion, there was a large range of disease tolerance found in the field experiments compared to previous investigations. The HAD-based disease tolerance seems to be mainly related to a large source capacity and a low sink capacity. However, the genotype ratings of high/low disease tolerance and associated physiological traits seem to vary according to the method of calculating tolerance. There were large differences in the ratio of visual symptoms-pathogen load between genotypes; even though this did not have a direct impact on disease tolerance or yield loss it could potentially be associated with increased metabolic costs.

Chapter 1: Introduction Wheat is the second most widely grown and consumed food crop of the world after rice, and is the staple food of around 35% of the world’s population. The present wheat production is about 749 million tons (FAO, 2016; http://www.fao.org/faostat) and to feed the world’s ever-growing population with annual growth rate of 2.6%, there will be a requirement to produce about 1040 million tons of wheat in 2020. To reach this target, it is crucial to keep the crop free from various biotic as well as abiotic stresses. In recent years, spot blotch caused by Bipolaris sorokiniana has emerged as a serious threat for wheat cultivation in warmer and humid regions of the world. It causes foliar spot blotch, root rot, black point on grains, head blight and seedling blight of wheat and barley. Estimates of yield losses due to spot blotch are reported to vary from 30-80% and can reach up to 100% under severe infection conditions. In spite of several efforts world over, no wheat variety highly resistant to spot blotch has been released for field cultivation. One of the main reasons for this is that the molecular mechanism behind resistance to spot blotch has not yet been fully understood. In order to develop measures to control plant diseases, it is very important to understand not only the characteristic features of the pathogen, but also the molecular mechanism behind the disease progression. With this purpose, the thesis encompasses the following objectives: Objectives of the study 1. To explore the mechanism of plant-pathogen interaction during spot blotch in susceptible and moderately resistant wheat varieties 2. To understand the mechanism of survival of Bipolaris sorokiniana on exposure to the fungicide propiconazole Chapter 2: Isolation and characterization of Bipolaris sorokiniana isolates from different geographical regions of India B. sorokiniana is a phytopathogenic fungus causing diseases in wheat, barley and other winter cereals. Previous studies involving large numbers of strains collected from around the globe suggest that B. sorokiniana exist as numerous forms of isolates varying in virulence and aggressiveness with specific and nonspecific interactions. B. sorokiniana has high morphological as well as pathological variations. We collected or isolated 12 strains of B. sorokiniana from three different wheat growing geographical regions of India. During microscopic examinations, some cultures were found to be polysporic and hence needed to be purified. Thus, monoconidial cultures were established for seven sporulating isolates of B. sorokiniana. These cultures were characterized at morphological level as well as by sequencing the ITS region of the isolates and confirmed to be B. sorokiniana. Like previous reports, our results also showed high morphological variability among the isolates. However, the morphological variation had no relationship with the geographical background. No correlation was observed between genetic similarity of the isolates and their geographical origin, concluding that the morphological characteristics expression is not conditioned solely by genes. Light microscopy and scanning electron microscopy of the spores showed several variations in conidial size, level of melanization and number of septa. For evaluation of disease reaction, the following reported methods of pathogen inoculation were attempted: leaf painting, sterile seed inoculation and inoculation at Zadok’s scale 12 stage. All these methods had some or other limitations for pathogenicity testing and hence another method, inoculation of germinated seeds, was developed. This method was found to be the best method for high throughput evaluation of pathogenicity as well as screening of germplasms. Our study showed that isolates from the same geographic region and morphological group could show differences in virulence levels. Chapter 3: Exploring the molecular interaction of wheat-Bipolaris sorokiniana during spot blotch disease Triticum dicoccum (emmer wheat) has superior organoleptic, therapeutic and nutritional qualities. However, dominance by high yielding hexaploid wheat varieties has restricted its cultivation to some niche areas in Europe and other regions including the peninsular India. T. dicoccum is resistant to various biotic stresses and rust diseases but highly susceptible to stripe rust and spot blotch. Spot blotch has become a major constraint in T. dicoccum cultivation in India. The hemibiotrophic disease cycle of this pathogen is observed only in the susceptible host. Interactive transcriptome sequencing is gaining importance in plant pathogen interaction studies and has enabled simultaneous analysis of expression of plant as well as pathogen genes. Similarly, next generation sequencing has enabled genome sequencing of organisms to a great extent. With the availability of the reference genome sequences from plants as well as pathogen, it has become much easier to align the reads from RNA-seq data and hence expression quantification. In order to explore the interaction, we performed global transcriptome analysis of spot blotch susceptible variety, DDK 1025 and a moderate resistant variety, Chirya 3 upon pathogen inoculation using Illumina HiSeq platform. To understand the infection process and mechanism of disease progression, we performed differential gene expression analysis of spot blotch susceptible variety, DDK 1025 upon pathogen inoculation. A time series comparative study was performed to understand the biotrophic (1 dpi, days post inoculation), early necrotrophic (4 dpi) and necrotrophic phase (6 dpi) responses. The numbers of differentially expressed genes (DEGs) from three stages were 1810, 1562 and 2908 individually. GO annotations were obtained using Blast2GO for 75.63%, 70% and 73.89% of these DEGs respectively. GO enrichment was performed using agriGo online tool (http://bioinfo.cau.edu.cn/agriGO/analysis.php) using Triticum aestivum transcript ID v2.2 as the reference. Biological processes associated with carbohydrate metabolic process, response to abiotic stress, photosynthesis, cell death, regulation of gene expression, secondary metabolic process and generation of precursor metabolites were enriched. Under molecular function category carbohydrate binding, catalytic activity, enzyme regulator activity, protein binding and hydrolase activity was enriched. Although cellular component distribution showed all cellular parts including endoplasmic reticulum, plastid etc., extracellular region was profoundly enriched. Since acceptable annotation of T. aestivum genome was not available, insights into functional annotation were achieved using blast against Oryza sativa japonica group using the STRING platform v10.5 (https://string-db.org/). Pfam enrichment was performed to gain comprehensive understanding about the gene families involved in the infection process. Pathways intricate to this interaction mechanism were explored by KEGG enrichment of DEGs using ClueGo (cytoscape plugin) using O. sativa blast hits. After several enrichments and annotations, major components involved in the interaction were recognized as glycolysis, phenylpropanoid biosynthesis, protein processing in endoplasmic reticulum, photosynthesis, glyoxylate and dicarboxylate metabolism, heat shock proteins, protein kinases and defense response genes like chitinases and hydrolases. Down-regulation of several defense responsive genes in biotrophic phase suggests the contribution of effector mediated susceptibility. Glutathione metabolism mediated regulation of glycolysis and pentose phosphate pathway was identified. Differential expressions of multiple components of ubiquitin mediated proteolysis emphasize their role in hormone signal transduction during spot blotch. Of these DEGs, 177 genes were differentially expressed across all the three time points irrespective of the phases. Co-expression analysis using k-means clustering showed six patterns. Annotations showed that these genes had activities like chitin catabolic process, defense response to fungus, Bowman-Birk proteinase inhibitor and phenylalanine ammonia-lyase. Further information about the significance of these genes during interaction with the pathogen needs to be revealed using over/under expression experiments. Likewise, to recognize the resistance phenomenon, we sequenced the transcriptome of spot blotch resistant (T. aestivum) hexaploid variety, Chirya 3 upon B. sorokiniana inoculation. Differential expression analysis was performed for three stages i.e. 1 dpi, 4 dpi and 6 dpi, which depicts the biotrophic, early necrotrophic and necrotrophic phases in the spot blotch susceptible variety. Our results showed that the number of upregulated genes was higher than downregulated genes. GO annotation was obtained for 64.38%, 66.6% and 64.25% genes from the DEGs. A higher number of genes were unannotated and were found to have significantly higher fold change expression. This suggests that these genes with unknown function could be novel defense responsive genes from wheat. Comparison of gene ontology enrichment showed that biological processes like photosynthesis and cell death were affected in susceptible variety but not in the resistant variety. Whereas, enhanced activity of extracellular proteinase inhibitors and peroxidases was observed in the resistant variety. Thus, early recognition and activation of defense pathways in resistant variety appears to hinder pathogen growth, survival and hence infection. The results from transcriptome sequencing analyses demand confirmation using other complementary techniques like the quantitative reverse transcriptase polymerase chain reaction (qRT PCR), which is an indispensable tool for gene expression analyses. The most adopted method for relative quantification of gene expression in qRT PCR is based on the DDCt method. However, accurate quantification by this method requires an appropriate internal reference gene with stable expression across all or most of the experimental tissues. Selecting an appropriate internal reference gene is very important to elucidate the target gene expression reliably. Several housekeeping genes including 18S rRNA, ACTIN, GAPDH and EF-1α have been proposed as standard reference genes for qRT PCR studies. However, in case of plant pathogen interaction analyses, selection of an appropriate reference gene is even more crucial due to the presence of RNA from both the plant as well as the pathogen in the infected tissues. As several of these genes are also present in the fungal pathogen genome, this could result in unintended cross amplification; which can cause improper quantification of the target genes. Hence, we aimed to identify a wheat gene with the most stable expression and unique primers, which would selectively amplify only the wheat gene and not the pathogen gene, providing accurate quantification of the target genes. Hence, we evaluated six previously reported genes with expression stability under different conditions using the wheat-Bipolaris sorokiniana system. We employed various statistical analysis methods, based on which, we identified two most stable genes, ubiquitin conjugation enzyme (ULE) and phytochelatin synthase (PCS) as the best reference genes for qRT-PCR based quantification in wheat pathosystem. We further confirmed the expression of several candidate defense genes in wheat using ULE as the reference gene. However, both the genes can be used either individually or together as internal reference genes. Chapter 4: Global gene expression analysis of Bipolaris sorokiniana after exposure to propiconazole Integrated disease management has been proposed to control the spot blotch disease. However, due to the unavailability of spot blotch resistant wheat varieties, the application of foliar fungicide is the most widely practiced measure. Propiconazole is a commonly used azole fungicide to manage the spot blotch disease in the field. However, due to its fungistatic mode of action, there is a possibility of emergence of fungicide resistant pathogen strains. Several mechanisms are reported for azole resistance in fungi. However, the strategies vary in different fungi. Resistance to the fungicide could be attributed to multiple molecular components in the fungus. Moreover, azoles have multiple modes of action out of which few are not explored yet. Global transcriptomics analysis of the pathogen after exposure to sub-lethal doses of the fungicide can reveal the mechanism of survival as well as the mode of action of the azoles in the fungi. Hence, a time series gene expression analysis was performed using RNA-seq. Transcriptome analysis using various tools showed overexpression of the target genes in the sterol biosynthesis pathway of the pathogen. In addition, this study also revealed altered expression of several metabolic pathways, transporters and stress regulators in the pathogen. The use of multiple analysis tools for transcriptomics analysis provided additional confidence on the observed results. The observed results were validated using qRT-PCR. We explored three strategies in B. sorokiniana against propiconazole stress: i) overexpression of target enzymes, ii) increased expression of transporter genes, and iii) expression modulation of stress responsive factors. This study revealed several novel putative targets such as ent-kaurene oxidase, ligninase lg6 precursor and spore germination protein. These genes help the fungi to overcome stresses and survive. Hence, the drugs targeting these genes can be developed, which are expected to impair the stress tolerance and hence survival of the pathogen. However, resistance is a polygenic phenomenon and to understand the functional contribution of each gene, knockout/knockdown studies are suggested. Chapter 5: Conclusions and Future Prospects Spot blotch is an emerging disease causing yield losses of economically important cereals. The worldwide distribution of the causal agent, B. sorokiniana makes it of global concern. In the present study, we aimed to explore the mechanism of interaction of the pathogen with spot blotch susceptible and resistant wheat varieties. Initially, we established monoconidial cultures of seven isolates of B. sorokiniana collected from three different wheat growing regions of India and characterized them at morphological and molecular level. Although pathogenicity cannot directly be correlated with morphology on culture media, melanization level might be considered an important aspect in determining the level of virulence. Effector mediated downregulation of innate immunity and delayed response by plants leads to successful establishment of the pathogen in susceptible variety. We found that differential expression of ubiquitin mediated proteolysis played a key role in development of the disease in susceptible variety. On the contrary, proteinase inhibitors and peroxidase secretion led to effective elimination of pathogen in the resistant variety. Genotypes with higher expression of these genes are likely to provide improved resistance against the spot blotch disease. As integrated disease management is a sustainable approach which also includes judicial use of fungicides to control the disease; we explored novel targets for developing efficient fungicides. However, essentiality of these genes for the pathogen survival needs to be confirmed through knock-out/down studies. Overall, this study helped in understanding the molecular paradigm of spot blotch disease in wheat. The outcome of this study will assist in advancement of controlling measures against spot blotch.<br>AcSIR

