Pyrenopeziza brassicae is a fungal pathogen that is the causative agent of Light leaf spot (LLS) disease that affects Brassicae species of plants. The disease manifests after a long period of symptomless growth during autumn and winter, with symptoms becoming visible in early spring in the UK. The early symptoms may be confused with frost damage, these include cracking of the leaf cuticle and discolouration of underlying tissue. Lesions appear surrounded by spore masses on various parts of the plant, which then turn necrotic. The disease causes devastating losses of 100-140M annually in the UK. Studying the genetic basis of resistance P. brassicae gives us a better understanding and opportunity at designing a B. napus cultivar better resistant against P. brassicae. This can aid to reduce the crop loss and therefore, improve economic losses and lowers global food loss.
This project was to better understand the B. napus cultivars resistance or susceptibility against P. brassicae. From previous glasshouse screening 4 most resistant and susceptible cultivars were short listed. They were short listed using 1-6 phenotype screening. Table 1 shows the B. napus cultivars and the disease score.
Table1: B. napus phenotype results under glasshouse conditions
|SWU Chinese 1
Our hypothesis was that P. brassicae DNA will be higher in cultivars with higher disease score. The aim of the project was to confirm if the phenotype and molecular level results correlate with each other. This was achieved by quantifying P. brassicae DNA from 4 susceptible (Cabriolet, Moana, Laser and Sansibar) and 4 resistant cultivars (Posh, cubs root, dwarf Essex and SWU Chinese 1).
The B. napus susceptible and resistant cultivars were grown under glass house conditions till 4-5 leaf stage. The plants were then spray inoculated with 105 P. brassicae spore suspension, inoculated leaves were collected 21 days post inoculation and incubated to increase sporulation. DNA from these leaf samples were extracted using DNAMITE plant kit (Sigma Aldrich) and assessed quality using nanodrop. Dilutions were then performed to bring all samples to a concentration of 20 ng/ml. Quantified P. brassicae DNA present in the samples using qPCR (Agilent Brilliant III UF MM SYBR QPCR low ROX kit).
The results show a correlation between resistance in B. napus and lower P. brassicae DNA present in the B. napus samples. This data suggests that resistant strains with low disease score appear to offer some resistance to P. brassicae. The strains POSH and cubs root show the lowest amounts of P. brassicae DNA present amongst samples. Crosses of these cultivars could be produced to create plants with higher levels of resistance to P. brassicae.
The results obtained were in congruence with the hypothesis. qPCR results showed DNA extracted from inoculated resistant strains (Posh, cubs root, dwarf Essex and SWU Chinese 1) showed significantly less P. brassicae DNA than the susceptible strains (Cabriolet, Moana, Laser and Sansibar).
It was a challenge to analyse some of the results, as the data exhibited a considerable amount of variability. Methods of DNA extraction were improved upon, and qPCR plating was optimised during experimentation in order obtain clearer results. In the process I gained excellent problem-solving abilities.
Over the course of my research, I learnt how to implement better experiment design, I was also able to come up with creative solutions to obstacles I faced during experimentation. Working alongside experienced scientists and hearing about their research solidified my interest in plant research and a better understanding of its impact.
I am grateful to the BSPP for giving me this amazing opportunity. I would also like to thank The University of Hertfordshire for their support, especially Ajisa Ali for mentoring me, and teaching me the skills necessary for this project, and Professor Henrik Stotz of the plant research lab for introducing me to this opportunity. As well as everyone at the research lab.
University of Hertfordshire
Figure1: Inoculated P. brassicae DNA quantification
Figure 3 above: Rehmat working on B. napus at The University of Hertfordshire lab (summer 2021)