Xanthomonas plant diseases: mitigating existing, emerging and future threats to UK agriculture

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Brassica cultivation is a multi-billion dollar industry worldwide and Xanthomonas is a bacterial plant pathogen pathogens that can infect more than 350 plant species including many brassicas. This multidisciplinary project brings together experts in microbiology, genomics, pathology, imaging, molecular biology and risk assessment to take one of the first large scale integrated studies of the plant pathogen Xanthomonas campestris (Xc), using approaches involving large scale genomic sequencing, in planta imaging and screening for pathogen resistance using a unique set of brassica diversity lines. The objectives are to enhance knowledge and support effective breeding and deployment of appropriate control strategies for Xanthomonas in the UK and other countries that face similar challenges.
Our central focus is on black rot of crucifers (Xc pv. campestris or Xcc for short) which can attack all cultivated brassicas. Xcc, is a seed-borne vascular (the tissue that allows water and nutrients to flow) disease and probably the most important disease of vegetable Brassicas (Brassica oleracea; e.g. cabbage, cauliflower, broccoli, Brussels sprouts and kale) in the world. Xcc also infects B. napus (oilseed rape, swede), B. juncea (mustards) and B. rapa (turnip, Chinese cabbage). It is therefore really important. Xcc can cause complete crop loss, though in the UK it usually leads to reduced quality and marketability.
There are two main problems with Xcc. First, as with most bacterial phytopathogens, there is no simple chemical interventions, unlike for fungal pathogens. Traditional bactericides such as copper oxychloride are being withdrawn from sale. Secondly, and one key driver of this proposal, there is virtually no resistance in commercial Brassica oleracea varieties - this has probably been bred out. So we are going back to wild relatives and are fortunate that Warwick's UK Vegetable Germplasm collection maintains more than 6000 brassica accessions. Obviously this is far too much to deal with, but we recently (with Defra funding) generated what we call Brassica diversity fixed foundation sets (DFFs). These are designed to capture the majority of the genetic variation in a population of homozygous lines which can then be screened for resistance to Xcc. This project will use DFFSs for Brassica oleracea, Brassica napus and also interfertile C genome crop wild relatives.
We are also fortunate to have the largest collection of Xanthomonas isolates in Europe (~ 1,000) as well as the UKs most knowledgable Xanthomonas pathologists Dr Joana Vincente on board.

We will sequence more than 900 Xanthomonas isolates from this collection so we can address some of the key challenges in the field, such as what makes a pathogen, what are the key genes a Xanthomonas pathogen needs to deploy to cause disease on specific plants and how the pathogen changes over time. A major output of this work will be a large set of diagnostic markers that can be used to identify specific pathovars of Xanthomonas, even in the field!
We will couple these studies to understanding how the pathogen infects and multiplies in the plant using two complementary imaging approaches, fluorescent/bioluminescent reporters and the plant endogenous chlorophyll fluorescence - which changes as disease progresses.
We also aim to ask the questions, can we use a beneficial soil fungus to protect from Xcc disease and can insect pests such as aphids transmit the disease in the field following feeding on infected plants?
One of the real concerns in a global community is incursions of pathogens. So aside from the diagnostic work proposed, we will also undertake a detailed risk assessment on a devastating Xanthomonas disease of maize, which is rapidly spreading in the US and causing over 50% of losses in some regions. This assessment will also consider risks to other crops considered potentially "at risk".