The research described in this týesis was focused on achieving a better understanding of the genetics of resistance of wheat to septoria tritici blotch (Mycosphaerella graminicola). Firstly, a detached leaf technique that could be a useful complement to field trials and an alternative to whole seedling assays in assessing cultivar resistance and investigating the genetics of the host-pathogen interaction was developed. Sets of inter-varietal substitution lines, developed at the John Innes Centre, involving known and possible sources of resistance to septoria tritici blotch, were tested with several single-pycnidium isolates in both the seedling and the adult plant stage. Two specific resistance genes were identified on chromosomes of `Synthetic 6x' and `Bezostaya 1'. A resistance gene, named Stb5, was identified using the M. graminicola isolate IP094269 and mapped on the short arm of chromosome 7D of `Synthetic 6x'. `Bezostaya I's specific resistance gene to IP0323 seems to be located in the same region as Stb6, and is indeed likely to be the same gene. `Bezostaya 1' and `Cappelle Desprez' also seemed to carry components for partial resistance. Triticum macha resistance to septoria tritici blotch on the other hand was both of a specific and isolatenon- specific nature. The specific components carried by T. macha seemed to be the Stb6 gene and an additional resistance gene, but it was not possible to identify their chromosomal location. To evaluate the relationship of heading date and plant height components to severity of septoria tritici blotch, an F6 single seed population of `Apollo' x `Thesee' was studied in natural conditions. Septoria tritici blotch levels were substantially lower in later-heading than in earlier-heading lines. Total plant height had comparatively little effect on disease severity, but increased distance between the two upper leaves increased disease levels. `Apollo' seems to carry partial resistance involving more than one QTL.

