Stagonospora nodorum is a pathogenic fungus of wheat causing Stagonospora nodorum blotch disease, an important disease in western Canada. S. nodorum produces a multitude of host selective toxins (HSTs), which when recognized by corresponding sensitivity gene in wheat results in a compatible interaction. In this study, novel HST-host sensitivity gene interactions were investigated. Two different putative HSTs were identified. SnTox3 was likely one of the HSTs present in S. nodorum isolate Swift Current culture filtrate as the chromosomal location of the compatible sensitivity gene corresponded to that of Snn3 locus. Another putative HST interacting with Tsn1 or a tightly linked sensitivity gene was identified from S. nodorum isolate Langham. SNOG_15679, a candidate gene for production of this putative HST was heterologously expressed in Pichia pastoris which caused chlorosis on a sensitive host. Additional tests will be required to confirm the bioactivity of putative novel HST(s) produced by isolate Langham. / May 2015

Stagonospora nodorum is a necrotrophic fungal pathogen, and the causal agent of stagonospora nodorum blotch of wheat. Despite the economic importance of this disease, the molecular basis of the pathosystem is poorly understood. The aim of this study was to investigate the interaction between metabolism and infection in this pathosystem, with particular reference to the metabolism of mannitol. In common with many fungi, the main metabolite produced by S. nodorum is the acyclic hexitol mannitol. Among the previously suggested roles for this compound is a role in pathogenicity. The metabolism of mannitol has been hypothesised as occurring in a cycle involving the enzymes mannitol 2-dehydrogenase (Mdh1) and mannitol 1-phosphate 5 dehydrogenase (Mpd1). A strain was created harbouring disruption constructs for both of these genes. The double mutant was unable to synthesise or catabolise mannitol, and was unable to sporulate. Addition of exogenous mannitol completely restored in vitro sporulation, and partially restored in planta sporulation. This demonstrated an essential role for mannitol in asexual sporulation. This is the first demonstrated role for this compound. A 13C NMR study of the wild type strain, the mdh1 and mpd1 single mutants, and mpd1mdh1 double mutant was undertaken to investigate carbon utilisation and cycling. Disruption of Mpd1 significantly altered the metabolite profile with the mpd1 mutants producing trehalose and glycerol in place of mannitol. Labelling patterns in the double mutant showed that scrambling of label can be explained by the triosephosphate isomerase triangle and pentose phosphate pathway. This suggests the contribution of mannitol to label scrambling has been overstated in previous studies. The evidence did not support the metabolism of mannitol in S. nodorum as occurring in a cycle, but rather as two separate pathways. A GC-MS analysis of diseased and non-diseased tissue from infected leaves, compared to non-infected and mock-inoculated leaves, could not detect any metabolites associated with a systemic host reaction to pathogen attack.

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. Very little is currently known about the molecular mechanisms required for pathogenicity of S. nodorum, despite its major impact on Australian agriculture. S. nodorum is a polycyclic pathogen. Rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate within 2-3 weeks. Several cycles of infection are needed to build up inoculum for the damaging infection of flag leaves and heads, sporulation is therefore a critical component of the infection cycle of S. nodorum; our aim is to determine the genetic and biochemical requirements for sporulation for development of control of the pathogen. Disease progression of S. nodorum on wheat cv. Amery was monitored by light microscopy to determine the time point when pycnidia development began. Early pycnidia development was evident 12 days post-infection. This information was used to guide a genomics and a metabolomics based approach to determine the requirements for sporulation in S. nodorum. The genomics approach utilised two cDNA libraries created from sporulating and non-sporulating cultures. EST frequency was used to determine highly expressed genes under the two developmental states. Gene expression from the most highly represented genes during sporulation were confirmed using quantitative PCR. A gene encoding an arabitol 4-dehydrogenase (Abd1), was mutagenised, in its absence sporulation was reduced by approximately 20%. The metabolomics approach isolated metabolites from both in planta infection and in vitro growth. Rapid changes in the abundance of metabolites were detected during the onset of sporulation. Key fungal metabolites identified include mannitol and trehalose. The concentration of both mannitol and trehalose increased dramatically in concert with pycnidia formation. Both mannitol and trehalose have also been linked to pathogenicity in filamentous fungi. Creation of deletion mutants of the gene encoding trehalose 6-phosphate synthase showed the synthesis of trehalose is required for full sporulation of S. nodorum in planta and in vitro.

