Exploiting next generation sequencing technologies to understand pathogenicity and resistance in Fusarium oxysporum

Lead Research Organisation: National Inst of Agricultural Botany
Department Name: Centre for Research

Abstract

Onion is an important horticultural crop which is cultivated by every agricultural nation in the world and is also the second most valuable vegetable crop in the world behind tomato. Onions are a staple crop in many countries and deliver a range of health benefits including anticarcinogenic, antithrombotic and antibiotic effects. Despite the value of the crop, research into breeding and genetic improvement is limited as few resources such as onion diversity sets, pure breeding lines and genomic information are available.

Diseases are one of the major constraints to onion production and one of the most important is caused by the soilborne plant pathogen Fusarium oxysporum. This fungus is diverse and has many different sub-species (formae speciales, f.spp.) which attack various crop plants. In onion, F. oxysporum f.sp. cepae (FOC) can infect plants at any stage causing a 'damping-off' symptom on seedlings and a basal rot on more mature plants and bulbs. This results in severe pre- and/or post-harvest losses and has been estimated to cost farmers in the UK in the region of £11M per annum. As FOC is a soilborne pathogen which produces long-lived spores that survive for many years, control approaches are difficult and have previously relied on the use of soil sterilisation / pasteurisation, drenches with fungicides or seed treatments. This approach has largely been unsuccessful, has undesirable environmental effects and is threatened by legislation governing restrictions in pesticide use. In the absence of effective control measures for Fusarium basal rot, identifying resistance in onion is extremely desirable but so far has been relatively unsuccessful. However, using a highly pathogenic FOC isolate in a rapid screening test we developed using onion seedlings, we have identified onion lines with much higher levels of basal rot resistance than current commercial cultivars.

In a collaborative project between the University of Warwick, East Malling Research and the intenational vegetable breeding company Nickerson-Zwaan, we propose to provide information, tools and resources which will lead to more effective and sustainable control of Fusarium basal rot, primarily through the development of FOC-resistant onion lines. This will benefit a wide range of stakeholders including breeders, growers and other researchers. The main outcomes of the project will be 1) the identification of FOC pathogenicity/effector genes which could be used as markers to distinguish this pathogen from other F. oxysporum f. spp. or non-pathogenic isolates, 2) the identification of FOC resistance loci and associated genetic markers in onion for use in future breeding programmes, 3) the production of new onion populations segregating for FOC resistance and pre-breeding onion lines to enable the development of basal rot resistant onion cultivars for the industry. Growers and the industry will clearly benefit from this research as the deployment of resistant cultivars will give them a more sustainable and attractive option for basal rot control in onion in the future. In addition, DNA markers associated with genes controlling FOC pathogenicity should also provide a platform for developing diagnostic and quantitative tests for the pathogen in soil, onion seed, sets and bulbs which will help farmers make decisions about disease risk and develop management options. Overall this means that the public will benefit from better quality onions grown with reduced pesticide inputs.

Technical Summary

This research will examine the genetic basis for pathogenicity of the basal rot pathogen Fusarium oxysporum f.sp. cepae (FOC) and resistance to this fungus in onion. The main objectives are to 1) to identify FOC pathogenicity/effector genes which could be used as markers to distinguish this pathogen from other F. oxysporum formae speciales or non-pathogenic isolates, 2) to identify FOC resistance loci and associated genetic markers in onion for use in future breeding programmes and 3) to produce pre-breeding FOC-resistant onion lines to enable the development of basal rot resistant onion cultivars for the industry. The project is split into four work packages (WPs). WP1 will use whole genome sequencing to understand the basis for FOC pathogenicity. We will sequence a set of F. oxysporum isolates from onion previously characterised as pathogenic or non pathogenic, compare with existing F. oxysporum genomes and identify effectors and other pathogenicity-related genes. In WP2 we will use genotype by sequencing to characterise individuals from an existing F2 onion population segregating for FOC resistance and create a genetic map. We will then identify SNPs linked to FOC resistance loci and convert them to breeder-friendly KASPar markers. In WP3 we will examine differential gene expression in interactions between pathogenic / non pathogenic Fusarium isolates and susceptible / resistant onion lines using RNA-seq and NanoString to further elucidate the genetic basis for these traits. Finally in WP4 we will generate new genetic resources segregating for FOC resistance with our industry partner Nickerson-Zwaan using the latest breeding techniques. These will involve FOC-resistant/FOC-susceptible double haploid lines crosses and FOC resistant non-DH individual plant/susceptible DH line crosses. The project will hence utilise data driven biology through the use of next generation sequencing approaches to identify and understand natural variation in genes controlling traits.

Planned Impact

Onions are grown in virtually every country in the world and have a production value of US$ 24,698M. However, considerable yield losses occur due because of Fusarium basal rot caused by F. oxysporum f.sp. (forma speciales) cepae (FOC), and in the UK this is equivalent to economic losses of approx £11M per annum. The major aim of this proposed research is to provide information, tools and resources which will lead to more effective and sustainable control of this disease. Primarily this will be through the development of genetic resources and markers to enable the breeding of FOC-resistant onions. However, we will also identify genetic markers to distinguish FOC from non-pathogenic F. oxysporum isolates or other f.spp. affecting different crops which will ultimately lead to tests for detection and quantification the pathogen. This integrated approach to basal rot control will therefore benefit a wide range of stakeholders and also addresses the BBSRC strategic priority area of sustainable crop production, particularly with respect to reducing chemical usage and waste.