Thesis (MSc.) -- Stellenbosch University, 1997.<br>ENGLISH ABSTRACT: An octoploid triticale was derived from the F1 of a Russian wheat aphid resistant rye, 'Turkey 77', and 'Chinese Spring' wheat. The alloploid was crossed (a) to common wheat, and (b) to the 'Imperial' rye to 'Chinese Spring' disomic addition lines. F2 progeny from these crosses were tested for Russian wheat aphid resistance and C-banded. Resistance was found to be associated with chromosome arm 1RS of the 'Turkey 77' rye genome. This initial work was done by MARAIS (1991) who made a RWA resistant, monotelosomic 1RS ('Turkey 77') addition plant available for the study. The F3 progeny of this monotelosomic addition plant was used to confirm the RWA resistance on chromosome 1RS. The monotelosomic addition plant was then crossed with the wheat cultivar 'Gamtoos', which has the 1BL.1 RS 'Veery' translocation. Unlike the 1RS segment in 'Gamtoos', the 'Turkey 77'- derived 1RS telosome did not express the rust resistance genes 5r31 and Lr26 which could then be used as markers. From the F1 a monotelosomic 1RS addition plant that was also heterozygous for the 1BL.1 RS translocation, was selected and testcrossed with an aphid susceptible common wheat, 'Inia 66'. Meiotic pairing between the .rye arms resulted in the recovery of five euploid, Russian wheat aphid resistant plants out of a progeny of 99 euploids. One recombinant also retained 5r31 and Lr26 and was allowed to self pollinate. With the aid of SOS-PAGE profiles, Russian wheat aphid resistant 1BL.1 RS translocation homozygotes were identified and it was possible to confirm that the Russian wheat aphid resistance gene was in fact transferred to the 1BL.1RS ('Veery') translocation. Two attempts were made to map the Russiar, wheat aphid locus or loci. (1) Telosomic mapping was attempted. For this purpose a plant with 2n = 40 + 1BL.1 RS + 1RS was obtained, and testcrossed with a Russian wheat aphid susceptible wheat. (2) A disomic, recombined 1BL.1 RS translocation line with Russian wheat aphid resistance but lacking the Lr26 and Sr31 alleles was crossed with 'Gamtoos' and the F1 testcrossed. The testcross in both strategies were done with 'Chinese Spring'. In the first experiment the Sr31 locus was located 10.42 map units from the Lr26 locus. The rust resistance data implied that the genetic distance estimates may be unreliable and therefore the laborious Russian wheat aphid resistance tests were not done. In the second experiment a Russian wheat aphid resistance gene was located 14.5 map units from the Lr26 locus. In the latter cross nonmendel ian segregation of the Russian wheat aphid resistance evidently occurred which implied that the estimated map distance may be inaccurate. It was also not possible to determine the number of genes involved from the data.<br>Digitized at 300 dpi Colour & b/W PDF format (OCR), using ,KODAK i 1220 PLUS scanner. Digitised, Ricardo Davids on request from ILL 25 April 2013<br>AFRIKAANSE OPSOMMING: 'n Oktaplo"lede triticale is gemaak vanaf die F1 van 'n kruising tussen 'n Russiese koringluis-weerstandbiedende rog, 'Turkey 77', en die koringkultivar 'Chinese Spring'. Die alloplo"led is gekruis met gewone broodkoring en met 'Imperial' rog/'Chinese Spring' disomiese addissielyne. Die F2 nageslag vanaf hierdie kruisings is getoets vir Russiese koringluisweerstandbiedendheid en C-bande is ook gedoen. Weerstand is gevind wat geassosieer is met die 1RS chromosoomarm van 'Turkey 77'. Hierdie oorspronklike werk is deur MARAIS (1991) gedoen en uit sy materiaal is 'n monotelosomiese 1RS ('Turkey 77') addissieplant beskikbaar gestel vir die huidige studie. Die F3 nageslag van hierdie monotelosomiese addissieplant is gebruik om die weerstand teen die Russiese koringluis op chromosoom 1RS te bevestig. Die monotelosomiese addissieplant is ook gekruis met die koringkultivar 'Gamtoos' wat die 1BL.1 RS-translokasie dra. Hoewel die 1RS segment van 'Gamtoos' die roesweerstandsgene, Sr31 en Lr26 uitdruk, is dit nie die geval met die 'Turkey 77' 1RS telosoom nie. Hierdie gene kon dus as merkergene gebruik word. Vanuit die F1 is 'n monotelosomiese 1RS addissieplant geselekteer wat ook heterosigoties was vir die 1BL.1 RStranslokasie. Hierdie plant is getoetskruis met 'n luisvatbare gewone broodkoring, 'Inia 66'. Meiotiese paring tussen die rogarms het daartoe gelei dat vyf euplo"lede Russiese koringluis-weerstandbiedende nageslag uit 99 euplo"lede nageslag geselekteer kon word. Een rekombinant het ook Sr31 en Lr26 behou en is toegelaat om self te bestuif. Met behulp van SDSPAGE profiele is Russiese koringluis-weerstandbiedende 1BL.1 RStranslokasie homosigote ge"ldentifiseer en kon bevestig word dat die weerstandsgeen vir die Russiese koringluis oorgedra is na die 1BL.1 RS ('Veery') -translokasie. Twee strategies is gevolg om die Russiese koringluislokus of -loci te karteer: (1) 'n Telosomiese analise is gedoen. 'n Plant met 2n = 40 + 1BL.1 RS + 1RS is verkry en met 'n luisvatbare koring bestuif. (2) 'n Gerekombineerde, disomiese plant met Russiese koringluis-weerstandbiedendheid maar sonder die Lr26 en Sr31 allele is gekruis met 'Gamtoos' en die F1 getoetskruis. Die toetskruisouer in beide die strategiee was 'Chinese Spring'. In die eerste eksperiment is die Sr31-lokus 10.42 kaarteenhede vanaf die Lr26-lokus gelokaliseer. Die raesdata het ge"impliseer dat onbetraubare genetiese kaarteenhede geskat sou word en daarom is die omslagtige Russiese koringluis weerstandsbepalings nie gedoen nie. In die tweede eksperiment is die Russiese koringluis-weerstandsgeen op 14.5 kaarteenhede vanaf die Lr26-lokus gelokaliseer. Nie-Mendeliese segregasie van die Russiese koringluis-weerstand in hierdie karteringseksperiment het ge'impliseer dat die berekende kaartafstand onakkuraat mag wees. Dit was ook nie moontlik om op grand van die data die aantal gene betrakke af te lei nie.

This PhD bridges the gap between the heterogeneity of plant diseases in the field and the emerging field of molecular pathology. It uses a three-way system consisting of the wheat host and two fungi. A range of techniques relating to plant pathology and histology as well as advanced molecular biology and cytology were used. The objective of the thesis was to study disease progress and pathogen interactions in wheat plants co-infected by two major fungi.

