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Lateral Flow Device (LFD) diagnostics for Phytophthora ramorum and Phytophthora kernoviae - PH0412

Description
Phytophthora ramorum (the causal agent of sudden oak death in California) and Phytophthora kernoviae pose a threat to UK native flora and are under statutory control. Current diagnosis involves sending suspect (symptomatic) plant material to central diagnostic laboratories for examination and testing. Current laboratory methods include culturing and microscopic examination followed by real-time PCR test for confirmation. Approximately 75% of samples received into the laboratory (many thousands) are found not be infected by Phytophthora species at all and only a proportion of the remainder are usually confirmed as Phytophthora ramorum or kernoviae. The ability to identify these two pathogens accurately in the field would generate obvious benfits through speed of response and savings in laboratory testing (particularly in prioritising only those samples likely to be positive and in need of laboratory confirmation).

Progress has already been made in developing simple disposable field tests (lateral flow devices: LFDs) which are currently used by the Plant Health & Seeds Inspectorate (PHSI) of Defra along with Forestry Commission staff. These tests are capable of indentifying Phytophthora species in general (genus specific) but are not capable of specifically identifying the two species of interest, although they will detect both of them. However this work has indicated that the approach is sound and that the end user (currently government inspection agencies) find them valuable and easy to use. Further studies are examining the use of field portable real-time PCR as a diagnostic tool for inspection agencies (EU PORTCHECK project). This approach is capable of identifying Phytophthora pathogens to the species level and is also very sensitive. However this approach relies currently on expensive capital equipment and trained staff. It is likely that this approach will be more suited to fixed inspection points such as ports of entry, rather than in multiple field or forestry locations for the present.

This project will concentrate on two approaches to achieving the diagnosis of Phytophthora infection, firstly, identifying the infection at the genus level, and secondly, species determination of Phytophthora ramorum and Phytophthora kernoviae using either an antibody based LFD apprroach or a molecular approach.

The current lateral flow devices rely on genus-specific monoclonal antibodies (produced from cell culture) to be able to distinguish Phytophthora species from other pathogens or physiological disorders. This project will seek to produce both genus-specific Phytophthora antibodies and species-specific monoclonal antibodies to both Phytophthora ramorum and Phytophthora kernoviae and then incorporate them into simple field test kits (LFDs). Genus-specific Phytophthora antibodies will be developed to ensure continuity of supply and to reduce costs. Specific monoclonal antibodies to P. ramorum and P. kernoviae, if achieved, will lead to immediate species determination on site. Raising of species specific antibodies comes with associated risks, but as this will run in parallel to the screening for genus specific antibodies, this will maximise the chances of success and minimise the costs involved. All these new tools will be extensively validated in the field with the help of government inspection agencies and other interested parties (including commercial and public users).

Work will also be carried out to see if an LFD approach using a genus-specific antibody, or even species-specific antibodies, could be combined with DNA-based laboratory methods to allow very simple laboratory confirmation of field-test positive results. Preliminary work has indicated that it is possible to send a positive genus-specific lateral flow device to a laboratory and then recover and amplify DNA from the device using PCR based methods. This would allow a simple and convenient way of permitting follow up confirmatory testing or further in-depth characterisation (e.g. DNA sequencing) of the pathogen at a central laboratory. This might be important to both confirm important samples or to investigate unusual or atypical disease (e.g. a suspect new host species or a more virulent plant response). It is anticipated that this will be considerably cheaper than using direct PCR on leaf material.

A suite of new species specific, easy to use test kits will significantly enhance the ability to identify trees and shrubs infected with Phytophthora ramorum and Phytophthora kernoviae in a rapid and cost effective manner. The tools will also be available and suitable for use by industry and other interested parties regardless of how policy develops.
Objective

1. Generate antigens from Phytophthora species for use as immunogens.

2. Screen both genus specific Phytophthora antibodies plus P.ramorum and P.kernoviae species specific antibodies.

3. Production of species specific lateral flow devices using antibodies generated.

4. Develop methods to use a genus specific lateral flow device for species specific laboratory molecular diagnosis.
Project Documents
• Final Report : Plant Health Report: Detection and identification of Phytophthora ramorum and Phytophthora kernoviae using simple field test kits   (787k)
• Final Report - Annex : Plant Health Report (Annex 1): Mechanical methods for improving LFD DNA extraction in support of the diagnosis of regulated Phytophthora species   (141k)
• Final Report - Annex : Plant Health Report (Annex 2): Evaluation of a Fera antibody-based prototype LFD for on-site diagnosis of Phytophthora   (333k)
Time-Scale and Cost
From: 2006

To: 2008

Cost: £102,898
Contractor / Funded Organisations
Central Science Laboratory
Keywords
Plant diseases              
Plant health              
Plants and Animals              
Fields of Study
Plant Health