Diagnostic: Development of Oomycete LFDs

Project summary:

In years one and two of the project, oomycete disease management tools will be developed for use on-site and by crop clinics for the effective management of oomycete diseases.  Several approaches will be used:

• A multiplex on-site test will provide generic probes to confirm the presence / absence of Pythium and Phytophthora in horticultural samples. If confirmed, the test will if present identify species / groupings considered pathogenic across crop sectors (Hardy Nursery stock, Protected Ornamentals, Protected Edibles and Soft Fruit). An additional test will be made available for the measurement of oomycete presence and viability for efficacy tests of disinfestation treatments such as chlorination, Pasteurisation, UV or slow sand biofiltration systems.

• A crop clinic test will measure and discriminate Pythium and Phytophthoraspecies commonly found to be pathogenic across UK-wide horticultural crop production systems. The crop clinic test will also provide information on the presence of mycoparasitic Pythium species (e.g. Pythium oligandrum, P. nunn) which may be important in biological control studies / precision application of treatments. 

Sampling procedures and translation of the test result will form a priority within the project. A key driver will be for the uptake of these tests by the industry to inform cohesive disease management strategies.  It is recognised that the test formats must deliver results in an accessible way that growers can readily interpret in terms of the disease implication for the cropping system involved. A strong grower / science based interface will therefore exist within four identified HDC sectors: Hardy Nursery stock, Protected Ornamentals, Protected Edibles and Soft Fruit. This cross-sectoral industry-focussed approach will operate throughout the life of the project. Digital media (for example You Tube Clips) will provide longevity and ease of access to information on test usage and interpretation. 

Throughout the life of the project comparison with current practice under field and clinic conditions will be used to evaluate and, where appropriate, validate the new tests developed.

Benefits to industry:

• Reliable and affordable detection and diagnosis are key to effective oomycete disease management. With increasing globalization, travel and the international trade in plants the risk of disease spread through inadvertent introduction is exacerbated.

• There is a reduction in broad spectrum fungicides which hold approval for use in the Horticulture Industry. Despite the development of the ‘off-label’ system for minor uses the number of pesticides that are currently approved for use on horticultural crops is under ever-increasing pressure. To utilise these efficiently and with optimal disease control performance there is a move away from routine pesticide application to targeted crop treatments (pesticides and biological) based on regular monitoring. This requires a greater depth of knowledge by producers and their staff to identify problems quickly. The ability to quickly diagnose many plant pathogens does not exist and simple, accurate, diagnostic tools for growers should be developed.

• Pathogen detection prior to infection or the development of symptoms invariably improves the efficacy of timed control measures and can significantly reduce disease epidemics and control treatment inputs.

• This project provides considerable scope for benefit in terms of early detection (i.e. ahead of symptom development and possibly even widespread infection) and targeted treatments (sanitization programmes, biological and/or chemical control). The predictive deployment of control measures will provide disease control systems that are sustainable with the use of lower-input farming systems Ultimately, financial benefit will be gained through improved quality and improved control procedures. The introduction of tests will also assist disease certification schemes. 

• As part of the Sustainable Use Directive (SUD) from 1st January 2014 growers are required to demonstrate that they have taken alternative IPM e.g. cultural and biological, measures to prevent pest & disease development before considering and to help justify conventional spray applications of insecticides and/or fungicides. The use of predictive tests will provide a significant step forward and help minimise the need for pesticide intervention.

• To provide industry understanding of the test formats and, their use for improved oomycete disease management strategies, the following approaches will be employed:

1. Regular visits (four times per annum) to identified nurseries/production facilities within each of four cross-sectoral areas.
2. Cross-sectoral workshops (to include ‘Hands on’ in Year 3) would run annually and throughout the life of the project. These will provide the industry with a background to oomycete diseases an understanding of the test formats and their use for improved oomycete disease management strategies.  
3. Web based visual media will be developed and made available to assist UK industry understanding of the test applications, test operation and interpretation of results with respect to disease management.
4. Technology developed in this project will be applicable to other diseases and  fundamental epidemiological and disease management studies

Aims and objectives:

Project aim:

To produce on-site, and laboratory tests which can be used to accurately monitor infective propagules of oomycetes in environmental samples (i.e. plants, growing media and water). The tests will operate in a multiplex format to identify oomycete presence and then specifically the pathogenic species / groups involved or propagule viability. The latter test (oomycete presence and viability) will be used in both on-site and laboratory confirmatory tests for the efficacy of disinfestation treatments, especially for water but also for growing media and circumstances where sterilants have been deployed to avoid or control disease outbreaks.