Bibliography: leaves 137-163. his is a comprehensive study of induced homoeologous recombination along most of the complete genetic length of two homoeologous chromosomes in the Triticeae (7A of common wheat and 7Ai of Agropyron intermedium), using co-dominant DNA markers. Chromosome 7Ai was chosen as a model alien chromosome because is has been reported to carry agronomically important genes conferring resistance to stem rust and barley yellow dwarf virus on its short and long arms, respectively.

This investigation covers genetics of rust resistance in common wheat and durum wheat. Stripe rust resistance in AUS26615 was conferred by three QTL and these were temporarily named; QYr.sun-1B, QYr.sun-3D and QYr.sun-6B. QYr.sun-1B represented the previously named APR gene Yr29. The other two QTL appear to be new. The detection of QYr.sun-3D in late sown experiment points to its better expression at relatively higher temperature regimes. The all stage resistance gene, YrAW12, carried by AUS26674, was shown to be Yr34 based on its co-segregation with Yr34-linked marker sunKASP_112 and similar seedling expression. AUS26674/Avocet S RIL population was genotyped with Yr18 and Yr29 linked markers, and responses of RILs carrying different combination of these loci were compared. The RILs carrying combination of YrAW12, Yr18 and Yr29 produced adult plant responses equal to the parent AUS266674. Among the two gene combinations, YrAW12 and Yr18 combination, produced adult plant stripe rust score 3, lower than the score 4 exhibited by other two dual combinations). Four RILs that lacked YrAW12, Yr18 and Yr29 displayed stripe rust response scores 5 to 7 indicating the presence of an additional APR locus in AUS266674. Eight leaf rust genes (Lr1, Lr13, Lr16, Lr24, Lr26, Lr27+Lr31, Lr37 and Lr73) and 11 stem rust resistance genes (Sr2, Sr8a, Sr8b, Sr9b, Sr9g, Sr17, Sr23, Sr24, Sr30, Sr31 and Sr38) were postulated singly or in different combinations among a set 85 genotypes. Nine and five lines, respectively, appear to carry uncharacterised leaf rust and stem rust resistance. Adult plant leaf rust responses ranged from 2 to 6, while stem rust scores varied from 2 to 8. Genetic analysis of stem rust resistance in a durum landrace AUS26677 indicated involvement of a single resistance gene, temporarily named SrAW4. SrAW4 was located on chromosome 4B.

T. aestivum ssp. spelta Iranian type has long been thought to potentially be the direct non-free threshing hexaploid progenitor. I evaluated a RIL population derived from a cross between CS and Iranian spelta accession P503 to identify loci suppressing free-threshabilty in P503. Identification of QTL associated with threshability in region known to harbor the Tg2A gene, and an inactive tg2D allele supported the hypothesis of Iranian spelta being derived from a more recent hybridization between free-threshing hexaploid and emmer wheat. Parastagonospora nodorum is an important fungal pathogen and secretes necrotrophic effectors that evoke cell death. In this research, a DH population segregating for Snn5 was used to saturate Snn5 region of chromosome 4B with molecular markers. The physical distance between Snn5 flanking markers was narrowed to 1.38 Mb with genetic distance of 2.8 cM. The markers developed in this study will provide a strong foundation for map-based cloning of Snn5.

