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

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 Traditional bactericides such as copper oxychloride are being withdrawn from sale.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".

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.

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.