Breeding for Fusarium head blight (FHB) resistance in wheat requires accurate and precise phenotyping. Additionally, breeding for FHB resistance requires an understanding of the pathogen population to which the host will be subjected. It is important to examine aspects of the competition between F. graminearum isolates that produce 3-acetyldeoxynivalenol (3-ADON) or 15- acetyldeoxynivalenol (15-ADON), including how infection by various isolates will affect the expression of resistance in wheat. The first objective of this study was to compare ranking of FHB reactions of ten wheat genotypes using three different macroconidial spray inoculation protocols and to determine if the protocols produce substantially equivalent results in terms of symptom development. The second objective was to examine the outcome of competition between 3-ADON and 15-ADON producing isolates of F. graminearum and to examine the effect inoculation with different isolate mixtures on FHB resistance in wheat genotypes 93FHB37 (resistant) and CDC Teal (susceptible).

Breeding for Fusarium head blight (FHB) resistance in wheat requires accurate and precise phenotyping. Additionally, breeding for FHB resistance requires an understanding of the pathogen population to which the host will be subjected. It is important to examine aspects of the competition between F. graminearum isolates that produce 3-acetyldeoxynivalenol (3-ADON) or 15- acetyldeoxynivalenol (15-ADON), including how infection by various isolates will affect the expression of resistance in wheat. The first objective of this study was to compare ranking of FHB reactions of ten wheat genotypes using three different macroconidial spray inoculation protocols and to determine if the protocols produce substantially equivalent results in terms of symptom development. The second objective was to examine the outcome of competition between 3-ADON and 15-ADON producing isolates of F. graminearum and to examine the effect inoculation with different isolate mixtures on FHB resistance in wheat genotypes 93FHB37 (resistant) and CDC Teal (susceptible).

Fusarium head blight (FHB) caused by Fusarium graminearum is a fungal disease of wheat that can result in severe yield losses and contaminate grain with deoxynivalenol (DON). Wheat cultivars with different levels of FHB resistance were combined with fungicides application to control FHB. Results showed that foliar fungicide Prosaro™ combined with moderately resistant cultivars greatly reduced the risk of FHB. Integrating fungicide application with moderately resistant cultivars can be an effective strategy in controlling FHB. Quantitative trait loci (QTL) for resistance to FHB related traits were analyzed using a double haploid population. Four QTL associated with FHB resistance was detected on chromosomes 2B, 2D, 4D and 7A. The QTL on chromosome 2B and 4D were found to reduce multiple FHB-related traits and were more frequently detected than QTL on chromosome 2D and 7A. QTL on chromosome 2B and 4D could be valuable for improving FHB resistance in wheat. / October 2015

Triticum aestivum (bread wheat) is an important cereal crop not only in Canada but also worldwide. The pathogen Fusarium graminearum is responsible for causing the disease fusarium head blight and generates yield loses and mycotoxin contaminated grains, including in wheat. A strategy used to mitigate this problem is through the production of FHB resistant wheat varieties by crossing strongly resistant germplasms from closely related wheat species. Thinopyrum elongatum is a wild wheat grass that carries genetic resistance to FHB on the long arm of its chromosome 7E (7EL). Previous work has developed genetic material by crossing Chinese Spring (CS) ph1b line with a CS-7E(7D) substitution line to facilitate introgression of 7E fragments from Thinopyrum into the 7D chromosome of wheat. In the first part of this project a genetic order for previously designed 7EL- and 7D- specific markers was proposed using IWGSC RefSeq v1.0 and was used to characterize the introgressed material from the above cross. Progeny from BC1F7 and BC1F5 families of different lineages were genotyped and phenotyped to characterize regions of introgression which were estimated to be at least 42 and 22 Mbp respectively. Gene expression analysis was also performed for selected 7EL genes. Results showed that the expression of selected 7EL genes present within the introgressed fragments were highly variable between the three families characterized as well as within families. It was also observed that the 7EL introgressed progeny had variable expression when compared to the addition line CS-7EL. Additionally gene expression analyses were also performed using 7D genes. These results showed that there was variation in 7D gene expression between the 7EL introgressed progeny and the controls CS-Fg and addition line CS-7EL-Fg. Possible explanations regarding the variation in gene expression includes differential methylation patterns, silencing of genes in the progeny, alteration of repetitive sequences or activation of transposable elements. Further research will be needed to test these hypotheses.

