Using phages as a precision tool to control pathogen abundance and virulence in the plant rhizosphere microbiome

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Plant pathogenic bacteria cause considerable economic losses to food production systems. The main reason for this is that hardly any effective control methods exist to mitigate this damage. In this proposal, we will develop a predictive framework and holistic understanding of how to control plant pathogenic Ralstonia solanacearum bacterium in the plant rhizosphere microbiomes by using bacteria-specific viruses, phages. We will focus on three key aspects that make phages especially useful tools for crop protection. First, we will explore the advantage of phage specificity and test if we can 'precision edit' microbiomes by selectively targeting only the pathogen. This is important not only for the efficacy but also for the safe use of phages as they should not cause collateral damage to the surrounding microbiome in the rhizosphere. Second, we will determine the evolutionary consequences of phage selection for pathogen competitiveness and virulence using metabolic modelling and direct experimentation. While it is likely that pathogens can rapidly evolve resistance to phages, this is often costly to the pathogen. Phages could thus be used as 'evolutionary tools' to weaken the pathogen by selecting resistance adaptations that incur high metabolic burden or impair virulence gene expression. Third, we will explore if phage-mediated changes in pathogen virulence and microbiome composition predictably alter plant immune responses having potential additional beneficial effects on the plant health. Here, we will move beyond pathogen-centric view to build a holistic understanding of community-level feedbacks by systematically exploring responses in plant gene expression when challenged with phage resistant and susceptible pathogens in the absence and presence of natural rhizosphere microbiome. Finally, the most promising phages will be selected for industrial manufacturing and their biocontrol efficacy and safety validated in greenhouse trials using potato. To achieve these goals, we will bring together an interdisciplinary research team consisting of academics and industry partners experienced in ecology and evolution of phage-bacteria-plant interactions, genomics, metabolic modelling and plant transcriptomics. The proposed research is innovative, timely and pushes the boundaries of traditional crop protection to precision control of plant pathogenic bacteria in the plant rhizosphere using phages.

Technical Summary

In this proposal, we will develop a predictive framework and holistic understanding of how to control plant pathogenic Ralstonia solanacearum bacterium in the plant rhizosphere microbiomes by using bacteria-specific viruses, phages. The aims of this project are: (A) to better understand the ecology and evolution of pathogen-phage-plant interactions in the plant rhizosphere, and (B) to use this information to develop, manufacture and validate a novel phage biocontrol application in collaboration with industry partners (iMEAN, APS Biocontrol and Fera). First, we will determine if phages can be used to 'precision control' pathogens in complex rhizosphere microbiomes and if phages are safe to the plants and non-pathogenic bacteria in the rhizosphere using lab and plant infection experiments (microbiological co-culture assays; 16S rRNA sequencing, pathogen-specific qPCR and infections using Microtom tomato model in plant growth chambers). Second, we will test if phages can be used as 'evolutionary tools' to select for less competitive and non-virulent pathogen genotypes using experimental evolution, genome-scale metabolic modelling (project partner: iMEAN) and direct validation linking mutations to pathogen competitiveness and virulence (microbiological assays for virulence trait expression; direct virulence measurements using Microtom; catabolism with Biolog assays; direct competition assays against fluorescently labelled ancestral strain). Third, we will explore if phage-mediated changes in pathogen virulence and microbiome composition predictably alter plant immune responses having potential additional beneficial effects on the plant health using plant transcriptomics (and RT-qPCR) and bacterial metatranscriptomics in factorially designed infection experiment. Finally, we will design, manufacture (project partner: APS Biocontrol) and validate phage biocontrol combinations in greenhouse experiments using potato (Project partner: Fera).

Planned Impact

Who will benefit and why?

1. General public. Education about plant-microbe interactions is important for the public understanding of how food can be produced and protected from pathogens environmentally friendly and sustainably in the future. To promote knowledge on phage-pathogen-plant interactions, we will deliver an exhibit for the University of York's annual Festival of Ideas (year 2), where we will showcase our research for the public in York city centre. We will focus on explaining what phages are, their role in medical, agricultural and natural microbial communities and their importance for crop protection and food production. We will also present our research at the yearly Pint of Science (https://pintofscience.co.uk/) in York (year 1), an event designed to encourage the public debate of science, and Soapbox Science (year 3), which publicises the work of women in STEMM.

2. Scientific community. We will organise a related research symposium on the topic in a large international scientific conference (ESEB, ISME, SMBE, AEM) during the project. This symposium will also be used as a basis to edit a special issue on the topic in MDPI Open Access Journal Life where Friman is 'Evolution' section Editorial Board member. Findings will also be communicated by all team members at scientific meetings. Target audiences will include biologists, agronomist, mathematical modellers and biotechnologists with interests in applied microbiome research. high impact journals with a cross-disciplinary readership will be targeted to publish findings in an open-access format. Press media releases will be written and discussed with the science communications team at York upon publication. Publication fees will be covered by York Open Access Fund (https://www.york.ac.uk/library/info-for/researchers/open-access/yoaf/).