Thesis ( MScAgric) -- Stellenbosch University, 1997.<br>ENGLISH ABSTRACT: Stem rust is considered the most damaging of the wheat rusts causing yield losses of more than 50% in epidemic years. Similarly, Russian wheat aphids (RWA) can be regarded as one ofthe most devastating insect pests of wheat. Yield losses due to R W A primarily result from a reduction in plant resources (sucking plant sap). Secondary losses are incurred by viruses transmitted during feeding. Mapping disease and insect resistance genes that are effective against prevailing pathotypes and biotypes of South Africa will optimize their utilization in breeding programmes. The wheat line, 87M66-2-l, is homozygous for a single dominant stem rust resistance gene located on chromosome lD. This stem rust resistance gene has been derived from Triticum tauschii accession RL5289 and is here referred to as Srtau. The aim of this study was to determine the chromosome arm involved. Following the chromosome arm allocation of Srtau, its possible linkage with the genes Rg2, Lr 21 , Sr X and Sr 33 was studied. A telosomic analysis has shown that Srtau is located on chromosome arm 1 DS and is linked to the centromere with a recombination frequency of 21 ± 3 .40%. Glume blotch and a heavy mildew infection of segregating families planted in the field in 1996 made the linkage study between Lr 21 (leaf rust resistance) and Rg2 (glume colour) impossible. However, estimated linkages of 9 ± 1.9 map units between Sr33 (stem rust resistance) and Srtau, ± 6 map units between Sr X (stem rust resistance) and Sr 3 3 and ± 1 0 map units between Sr X and Srtau suggested that SrX, Sr33 and Srtau are closely linked on I DS. Taking existing map data into consideration, it seems that the most likely order of the genes is: centromere - Srtau - Sr 3 3 - Sr X. A single dominant R W A resistance gene, Dn5, was identified in the T aestivum accession 'SA 463' and is located on chromosome 7D. The aim ofthis study was to determine the chromosome arm involved. The possible linkage of Dn5 with the endopeptidase locus, Ep-D1 b. and chlorina mutant gene, cn-D1, was then studied. Endopeptidase zymograms of 'SA 463' revealed two unknown polymorphisms. F 2 monosomic analyses involving the chromosomes 7 A, 7B and 7D were performed in an attempt to identify the loci associated with these polymorphisms. Dn5 was mapped on chromosome arm 7DL. A recombination frequency of60 ± 4.53% between Dn5 and the centromere suggested the absence of linkage. Linkage between Ep-Dl and cn-Dl could not be calculated as a result of similar isoelectric points of the 7DL encoded endopeptidases of the parental material studied. Recombination frequencies of32 ± 4.97% between Dn5 and EpDl and 37 ± 6.30% between Dn5 and cn-Dl were, however, encountered. The two novel endopeptidase alleles encountered in 'SA 463' were designated as Ep-Dle and Ep-Ald. A RWA resistance gene was transferred from the rye accession ' Turkey 77' to wheat and in the process the RWA resistant wheat lines 91M37-7 and 91M37-51 were derived. No rye chromatin could be detected in these plants following C-banding. The aim of this study was to determine (i) on which chromosome the gene(s) is located, and (ii) whether the resistance can be the result of a small intercalary translocation of rye chromatin. A monosomic analysis of the RWA resistance gene in 91M37-51 has shown that a single dominant resistance gene occurs on chromosome 7D. The use of rye-specific dispersed probes did not reveal any polymorphisms between the negative controls and RW A resistant lines 91M3 7- 7 and 91M37-51 which would suggest that it is unlikely that the resistance was derived from rye.<br>AFRIKAANSE OPSOMMING: Stamroes word as die mees vemietigende graanroessiekte beskou en het in epidemiese jare oesverliese van meer as 50% tot gevolg. Russiese koringluise is eweneens een van die emstigste insekplae van koring. Russiese koringluise veroorsaak oesverliese deurdat dit plantsap uitsuig en die plant van voedingstowwe beroof. Dit tree egter ook as 'n virusvektor op en kan so indirekte oesverliese veroorsaak. Kartering van siekte- en insekweerstandsgene wat effektief is teen die Suid-Afrikaanse patotipes en biotipes, sal hulle gebruik in teelprogramme optimiseer. Die koringlyn, 87M66-2-l , is homosigoties vir 'n dominante stamroes-weerstandsgeen wat op chromosoom ID voorkom. Hierdie weerstandsgeen is uit die Triticum tauschii aanwins, RL5289, afkomstig en word hiema verwys as Srtau. Daar is gepoog om te bepaal op watter chromosoomarm Srtau voorkom, waama sy koppeling met betrekking tot die gene Rg2, Lr21 , SrX en Sr33 bepaal is. 'n Telosoomanalise het getoon dat Srtau op chromosoom-arm 1 DS voorkom en gekoppel is aan die sentromeer met 'n rekombinasie-frekwensie van 21 ± 3.40%. Segregerende populasies wat in 1996 in die land geplant is, is hewig deur aarvlek en poeieragtige meeldou besmet en dit het die moontlike bepaling van koppeling tussen Lr21 (blaarroesweerstand) en Rg2 (aarkaffie kleur) belemmer. Koppelingsafstande van 9 ± 1. 9 kaart-eenhede tussen Sr 33 (stamroesweerstand) en Srt au, ± 6 kaart -eenhede tussen Sr X ( stamroesweerstand) en Sr 3 3 en ± 1 0 kaart -eenhede tussen SrX en Srtau is geraam en toon dat SrX, Sr33 en Srtau nou gekoppel is. Die waarskynlikste volgorde van die gene op lDS is: sentromeer- Srtau- Sr33- SrX. 'n Enkele dominante Russiese koringluis-weerstandsgeen, Dn5, is in dieT aestivum aanwins 'SA 463 ' ge"identifiseer en kom op chromosoom 7D voor. Die studie het ten doel gehad om te bepaal op watter chromosoom-arm Dn5 voorkom, asook wat die koppeling van Dn5 met die endopeptidase lokus, Ep-Dl, en die chlorina mutante geen, cn-Dl , is. Endopeptidase simograrnme van 'SA 463' het twee onbekende polimorfismes getoon. Die gene wat kodeer vir hierdie twee polimorfismes is met behulp van F2 monosoom-analises wat die chromosome 7 A, 7B en 7D betrek, gei:dentifiseer. Dn5 is op chromosoom 7DL gekarteer. 'n Rekombinasie-frekwensie van 60 ± 4.53% is gevind vir die sentromeer en Dn5 en dui op die afwesigheid van koppeling. Koppeling tussen Ep-Dl en cn-Dl kon nie bepaal word nie omdat die endopeptidase bande geproduseer deur die ouerlike materiaal wat in die studie gebruik is, nie met sekerheid in die nageslag onderskei kon word nie. Rekombinasie-frekwensies van 32 ± 4.97% tussen Dn5 en Ep-Dl en 37 ± 6.30% tussen Dn5 en cn-Dl is egter bereken. Dit word voorgestel dat daar na die twee onbekende endopeptidase-allele wat in 'SA 463 ' voorkom, verwys word as Ep-Dle en Ep-Ald. 'n Russiese koringluis-weerstandsgeen is uit die rog-aanwins, 'Turkey 77', oorgedra na koring en in die proses is die Russies koringluis weerstandbiedende lyne, 91M37-7 en 91M37-51 , geproduseer. Geen rog-chromatien kon egter met behulp van C-bande in hierdie lyne waargeneem word nie. Die doel van die studie was om te bepaal (i) op watter chromosoom die geen(e) voorkom, en (ii), of die Russiese koringluis weerstandsgeen die gevolg kan wees van 'n klein interkalere translokasie van rog- chromatien. 'n Monosoom-analise van die Russiese koringluis-weerstandsgeen in 91M37-51 het getoon dat 'n enkele dominante weerstandsgeen op chromosoom 7D voorkom. Rog-spesifieke herhalende peilers het geen polimorfismes tussen negatiewe kontroles en die Russiese koringluis weerstandbiedende lyne 91M37-7 en 91M37-51 getoon nie. Dit is dus onwaarskynlik dat die weerstand in die lyne uit rog verhaal is.

Several potential pathways exist for significant nitrogen loss from nitrogen fertilizer applied to winter wheat in Virginia. A number of materials have been previously identified which inhibit steps in one or more of these loss pathways. Ammonium polyphosphate, ammonium thiosulfate, dicyandiamide, potassium chloride, and monoammonium phosphate were chosen for use in the present study. They were mixed, singly and in combinations, with UAN solution or urea solution and spring top-dressed on soft red winter wheat. Experiments were conducted over two growing seasons at two locations each season. Nitrogen uptake was measured in mid-season and yield measured at harvest. In the second season, field measurement of ammonia volatilization was performed in the experiment utilizing urea solution. Addition of monoammonium phosphate significantly reduced volatilization from urea solution applied to winter wheat at both locations; however, weather conditions were such that total volatilization was low in this season, and the reduced losses were not reflected in increased nitrogen uptake or yield. None of the treatments significantly increased yield in any experiment, nor consistently increased mid-season nitrogen uptake.<br>Master of Science

Take - all disease ( caused by Gaeumannomyces graminis var tritici, Ggt ) can be suppressed by soil microorganisms after continuous monoculture of wheat ( take - all decline, TAD ). Fluorescent pseudomonads have been implicated in this suppression. Two strategies for controlling take - ail are the in situ development of disease suppressive soil, and / or the application of a biocontrol agent. However, TAD takes up to 10 years to develop after initially high levels of disease, and the performance of bacterial biocontrol agents has been inconsistent. It is not known what environmental factors select for disease antagonists. In this work the role of diseased root lesions in directing the evolution of a native pseudomonad community, and a model disease antagonist, Pseudomonas corrugate strain 2140 ( Pc2140 ) for increased disease suppression was investigated. This work shows that root lesions are a distinct niche, supporting increased populations of total aerobic bacteria ( TAB ), pseudomonads and Pc2140 ( compared to non - lesioned sections of diseased roots and healthy roots ). Lesions selected for fluorescent pseudomonads and pseudomonads which increase take - all severity. In. contrast, lesions selected for non - pseudomonads which decrease take - all, and healthy roots selected for non - fluorescent pseudomonads which decrease take - all. It was concluded that non - fluorescent pseudomonads and non - pseudomonads were important in reducing take - all, but not fluorescent pseudomonads. Pc2140 produced multiple variant phenotypes in vitro and on wheat roots which were altered in ( 1 ) their ability to inhibit pathogens in vitro and control take - all, and ( 2 ) GC - FAME and BIOLOG profiles to the extent that some variants were identified as different species. Different sets of phenotypes were produced in vitro and on roots. After 108 weeks culture of Pc2140 on root lesions and healthy wheat roots, variant colony types were generally slightly decreased in ability to reduce take - all, and reisolates with the wild type colony morphology were generally slightly increased in ability to reduce take - all compared to the ancestral Pc2140. This is the first report on the diversification of a pseudomonad biocontrol agent on roots, and has implications for the taxonomic identification and grouping of isolates based on phenotypic characteristics.<br>Thesis (Ph.D.)--Department of Crop Protection, 1998.

Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P.

