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The overall goal of the project was to bring together scientists, practitioners and official national bodies (NPPOs) to share knowledge on two important quarantine diseases of trees in Europe: Ceratocystis platani and Cryphonectria parasitica. The threats posed by Ceratocystis platani are often underestimated and even in countries where the disease already occurs, not much measures are taken to limit the spread of this devastating disease. The project contributed to: Improve knowledge on the distribution of Cryphonectria parasitica in European countries. It was shown that C. parasitica is present in Belgium and United Kingdom, but not in Ireland. In the Netherlands, the fungus was found several times on imported Castanea stakes/fences in 2018, but not in nurseries or public green. Develop a collection of C. parasitica isolates. The isolates were used to determine the population structure of C. parasitica in Europe. Evidence was found that in countries where the pathogen was relatively recently described (e.g. UK), the pathogen was introduced several times but has not spread widely, possibly due to suboptimal climatic conditions. Organise a test performance study (TPS) for the real-time PCR test from Pilotti et al. (2012) for the detection of C. platani in wood. Specifically, EvaGreen, SYBR Green and Taqman tests were validated. Standard curves confirmed that the tests have high amplification efficiency. For analytical sensitivity, all laboratories detected 15 and 3 fg C. platani gDNA, thus reproducing the previously reported detection threshold of the method. Testing blind samples yielded a 100% scoring sensitivity, accuracy and specificity in all the experiments, with an exception in the SYBR Green test for which results from one laboratory were nonconforming for the criterion of accuracy.

The main parts of the project consisted of testing new differential cultivars for pathotype identification (bioassays), and the generation of validation data for three molecular S. endobioticum detection and identification assays. As a bioassay, the partners in the project agreed upon the use of the Glynne-Lemmerzahl method to identify pathotypes. In the second year of the project (2013) a test performance study (TPS) was organised. Two different pathotypes were tested, pathotype 6(O1) and 18(T1), and five partners joined in. The cultivars Talent and Logo were chosen to replace cv Miriam in the actual set of differential cultivars (EPPO, 2004). In former research, cv Miriam proved to be less suitable for separating pathotypes 6(O1) and 18(T1), results were inconsistent. As a result, we concluded that cv Talent was the best replacement of cv Miriam in the actual set of differential cultivars. Also, the cultivar Gawin can be added to the set of differentials in a new version of the EPPO Diagnostic Protocol on Synchytrium endobioticum. An international test performance study was organised to generate validation data for three molecular S. endobioticum detection and identification assays: Boogert et al. (2005), van GentPelzer et al. (2010), and Bonants et al. (2015). Two TPS rounds were organised focussing on different test matrices: round 1: wart material, and round 2: winter spore suspensions. When using the assays for detection and identification of S. endobioticum in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. After applying a Ct cut-off value for the GentPelzer assay, all assays are regarded equal. When using the assays for detection and identification of S. endobioticum in winter spore suspensions, the Boogert and Gent-Pelzer assays significantly outperform the Bonants assay for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the Gent-Pelzer assay significantly outperforms the Boogert and Bonants assays and is regarded as the assay of choice when identifying S. endobioticum winter spores. The tests included in this TPS are regarded fit for purpose for routine testing of wart material and winter spore suspensions with ≥ 500 spores per sample., Report of the Euphresco project 'Diagnostic methods for Synchytrium endobioticum, especially for pathotype identification (SENDO)'

During a survey of Phytophthora diversity in natural ecosystems in Taiwan six new species were detected. Multigene phylogeny based on the nuclear ITS, ß-tubulin and HSP90 and the mitochondrial cox1 and NADH1 gene sequences demonstrated that they belong to ITS Clade 7a with P. europaea, P. uniformis, P. rubi and P. cambivora being their closest relatives. All six new species differed from each other and from related species by a unique combination of morphological characters, the breeding system, cardinal temperatures and growth rates. Four homothallic species, P. attenuate, P. flexuosa, P. formosa and P. intricata, were isolated from rhizosphere soil of healthy forests of Fagus hayatae, Quercus glandulifera, Q. tarokoensis, Castanopsis carlesii, Chamaecyparis formosensis and Araucaria cunninghamii. Two heterothallic species, P. xheterohybrida and P. xincrassata, were exclusively detected in three forest streams. All P. xincrassata isolates belonged to the A2 mating type while isolates of P. xheterohybrida represented both mating types with oospore abortion rates according to Mendelian ratios (4–33 %). Multiple heterozygous positions in their ITS, ß-tubulin and HSP90 gene sequences indicate that P. xheterohybrida, P. xincrassata and P. cambivora are interspecific hybrids. Consequently, P. cambivora is re-described as P. xcambivora without nomenclatural act. Pathogenicity trials on seedlings of Castanea sativa, Fagus sylvatica and Q. suber indicate that all six new species might pose a potential threat to European forests.

Oospores of Phytophthora ramorum were produced from intraspecific pairings between a European A1 and European or American A2 strains. Their viability was evaluated through colouration with tetrazolium bromide. The distribution of oospores in the different classes of colouration was similar to that found in other Phytophthora species (homothallic and heterothallic): most of the oospores stained purple, which corresponds to spores in dormancy. In order to produce single-oospore cultures, a method was developed to separate oospores from mycelium and chlamydospores. Germination of oospores was observed in 110, 250, 350 and 500-d-old cultures at a low proportion. Microsatellite marker analyses on oospore progenies revealed that the oospores resulted from hybridisation. More than 50 oospore progenies were characterised in terms of mating type, aggressiveness on Rhododendron leaves, and growth rate on two different media. The results are discussed in the context of pest risk analysis.

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., Scientific report of the Euphresco funded project 'Current and Emerging Phytophthoras: research supporting risk assessment and risk management' (CEP)