Commercial beneficiaries of the research will include plant breeders, seed producers and growers. The primary and immediate beneficiary will be Nickerson-Zwaan (NiZ) who is the industry partner in the project as they will have access to unique onion lines which they will utilise to develop elite FOC resistant cultivars. Their involvement and investment ensures that there is a direct route for delivery of the research through their existing global product development and marketing infrastructure. This process will be accelerated by the information, tools and resources generated in the project which include the identification of FOC resistance loci and associated genetic markers in onion, the production of new onion populations segregating for FOC resistance and pre-breeding onion lines. Although NiZ will have access to these resources first, these will also be made publicly available for the benefit o other onion breeders. Onion mapping populations, genome data and availability of other genetic resources in particular should facilitate breeding for other traits such as resistance to other disease and pests and daylength response. Markers for pathogenicity in Fusarium will also be an immediate output and could be informative for identifying effector-based resistance in other crops affected by F. oxysporum which will again benefit breeding companies.

Growers will benefit from the research as in due course they will have access to Fusarium resistant onion cultivars not previously available. This will give them a more sustainable and attractive option for basal rot management as chemical control of the disease is difficult, inefficient, becoming more undesirable and subject to loss of products through legislation. Hence there will be reduced losses and wastage due to bulb rotting in field or store with concomitant increased economic returns. The markers for Fusarium pathogenicity will also provide a platform for developing diagnostic and quantitative tests for the pathogen in soil, onion seed, sets and bulbs. This will also benefit growers and crop consultants as they will be able to determine the health of their planting material and select appropriate pathogen-free fields for onion growing. They will also be able to assess whether bulbs at harvest are FOC-free and hence make decisions about their storage capability.

The research towards developing onion lines resistant to onion basal rot and detection tests for FOC in this proposed research therefore represents two different approaches in developing a more sustainable integrated strategy to reduce Fusarium basal rot losses. The public will benefit by having access to high quality onions grown with reduced pesticide inputs. The research will also result in the generation of a wealth of data associated with onion and Fusarium genetics and eill hence benefit researchers and academics working in these areas.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/K020730/1 01/04/2014 31/01/2016 £102,276
BB/K020730/2 Transfer BB/K020730/1 01/02/2016 31/03/2017 £39,874
 
Description The soilborne fungus Fusarium oxysporum is a species complex that includes both pathogenic and non-pathogenic isolates. The many pathogenic forms of the fungus (known as formae speciales - f.spp.) are each adapted to particular host plants and cause some of the most devastating diseases of crop plants throughout the world. F. oxysporum f.sp. cepae (FOC) is the form that affects onion and is a major constraint to production globally with estimated losses in the UK of at least £11M per annum. FOC causes a basal rot which is most damaging as bulbs mature in the field or post-harvest in store. FOC also produces long-lived spores, which makes control extremely difficult, especially as traditional management strategies such as soil sterilisation/pasteurisation, fungicide drenches or seed treatments are often uneconomic or inefficient, can have negative impacts on the environment and are under threat or already banned through legislation. Plant resistance to basal rot in onion is therefore highly desirable, but so far successful onion cultivars have not been produced. One of the main aims of this HAPI project was to identify genetic markers associated with Fusarium basal rot resistance for use in breeding programmes using onion lines selected from a unique onion diversity set created at Warwick. A further aim was to identify the genetic basis for FOC pathogenicity which could allow the pathogen to be distinguished from other f. spp. or non-pathogenic F. oxysporum. High level resistance to FOC was confirmed in several onion lines using both seedling and mature onion plant tests. Onion populations segregating for resistance were also genotyped using more than 400 KASP markers and subsequent analysis has identified several associated quantitative trait loci and molecular markers. With refinement, these will be invaluable for use in breeding programmes to speed up selection of resistant onion lines. Our industrial partner Hazera Seeds is continuing to produce new double haploid onion populations segregating for resistance to help refine and improve the genetic analysis as well as pre-breeding material as a first step to producing an elite FOC-resistant onion cultivar. A set of 32 F. oxysporum isolates from onion were also extensively characterised through sequencing of housekeeping and putative effector genes as well as pathogenicity tests on onion seedlings and bulbs. For the first time we showed a clear association between the presence of seven of the fourteen known 'Secreted in Xylem' (SIX) genes (first described in F. oxysporum f.sp. lycopersici affecting tomato) and pathogenicity on onion. Non-pathogenic isolates did not contain any SIX genes. A subset of the FOC isolates was genome sequenced and analysis has shown that pathogenicity genes are clustered on lineage specific regions and several other putative effectors have also been identified. Furthermore, using an agar based system for inoculation of onion seedlings, we demonstrated that the SIX and other putative effector genes are all expressed 'in planta'. The development of molecular markers associated with FOC resistance and pre-breeding material in this project will facilitate the future development of new onion cultivars with resistance to basal rot disease. Furthermore, the presence / absence of SIX genes in FOC, and their unique sequence compared to other F. oxysporum f.spp. has allowed a specific PCR-based diagnostic to be developed for FOC through further AHDB funding in a related project. This allows FOC to be distinguished from non-pathogenic F. oxysporum and other f.spp. for the first time and will enable quantification of the pathogen on onion seeds, sets, bulbs or in soil. This approach is also being developed for other F. oxysporum f.spp affecting narcissus and column stocks. These developments have been of great interest to the industry and have been communicated through presentations and trade press articles. A paper describing the significance of SIX genes in FOC pathogenicity has been published as well as a further paper on FOC genome analysis.
Exploitation Route The FOC PCR diagnostic can potentially be used to assess pathogen levels in onion seeds, sets or bulbs and soil. This will help growers assess disease risk and be better able to manage basal rot. Similar diagnostic approaches based on pathogenicity gene targets may also be appropriate for other F. oxysporum f.spp. The identification of molecular markers appropriate for onion could be used by breeders to speed up the selection of new improved lines with FOC resistance.
Sectors Agriculture, Food and Drink