Doctor of Philosophy<br>Genetics Interdepartmental Program<br>Allan K. Fritz<br>Wheat diseases cause significant economic losses every year. To ensure global food security, newly released cultivars must possess increased levels of broadly-effective resistance against wheat pathogens, acceptable end-use quality, and high yield potential. Genetic host resistance stands out from other management strategies as the most viable option for controlling diseases. New genotyping platforms allow whole genome marker discovery at a relatively low cost, favoring the identification of novel loci underlying traits of interest. The work presented here describes genomic approaches for mapping and predicting the resistance to Fusarium head blight (FHB) and wheat rusts. The first study used biparental mapping to identify quantitative trait loci (QTL) associated with Fusarium head blight (FHB) resistance. A doubled haploid population (DH) was originated from a cross of Everest and WB-Cedar, which are widely grown wheat cultivars in Kansas with moderately resistant and moderately susceptible reactions to FHB, respectively. We confirmed that neither of the parents carry known large-effect QTLs, suggesting that FHB resistance is native. Eight small-effect QTLs were identified as associated with multiple mechanisms of FHB resistance. All QTLs had additive effects, providing significant improvements in levels of resistance when they were found in combinations within DH lines. In the second study, a genome-wide association mapping (GWAS) and genomic selection (GS) models were applied for FHB resistance in a panel of 962 elite lines from the K-State Wheat Breeding Program. Significant single nucleotide polymorphisms (SNPs) associated with the percentage of symptomatic spikelets were identified but not reproducible across breeding panels tested in each year. Accuracy of predictions ranged from 0.25 to 0.51 depending on GS model, indicating that it can be a useful tool to increase levels of FHB resistance. GWAS and GS approaches were also applied to a historical dataset to identify loci underlying resistance to leaf and stem rust at seedling stage in a panel of elite winter wheat lines. Infection types of multiple races of wheat rusts from the last sixteen years of the Southern Regional Performance Nursery (SRPN) were used in this study. A total of 533 elite lines originating from several breeding programs were tested in the SRPN during this period of time. GWAS identified significant SNP-trait associations for wheat rusts, confirming the effectiveness of already known genes and revealing potentially novel loci associated with resistance.

This study was part of a larger pre-breeding effort to develop new parental materials carrying useful genes for disease resistance and adaptation. Firstly, marker-assisted backcrosses were employed to transfer and pyramid combinations of disease resistance genes (Fhb1, Lr34, Lr53, Sr2, Sr26, Sr39, and Sr50) and a reduced height gene (Rht-B1b) into the variety Norstar. Following the third backcross to Norstar, the various backcrossed progenies were inter-mated to derive progenies having combinations of Fhb1 and Rht-B1b plus the targeted leaf and/or stem rust resistance genes. Five NILs (each carrying Fhb1 and Rht-B1b) that differ for the leaf and stem rust resistance genes they possess were recovered. Secondly, a mapping study using the 9K Illumina Infinium iSelect wheat assay was conducted with a F2 mapping population developed by crossing Lr59-25 (0306/2*CSph1b//CSN1AT1B/3/Thatcher) and Superb. Lr59 was mapped 0.5cM distally from the co-segregating SNPs IWA1495, IWA6704, IWA2098 and IWA969 on wheat chromosome arm 6BS.<br>North Dakota Wheat Commission<br>Minnesota Wheat Research and Promotion Council<br>U.S. Wheat and Barley Scab Initiative (USWBSI)

Wheat is the most widely grown crop in the world and as such, is an essential source of energy and nutrition. The challenges that breeders presently face is to increase production to feed the rising population of the world, while also accounting for climate change, pollution, water and environmental stresses. As genetic uniformity of modern cultivars has increased vulnerability to pests and diseases, the wild relatives of wheat offer a rich source of genetic diversity and stress tolerance traits, that can be harnessed and transferred in to modern wheat. In this study, we used array-based genotyping to explore genetic diversity in 385 domesticated and non-domesticated lines of wheat and their wild relatives. Genetic characterization using the wheat 90K array, and subsequent filtering and validation mapped 9,570 single nucleotide polymorphic markers onto the wheat reference genome. Phylogenetic analyses illustrated four major clades, clearly separating the wild species from the domesticated, and the ancient Triticum turgidum species from modern T. turgidum cultivars. Using this diverse germplasm, a genome-wide association study (GWAS) was performed for leaf rust, the most widespread rust disease of wheat. Identification of novel sources of resistance is necessary to maintain disease resistance and stay ahead in the plant-pathogen evolutionary arms race. GWAS was conducted using eight statistical models for infection types against six leaf rust isolates and leaf rust severity rated in field trials for 3-4 years at 2-3 locations in Canada. Functional annotation of genes containing significant quantitative trait nucleotides (QTNs) identified 96 disease-related nucleotide associated with leaf rust resistance. A total of 21 QTNs were in haplotype blocks or within flanking markers of at least 16 known leaf rust (Lr) resistance genes. The remaining significant QTNs were considered loci that putatively harbor new Lr resistance genes. Future efforts to validate these loci will help understand their role in disease resistance and promote their utility for marker-assisted selection in pre-breeding.

The responses of 92 barley genotypes to selected P. hordei pathotypes was assessed in greenhouse tests at seedling growth stages and in the field at adult plant growth stages to determine known or unknown resistances. On the basis of multipathotype tests, 35 genotypes were postulated to carry Rph2, Rph4, Rph5, Rph12, RphCantala alone or combinations of Rph2 + Rph4 and Rph1 + Rph2, whereas 52 genotypes lacked detectable seedling resistance to P. hordei. Five genotypes carried seedling resistance that was effective to all pathotypes tested, of which four were believed to carry uncharacterised resistance based on pedigree information. Field tests at adult plant growth stages indicated that while 28 genotypes were susceptible, 57 carried uncharacterised APR to P. hordei. Pedigree analysis indicated that APR in the test genotypes could have been derived from three different sources. The resistant responses of seven cultivars at adult plant growth stages were believed to be due to the presence of seedling resistance effective against the field pathotypes. Genetic studies conducted on 10 barley genotypes suggested that ‘Vada’, ‘Nagrad’, ‘Gilbert’, ‘Ulandra (NT)’ and ‘WI3407’ each carry one gene providing adult plant resistance to P. hordei. Genotypes ‘Patty’, ‘Pompadour’ ‘Athos’, ‘Dash’ and ‘RAH1995’ showed digenic inheritance of APR at one field site and monogenic inheritance at a second. One of the genes identified in each of these cultivars provided high levels of APR and was effective at both field sites. The second APR gene was effective only at one field site, and it conferred low levels of APR. Tests of allelism between resistant genotypes confirmed a common APR gene in all genotypes with the exception of ‘WI3407’, which based on pedigree information was genetically distinct from the gene common in ‘Vada’, ‘Nagrad’, ‘Patty’, ‘RAH1995’ and ‘Pompadour’. An incompletely dominant gene, Rph14, identified previously in an accession of Hordeum vulgare confers resistance to all known pathotypes of P. hordei in Australia. The inheritance of Rph14 was confirmed using 146 and 106 F3 lines derived from the crosses ‘Baudin’/ ‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks from F3 lines using diversity array technology (DArT) markers located Rph14 to the short arm of chromosome 2H. Polymerase chain reaction (PCR) based marker analysis identified a single simple sequence repeat (SSR) marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cM in the populations ‘Baudin’/ ‘PI 584760’and ‘Ricardo’/‘PI 584760’, respectively. Seedlings of 62 Australian and two exotic barley cultivars were assessed for resistance to a variant of Puccinia striiformis, referred to as BGYR, which causes stripe rust on several wild Hordeum species and some genotypes of cultivated barley. With the exception of six Australian barley cultivars and an exotic cultivar, all displayed resistance to the pathogen. Genetic analyses of six Australian barley cultivars and the Algerian barley ‘Sahara 3771’, suggested that they carried either one or two major seedling resistance genes to the pathogen. A single recessive seedling resistance gene, Bgyr1, identified in ‘Sahara 3771’ was located on the long arm of chromosome 7H and flanked by restriction fragment length polymorphism (RFLP) markers wg420 and cdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using a doubled haploid population derived from the cross ‘Clipper’/‘Sahara 3771’ identified two major QTLs on the long arms of chromosomes 3H and 7H that explained 26 and 18% of total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to the seedling resistance gene Bgyr1. The second QTL was concluded to correspond to a single adult plant resistance gene designated Bgyr2, originating from cultivar ‘Clipper’.

