Abscisic acid (ABA) has been well defined as an important stress hormone in plants. The signaling pathway of ABA involves a family of pyrabactin resistant-like-1 PYR/PYL/RCAR receptors (PYL receptors) that bind ABA and form a complex with a protein phosphatase 2C (PP2C) family member resulting in downstream signaling events. The ABA receptor family has been well characterized in the model dicot Arabidopsis thaliana and more recently this characterization has branched out into cereals Oryza sativa (rice) and Hordeum vulgare (barley), as well as the monocot model plant Brachypodium distachyon and Fragaria vesca (strawberry). The analysis of these characterized ABA receptors and the use of online databases has allowed the identification of multiple putative ABA receptors in Triticum aestivum (wheat). ABA has been historically called a positive effector. Overexpression of proteins in the ABA signalling pathway or exogenous application of ABA is known to cause an increase in drought, cold, and salt tolerance. More recently ABA has been linked to increased fungal susceptibility in several plants. The role ABA plays in the biotic stress response is still largely unexplored. The focus of this project was to identify and characterize a putative wheat ABA receptor through bioinformatics and an in vitro enzyme activity assay, and use virus induced gene silencing (VIGS) to test what role this receptor plays in fugal susceptibility. A total of 13 putative ABA receptors were located, nine of which are unique between the wheat subgenomes. One receptor TaPYL5.1 was recombinantly expressed, purified, and confirmed as an ABA receptor through a phosphatase based enzyme activity assay. A receptor with high sequence identity to TaPYL5.1, TaPYL5.2A, was targeted for plant trials because the TaPYL5.1 plasmid sequence was codon optimized. A VIGS approach was used to knock down TaPYL5.2A in planta. The TaPYL5.2A knockdown plants were found to have an increased resistance to Fusarium Head Blight progression in the early stages of the disease. In conclusion, wheat ABA receptors were successfully identified and an important correlation between decreased receptor levels and increased early Fusarium Head Blight resistance was found. This correlation however was not easily reproducible due to the severity of coupling VIGS with Fusarium Head Blight, and should be followed up with additional studies looking at the broader family of wheat ABA receptors.

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

No description available.

The fungus Fusarium sacchari (Butler) Gams causes stem rot in sugarcane especially in association with the stem borer Eldana saccharina Walker. Sugarcane plants tolerant to F. sacchari PNG40 were obtained by chemical mutagenesis and in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF), followed by selection in the greenhouse. Somaclonal variants tolerant to F. sacchari PNG40 CF were established by treatment of calli with ethyl methanesulphonate (EMS) and various selection treatments. Investigations were conducted to test the effect of varying CF concentrations and the culture developmental stages (embryo maturation, embryo germination and plantlets) that were most effective in screening calli and plants. Incorporation of CF (0-100 ppm) in the media, at either embryo maturation or germination stages, resulted in significant callus necrosis, and consequent decreased plantlet yield. The highest callus necrosis of 95.55 ± 0.9 % and the lowest plant yield of 1.4 ± 0.45 plants/0.2 g were obtained after inclusion of 100 ppm CF in the germination medium compared with 61.5 ± 3.8 % and 43.8 ± 5.6 plants/0.2 g in the maturation medium, respectively. Exposure of whole plants with trimmed roots to 0-1500 ppm CF resulted in inhibition of root re-growth, with the 1500 ppm CF treatment having the greatest negative effect. Subsequent treatments involved immersing in vitro plantlets in varying concentrations of F. sacchari conidial suspensions. This resulted in 33.3 % and 100 % mortality with 103 and 105 conidia/ml treatments, respectively. Control and EMS-treated calli and potentially tolerant regenerated plants were selected using the established CF and inoculation treatments. Plants from EMS treatments displayed more varying root length. More plants with increased root growth, in the presence of CF, were produced from these treatments than from non-EMS treatments, indicating the ability of EMS to induce somaclonal variation. These putative tolerant plants were inoculated with PNG40 and those selected using CF in vitro were symptomless whilst the positive controls (plants unexposed to CF) were symptomatic. Re-isolation of Fusarium from the inoculated plants and identifying isolates as PNG40 using ISSR analysis confirmed tolerance of the asymptomatic plants and the fungus as the causal agent of the observed symptoms. This confirmed that tolerance to CF correlates to tolerance to F. sacchari PNG40. Future work includes testing stability of tolerance in the field and after sexual reproduction, and use of this protocol to produce plants that permit endophytic PNG40 colonisation towards biological control of E. saccharina. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