Technical Summary

X. campestris pv. campestris, one of the most important brassica diseases worldwide, causes black rot, particularly on B. oleracea, where resistance is rare. This multidisciplinary project draws upon risk assessment, pathology, bioinformatic, molecular, imaging in a detailed analysis of Xc genomics, Xcc infection dynamics and screens for new Xcc resistance that can be ultimately be deployed in the field. There are two notable strengths to this proposal. The unique Warwick HRI collection of ~1,000 Xanthomonas isolates, curated and largely collected by Joana Vincente who spearheads the pathology component and who described the first six races of Xcc and Warwicks unique collection of brassica diversity fixed foundation sets (DFFs).
To address specific biological questions outlined in the proposal we will sequence ~960 Xc isolates, including 771 Xcc isolates and multiple isolates of the apoplastic pathogen Xc. raphani. Candidate genes, variants and other genetic elements that are associated with race and infection lifestyle will be used in a variety of assays including generating diagnostic markers, with in field utility, including identification of potential threats such as X. fragariae, X. nasturtii and th X. vasicola pv. vasculorum, supporting our pathogen risk work programme.
Core and variant genes will be delivered in planta, singly and in combination via Xc or P. syringae using chlorophyll imaging to quantify their contribution to virulence, resistance, or as our preliminary data suggests, masking other effector activity. We will also image Xc infections undertaken using new bioluminescence and fluorescent reporters whose emission sits between the Xanthamonadin and chlorophyll emission spectra.
In parallel, we will screen Brassica oleracea, Brassica napus DFFs and also the interfertile C genome crop wild relatives DFF as well as revisiting informative historical mapping lines to identify loci showing race specific or broad spectrum resistance to Xcc.

Planned Impact

Who will benefit from this research?

This project targets UK brassica growers as the ultimate beneficiaries by providing the genetic and genomic knowledge to facilitate new understanding of Xanthomonas virulence mechanisms, how virulence emerges and to identify new sources of Xanthomonas resistance to incentivize breeding companies to use these resources.
The work targets, black rot of crucifers caused by Xanthomonas campestris pv. campestris, a disease that regularly causes loses in brassica UK production, and comparisons with related pathovars, will have impact in the UK producers and related industry including seed and transplant producers. Strategies for using new sources of resistance will lead to the development of new cultivars and the work on Xanthomonas genomics should lead to improved methods of diagnostics that will be available to growers and industry in general.

Additional potential benefits to UK growers.
One of the real concerns of UK growers is incursions of pathogens. Xanthomas disease of maize, a devastating disease in US is also a major concern of UK maize growers. In addition to the diagnostic work proposed which will monitor the spread of the disease in maize fields around the country, we will also undertake a detailed risk assessment which will help maize growers prepare for a future disease outbreak. The knowledge on Xanthomonas diseases of strawberry, watercress and maize, will be important to avoid the introduction and spread of these pathogens in UK crop production. The findings will specifically inform the Defra Plant Health Risk Register and if phytosanitary measures are justified against X. vasicola pv. vasculorum.
Training:
The project will help train the next generation of phytobacterial workers. The UK expertise in plant pathology and in particular bacterial plant diseases has been reduced in the past 10-20 years and the work proposed here will contribute to strengthen teams working at mainly at three institutions (University of Exeter, Fera and University of Warwick) and, critically, will provide training for new students and researchers.
The combination of skills in Plant Pathology, genetics, plant-pathogen interactions and bioinformatics will be essential to provide new knowledge on Xanthomonas pathogens and their hosts. We will use resources available at Warwick and Fera including bacterial collections and plant selections. We will also add to these invaluable collections.
Implementation: The PIs have excellent track records in presenting in scientific conferences and are very well connected within relevant UK and worldwide academic communities. Our track record and extensive network of colleagues and collaborators, in different disciplines of plant biology, will ensure the effective communication of our research output. We will publish our findings in high impact open access journals and we will present our findings at national and international meetings. We will also disseminate our research findings through our project website, social media, news-and-view style articles podcasts.
Genomic data: Sequencing data generated will be made publicly available in relevant databases. This proposal will also contribute to maintain and invest in facilities and new equipment that is necessary for keeping large collections of bacterial isolates and for imaging. These will also be available for other researchers to develop additional studies.
Outreach: We will engage with producers, agronomists and industry groups through site visits and through attendance of meetings and events as described in pathways to impact. In addition, we will also attend events aimed at the general public and will raise awareness on the importance of crop production in the UK and importance of putting in place strategies to reduce the impact of plant bacterial diseases - in particular, the introduction of new diseases should be avoided through testing of seeds and planting materials.