The on-site kits will provide simple, robust and inexpensive testing for early detection of oomycete fruit, root and collar rot pathogens by growers and consultants. Development of laboratory processes will enable plant clinics to provide high throughput screening of environmental samples (to include plant material). There will be capability to measure Pythium and Phytophthora to species level and to differentiate between mycoparasitic (potential biocontrol agents), non-pathogen saprobe and crop specific pathogenic species.

Application of these tests will be developed for their integrated use within disease avoidance management and control systems. Commercial ‘do it yourself’ test kits would be made available at the end of the project to the UK horticulture industry. To encourage industry uptake of the technology UK-wide ‘hands-on’ workshops will be run during Years 2 and 3 of the project. Web-based visual media will be made available to assist industry understanding of the test applications, test operation and interpretation of results with respect to management of disease. Technology developed in this project will greatly enhance the capacity for accurate and rapid assessment of samples, especially water, and will be applicable to fundamental epidemiological studies and thus facilitate longer-term improvements in cultural practice.

Project objectives:

A)  Develop inexpensive on site antibody-based diagnostic tests to measure in a multiplex format oomycete presence (Pythium and Phytophthora) and when present key crop-specific oomycete pathogens:

• Produce antibody probes with reliable genus specificity to Pythium and Phytophthora

• Produce monoclonal antibody probes to key plant pathogen clade groupings within the Phytophthora genus, selected for their relevance to UK-wide horticultural production, and in some instances to specific commodities, providing a panel of probes of industry-wide application. The antibodies would be raised to the following representative species:   P. cryptogea, P. cactorum, P. fragariae.

• Produce a similar representative panel of monoclonal antibody probes to plant pathogen clade groupings of Pythium species. These would not be limited to but include:  P. aphanidermatum, P. deliense P. debaryanum, P. sylvaticum, P. irregulare, P. intermedium, P.ultimum and P. splendens

B)  Develop an on-site test to differentiate between viable and non-viable oomycete propagules. This approach is essential for effective confirmatory efficacy tests for disinfestation treatments as a generic probe for viable oomycete propagules can test efficacy even when pathogen spores are not present because there are a large number of commonly-found oomycetes in water and growing media that should be removed by effective treatments in the same way as pathogenic species and can thus be used as indicators.

• Produce antibody probes with reactivity to commonly isolated waterborne oomycetes  (Pythium, Phytophthora, Saprolegnia and Aphanomyces)

• Identify and by recombinant protein sequence expression produce and isolate an external protein complex associated with oomycete zoospore viability. Develop a monoclonal antibody probe  to the protein complex for   test development to discriminate and measure viable  oomycete propagules (Pythium Phytophthora, Saprolegnia, Achlya and Aphanomyces species) in environmental samples

C)  Develop, using existing published molecular sequences, a MAGPIX DNA-based ELISA array. This test format would be for plant clinic use to measure and discriminate key Pythium and Phytophthora species associated with UK horticultural production. This would include the measurement of mycoparasitic species for application within biological control programmes. The test format would be transferrable to a membrane hybridization macro array.

The developed antibody probes (A) would also be evaluated for use in a simple ELISA format for use by crop clinics to differentiate between and quantify species present in a sample.

D) Provide strong cross-sectoral industry presence for uptake of the tests developed in A, B and C. Ensure that the applications, strengths and limitations of each test are well understood within the horticultural industry. This would include knowing when samples are best sent away for more detailed laboratory assessment and what might be reasonably gained from this. Develop a media support (you tube style clips, understanding the test, the application it is suited to and implication of test results) to provide technical assistance with test applications post project completion.