<i>Trichoderma harzianum </i>and <i>Aneurinobacillus migulanus </i>were tested separately and in combination as potential biological control agents against <i>F. oxysporum </i>f. sp. <i>gladioli </i>on gladiolus in soil-less culture.  The major secondary metabolites produced by <i>Trichoderma harzianum </i>against <i>F. oxysporum </i>f. sp. <i>gladioli </i>in the treated gladiolus corms as well as in the potato dextrose broth were studied.  The impact of re-application times on the effectiveness of <i>Trichoderma harzianum </i>and <i>Aneurinobacillus migulanus </i>against gladiolus corm rot and Gladiolus production in soil-less culture was examined. Results showed that <i>T. harzianum </i>proved to use different mechanisms against <i>F. oxysporum </i>f. sp. <i>gladioli </i>depending on  the inoculation method. <i>T. harzianum </i>and <i>A. migulanus </i>reduced host defence enzymes.  Also, they increased GA ₃ and IAA content in the corms.  SEM showed that the suppressive mechanisms of <i>T. harzianum </i>and <i>A. migulanus </i>differed <i>T. harzianum </i>appeared to operate through a combination of antibiosis and substrate competition, 6-n-pentyl-6H-pyran-2-one (6PP) and harzianic acid (HA) were found as the main metabolites secreted by <i>T. harzianum.  </i>A significant positive correlation was revealed between qPCR and CFU for both <i>T. harzianum </i>and <i>F. oxysporum </i>f. sp. <i>gladioli.  </i>Multiple applications of <i>T. harzianum </i>and <i>A. migulanus </i>re- application was a successful strategy for suppression of <i>F. oxysporum </i>f. sp. <i>gladioli.  </i>Briefly, <i>T. harzianum </i>was more effective than <i>A. migulanus </i>in <i>F. oxysporum </i>f. sp. <i>gladioli </i>suppression and Gladiolus growth enhancement in soil-less culture. The mixing strategy between <i>T. harzianum </i>and <i>A. migulanus </i>failed.

The activity of plant 9-lipoxygenases (LOXs) influences the outcome of Arabidopsis thaliana interaction with pathogen and insects. Evidence provided here indicates that in Arabidopsis, 9-LOXs facilitate infestation by Myzus persicae, commonly known as the green peach aphid (GPA), a sap-sucking insect, and infection by the fungal pathogen Fusarium graminearum. in comparison to the wild-type plant, lox5 mutants, which are deficient in a 9-lipoxygenase, GPA population was smaller and the insect spent less time feeding from sieve elements and xylem, thus resulting in reduced water content and fecundity of GPA. LOX5 expression is induced rapidly in roots of GPA-infested plants. This increase in LOX5 expression is paralleled by an increase in LOX5-synthesized oxylipins in the root and petiole exudates of GPA-infested plants. Micrografting experiments demonstrated that GPA population size was smaller on plants in which the roots were of the lox5 mutant genotype. Exogenous treatment of lox5 mutant roots with 9-hydroxyoctadecanoic acid restored water content and population size of GPA on lox5 mutants. Together, these results suggest that LOX5 genotype in roots is critical for facilitating insect infestation of Arabidopsis. in Arabidopsis, 9-LOX function is also required for facilitating infection by F. graminearum, which is a leading cause of Fusarium head blight (FHB) disease in wheat and other small grain crops. Loss of LOX1 and LOX5 function resulted in enhanced resistance to F. graminearum infection. Similarly in wheat, RNA interference mediated silencing of the 9-LOX homolog TaLpx1, resulted in enhanced resistance to F. graminearum. Experiments in Arabidopsis indicate that 9-LOXs promote susceptibility to this fungus by suppressing the activation of salicylic acid-mediated defense responses that are important for basal resistance to this fungus. the lox1 and lox5 mutants were also compromised for systemic acquired resistance (SAR), an inducible defense mechanism that is systemically activated throughout a plant in response to a localized infection. the lox1 and lox5 mutants exhibited reduced cell death and delayed hypersensitive response when challenged with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv tomato. LOX1 and LOX5 functions were further required for the synthesis as well as perception of a SAR-inducing activity present in petiole exudates collected from wild-type avirulent pathogen-challenged leaves. Taken together, results presented here demonstrate that 9-LOX contribute to host susceptibility as well as defense against different biotic stressors.