A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

No description available.

Master of Science / Department of Agronomy / Allan Fritz / Fusarium head blight (FHB) is one of the most devastative diseases in wheat. Growing resistant cultivars is one of the most effective strategies to minimize the disease damage. Huangcandou (HCD) is a Chinese wheat landrace showing a high level of resistance to FHB spread within a spike (type II). To identify quantitative traits loci (QTL) for resistance in HCD, a population of 190 recombinant inbred lines (RILs) were developed from a cross between HCD and Jagger, a susceptible hard winter wheat (HWW) released in Kansas. The population was evaluated for type II resistance at the greenhouses of Kansas State University. After initial marker screening, 261 polymorphic simple-sequence repeats (SSR) between parents were used for analysis of the RIL population. Among three QTL identified, two from HCD were mapped on the short arms of chromosomes 3B (3BS) and 3A (3AS). The QTL on the distal end of 3BS showed a major effect on type II resistance in all three experiments. This QTL coincides with a previously reported Fhb1, and explained 28.3% of phenotypic variation. The QTL on 3AS explained 9.7% of phenotypic variation for mean PSS over three experiments. The third QTL from chromosome 2D of Jagger explained 6.5% of phenotypic variation. Allelic substitution using the closest marker to each QTL revealed that substitution of Jagger alleles of two QTL on 3AS and 3BS with those from HCD significantly reduced the PSS. HCD containing both QTL on 3AS and 3BS with a large effect on type II resistance can be an alternative source of FHB resistance for improving FHB type II resistance in wheat. Besides, meta-analyses were used to estimate 95% confidence intervals (CIs) of 24 mapped QTL in five previously mapped populations derived from Chinese landraces: Wangshuibai (WSB), Haiyanzhong (HYZ), Huangfangzhu (HFZ), Baishanyuehuang (BSYH) and Huangcandou (HCD). Nineteen QTL for FHB type II resistance were projected to 10 QTL clusters. Five QTL on chromosomes 1A, 5A, 7A, and 3BS (2) were identified as confirmed QTL that have stable and consistent effects on FHB resistance and markers in these meta-QTL regions should be useful for marker-assisted breeding.

