Quantitative assessment and genetics of the interaction between the light leaf spot pathogen Pyrenopeziza brassicae and its oilseed rape host Brassica napus

Pyrenopeziza brassicae is a pathogenic fungus responsible for light leaf spot disease, one of the most important diseases affecting winter oilseed rape (Brassica napus) crops across the UK and Europe, currently considered the most economically damaging disease affecting the UK crop. Current cultural and chemical control practices are insufficient to manage epidemics of the disease, therefore genetic host resistance against P. brassicae has become of increasing importance as an effective control strategy. The aim of this project was to expand the existing knowledge on B. napus-P. brassicae pathosystem and explore the genetic basis of host resistance, focusing on quantitative disease resistance (QDR). To study the interaction of the B. napus - P. brassicae pathosystem, it is fundamental to establish a reliable method to detect and quantify the phenotypical changes related to the colonisation and determine the severity of the disease. Currently, the strategies used to assess this are based on the presence of acervuli on the surface of the leaves, visually measuring the percentage of area covered by the sporulating structures, classifying the disease severity on a 1-6 scale. Visual assessment, however, can lead to error; therefore, four different quantitative phenotyping strategies were tested and compared: acervuli quantification, spore counting, WAC assay and qPCR. Different B. napus cultivars that differed in their resistance response to P. brassicae were grown in controlled environment conditions and inoculated with a fungal pathogen population. Harvested samples were assessed using visual assessment and the four quantitative phenotyping techniques, and the results statistically compared. Quantitative techniques yielded better results than visual assessment, showing higher accuracy and an improved ability to distinguish between resistant phenotypes. Quantitative PCR produced the most significant results, followed by WAC assay, spore counting and acervuli quantification, in that order. A previous study identified the BAHD acyltransferase BnHAT1 gene as potentially involved in susceptibility to P. brassicae (Fell et al., 2023). This current study aims to characterise the function of this candidate gene in relation to host resistance. To test this, TILLING mutant line (D167N) with SNP mutation in the gene of interest and wild type (WT) individuals were grown under controlled environment conditions, inoculated with a P. brassicae population and phenotyped. A similar experiment was performed using a segregating TILLING population with homozygous individuals that carried a SNP mutation, homozygous individuals with the WT allele and heterozygous individuals with both alleles. The results of these experiments concurred at showing that mutated individuals, in which the BnHAT1 gene had been disrupted, were significantly more resistant, supporting the hypothesis that the BnHAT1 gene is associated with susceptibility to the pathogen. This was complemented with the RNA-seq analysis of a gene expression experiment that showed that the expression of BnHAT1 was significantly higher in susceptible cultivars than in resistant lines. The RNA-seq analysis also revealed differentially expressed genes in the transcriptome that could potentially be associated with resistance or susceptibility to the pathogen, and that are worth further study. To study the genetics of QDR in order to identify QTLs potentially associated with resistance or susceptibility to P. brassicae, a bulked segregant analysis was done. A cross between a resistant and a susceptible cultivar, Lipid and Abukuma, respectively, resulted in an F1 hybrid that was selfed to produce an F2 population. The individuals from these populations were grown under glasshouse conditions in batches and inoculated with a fungal population of P. brassicae. The response of F1 to the pathogen was closer to that of the susceptible parent Abukuma, therefore it could be inferred that susceptibility to the pathogen is inherited as a dominant trait. The screening of the F2 individuals showed a continuous distribution of the disease severity levels, which did not fit Mendelian ratios, proving that QDR was associated with resistance, rather than R-genes. Finally, the extreme phenotypes of the F2 individuals were selected and pooled for a bulked segregant analysis, that revealed two QTL regions on chromosomes A01 and A06, associated with resistance to P. brassicae, and a QTL region on chromosome A07, associated with susceptibility, which had never been reported and could be a novel source of susceptibility to the pathogen. Overall, this research discovered different sources of resistance and susceptibility to P. brassicae in the B. napus genome, all linked to QDR rather than to R-mediated resistance. These could be used to control the spread of light leaf spot epidemics by including them in breeding or bioengineering programs to produce resistant cultivars, which would be highly beneficial for farmers and breeders, as well as reduce the environmental impact.