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

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 international 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.

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.

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 of 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.