Project Outline

In the UK and much of Europe, tree populations are being devastated by the frequent emergence of new pathogens. This has naturally caused concern both within the scientific community and forestry industry, but also within the public¹. Cherry is both an important commercial tree for fruit production, as well as a valued timber tree. Bacterial canker infects all tissues including leaves, stem, branches and fruits. Infections in the stem (canker) are the most dangerous as these can lead to irreversible damage to the vascular systems, cutting off water and nutrient flow that eventually leads to extensive tree damage and death, causing up to 75% losses. It is caused by at least three distinct clades of Pseudomonas syringae, a bacterial pathogen of many important crop species with more than 100 pathovars (pv), which are specialised to particular plant hosts. These are P. syringae pv. syringae (Pss), P. syringae pv. morsprunorum (Psm) race 1 and Psm race 2. A lack of genetically resistant varieties and effective control measures have hindered progress in combating this disease. There is therefore an urgent need to develop measures to either prevent the spread of the pathogen or develop control of the disease².

A potential strategy for treating bacterial diseases is the development of phage biopesticides (phage therapy). Phage therapy approaches have been used for the control of both human/animal and plant pathogens. Phages are viruses that infect and kill bacteria. Phages are natural “products” already present in the environment; some phages have very narrow host ranges, thus specifically targeting only the pathogen and not the beneficial microbiome; phages can rapidly evolve, helping with the directed evolution of new genotypes and providing an element of in situ sustainability to potentially overcome bacterial resistance in real time. In plants, phage cocktails have been approved for use in agriculture and proven successful in treating and preventing plant diseases e.g. bacterial fire blight of apple. Moreover, recent advances in our knowledge of the evolutionary ecology of bacteria-phage interactions strongly suggest that phage therapy could be successfully used to treat cherry canker^3,4.

We have identified a range of phage isolates that infect Pss and Psm strains, including new phage clades and developed all the methodology for characterising them (e.g. purification and enrichment, host range analysis, killing curves, genome sequence analysis). In vitro studies show that individual phages can rapidly reduce bacterial population numbers, but bacterial resistance to phage infection quickly emerges⁵. To understand this process further, we have employed an initial experimental evolution approach to track the bacterial population through time and identify the changes occurring in both the bacterium and phage populations. Genome sequencing of the coevolved bacteria show that there are resistance mechanisms in bacteria that enable them to resist phage infection. However, we still do not know how efficient phage therapy is, for example how phage is spread in the plant environment, whether it disperses, thus how far, for how long it remains viable. Phage engineering is instrumental in understanding these questions and paving way toward potential use of phages in crop settings, therefore protecting against and preventing antimicrobial resistance in crop disease management strategies.

Methodologies

How efficient phage therapy is in plants, whether they remain within the plant tissue or disperse?

Fluorescence-labelling of phages (green fluorescent protein) and bacteria (Red fluorescent protein) will be applied to cherry leaf via spray method. The hypothesis being tested here is that phages remain within plant tissue after reducing the population of their target pathogen. Fluorescence imaging of cherry leaves will be performed with a CF Imager (Technologica Ltd.) and confocal microscopy. Live cell imaging will be captured on a Zeiss LSM 880 confocal microscope. Immunofluorescence microscopy will be undertaken to visualize the association of phages with both their bacterial host and the plant tissues. These are all established protocols being used at the phage science group at University of Warwick^6,7. This will enable to view phage dynamics over time rather than using a culturing method or PCR approach which is only quantitative, but not spatial.

Does phage engineering increase their efficacy?

By identifying the genetic mutations in phage-bacteria interaction and the phage receptors, phages will be engineered to overcome the resistance in bacteria. We will use the homologous recombination, followed by marker-based methods for the development of an efficient system of engineering and constructing phage mutants. Phage mutants that target receptors on bacterial cell surface will be generated⁸. This will help to develop phages that bacteria cannot develop resistance to and therefore can be used in phage cocktail design.

Does diversity of microbial community differ under phage application?

Impact of phage application on leaves microbial community will be tested by 16S/ITS rRNA amplicon next generation sequencing to catalogue resident bacterial/fungal OTUs and communities and to identify the impact. This will help with designing phage cocktail for bacterial disease management.

References

1. Cazorla et al. 2002 Phytopathol 92:909
2. Hulin et al. 2018 Plant Pathol 67:1177
3. Koskella et al. 2013 Viruses 5:806
4. Koskella et al. 2012 Proc Royal Soc B 279:1896
5. Rabiey et al. 2020 Microb Biotechnol 13:1428
6. Breen, S., et al. 2022 Plant Cell Environ 45:3001-3017
7. Dhanoa et al. 2022 Access Microb. 4(5)
8. Joshua et al. 2023 ACS Synthetic Biology, 12:2094-2106

Techniques

Molecular work will include DNA isolation, PCR, whole genome sequencing, amplicon sequencing of bacteria and fungi using 16S/ITS rRNA gene and data analysis will be conducted. Microbiology skills will include culturing bacteria and phages and testing their interaction.