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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.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but it causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica, and Nerium oleander. The host range of the pathogen can vary, depending on the subspecies and even the strain. The availability of fast and reliable diagnostic tools is indispensable in management strategies to control diseases caused by X. fastidiosa. To improve the reliability of the TaqMan assay, currently widely used in surveys, a triplex TaqMan assay was developed in which two specific and sensitive TaqMan assays, previously designed for X. fastidiosa, were combined with an internal control. The triplex assay exhibited the same diagnostic sensitivity as the simplex assays. In addition, the usefulness of a metagenomic approach using next-generation sequencing (NGS) was demonstrated, in which total DNA extracted from plant material was sequenced. DNA extracts from plant material free of X. fastidiosa, from artificially inoculated hosts plants or from naturally infected plants sampled in France, Spain, and Italy, or intercepted in Austria and the Netherlands, were analyzed for the presence of X. fastidiosa using the metagenomic approach. In all samples, even in samples with a low infection level, but not in the pathogen-free samples, DNA reads were detected specific for X. fastidiosa. In most cases, the pathogen could be identified to the subspecies level, and for one sample even the whole genome could be assembled and the sequence type could be determined. All results of NGS-analyzed samples were confirmed with the triplex TaqMan polymerase chain reaction and loop-mediated isothermal amplification.

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.

The obligate biotrophic pathogen Puccinia horiana is the causal agent of chrysanthemum white rust. Although P. horiana is a quarantine organism, it has been able to spread to most chrysanthemum-producing regions in the world since the 1960s; however, the transfer routes are largely obscure. An extremely low level of allelic diversity was observed in a geographically diverse set of eight isolates using complexity reduction of polymorphic sequences (CRoPS) technology. Only 184 of the 16,196 contigs (1.1%) showed one or more single-nucleotide polymorphisms (SNPs). Thirty-two SNPs and one simple-sequence repeat were translated into molecular markers and used to genotype 45 isolates originating from North and South America, Asia, and Europe. In most cases, phylogenetic clustering was related to geographic origin, indicating local establishment. The European isolates mostly grouped in two major populations that may relate to the two historic introductions previously reported. However, evidence of recent geographic transfer was also observed, including transfer events between Europe and South America and between Southeast Asia and Europe. In contrast with the presumed clonal propagation of this microcyclic rust, strong indications of marker recombination were observed, presumably as a result of anastomosis, karyogamy, and somatic meiosis. Recombination and transfer also explain the geographic dispersal of specific markers. A near-to-significant correlation between the genotypic data and previously obtained pathotype data was observed and one marker was associated with the most virulent pathotype group. In combination with a fast SNP detection method, the markers presented here will be helpful tools to further elucidate the transfer pathways and local survival of this pathogen.

In several crops, their quality and condition is very important. The products are relatively expensive and plant diseases can destroy valuable harvests in a very short time. The presence or absence of plant pathogens and pests, therefore, needs to be accurately tested. For decades, identification and detection methods have been designed to demonstrate that starting material for further propagation and also certified bulbs, tubers, cuttings, and plants are free of many plant pathogens. Every year, millions of plants are tested, including approximately one and a half million bulbs. Despite the ease of low-cost, fast, and well-automated test methods, such as ELISA, more and more molecular-based techniques are used the last decade. These techniques are, generally, more specific and more sensitive. These new tests are also necessary because plant pathogens are often latent (asymptomatic) present in the material to be tested. The Netherlands has a high in—and export of plants and plant products. These large trading volumes may harbour many plant pathogens. The European Commission has drawn up a list of unwanted plant pathogens (quarantine organisms). This list contains more than 300 different organisms (viruses, bacteria, fungi, phytoplasmas, nematodes and insects). Each EU-country has to control the in—and export traffic of plants and plant products for the introduction of these unwanted organisms. In the Netherlands, this task is performed by four inspection services: Naktuinbouw (Netherlands Inspection Service for Horticulture), NAK (The Dutch General Inspection Service), BKD (Inspection Service for Flowerbulbs) and KCB (Quality Control Bureau). The NVWA (Dutch Food and Consumers Authority) is the NPPO (National plant protection organisation) in the Netherlands and supervises the inspection services. Many different molecular methods are used daily for the detection and identification of many plant pathogens and plant diseases.

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 the fungal agent causing potato wart disease. Because of its severity and persistence, quarantine measures are enforced worldwide to avoid the spread of this disease. Molecular markers exist for species-specific detection of this pathogen, yet markers to study the intraspecific genetic diversity of S. endobioticum were not available. Whole-genome sequence data from Dutch pathotype 1 isolate MB42 of S. endobioticum were mined for perfect microsatellite motifs. Of the 62 selected microsatellites, 21 could be amplified successfully and displayed moderate levels of polymorphism in 22 S. endobioticum isolates from different countries. Nineteen multilocus genotypes were observed, with only three isolates from Canada displaying identical profiles. The majority of isolates from Canada clustered genetically. In contrast, most isolates collected in Europe show no genetic clustering associated with their geographic origin. S. endobioticum isolates with the same pathotype displayed highly variable genotypes and none of the microsatellite markers correlated with a specific pathotype. The markers developed in this study can be used to assess intraspecific genetic diversity of S. endobioticum and allow track and trace of genotypes that will generate a better understanding of the migration and spread of this important fungal pathogen and support management of this disease.

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.

This chapter describes pesticide-free strategies to minimize the risks of root-borne diseases in soilless systems, since these strategies are unique for soilless plant growing systems. Recirculation of the nutrient solutions offers an ideal opportunity for the detection of microorganisms. Recirculation of nutrient solutions also allows treatment of pathogens in it. Furthermore, the start with fresh and pathogen-free substrates is a challenge for the design of microbiologically optimized growth media which are more suppressive against plant diseases. This chapter also deals with detection of pathogens in the nutrient solution. Recent technical developments of detection techniques allow the design of specific and quantitative detection of pathogens in nutrient solutions. Following this, the study describes the development of a microbially balanced system, under which, it presents the concept of balancing populations of various microbes so as to keep them all under better control. It also provides an understanding of the current knowledge about microbes in soilless production systems, application of biocontrol agents, factors stimulating a more balanced microbial system, as well as disease suppression by various substrates. Following this, it states that the challenge is to combine the available strategies into environmentally and economically sound soilless plant production systems with low risks for pathogen outbreaks, and soilless systems have the potential of creating a balance between a pathogen-free start and a suppressive microflora. Finally, this chapter provides examples of such combined strategies and presents an outlook to the future.

By use of steady state and time-resolved fluorescence techniques Forster energy transfer between the cofactors, FAD and FMN, in cytochrome-P450 reductase is observed. Red-edge spectroscopy allowed us to assign the actual rate constant of transfer as apparent in the anisotropy decay of the holoenzyme. The same approach was used in biflavinyl compounds entrapped in polymethylmetacrylate. As anticipated the fluorescence depolarisation of this system showed a great similarity with the enzyme system. From the results the interflavin distance in the enzyme was estimated between 1.6 and 2.4 nm.