This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Pseudomonas syringae (Ps) is a common plant pathogen which can cause diseases in a wide range of plants. It has high genetic diversity corresponding to the production of various effectors and/or toxins, which allows different Ps strains to infect different hosts including a variety of economically important crops such as cherry, plum, and kiwifruit. As Pseudomonas syringae infection leads to losses in agricultural production, it is a challenge and necessity to understand Ps genomic diversity so that effective means of control can be designed.
Our project focuses on the diversity of Pseudomonas syringae responsible for bacterial canker in Prunus spp. By repeated sampling of Ps strains from cherry and related plants in orchards and woodlands at multiple sites around the UK (Southwest, Kent, Scotland, Hereford), we aim to investigate the genetic variations of Ps both spatially and temporally via repeated sampling and high throughput sequencing. Bacteria isolated from leaf and shoot samples, after being identified as Ps by PCR, will be sent for whole genome sequencing to detect for pathogenicity genes encoding type III effectors and/or toxins. 16S/18S amplicon sequencing will be applied to investigate the microbiome i.e., bacteria and fungi in the leaf and shoot samples.
During my internship at NIAB, I participated in the June sampling trip to Scotland (Figure 1A), followed by bacterial isolation, purification, and storage. Bacteria on leaf and shoot surfaces were isolated by phosphate-buffered saline (PBS) wash and grown on King’s B agar (KBA) plates containing 2 antimicrobials, namely cycloheximide and cephalexin, for selection against fungi and other bacteria respectively (Figure 1B). Two individual colonies with representative Ps colony morphology were then selected from each plate for purification by streaking on Luria-Bertani agar (LBA) plates containing the same antimicrobials. Purified single colonies were inoculated into liquid broth and stored in glycerol under -80℃ for further characterisation. Polymerase chain reactions (PCR) using a set of Ps species-specific oligos were performed to differentiate Ps from other bacteria. I characterised the strains isolated in May 2021. As shown in Figure 2A, an amplicon of 144bp is observed for a Ps strain. A subset of the verified Ps strains can then be whole genome sequenced. Some phylogeny-specific primers were also used to identify the phylogeny groups of these Ps strains (Figure 2B).
In addition to the analysis of recently collected strains, pathogenicity tests were performed on a series of deletion gene mutants of Pseudomonas syringae pv. syringae (Pss) 9644, a well-known cherry pathogen strain (Figure 3A). These experiments clarify the importance of different groups of effectors/toxins in Pss bacterial invasion and establishment in cherry leaves. Bacteria suspensions adjusted to 2×106 CFU/ml were inoculated into healthy detached cherry leaves by infiltration using 1ml needle-less syringe. Symptoms were scored in a range of 0-5 according to lesion occupancy, and leaf discs of the inoculation sites were harvested and homogenised for bacterial population count 0, 3, 6 days post inoculation. 10μl extracts were spotted on LBA plates (with 10-fold dilution series for 3 and 6dpi), and numbers of individual colonies were counted after 2-overnight incubation (Figure 3B).
The undergraduate vacation bursary project has been a wonderful experience for me to learn the topic I am interested in and wider missions in the area, as well as practising my field/lab skills and making new friends. I would like to thank BSPP and my supervisor Dr Ziyue Zeng and Ms Andrea Vadillo for offering me the chance to improve my research ability and advising me in critical thinking. The intriguing processes of problem solving in research will prompt me to pursue further study of plant pathology in the future.
Figure 1 ABOVE: (A): Sampling in the field and (B) isolated bacterial colonies on KBA plates.
Figure 2: (A) PCR on bacteria isolated from 2021 May Kent woodland samples using Ps-specific primers (PCR product = 144bp i.e., second lowest band right above primer dimers). Pss 9644 was used as a positive control (+ve); the three negative controls are no template (-ve) and two non-syringae fluorescent Pseudomonas spp. (12G, 12H). Multiple unspecific binding patterns reflect high genetic variation of Ps and other environmental bacteria isolated from samples.
(B) Multiplex PCR on 2021 May southwest woodland samples with Ps-specific primers, PG1 specific primers (321bp product, see white boxes), and PG3-specific primers (837bp product, see white boxes). Positive controls are Pss 9644 (12E), a PG3 strain R1 5244 (12F), and a PG1 strain R2 Leaf (12G), and the negative (-ve) control has no sample.
(B) Some representative plates showing pathogenicity test results. r3 = 3rd biological repeat of bacteria colony from which the suspension was made, 3d = 3 days post inoculation, 18.1 = strain 18 inoculated on 1st biological repeat of host leaf, rows = dilution series, columns = technical repeats.