Les maladies du blé causées par Fusarium, comme la brulure de l’épi (FHB) et la pourriture de la tige (FCR), entrainent une réduction de production, de la qualité du blé et des problèmes de sécurité alimentaire liés à la présence de mycotoxines affectant la santé de l’Homme et des animaux: la plus représentée étant le déoxynivalénol (DON). Le DON est un inhibiteur de la synthèse protéique qui agit durant l’infection comme un facteur de virulence. La glycosylation du DON en D3G (DON-3-O-glicoside) catalysée par des UDP-glycosyltransférases (UGTs) est le principal mécanisme de protection des plantes contre sa toxicité. Dans ce travail, nous avons démontré que la détoxification du DON par l’UGT confère une résistance à large spectre contre les champignons produisant DON F.graminearum et F.culmorum. Nous avons produit des plants de blé dur exprimant de manière constitutive le gène HvUGT13248 (Ubi-UGT) et des plants de blé panifiables exprimant ce gène au niveau du tissu floral (Lem-UGT). Les plants Ubi-UGT ont montré une réduction significative des symptômes de FHB durant les stades précoces et médians de l’infection, et de FCR à tous les stades de l’infection. De plus, les plants Lem-UGT ont montré une corrélation entre les niveaux d’expression de l’UGT et de protection observée. Finalement, nous avons démontré que la pyramidation des gènes associés à des mécanismes de résistance différents peut renforcer la résistance de l’hôte à l’infection. Des plants de blé ont été générés exprimant à la fois l’enzyme HvUGT13248, et des inhibiteurs de glycosidases: AcPMEI ou PvPGIP2, impliqués dans la dégradation de la paroi cellulosique, et qui ont montré une résistance accrue à la FHB. / Fusarium diseases, including Fusarium head blight (FHB) and Fusarium crown rot (FCR) represent major agricultural problems worldwide, causing reduction of grain yield and quality and food safety. In particular, grain contamination by Fusarium mycotoxins, mainly deoxynivalenol (DON), is responsible for health problems in humans and animals. DON is a protein synthesis inhibitor, acting as a virulence factor during pathogenesis. The principal mechanism involved in enhancing plant tolerance to DON is glycosylation, forming DON-3-β-D-glucoside (D3G), performed by specific UDP-glucosyltransferases (UGTs). In this work, we demonstrated that DON-detoxification by UGT confers a broad-spectrum resistance against the DON-producing fungi F. graminearum and F. culmorum, characterized by different time of infection and target organs. We produced transgenic durum wheat plants (Ubi-UGT) constitutively expressing the barley HvUGT13248 and bread wheat plants (Lem-UGT) expressing HvUGT13248 in flower tissues. Ubi-UGT plants revealed significant reduction of FHB symptom, during early-mid stages of infection, and of FCR symptom, throughout the infection timing. The floral-specific expression highlighted a dose-dependent efficacy of the UGT detoxification mechanism. In addition, we demonstrated that pyramiding of genes controlling different resistance mechanisms can further reinforce the host response by stacking transgenes controlling the DON-to-D3G conversion and the inhibition of cell wall degrading enzymes by glycosidase inhibitors in the same wheat genotype. We obtained plants expressing HvUGT13248 and AcPMEI or HvUGT13248 and PvPGIP2, which exhibited increased FHB resistance.

Doctor of Philosophy / Department of Agronomy / Guihua Bai / Growing resistant cultivars is an economically effective method to control wheat disease Fusarium head blight (FHB) caused by Fusarium graminearum. Ninety-five wheat lines mainly from China and Japan were evaluated for resistance to initial infection (type I), spread of symptoms within a spike (type II), and deoxynivalenol (DON) accumulation in infected grains (type III). Most of lines were resistant or moderately resistant, 15 lines had DON content lower than 2 ppm and six lines showed a high level of resistance for all the three types. Deoxynivalenol content was significantly correlated with type II, but not type I resistance. Fifty-nine of the ninety-five lines were evaluated for genetic diversity on the basis of amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSRs). Genetic relationships among these lines were consistent with pedigrees and their geographic distribution. Chinese lines had broader genetic diversity than Japanese lines. Sumai 3 is a widely used Chinese variety for FHB-resistant breeding in the US and elsewhere. Haplotype patterns of the SSR markers linked to FHB resistance quantitative trait loci (QTL) on chromosomes 3BS, 5AS and 6BS of Sumai 3 indicated that only a few Sumai 3 derivatives carry all of these Sumai 3 QTL. SSR data also suggested that these QTL in Sumai 3 were derived from Chinese landrace Taiwan Xiaomai. Some highly resistant lines may carry novel QTL for FHB-resistance QTL, and need further investigation. A mapping population of 139 recombinant inbred lines derived from the cross of Wangshuibai (resistant Chinese landrace)/Wheaton (susceptible cultivar) was genotyped with more than 1300 SSR and AFLP markers. Five QTL for type I resistance were detected on chromosome arms 3BS, 4BS, 5DL, 3AS, and 5AS; seven QTL for type II resistance on 3BS, 1AL, 5AS, 5DL, 7AL, and 3DL; and seven QTL for type III resistance on 3BS, 5AS, 1AS, 5DL, 1BL, and 7AL. These QTL together explained 31.7%, 64%, and 52.8% of the phenotypic variation for FHB type I, II, and III resistance, respectively. QTL on 5AS, the distal end of 3BS, and 5DL contributed to all three types of resistance. FHB resistance QTL identified in Wangshuibai can be used in developing wheat cultivars with enhanced FHB resistance by pyramiding FHB resistance QTL from other sources.