Fusarium head blight (FHB) continues to threaten the economic sustainability of wheat and barley production in Canada and worldwide. The overall goal of this thesis is to expand our current knowledge of the FHB pathogen, Fusarium graminearum and its trichothecene chemotype diversity. Continuous monitoring of trichothecene chemotypes may well inform on the potential risk and the type of Fusarium populations present in a given region. Fusarium populations in Winnipeg and Carman, Manitoba were examined using chemotype as a marker in the field. Rapid expansion of the 3-acetyldeoxynivalenol (3-ADON) chemotype was observed in Winnipeg and Carman. 3-ADON chemotype is consistently found at high frequencies over the previously common 15-acetyldeoxynivalenol (15-ADON) chemotype, suggesting that the shift in pathogen populations is continuing. This study provides the first evidence on the presence of nivalenol (NIV) producing F. cerealis strains in winter wheat in Manitoba, Canada. Therefore, discovery of NIV producing F. cerealis in wheat poses a serious concern for the wheat industry in Canada. Phylogenetic, chemotypic, phenotypic, and pathogenic abilities of 150 strains of F. graminearum species complex (FGSC) from eight countries were investigated. Type and amount of trichothecenes produced by a strain are key factors in determining the level of aggressiveness of that strain regardless of its species origin. The sequence variations of TRI8 gene in different species in the FGSC were examined as Fusarium species may produce different types of trichothecenes depending on differences in the core trichothecene (TRI) cluster genes. The TRI8 haplotypes did group according to chemotype rather than by species, indicating that 3-ADON, 15-ADON and NIV chemotypes have a single evolutionary origin. Comparison of TRI gene expression demonstrated that accumulation of TRI transcripts was higher in 3-ADON producing strains compared to 15-ADON and NIV strains. The presence of masked mycotoxins deoxynivalenol-3-glucoside (D3G) in food and feed is an increasing concern. Canadian spring wheat cultivars inoculated with different chemotypes produce D3G upon Fusarium infection and moderately resistant/intermediate cultivars showed higher D3G/DON ratio compared to susceptible cultivars. / October 2016

Doctor of Philosophy / Department of Plant Pathology / Christopher R. Little / Understanding the genetic structure of fungal pathogens enables the prediction of evolutionary forces that drive pathogen evolution, which assists informed decision-making regarding disease management. The genetic structure of Fusarium thapsinum and F. andiyazi, two important pathogens that cause grain mold and stalk rot of sorghum (Sorghum bicolor), are little understood. The genetic structure and pathogenicity of a F. thapsinum population from sorghum in Kansas were evaluated with amplified fragment length polymorphisms (AFLPs), vegetative compatibility groups (VCGs), sexual cross-fertility, and seedling pathogenicity. Two sympatric populations and a genetically intermediate "hybrid" group were identified in Kansas. Seedling pathogenicity of strains ranged from non-pathogenic to pathogenic, which may be partially attributable to genetic variability in the F. thapsinum populations. Genetic relatedness between populations of F. thapsinum from sorghum in Kansas, Australia, Thailand, and three African countries (Cameroon, Mali, and Uganda) were evaluated with AFLP markers and sexual crosses. Genetic diversity was high in all locations, but female fertility is very low. These results are consistent with the hypothesis that both sexual and asexual modes of reproduction are important components of the life cycle of F. thapsinum in these populations. More strains from Kansas and Africa were available for analysis than from Australia and Thailand, so the Kansas and Africa populations dominated the genetic structure observed. The two smaller populations from Australia and Thailand were more closely related to the Kansas population than they were to the African population. The three non-African populations contained information from the African population and from other, as yet unidentified, source population(s). Identifying the population(s) from which this genetic diversity originated is an important unanswered question. Stalk rot of sorghum was evaluated by inoculating stalk rot sensitive and stalk rot resistant sorghum lines with six genetically diverse F. thapsinum strains from Kansas under field and greenhouse conditions. One susceptible line (Tx7000) and two resistant lines (SC599 and BTx399) were evaluated in the field but only Tx7000 and SC599 were evaluated in the greenhouse. Disease severity was measured by major lesion length and the number of nodes crossed by the lesion. There were differences in aggressiveness amongst the F. thapsinum strains in both the greenhouse and field evaluations. This study provides the first evidence for differences in stalk rot aggressiveness amongst F. thapsinum strains and highlights the importance of challenging germplasm with well-characterized strains that represent the genetic spectrum of the entire population. The genetic diversity within F. andiyazi populations and some closely related strains was evaluated with AFLP markers. Phylogenetic and STRUCTURE analyses of the AFLP markers grouped the 81 F. andiyazi strains into three distinct clusters. The clusters were not based on the geographic origin of the strains. These results indicate the presence of at least one and possibly two undescribed sister taxa of F. andiyazi. More work is needed to further characterize these sister species of F. andiyazi and to understand their role in sorghum pathogenicity. There is genetic variation in global populations of F. thapsinum and the observed variation could be associated with variation in both seedling and adult plant pathogenicity. The study of F. andiyazi populations validated the need to properly identify and characterize Fusarium spp. associated with sorghum from different regions of the world.

