Report Open Access
Turner, Judith; Riccioni, Luca; Heungens, Kurt; Meffert, Johan; Braganca, Helena; Webber, Joan; Choiseul, James; Schlenzig, Alexandra; Talgø, Venche; Stromeng, Gunn Mari; Campos Abad, Paloma; Perez Sierra, Ana; McCracken, Allistair; Gusina, Valentina; Lasner, Helena; Brandt, Linda
This EU-wide EUPHRESCO project brought together partners from nine countries in project meetings and workshops to exchange knowledge on diagnostic methodologies for Phytophthora species and facilitate the standardisation of protocols across Europe. Validation work on in-field diagnostic tests highlighted the effectiveness of the use of lateral flow devices (LFDs) to identify plants infected by Phytophthora species. However, the in-field PCR and LAMP tests, which were at that point still undergoing development and validation, showed a much lower rate of detection compared to the results from laboratory isolation and identification. Newly developed and validated real-time LAMP assays on the Genie II were demonstrated to be effective for detection of a range of Phytophthora species in different hosts and tissue types. The use of alkaline PEG extraction greatly increased the potential for use of LAMP-based testing outside the laboratory in a range of scenarios. This has led to the development of reagents in a kit format to increase accessibility and convenience for a range of potential end-users. Since the end of this project, the P. ramorum and P. kernoviae LAMP assays have been developed into kits, and the potential now exists for the other assays described in this report to be made available in the same way. Novel LAMP assays were developed and validated for use in detection of P. austocedrae, P. lateralis and P. pseudosyringae in the field. A generic LAMP assay for detection of Phytophthora species was also developed for use in any future outbreaks of Phytophthora species of quarantine importance. Analyses of the genotypes of 65 isolates of P. ramorum confirmed the existence of a group of isolates unique to the UK which continue to diversify as the pathogen spreads in the natural environment. No geographical or host specificity was detected for these groups although continuing diversification is a sign of growth and spread of the disease. In contrast, genotypic variation within a small number of isolates sourced from Portugal was very limited except for a single isolate whose genotype suggested that this isolate may have moved in trade between Spain and Portugal. This illustrates the capability of these analyses in detecting pathways of spread for the rarer genotypes and can be useful in calculation dispersal distances and potential rates of spread of pathogens. Clear distinctions were seen between UK and New Zealand isolates of P. kernoviae with no intermixing of genotypes between countries indicating no direct connection between the two populations and no evidence for NZ being the source of introduction of P. kernoviae to the UK. Genetic diversity was greater within the New Zealand population of P. kernoviae compared to that found in the UK suggesting that the population in the UK is more recent in its introduction. Extensive research and analysis from the UK on risk assessment and disease management in natural outbreaks have been shared with national representatives from nine EU countries, completing objectives for knowledge transfer and facilitating capabilities for response to any future outbreaks of Phytophthora species across Europe. An improvement in efficacy, versatility and provenance of diagnostic methodologies as outputs from this project could significantly enhance the development of improved surveillance strategies and hence aid timely risk analysis for current outbreaks as well as deliver generic approaches for future outbreaks caused by Phytophthora species. In addition, outputs from genotyping using microsatellite analysis, 454 and Illumina sequencing of P. ramorum isolates could inform risk assessments for epidemic development and assist in identification of pathways of disease entry and spread.