 
Description Further development is currently being pursued through an AHDB project. In this project we built upon the knowledge from this HAPI programme of which key effectors tag Fusarium oxysporum fsp. cepae in soils. This allowed (for the first time ) an NGS based diagnostic of FOC that tags effectors at the same time as tagging many other microbes in the soil and charting their relative abundance. This lays the foundation for modelling approaches to predict Fusarium diseases in real-world situations.
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Developed application with AHDB
Amount £309,921 (GBP)
Funding ID POBOF 452 
Organisation Agricultural and Horticulture Development Board 
Sector Public
Country United Kingdom
Start 04/2017 
End 11/2018
 
Description Apple canker workshop at Yangling A&F University 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attendance and contribution at a workshop associated with BBSRC China Partnering funding on apple canker research. This allowed presentation on Apple canker research at NIAB EMR, involvement in discussions on European and Asian apple cankers and also highlighting genomic research performed across other projects at NIAB EMR associated with development of molecular diagnostics. Genomic work performed on apple canker at NIAB EMR led to discussions on joint publications between groups. Discussion on Fusarium and how we have developed molecular diagnostics from that work led to discussion on further research projects.
Year(s) Of Engagement Activity 2018
 
Description Food secutiry for vulnerable populations: the fungal threat 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation and group discussion under the Researcher Links scheme. This was a joint meeting funded by the British Council and FAPESP in Ribeirao Preto, Brazil on 11-14 September 2018. The workshop was coordinated by Simon Avery (Univ. Nottingham) and Gustavo Goldman (Univ. Sao Paulo), and had contributions from other experienced researchers. My presentation covered work on Fusarium, Phytophthora, Verticillium and Alternaria at NIAB EMR, raising the profile of the institute and of the funded genomic research that we have been performing. This led to discussions on future research projects and possible new joint-funded grants.
Year(s) Of Engagement Activity 2018
 
Description Practical Course on Plant Pathogen Diagnostics: From Symptoms to Sequence 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Hosting of a workshop on pathogen diagnostics and identification for researchers, agronomists, farm managers and all members of public interested in plant diseases.
This workshop covered:
Theoretical and practical demonstrations of core plant pathogen diagnostics skills
Diseases on annual and perennial crops
The latest developments in molecular detection and classification of pathogens
High throughput detection and imaging tools for large scale environmental monitoring

In addition to organisation and running of the workshop, a presentation was given highlighting how BBSRC funded research using next generation sequencing is influencing Fusarium identification, diagnostics and control.
Year(s) Of Engagement Activity 2018
URL http://www.emr.ac.uk/events/practical-plant-pathogen-diagnostics/
 
Description Presentation at AHDB CFC open evening 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation at the Cut Flower Centre's (CFC) open evening, highlighting challenges faced by industry and research being done to address these problems. We gave one of the four presentations of the evening describing the use of genomic approaches to develop new diagnostic tests for Fusarium diseases. This gave business representatives and professional practitioners greater understanding of how funding basic science can lead to applied benefits to production. Trade media documented the event leading to coverage in trade magazines and impact on a wider audience beyond the ~100 delegates in the audience.
Year(s) Of Engagement Activity 2018
URL https://horticulture.ahdb.org.uk/event/cut-flower-centre-open-evening-2018
 
Description The Third International Horticulture Research Conference (Nanjing, China) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Third International Horticulture Research Conference brought together international researchers conducting basic research on horticultural crops, to showcase the latest research findings. Andrew Armitage presented a poster at the conference titled "Lineage specific regions in the onion basal rot pathogen Fusarium oxysporum f. sp. cepae" winning runner up poster prize.
Year(s) Of Engagement Activity 2016
URL http://www.hortres-conference.org/uploadfiles/The%20Third%20International%20Horticulture%20Research%...