Doctor of Philosophy<br>The responses of 92 barley genotypes to selected P. hordei pathotypes was assessed in greenhouse tests at seedling growth stages and in the field at adult plant growth stages to determine known or unknown resistances. On the basis of multipathotype tests, 35 genotypes were postulated to carry Rph2, Rph4, Rph5, Rph12, RphCantala alone or combinations of Rph2 + Rph4 and Rph1 + Rph2, whereas 52 genotypes lacked detectable seedling resistance to P. hordei. Five genotypes carried seedling resistance that was effective to all pathotypes tested, of which four were believed to carry uncharacterised resistance based on pedigree information. Field tests at adult plant growth stages indicated that while 28 genotypes were susceptible, 57 carried uncharacterised APR to P. hordei. Pedigree analysis indicated that APR in the test genotypes could have been derived from three different sources. The resistant responses of seven cultivars at adult plant growth stages were believed to be due to the presence of seedling resistance effective against the field pathotypes. Genetic studies conducted on 10 barley genotypes suggested that ‘Vada’, ‘Nagrad’, ‘Gilbert’, ‘Ulandra (NT)’ and ‘WI3407’ each carry one gene providing adult plant resistance to P. hordei. Genotypes ‘Patty’, ‘Pompadour’ ‘Athos’, ‘Dash’ and ‘RAH1995’ showed digenic inheritance of APR at one field site and monogenic inheritance at a second. One of the genes identified in each of these cultivars provided high levels of APR and was effective at both field sites. The second APR gene was effective only at one field site, and it conferred low levels of APR. Tests of allelism between resistant genotypes confirmed a common APR gene in all genotypes with the exception of ‘WI3407’, which based on pedigree information was genetically distinct from the gene common in ‘Vada’, ‘Nagrad’, ‘Patty’, ‘RAH1995’ and ‘Pompadour’. An incompletely dominant gene, Rph14, identified previously in an accession of Hordeum vulgare confers resistance to all known pathotypes of P. hordei in Australia. The inheritance of Rph14 was confirmed using 146 and 106 F3 lines derived from the crosses ‘Baudin’/ ‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks from F3 lines using diversity array technology (DArT) markers located Rph14 to the short arm of chromosome 2H. Polymerase chain reaction (PCR) based marker analysis identified a single simple sequence repeat (SSR) marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cM in the populations ‘Baudin’/ ‘PI 584760’and ‘Ricardo’/‘PI 584760’, respectively. Seedlings of 62 Australian and two exotic barley cultivars were assessed for resistance to a variant of Puccinia striiformis, referred to as BGYR, which causes stripe rust on several wild Hordeum species and some genotypes of cultivated barley. With the exception of six Australian barley cultivars and an exotic cultivar, all displayed resistance to the pathogen. Genetic analyses of six Australian barley cultivars and the Algerian barley ‘Sahara 3771’, suggested that they carried either one or two major seedling resistance genes to the pathogen. A single recessive seedling resistance gene, Bgyr1, identified in ‘Sahara 3771’ was located on the long arm of chromosome 7H and flanked by restriction fragment length polymorphism (RFLP) markers wg420 and cdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using a doubled haploid population derived from the cross ‘Clipper’/‘Sahara 3771’ identified two major QTLs on the long arms of chromosomes 3H and 7H that explained 26 and 18% of total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to the seedling resistance gene Bgyr1. The second QTL was concluded to correspond to a single adult plant resistance gene designated Bgyr2, originating from cultivar ‘Clipper’.

The theory and the practical application of the simulation of root infection of winter wheat by the take-all fungus, <i>Gaeumannomyces graminis</i> var. <i>tritici</i>, are critically evaluated with respect to field epidemics and to infection of seedlings within controlled environments. Several simple models for disease progress in field epidemics are evaluated with respect to field data, including a generalized logistic equation and systems of simple non-linear differential equations, with and without algebraic solutions. An investigation is made of disease heterogeneity in the field and transect data derived from sampling 11,000 plants are analysed for the presence of significant pattern. The effect of the observed spatial heterogeneity on the precision of field data is also empirically investigated. The use of a controlled-environment experiment to model the effect of volunteer infestation on inoculum survival in the field is demonstrated, and a simple model is used to quantitatively estimate the effect of volunteer infestation on inoculum multiplication. Data for a seedling disease epidemic are simulated by three mathematically and computationally diverse simulators derived from a single underlying theoretical model. The first is a complex simulator written in FORTRAN and run on a mainframe computer which resolves the infection process into a number of detailed submodels. The second simulator is written in BBCBASIC and 6502 machine code and makes use of a discrete root map to hold information on host growth and infection. In the third simulator the model is expressed as a series of rate equations and is run on a simulation package on the BBC microcomputer. The simulation techniques used are discussed and evaluated with respect to model development and the descriptive accuracy of the simulators. In conclusion a strategy is proposed for the development of a comprehensive model for field epidemics of take-all by means of controlled-environment experimentation.

Take-all, caused by the soil-borne ascomycete fungus Gaeumannomyces graminis var. tritici, (Ggt), is a root disease that devastates wheat production worldwide. Current control measures consist of partially effective chemical seed dressings and cultural methods such as crop rotation. There is currently no genetic control of the disease. The first aim of this PhD project was to characterise a range of diploid and hexaploid wheat germplasm that possess a promising level of take-all resistance under field conditions. Both above and below ground phenotyping was carried out and soil moisture probes were used to evaluate upper root function for a range of hexaploid varieties. A diploid Triticum monococcum MDR037 (S) X MDR046 (R) mapping population was screened and revealed a good spread in susceptibility to take-all across two field seasons. The population has subsequently been genotyped and genetic analyses will be carried out to explore the genetic basis of resistance. Phialophora fungal species, belonging to the genus Gaeumannomyces, colonise wheat roots but do not destroy the vascular tissue and have previously been found to suppress take-all disease. In the second approach to control Ggt, winter wheat varieties on the AHDB Recommended List (RL) were screened for their ability to build-up natural populations of Phialophora fungi in the field. Differences were revealed in their potential to build-up Phialophora spp. under a first wheat crop. A Phialophora isolate collection was gathered and draft genomes were sequenced, assembled and annotated for the three Phialophora spp. found in UK soils. Preliminary analysis suggests that considerable polymorphism may exist between homologous genes found in all three species. These findings provide a novel contribution to the potential of these two differing control mechanisms against take-all disease.

