Concepts and methodologies

General concepts and methodologies

Concept

The overall concept of the project is to seek European solutions to solve challenges caused by wheat rust disease (yellow-, leaf- and stem rust) - emerging pathogens which spread across national borders and affect major food crops in EU member states. This involves inter-disciplinary collaboration between plant pathology, breeding, agronomy, agro-chemistry, molecular genetics and Information and Communication Technologies (ICT), i.e., to increase the expertise and efficiency in rust monitoring within these stakeholder networks. Research activities will interact at all these steps and will involve two-way communication with stakeholders, recognizing their expertise to resistance breeding, disease surveillance and pathogen sampling. IPM-based disease management will involve project partners as well as larger lead users by linking with pre-existing stakeholder networks within plant breeding, value-for-cultivation-and-use (VCU) trials, agricultural advisory services and the agrochemical industry

Development of the European early warning system for wheat rust is based on  a multi-actor approach, which takes into account new knowledge about drivers for spread and establishment of new rust races and their implications for disease prevention and control. The system will enable access to shared facilities and it will be adopted and validated in 5 case study regions.

The proposed early warning system will be implemented via case studies in five target regions, which serve two main purposes: i) development and validation of the early warning system in collaboration with a wide array of stakeholder groups, ii) engagement of agricultural advisers and ultimate end-users (farmers), involving them in disease surveillance and rust sampling, and getting valuable information about potential barriers in adopting specific recommendations.

The case study approach will allow us to investigate the threats of invasive rust races at the regional scale and suggest IPM-based prevention and control options adjusted to the relevant agronomical practices and environmental conditions in the considered region. Each case study region will host at least one IPM trial and engage stakeholder groups in workshops and Open Field Days. Feed-back from the case study region activities will contribute to the development of the proposed early warning system.

Aarhus University hosts the databases and web tools via the Wheat Rust Toolbox IT platform, which feeds into associated websites like RustTracker (http://rusttracker.cimmyt.org/), EuroWheat developed in the ENDURE network (http://www.endure-network.eu/), and the GRRC web site (wheatrust.org). The Toolbox is a data management and analysis system that also generates maps and charts, which are disseminated via external web sites. RustWatch will make use of the Wheat Rust Toolbox and disseminate results and early warnings via www.EuroWheat.au.dk. In conclusion, most rust research within the EU as well as national and international based project activities on rust surveillance and dissemination have had participation of RustWatch partners. New activities in RustWatch will build on the results and lessons learned from these previous projects, including EU funded projects, which ensures progression and that duplication can be avoided.

Methodology

Prevention and control of rust fungi is highly dependent on the current and future composition of the rust pathogen populations, which are dynamic due to pathogen evolutionary forces within Europe, coupled with the spread of new races from non-European countries. Regular pathogen monitoring and easy-to-use diagnostic tools are therefore essential. A very high priority is increased, timely access for lead users within the plant breeding, agrochemical, extension and VCU testing sectors to information about pathogen race and genotype, as well as access to physical facilities and expertise to support host resistance phenotyping. The concept of shared facilities for improved access to the four rust diagnostic lab facilities with long-term expertise (UK, France, Germany and Denmark), and the possibility for submission of rust-infected wheat from countries without national rust diagnostic capacity, will allow rapid, efficient diagnosis of new, invasive races at the earliest possible time.

The work is organised in 5 WP’s.

WP1 Methodologies

In WP1, new molecular genotyping tools and procedures, improved race phenotyping, quantification of pathogen aggressiveness and adaptation to different temperature regimes, and extending sampling areas beyond countries with national rust diagnostic labs (in collaboration with WP2 and WP3) make up the cornerstones for understanding the biology and drivers of the wheat rust populations in Europe. Five types of activities will be undertaken, i.e., 1) molecular genotyping and virulence phenotyping of isolates from countries without access to national rust diagnostic labs, 2) alignment of results between existing national rust diagnostic labs (activities largely funded by national projects) based on exchange of reference isolates and methodology, 3) assessment of pathogen aggressiveness and adaptation to warmer temperatures for selected strains, and 4) molecular genotyping of samples of rust-infected leaves of Berberis vulgaris, the sexual host of Pst and Pgt. The use of race phenotyping based on differentials will be integrated with new molecular genotyping procedures, which allows rapid detection of novel genotypes and unique resolution of population genetic grouping. We will test the potential of innovative new amplicon sequencing techniques and mobile sequencing platforms for rapid on-site detection of rust strains. The main effort will be devoted to yellow rust, but in view of the recent stem rust epidemics in Southern Italy (2016) and Russia (2015, 2016), and frequent epidemics of leaf rust in Central and Southern Europe, these pathogens will also be included in the research.

The most appropriate markers will be used as basis for population genetic analyses of new races of yellow and stem rust in the context of at least 1500 reference isolates of non-European origin available at GRRC and INRA, and allow conclusions about i) where new invasive rust races are coming from, ii) the processes generating pathogen diversity and, iii) spore transmission and long-distance spread to Europe.

Less knowledge is available about Pt populations in Europe, so the first immediate task will be to analyse genetic variability and composition of the European Pt population based on stock isolates collected in recent years by different RustWatch partners. INRA will lead this activity.