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The thesis deals with important pathogens affecting winter wheat, in particular the speckled glume and leaf blotch on wheat(Phaeosphaeria nodorum), septoria leaf blotch on wheat(Mycosphaerella graminicola)and tan spot on wheat(Pyrenophora trtici-repentis. It outlines the biology, symptoms that appear on wheat plants, and economic importance of these diseases and emphasizes the climatic conditions needed for their development and spread .The conclusion sumarizes the occurence data of the above-mentioned 3 diseases from 3 production(corn, beetroot and potato) areas and 4 districts (2 Moravian and 2 Bohemian districts per each production area) in the period of 1971 to 2010 and compares their frequency of occurrence. That work also includes the occurrence data of the diseases from 2012 when their occurrence on winter wheat was monitored at the training experimental station of the Mendel University in Brno in Žabčice.

Stagonospora nodorum blotch (SNB), is a major foliar disease of wheat in the mid-Atlantic U.S., is caused by the necrotrophic fungus Parastagonospora nodorum. SNB is managed using cultural practices, resistant varieties, and foliar fungicides. There are increasing trends of severity and incidence of SNB in Virginia and the surrounding mid-Atlantic region, but it is not known if changes in the pathogen population are contributing to this trend. The overall goal of this research was to 1) determine the occurrence of quinone outside inhibitor (QoI) resistance in Virginia populations of P. nodorum infecting wheat, 2) quantify the distribution of G143A mutations conferring fungicide resistance in Virginia populations of P. nodorum, and 3) characterize genetic diversity of P. nodorum populations in Virginia and assess influences of cultivars and environments on population structure and SNB severity. For Objective 1, QoI resistant isolates of P. nodorum were identified from Virginia wheat fields, and this was the first report of QoI resistant P. nodorum in the United States. The G143A substitution in the cytochrome b gene of P. nodorum was associated with reduced QoI sensitivity, and in Objective 2, a state-wide, two-year survey of P. nodorum populations in Virginia determined that the G143A mutation was widespread in the state and among sampled fields the frequency ranged from 5-32% (mean = 19%). For Objective 3, P. nodorum was isolated from five different wheat cultivars across seven locations over two years in Virginia. SNB severity varied by cultivar but greater differences in disease severity were observed among locations and years suggesting environment plays an important role in SNB development. Among the necrotrophic effector (NE) genes examined, SnTox1 was predominant followed by SnTox3, and frequencies of NE genes did not vary by cultivar or location. P. nodorum populations in Virginia had high genetic diversity, but there was no genetic subdivision among locations or wheat cultivars from which individuals were isolated. Results also indicated that the P. nodorum population in Virginia undergoes a mixed mode of reproduction, but sexual reproduction made the greatest contribution to population structure. Overall, this work provides insights into the population biology of P. nodorum in Virginia and information on variability in fungicide sensitivity and cultivar susceptibility to SNB that has implications for the current and future efficacy of fungicides and host resistance for management of SNB. / Doctor of Philosophy / Wheat (Triticum aestivum L.) is one of the major cereal crops grown worldwide for food, feed, and other products. However, yields of this crop are often limited by fungal diseases including Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum. Increasing trends of severity and incidence of SNB may be due to reduced sensitivity of P. nodorum to fungicides or increased virulence of P. nodorum populations on commonly grown cultivars. Fungicides such as quinone outside inhibitors (QoIs) are one of the major classes of fungicides used for disease control and G143A substitution is the most common point mutation associated with complete resistance to QoIs. Therefore, the overall goal of this research was to better understand genotypic and phenotypic variation in Virginia populations of P. nodorum in the context of fungicide sensitivity and susceptibility of wheat cultivars to SNB. The specific objectives were to 1) determine the occurrence of quinone outside inhibitor (QoI) fungicide resistance in Virginia populations of P. nodorum infecting wheat, 2) quantify the distribution of G143A mutations conferring QoI fungicide resistance in Virginia populations of P. nodorum, and 3) characterize genetic diversity of P. nodorum populations in Virginia and assess influences of cultivars and environments on population structure and SNB severity. Results from this research indicate that QoI fungicide resistance occurs in Virginia populations of P. nodorum due to a target site mutation (G143A substitution in the cytochrome b gene), and this mutation is widespread and relatively common in Virginia wheat fields. Based on a multi-year multilocation study, P. nodorum populations were genetically diverse, but there was no genetic subdivision among locations or wheat cultivars. SNB severity varied by location and cultivar, but disease severity was greatest at site-years with moderate springtime temperatures and high rainfall. Overall, this work contributes to a better understanding of P. nodorum populations including the current efficacy of fungicides and host resistance for management of SNB in the region.