Publications

10 25 50
 
Description There have been multiple outputs - as expected from a largish multidisciplinary grant looking at one of the most important crop bacterial pathogens globally.
These can be broken down into 4 key outcomes.
1) sequenced > 9000 accessions of the Xanthomonas plant pathogen
2) reclassification, new Xanthomonas species identified
3) screened > 300 brassica lines to identify and characterise resistance markers to the two most important black rot races in Europe - Race 1 & Race 4.
4) Undertook Plant risk Assessments for possible threats of Xanthomonas to Maize, Strawberry, Watercress and Wallflower
5) Developed a novel reporter construct that enabled, for the first time, monitoring of Xanthomonas campestris spread from the infect leaf, through the inflorescence meristem, to the flower and into the seed. Demonstrating that the bacterial survived in the seed
Exploitation Route Established interactions with Industry (Seed Companies) for Disease Resistance/Xanthomonas Genome Sequencing/Gene editing of Brassica
Sectors Agriculture

Food and Drink

URL https://bacterialplantdiseases.uk/curious-about-cabbages-visiting-lincolnshire-seed-companies/
 
Description Developed Plant Risk Assessments for potential impact of Xanthomonas on Strawberry and Maize and concluded; X. vasicola pv. vasculorum is a threat for maize production in Europe X. fragariae is probably of minor importance but a possible threat because popular strawberry varieties are susceptible. Showed for the first time that Xanthomonas campestris can move from infected leaves to seed, thus proof local infection can lead to "dirty" seed. This has biosecurity implications and discussions how this can be used in the seed industry are ongoing. Developed diagnostic primers for identifying Xanthomonas spp and Xanthomonas campestris pv. campestris races. At time of writing developing diagnostics for X. vasicola pv's and Xanthomonas campestris pv's the former to support the biosecurity threat identified in the PRA for maize and the latter for two interest companies, Elsoms seed (UK) and Sakata (Japan).
First Year Of Impact 2024
Sector Agriculture, Food and Drink
Impact Types Policy & public services

 
Description Chancellor's International Scholarship
Amount £120,000 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 01/2022 
End 12/2025
 
Description Daphne Jackson Fellowship
Amount £45,543 (GBP)
Organisation University of Surrey 
Department Daphne Jackson Trust
Sector Charity/Non Profit
Country United Kingdom
Start 11/2020 
End 11/2023
 
Description PhytoBacExplorer: A Phylogenomic Resource for the Phytobacterial Community
Amount £463,477 (GBP)
Funding ID BB/W019183/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2022 
End 06/2027
 
Description Univeristy of Warwick MIBTP
Amount £120,000 (GBP)
Funding ID BB/T00746X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2021 
End 10/2025
 
Title Novel tool to image Xanthomonas infection 
Description Xanthomonas campestris pv. campestris cell lines with a dual reporter for whole plant imaging in real time vascular infection (temporal-spatial) and an mScarlet reporter (unlike GFP/YFP works in Xanthomonas) to look at tissue, cellular localisation of the infecting bacteria. Reporter is modular so can be used in other Xanthomonas spp/pathovars providing the promoter is suitable. 
Type Of Material Cell line 
Year Produced 2023 
Provided To Others? Yes  
Impact First visualisation of systemic movement of the vascular pathogen, Xanthomonas campestris pv. campestris from a hydrathode infection to a developing seed and subsequent validation that it is seed bourne - which is most likely the primary cause of seed contamination in the field. 
 
Title Pipeline for automated annotation of Xanthomonas genomes 
Description A beta pipeline has been developed for the automated annotation of sequenced Xanthomonas genomes. This will be improved over the course of the next 12 months. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact N/A yet. 
 
Title Genome sequencing of Xanthomonas campestris pv. campestris 
Description Xanthomonas campestris pv. campestris is a gram-negative bacterium that causes black rot disease in cruciferous plants, including cabbage, cauliflower, and broccoli. It is a plant pathogen that affects the vascular system of the host plants, leading to severe damage and economic losses in agricultural settings. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Provides a comprehensive set of Xcc genomes to develop diagnostic primers and used in publications to validate taxonomic reclassification 
URL https://www.ncbi.nlm.nih.gov/bioproject/982295
 
Title Genome sequencing of Xanthomonas fragariae 
Description Xanthomonas fragariae is a bacterial pathogen that causes strawberry angular leaf spot disease, which is characterized by the appearance of angular or V-shaped water-soaked lesions on the leaves of strawberry plants. These lesions may turn brown or dark red as the disease progresses. The bacteria are typically spread through splashing water, wind, or contaminated tools and equipment. The infection can lead to reduced plant vigor, stunted growth, and yield losses in strawberry crops. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Provides a dataset for develop bespoke diagnostic primers against strawberry - identified as a possible threat to UK strawberry industry. 
URL https://www.ncbi.nlm.nih.gov/bioproject/985246
 
