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Identification and classification of members of the Ralstonia solanacearum species complex (RSSC) is challenging due to the heterogeneity of this complex. Whole genome sequence data of 225 strains were used to classify strains based on average nucleotide identity (ANI) and multilocus sequence analysis (MLSA). Based on the ANI score (>95%), 191 out of 192(99.5%) RSSC strains could be grouped into the three species R. solanacearum, R. pseudosolanacearum, and R. syzygii, and into the four phylotypes within the RSSC (I,II, III, and IV). R. solanacearum phylotype II could be split in two groups (IIA and IIB), from which IIB clustered in three subgroups (IIBa, IIBb and IIBc). This division by ANI was in accordance with MLSA. The IIB subgroups found by ANI and MLSA also differed in the number of SNPs in the primer and probe sites of various assays. An in-silico analysis of eight TaqMan and 11 conventional PCR assays was performed using the whole genome sequences. Based on this analysis several cases of potential false positives or false negatives can be expected upon the use of these assays for their intended target organisms. Two TaqMan assays and two PCR assays targeting the 16S rDNA sequence should be able to detect all phylotypes of the RSSC. We conclude that the increasing availability of whole genome sequences is not only useful for classification of strains, but also shows potential for selection and evaluation of clade specific nucleic acid-based amplification methods within the RSSC.

Numerous Phytophthora and Pythium disease outbreaks have occurred in Europe following inadvertent introduction of contaminated ornamental plants. Detection and identification of pathogens are crucial to reduce risks and improve plant biosecurity in Europe and globally. Oomycete diversity present in roots and compost was determined in 99 hardy woody plants bought from nurseries, retailers and internet sellers, using both isolations and molecular analyses. Oomycete DNA was quantified using real-time PCR of environmental DNA from the plants using three loci: ITS, trnM-trnP-trnM and atp9-nad9. At least one oomycete species was isolated from 89.9% of plants using classical techniques. In total, 10 Phytophthora spp., 17 Pythium spp. and 5 Phytopythium spp. were isolated. Oomycetes were isolated from 86% of asymptomatic plants, but real-time PCR demonstrated that oomycetes were associated with all plants tested. More oomycete DNA occurred in composts in comparison with roots and filters from baiting water (a mean of 7.91 ng g−1, 6.55 × 10−1 ng g−1 and 5.62 × 10−1 ng g−1 of oomycete DNA detected in compost with ITS, trnM and atp9 probes, respectively), the ITS probe detected the highest quantities of oomycete DNA. No significant differences were found in quantities of oomycete DNA detected using real-time PCR in plants purchased online or from traditional retailers.

The Ralstonia solanacearum species complex (RSSC) can cause bacterial wilt in a wide variety of plant species, including a number of ornamental glasshouse crops. Recently in Europe, ornamental rose plants for the production of cut flowers and propagation materials have been strongly affected by Ralstonia pseudosolanacearum , phylotype I, biovar 3. To test for the presence of the pathogen in the glasshouse, sampling of water from a drainage gutter or well may be an efficient strategy since it is known that RSSC can be released from infected root systems in the water. A protocol was developed to detect low densities of R. pseudosolanacearum in drain water collected from rose growers. Drain water was filtered through a bacterial filter, the filtrate was collected and target bacteria enriched for 48 h in Semi‐selective Medium South Africa (SMSA) broth supplemented with sterilized tomato plant extracts. DNA extracted from the enrichment broth was analysed using a TaqMan test in a duplex format, based on specific egl sequences of RSSC and the use of an extraction and amplification control. The optimized protocol had a detection level of ≤1–10 colony forming units of R. pseudosolanacearum in drain water.

The ability of National Plant Protection Organizations (NPPOs) and their affiliated laboratories to quickly and reliably detect and identify organisms is critical for the effective performance of phytosanitary measures. Plant pest diagnostics is also essential to support the phytosanitary certification of consignments of plants and plant products exported from the EU. Access to reference material has been identified as a limitation on the ability of laboratories to develop and validate diagnostic tests and ensure the reliability of diagnostics. The EU FP7 Q-collect project worked to establish the state of the art of current phytosanitary collections, to identify gaps and propose minimum quality standards, to facilitate access to specimens and to design and build networks of reference collections. The main results of the project are presented in this paper.

This paper describes the database Q-bank (www.q-bank.eu). This freely accessible database contains data on plant pathogenic quarantine organisms to allow fast and reliable identification. Development of accurate identification tools for plant pests is vital to support European Plant Health Policies. Council Directive 2000 / 29 /EC lists approximately 300 entries representing a large number of species (e.g. non-European Tephritids contains approximately 3500 species) for which protective measures, against introduction and their spread within the European Community, need to be taken. The risk of introduction of pests into the European Union is increasing because of the increase in the volumes, commodity types and origins of trade, the introduction of new crops, the continued expansion of the EU, the numbers of international travellers and the impact of climate change. Identifying pests (in particular new emerging pests) requires staff with specialised skills in all disciplines (mycology, bacteriology etc.), which is only possible within large centralised laboratory facilities. Expertise in taxonomy, phytopathology and other fields in plant health, which are vital for sustaining sound public policy on phytosanitary issues, are under threat. Sharing knowledge on regulated and non-regulated pests is necessary to manage a cost-effective and efficient plant health system in the context of expanding globalisation of trade in plant material

Synchytrium endobioticum is a severe pathogen of potato causing wart disease. For this obligate fungus many pathotypes exist, which are pathogenic or non-pathogenic to different cultivars of potato. To determine to which pathotype an isolate belongs, two biological assays are used on a set of different potato cultivars: the Spieckermann and the Glynne-Lemmerzahl test. A differential set of cultivars has been recommended by EPPO (European Plant Protection Organization) for these tests. Drawbacks of these tests are that it can take up to several months to score the interaction and results are difficult to score and also often not conclusive. Therefore, possibilities were investigated to look for molecular markers for pathotype specificity. An extremely low level of diversity was observed in a set of eight isolates using CRoPS™ (Complexity Reduction of Polymorphic Sequences) technology. Only 191 sequence polymorphisms were found in 14,660 contigs representing approximately 195 Kb of sequence for eight isolates. Nine sequence polymorphisms could be related to pathotype specificity. Two of these polymorphisms were transferred into a real-time TaqMan PCR assay to discriminate between pathotype specific groups. Validation of the assays was performed using multiple isolates from different countries for which the bioassay had been performed.

In 2009 a new three year EU funded project (QBOL) started on DNA barcoding of important plant pests. An international consortium of 20 partners (universities, research institutes, and phytosanitary organizations) from around the world, coordinated by Plant Research International (Wageningen, the Netherlands), will collect DNA barcodes from many plant pathogenic quarantine organisms, store these sequences in a database accessible over the internet, develop a DNA bank and train end-users. All these activities should help National Plant Protection Services in the correct identification and detection of plant pathogenic quarantine organisms. © 2010 The Authors. Journal compilation © 2010 OEPP/EPPO.