Detection and diversity of Zymoseptoria tritici and its effectors from wheat field sites

Fungal pathogens are causal agents of several diseases in plants, specifically in cereals. Such pathogens are a threat for food security and cause economic and yield losses every year. These organisms spread through airborne spores and secrete a large number of small proteins called effectors into the host plant to successfully colonise it. The wheat fungal pathogen Zymoseptoria tritici is one of the major threats for its high genomic plasticity, it has the capability to overcome host defences, and spreads easily though wind and rain splashes. In this thesis, a metagenomic approach for recognition of cereal fungal pathogens from airborne spores was investigated and compared with a more traditional microscopic method. A total of 150 different species within 30 genera of cereal fungal pathogens were recognised by the metagenomic approach while the microscopy was able to recognise only 20 genera / species. Microscopic bi-hourly results for Z. tritici and Fusarium spp. ascospores release were also used for comparison with weather data. A positive correlation was found between ascospore release and air relative humidity, and a negative correlation between Z. tritici ascospore release and air temperature. Furthermore, the effector gene candidate Zt-11 of Z. tritici was characterised in-planta for its role in virulence using Agrobacterium mediated gene deletion in Z. tritici IPO323 strain. Meanwhile, a novel DNA extraction method was established using sonication from various tissues for downstream PCR analysis, this method was used for screening of effector gene knockout strains. The Zt-11 effector was discovered to be involved in virulence, knockout mutants showed a delay in disease symptoms, lower and smaller pycnidia number, and a decrease in number of asexual pycnidiospores. An associated population genomic study showed that the Zt-11 gene is highly polymorphic and under positive selection. Lastly, a target enrichment sequencing experiment from airborne ascospores on spore trap tape samples, specifically targeting 299 Z. tritici effector gene candidates, was successfully designed, and executed. From the effector enrichment results, the pangenome of Zt-11 was investigated for polymorphisms, finding a greater variety of mutations in the ascospores population than in the field isolates population.