Title Genome sequencing of Xanthomonas vasicola 
Description Here we sequence genomes of two further strains of X. vasicola pv. musacearum from Africa plus an additional X. vasicola pv. vasculorum from Madagascar, which is geographically distinct from previously sequenced strains of this pathovar 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact X. vasicola pv. vasculorum has been diagnosed through the project as a threat to UK maize production. Most of the sequenced isolates sequenced in this project that were virulent on maize were monophyletic. These three strains are important for diagnostic validation and additionally, the X. vasicola pv. musacearum sequences support development of X. vasicola.pv diagnostics. 
URL https://www.ncbi.nlm.nih.gov/bioproject/743420
 
Description Genomic characterisation of Serbian Xanthomonas isolates 
Organisation Institute for Plant Protection and Environment
Country Serbia 
Sector Public 
PI Contribution Established a collaboration with Serbia's foremost phytobacteriologist, TP,
Collaborator Contribution TP and her team have supplied more than 200 pathotyped strains of Xanthomonas from a variety of crop and tree spp to supplement our Warwick collection. This invaluable resource covers a geographical as well as host range absent from out collections, thus adding considerable value to our phylogenomic analyses.
Impact Too ealry - currently preparing for sequencing at time of writing
Start Year 2022
 
Description Japanese Xanthomonas collaboration 
Organisation Sakata Seed
Country Global 
Sector Private 
PI Contribution Following a BBSRC Training Grant FTMA4 'Opportunities for researcher and stakeholder mobility within the Bacterial Plant Diseases programme' award involving visits to elsoms seed s and Sakata seeds in Lincolnshire we were subsequently contacted by the Sakata seed company, one of the largest suppliers of Brassica seed to the UK. They want to collaborate and even pay for race ype screening. Development of MTA;s is in progress for the following; Whole genome sequencing of 20 Xanthomonas campestris campestris (Xcc) races from Japan; Providing Sakata with our core Xcc race typing collection; Providing Sakata with our Brassica lines fro race typing their field Xcc isolates.
Collaborator Contribution Our partners were proactive in approaching us to develop this collaboration based on the tools directly developed in this project. They realised the enormous potential of these and this was based on their assessment that climate change in Japan (in particular) would exacerbate the threat of Xcc to Japanese agriculture.
Impact Just established
Start Year 2024
 
Description Xanthomonas bacterocins 
Organisation University of Glasgow
Department School of Life Sciences Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution We are working collaboratively with Dr Joel Milner to identify novel bacterocins produced by Xanthomonas as well as test other bacterocins (predominately those produced by Pseudomonads) for their ability to kill agronomically important Xanthomonas phytopathogens, both from a collection of pv. campestris and pv. raphinii isolates.
Collaborator Contribution The partner undertakes the screening with the purified bacterocins (blinded) and we undertake the genomic mining for novel bacterocins with our unique collection of sequenced Xanthomonas isolates.
Impact We have, with BBSRC FTMA visited Glasgow and with their expertise sorted out methodology and even got activity with one of the 4 bacteriocins we have cloned and expressed to date.
Start Year 2020
 
Description Conference presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presented at the BSPP Presidential Meeting, Our Plants Our Future on the Bacterial Plant Diseases Initiative and the Xanthomonas Threats progress.
Year(s) Of Engagement Activity 2021
 
Description Conference presentation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Study participants or study members
Results and Impact Presentation of results to date in the Xanthomonas Threats project at the annual Bacterial Plant Diseases Initiative meeting
Year(s) Of Engagement Activity 2023
 
Description Invited Talk BSPP Birmingham UK, 8th September 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Conference presentation (and posters; https://zenodo.org/records/8330865
https://zenodo.org/records/8330893 ; https://zenodo.org/records/8392974; https://zenodo.org/records/8390337) at the British Society of Plant Pathogens Metting
Year(s) Of Engagement Activity 2023