2024-W15 Development of phage biopesticide to control Xanthomonas campestris pv. campestris causing black rot in Brassicas

Project highlights

Isolation of phages against Xanthomonas species
Characterisation of phages most effective as biocontrol agents
Development of phage cocktail to treat brassica black rot

Overview

The diverse gram-negative bacterial genus Xanthomonas causes disease on more than 400 plant species, including trees, and often causes economically significant losses in agriculture. Xanthomonas campestris pv. campestris (Xcc) causes disease in a large number of economically important vegetable Brassica crops, such as cauliflower, cabbage, and broccoli and a number of other cruciferous crops, ornamentals and weeds, including the model plant Arabidopsis thaliana (Vicente and Holub, 2013). Xcc comprises eleven races, of which Race 1 and Race 4 are considered the most common and destructive for Brassica crops (Lu et al., 2021). Xcc is a vascular pathogen, colonising via hydathodes or wounds on the plant and forms biofilms in the vascular system, where it releases degradative extracellular enzymes and virulence factors (Papaianni et al., 2020).

The control of black rot is difficult and currently relies on the use of pathogen-free planting material and the elimination of other potential inoculum sources (infected crop debris and cruciferous weeds) (Vicente and Holub, 2013). One potential alternative method to control bacterial diseases is through bacteriophage biocontrol. Bacteriophages (phages) are viruses that infect bacteria and represent one of the most abundant organisms in the biosphere. Phage are almost always highly specific to their target bacterial host, hijacking its replication machinery to complete their replication, leading to lysis of their bacterial host, without directly causing harm to plants or animals. The potential of phage against Pseudomonas syringae pathovars actinidiae, aesculi and syringae have been explored as potential biocontrol agents (Di Lallo et al., 2014, James et al., 2020, Rabiey et al., 2020).

We aim to develop a novel phage-based biocontrol for use in Brassicas (cabbage, broccoli, cauliflower, kale) affected by Xcc black rot disease.

Host

University of Warwick

Theme

Climate and Environmental Sustainability

Supervisors

Project investigator

Dr Mojgan Rabiey, School of Life Sciences, University of Warwick, [email protected]

Co-investigators

Prof Murray Grant, School of Life Sciences, University of Warwick, [email protected]

How to apply

Each host has a slightly different application process.
Find out how to apply for this studentship.
All applications must include the CENTA application form.
Choose your application route

Methodology

Collection of bacteria and phages from brassicas field. Wellesbourne curates a well-characterised set of Xcc isolates (>600). Phages will be isolated following our established methods (Rabiey et al., 2020). Phage characterization will be done (electron microscopy and genome sequencing). Host specificity of isolated phages will be tested, initially on an Xcc diversity set based on whole genomic sequence of 600 Xcc isolates.
Bacteria-phage interaction coevolution study. Phages will be co-evolved with Xcc to determine whether bacterial resistance emerges. Both single phage isolates and cocktails of natural isolates and evolved phages will be tested, monitoring any antagonistic or synergistic effects that emerge from these studies and prioritising phage for efficacy studies.
Testing the efficacy of phage for controlling disease. These selected phages will be optimized in terms of production environment (e.g. temperature, MOI, time) with the aim of producing maximal titres for commercial applications. The best phage mixtures will be assessed for Xcc control in controlled environment containment facilities across a wider spectrum of the Wellesbourne collection, including other Xantghomonas species.

Training and skills

Students will be awarded CENTA2 Training Credits (CTCs) for participation in CENTA2-provided and ‘free choice’ external training. One CTC equates to 1⁄2 day session and students must accrue 100 CTCs across the three years of their PhD.

The student will receive specialist training for this multidisciplinary project, encompassing fieldwork, microbiology, electron microscopy, genomics, data management and statistical analysis and interpretation of large and complex data sets.

The student will be supported to develop these skills within the SLS at Warwick, allowing the student to excel in these key aspects of data acquisition, analysis and dissemination and to build important networks.

The supervisory team is multi-disciplinary and highly experienced, based in excellent, well-equipped institution, and will provide comprehensive support for the student across all aspects of the project. S/he will also have access to unique imaging tools and the PhytoBac Explorer bacterial genomics database to undertake comparative studies between effective and tolerant phage to identify potential toxin/antitoxin components.

Further details

Further details on how to contact the supervisor for this project and how to apply for this project can be found here:

For any enquiries related to this project please contact Dr Mojgan Rabiey, School of Life Sciences, University of Warwick, [email protected].

To apply to this project:

You must include a CENTA studentship application form, downloadable from: CENTA Studentship Application Form 2024.
You must include a CV with the names of at least two referees (preferably three) who can comment on your academic abilities.
Please submit your application and complete the host institution application process via: https://warwick.ac.uk/fac/sci/lifesci/study/pgr/studentships/nerccenta/ Complete the online application form – selecting course code P-C1PB (Life Sciences PhD); from here you will be taken through to another screen where you can select your desired project. Please enter “NERC studentship” in the Finance section and add Nikki Glover, [email protected] as the scholarship contact. Please also complete the CENTA application form 2024 and submit via email to [email protected]. Please quote CENTA 2024-W15  when completing the application form.

Applications must be submitted by 23:59 GMT on Wednesday 10th January 2024.

Possible timeline

Year 1

Field experimental design and field data/sample collection training will be done. Samples will be collected from Brassicas fields. Phages will be isolated from the samples. Phage characterisation will be done, including killing curve assay, host range assay, growth rate assay, whole genome sequencing, and temperature assay.

In parallel develop the initial diversity screening panel that maximises isolates with geographic and genomic diversity across the key Races, 1 and 4.

Year 2

Isolated phages will be coevolved with the pathogen and the genetic mechanism of phage-bacteria interaction and evolution will be tested by whole genome sequencing. The phages will be tested in planta on brassicas species.

Year 3

The impact of phages on other microbiota will be tested in planta, by DNA extraction and 16S and ITS sequencing. Data analysis of the sequencing data and year 1 and 2 experiments undertaken.

Final analysis of the data, explore stakeholder interest and possibility of dual pahge/bacteriocin treatments. Writing up of manuscripts and thesis completion.