Infection by Pseudomonas syringae is the lead cause of bacterial canker, a disease affecting the global industries of cherries, plums and other Prunus species. P. syringae are rod-shaped, Gram-negative bacteria that contain huge levels of species complexity with great variation in pathovars and strains. The goal of my project was to investigate the species complexity of environmental Pseudomonas populations, aiming to provide insights into the relationship between pathogen and host in a multitude of ways.
In order to isolate, identify and characterise P. syringae populations, over 2000 leaf and shoot samples were collected from cherry trees and related species in four locations across the UK – Scotland, Hereford, Kent and the South-West. Both orchards and woodlands were sampled in order to determine whether the cultivation of crops in orchards had any effect on the Pseudomonas populations present. Samples were taken at different times throughout the growing season of the plants, in order to help construct a temporal analysis of the microbiome of these different areas. I worked in conjunction with a larger, multi-year project which would analyse the temporal variation in Pseudomonas populations in greater depth.
Whilst Pseudomonas displays some distinct morphological traits, such as fluorescence, the high species complexity and colony morphology required the development of a protocol that could assess the cultures and accurately identify which ones were actually Pseudomonas. We developed a multiplex PCR system that could simultaneously identify and characterise the bacterial strains. The positive control test was generated by using forward and reverse RpoB primers, amplifying a region of the β-subunit of bacterial polymerase which should be found across all bacteria. PsGenus primers were used to amplify a region that is highly conserved within the Pseudomonas genus, allowing us to distinguish between Pseudomonas and other environmental strains with similar morphology at a genetic level. In order to determine whether the strains were potentially pathogenic, we attempted to find evidence of a type three secretory system present within the bacteria. This required the testing of multiple primers on the model pathogenic strain Pss9644, eventually deciding upon HrcC primers, which amplify conserved structural component of the type three secretory system. Known colonies of pathogenic and non-pathogenic Pseudomonas, and non-Pseudomonas colonies such as Escherichia coli were used as controls to ensure the multiplex PCR system we developed remained accurate and reliable.
The development of this multiplex PCR protocol enabled rapid characterisation of different unknown bacterial colonies, enabling 96 samples to be analysed simultaneously during each PCR cycle. This work set the benchmark for the remainder of the multi-year project and should allow for greater quantities of bacteria to be sampled, providing an even greater understanding of the microbiomes of different areas. I applied for the BSPP summer studentship as I was unable to experience practical lab sessions during the lockdowns, and this project was an incredible way of enabling me to utilise my knowledge in a practical fashion and increasing my confidence in lab. I now have a much clearer understanding of what I would like to do in my post-university career, and it has provided me with unrivalled experience in my field of interest. I want to thank NIAB for hosting me, Dr Ziyue Zeng for her guidance, and the BSPP for their generosity and enabling this experience to occur.
Alex Seward
University of Leeds
Multiplex PCR for bacterial identification and characterisation. Multiplex PCR using oligoes for rpoB gene (750 bp) for indicating successful PCR for each sample, genus specific oligoes (620 bp) for identifying Pseudomonas and oligoes for the hrcC gene for detecting Type 3 Secretion System of potentially pathogenic strains.