Plants are inhabited by diverse species of bacteria and fungi, which affect plant health and fitness. Endophytes are bacteria or fungi that live within plant host tissues without causing symptoms of disease, and mediate important plant traits in agriculture, such as nutrient acquisition, disease resistance, and abiotic stress tolerance. However, we know little about the general ecology of endophytes, including which factors determine their compositions within plants. Seedborne transmission may represent an important source of bacterial and fungal endophytes, which can significantly impact the plant microbiome and plant traits. However, seeds are also a vehicle for transmission of plant pathogens. Seeds are commonly treated to control against seedborne pathogens, and increasingly bacteria and fungi are inoculated onto seeds to serve as biological control against pathogens. My dissertation explores the theoretical and applied ecology of seedborne endophytes of maize, including their interactions with pathogenic Fusarium fungi, and with seed treatments designed to control Fusarium. In Chapter II, I examine factors that affect the transmission of seedborne fungal endophytes and Fusarium into maize seedlings, including the influence of soil microbiota, and the impact of disinfection and biological control seed treatments. In Chapter III, I determine the long-term effects of seed disinfection and biological control inoculants on maize bacterial and fungal endophytes and Fusarium pathogenicity across three different farms. In Chapter IV, I recruit maize seed growers across the Pacific Northwest and U.S. to participate in a broadscale study of seedborne endophytes. Across submitted seed samples, I find commonly occurring seedborne endophyte taxa, and delineate how maize varieties interact with environmental factors to affect the composition and diversity of seed-associated endophytes in seeds. Throughout these chapters, I explore the potential applications of seedborne endophytes in agriculture, particularly as a source for biological control against Fusarium in maize, and speculate how seed treatments can have significant, lasting impacts on the plant microbiome.

Para prevenir que el consumidor rechace las bebidas deterioradas por mohos, es importante evaluar la probabilidad de que una determinada bebida sea deteriorada microbiológicamente antes que alcance su vida útil. Los modelos matemáticos, que son capaces de predecir el efecto de diferentes factores sobre el crecimiento fúngico, mejoran la toma de decisiones de la vida útil y optimizan las medidas de control durante la producción, siendo de gran utilidad como herramientas efectivas en el control de calidad de la industria de bebidas. Por esta razón, el objetivo principal de esta investigación fue usar el modelo logístico de la microbiología predictiva para determinar la probabilidad de crecimiento de Fusarium oxysporum en la bebida isotónica sabor mandarina, frente a dos condiciones de temperaturas de almacenamiento: 20 y 30 °C y a dos cargas de inóculo: 100 y 101 esporas/100 mL de bebida isotónica. Los resultados mostraron que el deterioro ocurrió más rápido a 30 °C con una carga de 101 esporas/100 mL de bebida isotónica (con un RMSE “Raíz del Error Cuadrático Medio” = 5.9196) que a 20 °C con 100 esporas/100 mL de bebida isotónica (RMSE = 5.4397) y el tiempo al crecimiento de Fusarium oxysporum decrece en función del incremento tanto de la temperatura de almacenamiento como del tamaño del inóculo. El menor valor de t10% (67.04 horas), que representa el tiempo que da la alerta a la industria de bebidas para recuperar los productos del mercado por un caso de contaminación, se obtuvo con un tratamiento de 101 esporas/100 mL de bebida isotónica a 30 °C. Este estudio logró demostrar que incluso a muy bajas concentraciones de inóculo, la bebida isotónica es deteriorada fácilmente por Fusarium oxysporum y el modelo logístico proporciona a la industria de bebidas un método rápido y de bajo costo para estimar el efecto de la temperatura de almacenamiento y tamaño del inóculo en la bebida isotónica. / Tesis