WP2 Methodologies

WP2 will develop new strategies and procedures for host resistance phenotyping of wheat varieties and breeding lines by integrating in-vivo assays utilizing a panel of rust races and SNP analyses based on the design of a primer set, which will be dedicated to rust R-Gene genotyping. The underlying development of SNP assays targeting up to 96 rust R-genes is done by INRA-GDEC, and will be made available for all interested RustWatch partners. The latter is considered to greatly enhance capacity for the characterization of rust resistance in commercial wheat as well as in breeding lines (up to several 100s of entries per year). The characteristics of different sources of resistance will be investigated by new microscopic methods at the cellular level. Different categories of resistance phenotypes based on host responses will be identified, allowing plant breeding to combine such resistances in individual varieties, thereby increasing diversity for rust resistance. This is expected to increase time and area, where new varieties will maintain adequate levels of resistance for preventing new rust epidemics (i.e., increasing ‘durability of resistance’ sensu Roy Johnson (Johnson, 1984)). New inoculation procedures under quarantine conditions will allow assessment of specific (qualitative) and partial (quantitative) resistance to new races before they become widespread in Europe, allowing rapid, precise identification of varieties that may be particularly vulnerable to new invasive races detected in WP1. WP2 will take advantage of the ongoing genomic revolution in wheat by integrating information about cloned and putative R-genes, and sequence based markers associated with rust resistance, taking R-gene deployment (i.e., host diversification) into a new era where we will be able to monitor and suggest host resistance diversification at the regional/national landscape level.

WP3 Methodologies

WP3 aims to build up and develop stakeholder networks, enabling access to shared facilities in WP1 and WP2, and validating the new early warning system in 5 case study regions, representing different agro-ecological features with respect to the climate and to the average usage of fungicides. Pre-existing stakeholder networks and services within plant breeding, agrochemical industry, extension services and VCU approval in cereals will be used to assist rust surveillance and sampling. A series of field trials in at least six regions will be used for validation of specific solutions for rust management following the principles laid down in IPM Directive 2009/128/EC, e.g., using cultural methods, partly resistant varieties and variety mixtures, and synthetic fungicides and bio-pesticides with different levels of input and potentially new active ingredients. Strategies without synthetic fungicides should be appealing to both organic and conventional farmers. This WP will also develop and implement a unique concept of shared facilities for improved utilization and access to field facilities in Europe. It will also establish a system of field trialling in the “centre of diversity” of the Pst fungus in the Himalayas, where European wheat varieties and breeding lines can be tested in a ‘worst-case’ scenario with maximum pathogen variability. In turn, samples from high-yielding breeding lines and varieties with promising rust resistance under European conditions, which may be susceptible to rust in Pakistan, will be selected for genotyping and race typing (WP1) to specifically evaluate the disease vulnerability of such lines. A set of at least 6 wheat lines differentiating Pst races will be planted at VCU trial sites across Europe to facilitate representative sampling of wheat rust populations across Europe, to be tested WP1. The five case study regions will also be used to validate the new early warning system for wheat rusts , and feed back from lead users and end user farmers will be taken into account. Hands-on workshops and training in disease assessment, sampling, analysis and use of tools and services generated in the project will also be organized in the context of case study regions with inputs from participants in WP1, WP2 and WP4. Specific tasks and key stakeholder representatives have been appointed to facilitate communication with non-partner companies and organizations (stakeholder network coordinators) allowing unusual rust disease patterns at any time and location to be recorded and samples to be collected and diagnosed. This will ensure the implementation of new solutions for prevention and control of rust diseases beyond project partner organizations and companies, reflecting a true multi-actor approach.

WP4 Methodologies

Data emerging from WP1, WP2 and WP3 will generally be handled via tools for data management and displays developed in WP4. The disease surveillance data and race and genotypic data will be stored and analyzed via the Wheat Rust Toolbox, which has been developed to host data from projects within the Borlaug Global Rust Initiative (www.globalrust.org). This approach will allow analyses of new pathogen population genetic data from this project in a global context. New web tools and smartphone applications will be developed for management, analysis and display of data including results from case study regions. All these tools and applications will be generic and they can at any time be adopted by any country wishing to be included in the early warning system. RustWatch will integrate with existing national surveillance databases, harmonize data and show information in a pan-European context on maps and charts. RustWatch will adapt and offer a population genetics analysis platform for molecular data (e.g., the Shiny-R poppr) developed by Aarhus University and link with relevant bioinformatics databases and web tools developed by partners in the UK. All relevant results, protocols, guidelines, factsheets, alerts, maps and charts will be integrated as a web-based early warning system on EuroWheat.au.dk. In the case study regions, the relevant maps and charts will be integrated into regional web sites, where end-users normally seek information. All stakeholder networks, case study partners and outputs from shared facilities will be included in the development of all components of the early warning system based on a multi-actor approach. The RustWatch Toolbox will also allow access to an interactive training module for simulations of pathogen dynamics as influenced by e.g., host resistance characteristics, area of deployment of host resistance, pathogen virulence and aggressiveness and expected number of generations per year.

WP5 Methodologies

WP5 will deliver efficient coordination and overall management and facilitate integration between WPs. Project activities, results and communication within the individual stakeholder networks within plant breeding, agrochemical industry, extension services and VCU testing will be coordinated via a network representative, and task leaders have been assigned for individual tasks. Based on the outcome of the research in the project, WP5 will contribute to implementation of the EU plant health policies by (i) developing revised criteria for the definition of important non-quarantine pests in the EU, (ii) identifying routes for the transmission of such pests to Europe, (iii) proposing cost-effective measures to restrict their spread into and within Europe, (iv) raising awareness about agricultural and economic risks of the spread of new rust genotypes and (v) contributing information about plant health risks to support integrated pest management.  Decisions in the project will be implemented via an executive committee (ExCom) consisting of the project coordinator, WP leaders and representatives of the individual stakeholder networks