Pseudomonas syringae is a rod-shaped gram-negative bacterium that has been found to infect many important crop plants such as tomato and soy. The devastation caused by P. syringae leads to a great amount of economical strain on many crop species. Therefore, strategies developed to address P. syringae are of paramount importance. Its primary mode of transmission is through batches of infected seeds. This summer during my studentship I focused on optimising a method of seed infection and seed treatments for seeds from economically important crop.
The method of seed infiltration was based on the traditional vacuum infiltration method. To optimise the vacuum infiltration method in both tomato and soybean seeds, adaptations had to be made. For example, contamination of the Kings Broth agar plates was reduced via the method of seed sterilisation. To further minimise contamination we plated out P. syringae on Kings broth plates supplemented with a low level of vancomycin. Different atmospheric pressures were used during the infiltration phase and the efficiency of the infections determined by crushing seeds to release the bacteria and tittering out bacterial levels in the seeds. It was found that -1 bar of pressure was the optimal pressure during the vacuum infiltration process. The optimum concentration of inoculum for each P. syringae strain was tested and it was found that the level of bacteria in the seeds didn’t increase much when increasing the concentration of the infiltration inoculum.
After successfully optimising the infiltration of P. syringae in seeds, we then measured the growth kinetics of bacteria in soybean seeds. 24 hours post infection the bacterial load in soybean increased by 7.6-fold and after 48 hours grew 28-fold. This confirmed that I successfully optimised the seed infiltration assay with P. syringae strains in soybean.
The next step is to treat the infected seeds with antimicrobial compounds. First, we tested kanamycin, a highly potent antibiotic known to kill the P. syringae. We soaked seeds in kanamycin and water and measured the bacterial load immediately after soaking and 24 hours after soaking. Seeds treated with kanamycin had 10-fold less bacteria recovered from them compared to the water treated seeds. We also tested a new narrow spectrum biological agent that is being currently characterised in the lab and it showed that there was a 5-fold reduction. Future work should focus on seed infections in other economically important crop seeds and testing the efficacy of antimicrobial agents being researched in the lab.
Not only did I successfully optimise methods used in my project during my placement, but I also learned other valuable lessons, such as the importance of a well-kept lab book and time management with respect to my research project. My project was an invaluable experience that has deepened my interest in studying plant pathology and working with plant models. It has taught me many transferable skills to enable me to do my upmost and feel confident in any academic setting. I extend my gratitude to BSPP for funding my project and for allowing me to undertake one of the best experiences of my undergraduate career to date. I would like to thank Dr Joel Milner and William Rooney for their enthusiasm during my project and the exciting opportunity.
University of Glasgow