3. Crop protection and microbial inoculant industry. Friman currently collaborates with two agri-food companies, DLF Trifolium (DK) and Legume Technology (UK), through a large Danish research project, NCHAIN, to improve cattle feed production by matching specific nitrogen-fixing rhizobium bacteria with high-yield clover cultivars (PhD project supervision at York). Commercial Rhizobium inoculants are currently sold in peat carrier mixture that gives the inoculant a shelf life of two months. In the proposed project, we will develop a new approach for storing and disseminating phage products with APS Biocontrol (UK). Specifically, we will trial new lyophilisation methods to turn phages into a powder format, which can be packed in sachets and mixed with water before application. Friman already has a non-disclosure agreement in place with APS Biocontrol and meetings will be set-up with the University of York intellectual property & legal (IPL) team to assess the potential need for patents during the project.

4. The Government and Agriculture policymakers. York is the institutional lead in the N8 AgriFood initiative, an HEI- and government-funded initiative to connect academic research with wider society including businesses, policymakers and NGOs. We will work with N8 AgriFood Knowledge Exchange Fellows who are industry specialists embedded within our research teams. We will also liaise with the head of Agricultural Development at the Soil Association and present our results in one of their knowledge transfer events and develop existing links with Fera, Defra, CHAP and SASA. All activities will be advertised, disseminated and reported through the existing N8 communication mechanisms and dedicated stakeholder engagement events during the project.
 
Description Key findings (Updated in Spring 2022):

- Creation of phage bank (70 environmental phages from the UK and China) and determination of their host specificity against 400 pathogen isolates (identification of suitable phages for biocontrol)
- Creation of synthetic rhizosphere bacterial communities from natural bacterial isolates from potato, kale, maize, pea and wheat rhizosphere soils
- Sequencing of phage genomes to characterise difference at the genetic level (March 2022)
- Creation of metabolic model for a model R. solanacearum phylotype 2 potato strain in collaboration with project collaborator, iMEAN biotech (September 2020-ongoing); creation of a regulatory model for Tomato host plant (Spring 2022-ongoing)
- Extensive bioinformatics analysis to screen our R. solanacearum pathogen collection for the presence of phage defences systems, prophages and insertion sequences to understand how these mobile genetic elements might affect its fitness and virulence (and biocontrol using lytic phages)
- Greenhouse experiments to validate and screen phages that are effective at constraining bacterial wilt infection
Exploitation Route N/A
Sectors Agriculture, Food and Drink,Environment

 
Description Microbiology Society Harry Smith Vacation Studentship 2020
Amount £2,720 (GBP)
Organisation Microbiology Society 
Sector Learned Society
Country United Kingdom
Start 06/2020 
End 08/2020
 
Description Royal Society of Biology Plant Health Undergraduate Studentship
Amount £2,500 (GBP)
Organisation Royal Society of Biology (RSB) 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2021 
End 08/2021
 
Description Society for Applied Microbiology Summer Studentship
Amount £2,500 (GBP)
Organisation Society for Applied Microbiology 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2021 
End 08/2021
 
Description APS Biocontrol 
Organisation APS Biocontrol
Country United Kingdom 
Sector Private 
PI Contribution We are working together with APS Biocontrol to spray dry phages into powder, which could allow the manufacturing of phage products with good shell life and an easy route for application when mixed in irrigation water.
Collaborator Contribution We are at the early stages of collaboration; during the project, APS Biocontrol will test several storaging strategies for the therapeutic phages we have identified and tested.
Impact N/A
Start Year 2022
 
Description Collaboration with iMEAN 
Organisation iMEAN
Country France 
Sector Private 
PI Contribution iMEAN biotechnology is external collaboration in this project. Thus far, we have together developed genome- and cell-scale metabolic models for the UK-specific Ralstonia solanacearum bacterial pathogen. These models can be used to predict the effect of different mutations for the fitness and virulence of the pathogen
Collaborator Contribution See above
Impact N/A
Start Year 2020
 
Description Collaboration wth FERA Science Ltd 
Organisation Fera Science Limited
Country United Kingdom 
Sector Public 
PI Contribution Our research team has screened suitable phages to be used in the biocontrol of plant pathogenic Ralstonia solanacearum bacterium. These phages will be then combined into phage biocontrol combinations, whose efficiency will be tested in greenhouse experiments at Fera.
Collaborator Contribution We are currently in a process or finalising collaboration contract with Fera and have initially booked greenhouse sapce for experiment this Spring and Autumn.
Impact N/A
Start Year 2020
 
Description Interview for FEMS 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview at the Danish Microbiological Society (DMS) Congress in Copenhagen by Eleni Koursari, FEMS Science Communications Officer to talk about the phage therapy after his lecture: ''Using Ecology and Evolution to Design Effective Phage Therapeutics"
Year(s) Of Engagement Activity 2021
URL https://www.youtube.com/watch?v=1ORA0FPLfjc
 
Description Interview in 'Phage Phriday' program in clubhouse platform to discuss about phage training 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview in 'Phage Phriday' program in clubhouse platform to discuss about phage training
Year(s) Of Engagement Activity 2021
URL https://www.youtube.com/watch?v=adNGCHX_4zY