Oats for the future: deciphering potential of host resistance and RNAi to minimise mycotoxin contamination under present and future climate scenarios

Mycotoxins are natural contaminants of a wide range of staple foods produced by fungi under conducive environmental conditions. The consumption of mycotoxin contaminated foods can result in several acute and chronic diseases in humans and animals. Oats, which have become more popular because if their health benefits can be infected by a Fusarium species, F.langsethiae, which can contaminate oats with the mycotoxins T-2/HT2 toxins. The EU has maximum recommended levels for these two toxins in place. F.langsethiae infects oats during ripening but produces no visible symptoms and is thus difficult to assess except by measuring the mould biomass in the ripening oat grain or by analytically quantifying the amounts of T2/HT-2 present. Limited work has been done on screening oat cultivars for resistance to F.langsethiae infection. Climate change (CC) scenarios has been suggested to probably exacerbate fungal infection and mycotoxin production partially via increases in pest reproduction and damage to ripening crops. Thus there is interest in understanding the relationships between interacting factors of CC including elevated temperature (+2-4oC), x2 or x3 existing CO2 concentrations (400 ppm vs 800 and 1200 ppm CO2) and episodes of drought stress. Very little information is available on how these interacting CC factors impact on infection by this Fusarium species and contamination of oats with the associated mycotoxin. This project is a collaborative project between Cranfield University in the UK and University College Dublin in Ireland to jointly address the issues of a better understanding of the relationship between this important fungal pathogen on different oat cultivars, the impact that CC factors will have on infection and mycotoxin contamination and the development of some novel methods for blocking the production of the mycotoxins.
The objectives can be summarised as being (a) In vitro examination of the effect of interacting environmental factors on growth and expression of biosynthetic gene clusters of F.langsethiae during colonisation of oat-based media and oat grains and on T-2/HT-2 toxin production by strains from the UK and Ireland. The effect of interacting CC factors the life cycle of the pathogen will be quantified. RNA-Seq technology will be used to study the gene regulation, focusing on the trichothecenes type A gene cluster for the first time; (b) a range of UK and Heritage Irish oat lines will be screened for distinct gene expression profiles relevant to differential mycotoxin contamination and potential resistance using sequencing approaches; (c) 'In planta' studies using two heritage and UK oat CVs will examine CC factors on plant/fungus gene expression patterns and differential mycotoxin contamination using non-acclimatised and acclimatised strains of F.langsethiae. The effects on shifts/fluctuations in T-2/HT-2 toxin production will be quantified; (d) the relative competitiveness of F.langsethiae and other Fusarium species such as F.graminearum and F.sporotrichioides and oat mycobiota will be examined under normal agronomic and CC conditions in in vitro and in planta studies on dominance at a molecular, colony and ecosystem level; and (e) minimisation strategies using interference RNA technology and novel delivery systems to interfere with the key TRI genes involved in T-2/HT-2 production. In planta trials will be carried out to test control efficacy of toxin production under normal agronomic and CC conditions for the first time.

This project focuses on the oat:Fusarium pathosystem, to understand the genetic diversity in UK and Irish heritage oat cultivars and unveil the genetic traits related to resistance to F.langesthiae infection and T-2/HT-2 toxin contamination as well as determining genome sequences and trancriptome analyses under existing and future climates change scenarios and develop novel control strategies using RNAi approaches. The project will screen a range of UK recommended husked and naked cultivars and heritage Irish oat cultivars to F.langsethiae using a combination of toxin profiling, genotyping by sequencing and RNAseq. Potential lines with differential sensitivity/resistance to mycotoxin accumulation will be identified by examining pathways/genes associated with reduced mycotoxin loads. Acclimatisation of UK and Irish strains of F.langsethiae by repeated culturing under forecasted CC conditions expected in the next 25-50 years (10 generation; Vary et al., 2015) will be used to compare infection of oat cultivars by using molecular signals involved in infection of oat cultivars and relate this to TRI gene expression and T-2/HT-2 mycotoxin contamination. This will facilitate a better understanding of the impact of CC on oat cultivation and the routes for better resilience by integration of molecular, ecophysiological and phenotypic trichothecene production in vitro and in planta for this cereal pathosystem. Minimisation strategies for T-2/HT-2 toxin contamination of oats will use RNAi approaches using quantum dots as carriers to inhibit key TRI genes (e.g. the TRI5 regulatory gene) involved in the biosynthesis of trichothecene mycotoxins. This project will benefit from the complimentary pre-harvest molecular/epidemiological and cultivar genotyping and RNA sequencing expertise and CC facilities at UCD, and the Cranfield facilities and expertise in molecular ecology, ecophysiology, metabolomics and prevention strategies.

The key pathways to impact are via national UK and Irish beneficiaries and in Europe and Internationally. This will be achieved through meaningful engagement with key stakeholders and beneficiaries, as described below.
Stakeholders involved in farming and the food security agenda
It will produce valuable new data on the impact of climate change scenarios on differential mycotoxin contamination of oats. Engagement with stakeholders involved in the farming and the food security agenda will ensure a better understanding of the effect of climate change factors in which mycotoxigenic fungi and mycotoxins may become dominant and the impact that this will have on downstream food and feed chains. A number of initiatives are planned in this regard:
1. We will provide summary information on oat cultivars which may have better resistance to F.langsethiae and T-2/HT-2 toxins and the boundary conditions over which they may be produced in terms of both growth and toxin production. Thus, summary moisture sorption isotherms indicating the moisture contents and temperatures which represent low and high risk where recommended limits may be exceeded during ripening, drying of harvested grain and storage will be provided. These will be made available via the project web site and directly to R&D managers in the Food Standards Agency (FSA), Agricultural and Horticultural Development Board (AHDB), British Oat and Barley Millers Association (BOBMA), UK and Irish Millers Association (Nabim), European Millers Association (EOM), European Flour Millers Association (GAM) as Summary documents after 18, 36 and 48 months of the project..
2. We will ensure that there is effective dissemination of the science via contacts in the grain industry in the UK and Ireland (AHDB, BOBMA, Nabim), in Europe (GAM, European Millers Association) and via the farming press to notify stakeholders of the key useful messages which come out of this project which can be practicably beneficial. This includes a minimum of two articles per annum in publications such as Farmers Weekly, The Agronomist, Food Engineering and Ingredients, New Food Magazine. An example of similar activity is an article entitled: Mycotoxins, food security and climate change: do we know enough? This will be published in the February, 2016 issue of Microbiology Today.
3. After the appropriate scientific publications (as described in the data sharing assessment) we will make the genome sequence of F.langsethiae publicly available in the project website which will be hosted by Cranfield University but linked to UCD for access by interested stake holders nationally and internationally. This will be beneficial for researchers to compare their genomes or find individual gene similarities. This will open further pathways for research and will contribute to the general knowledge of mycotoxigenic fungal species.
4. After 24 and 48 months we will provide press releases on the mid-term and final summary outcomes and the use of the data for better mycotoxin management in the context of oats and climate change and the food security agenda in the farming press and the stakeholders detailed above.
Legislators and policy makers: 1. We will provide underpinning research results for future legislative developments. The strategies developed in this project can be applied to other pathosystems (e.g. Fusarium graminearum & Aspergillus flavus-maize, F.graminearum-wheat), which will be beneficial in the UK/Ireland and EU generally where information can assist in framing future legislative limits on different mycotoxins. The methodology/models developed in this project will help to inform legislators examining new emerging risks. 2. Inform the Contaminants Division of the FSA, EFSA and JECFA on the results in the context sustainable food production and food security.