Wheat is one of the first domesticated and cultivated crops together with barley. Rust diseases are considered as one of the major threats to wheat production in almost all of the wheat growing areas worldwide and disease control relies mainly on the utilisation of genetic resistance. The general objective of this doctoral study is to reduce the risk of yield loss due to rust diseases by providing genetic knowledge on wheat-rust pathosystems at the molecular level. In this thesis, integrated efforts have been devoted to i: understand rust pathogen populations at the molecular level; ii: characterise genetic resistance in the host; iii: understand the functional mechanisms underlying the plant-microbial interaction; and iv: to inform the effective deployment of characterised genetic resistance. In Chapter 3, the relationship between virulence and molecular patterns using SSR markers on selected isolates of Puccinia graminis f. sp. tritici (Pgt) collected in Australia during a 52 year period was examined and an evolutionary pathway involving these isolates was proposed. In Chapter 4 and 5, I focused on the stem rust resistance locus Sr26, derived from Thinopyrum ponticum (tall wheat grass), as an excellent example of the successful utilisation of genetic resistance from a wild relative of a crop plant that remains one of the few durable sources of resistance currently effective against all known field isolates of Pgt. I undertook the cloning of Sr26 by MutRenSeq pipeline. A candidate gene of Sr26 was successfully identified and I demonstrated that this gene candidate encoded a CNL (CC-NB-ARC-LRR) type of R protein. In Chapter 6, candidate genes were successfully identified for the stripe rust resistance genes Yr7, Yr5, and YrSP. Chapter 7 comprised the addendum of Chapter 6, the complementary transgenic validation for Yr5 and YrSP were provided.

Doctor of Philosophy<br>Department of Plant Pathology<br>William W. Bockus<br>James P. Stack<br>Wheat blast, caused by the Triticum pathotype of Magnaporthe oryzae (MoT), is a serious disease of wheat causing yield failures and significant economic losses during epidemic years in Brazil, Paraguay, and Bolivia. Although outbreaks occur only sporadically, wheat blast is considered a major disease affecting wheat production in South America and may be a threat to the wheat crop in the United States. Wheat is a major crop in the U.S. and wheat exports from the U.S. are important to food security of several countries around the World. Thus, it is important to understand the potential for MoT entry and establishment into the U.S. and to test U.S. wheat cultivars for susceptibility to MoT. The hypotheses of this research project were a) importing wheat grain from Brazil does not pose a risk for MoT establishment in the U.S., and b) resistance to MoT head infection does not exist in U.S. hard red winter wheat elite cultivars. Quantitative pathway analysis models were used to estimate the risk of MoT entry and establishment, in the coterminous U.S. and in a more targeted area within southeast North Carolina, via the importation of wheat grain from Brazil. The pathway model predicted that significant risk for MoT entry and establishment exists in some areas of the U.S. However, in approximately 60% of the coterminous U.S. winter wheat production areas the risk of MoT establishment was estimated to be zero. With respect to winter wheat growing areas in the U.S., conditions for MoT establishment and wheat blast outbreak occur only in small, restricted geographic areas. A higher resolution pathway analysis based on a ground transportation corridor in North Carolina indicated that conditions for MoT establishment exist seven out of ten years. Among U.S. cultivars tested, a continuum in severity to head blast was observed; cultivars Everest and Karl 92 were highly susceptible with more than 90% disease severity, while cultivars PostRock, Jackpot, Overley, Jagalene, Jagger, and Santa Fe showed less than 3% infection.

Bread wheat (Triticum aestivum) is a vital crop that supplies ~20% of globally consumed calories. However, fungal pathogens can devastate production in wheat growing regions and pose a major threat to food security. Pathogens can rapidly evolve to overcome genetically resistant varieties so there is much focus on the study of disease resistance genes (R genes) to combat this threat. Cloning specific R genes has been a major effort as the immense size and complexity of the wheat genome means it can take a decade or more to isolate a single R gene using classic map-based methods. However, the advancement of next-generation DNA sequencing (NGS) has significantly reduced the time and resources required for R gene cloning, allowing more research into their biology and evolution. Most R genes encode immune receptor proteins known as nucleotide-binding leucine rich repeats (NLRs). The technique known as mutagenesis and R gene enrichment sequencing (MutRenSeq) targets NLRs and was used to clone three distinct R genes in wheat, Pm1 conferring resistance to powdery mildew (Blumeria graminis), Lr13, conferring resistance to leaf rust (Puccinia triticana), and Sr6, conferring resistance to stem rust (P. graminis). These genes reside in regions of the genome that did not permit map-based cloning methods. A bioinformatic protocol was developed to enhance the outcomes of target identification from sequencing data. Candidates were identified for each gene and genetic validation was performed to confirm their identity. Further work explored the unique aspects of these genes which are associated with hybrid necrosis (Lr13), chromosomal rearrangement (Pm1) and temperature sensitivity (Sr6). This work highlights the functional and genetic diversity of NLRs and demonstrates cloning of R genes from complex genomes is no longer a major bottleneck in pursuing novel sources of disease resistance and a deeper understanding of host-pathogen interactions.

Fusarium ear blight (FEB) is a disease of wheat and small grain cereals, caused by the fungi Fusarium culmorum and Fusarium graminearum. The disease causes premature bleaching of spikelets and shrivelling of the grain can result in a direct yield loss. Mycotoxins such as deoxynivalenol produced by the fungus can reduce grain quality. Breeding for resistant wheat cultivars is considered one of the best control options. Previously identified resistance has been reported in the Chinese genotype Sumai 3. The principal aim of this project was to identify novel sources of resistance to FEB. Twenty four wheat genotypes were evaluated for resistance to FEB in this project. Molecular markers linked to previously identified QTL from Sumai 3 conferring resistance were used to confirm their absence in the genotypes under investigation, and revealed that none of the genotypes under investigation contained all of the QTL for resistance. Field trials conducted over two years screening for resistance demonstrated that, although not statistically similar to Sumai 3, levels of disease were below 10% in some of the Chinese genotypes. Follow up experiments using reporter strains of Fusarium graminearum explored the accumulation of fungal biomass and the expression of the gene Tri5, which is essential for DON biosynthesis. Fungal biomass levels were not significantly different between genotypes; however expression of the Tri5 gene was significantly lower in the genotype Alsen. A previously developed wheat leaf seedling bioassay was also explored. Scanning electron microscopy revealed the presence of fungal hyphae in advance of the visible lesion during the infection course of F. culmorum. Inoculation with a Tri5 mutant strain of F. graminearum demonstrated that a lack of mycotoxin production altered the lesion type. This project has successfully identified potential novel resistance mechanisms and the future prospects for the control of this disease are discussed.