Search

In this study, we report on the development of two quantitative PCR assays for the detection and quantification of the soilborne plant pathogen Phytophthora cactorum causing root and crown rot of strawberry. The assays rely on the use of SYBR Green I chemistry and use the internal transcribed spacer region 2 (ITS2) and the Ras-related protein gene Ypt1 as detection markers. An extensive list of Phytophthora isolates was included in the study to evaluate assay specificity. High specificity was obtained when Ypt1 was used as detection marker, with cross-reactions only occurring with the closest relatives of P. cactorum, including P. hedraiandra, P. idaei and P. pseudotsugae. In contrast, the ITS-based assay was less specific, resulting in a larger number of cross-reactions (eight species when tested at 10 pg DNA). In sensitivity tests, P. cactorum DNA was detected down to 10 fg using the ITS-based assay, while the limit of detection (LOD) for the Ypt1-based assay was 1 pg DNA, irrespective of the presence of background DNA from strawberry or soil. When strawberry, growth substrate and soil samples were spiked with a 10-fold dilution series of P. cactorum zoospores, the LOD for the ITS-based assay was 10 zoospores g−1 plant material, growth substrate or soil. The limit of quantification (LOQ) of the assay was 109 zoospores g−1 plant material, 15.8 zoospores g−1 growth substrate, and 106 zoospores g−1 soil. For the Ypt1-based assay, the LOD and LOQ values were 1000 and 5910 zoospores g−1 plant material, 1000 and 1818 zoospores g−1 growth substrate, and 1000 and 3823 zoospores g−1 soil, respectively. In terms of detection, analysis of field samples suggested that the Ypt1-based assay almost performed equally well compared to the ITS-based assay, especially for plant and growth substrate samples. Further, our results showed that both qPCR assays outperformed classical plating, illustrating their power to be used for diagnostic purposes. Interestingly, P. cactorum was also detected in soil and growth substrate samples from areas with no symptoms, suggesting that the assays can also aid in early pathogen detection before the disease manifests.

Background Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus. We specifically used the pangenome to investigate genetic differences between virulent and avirulent strains of P. brasiliense, a potato blackleg causing species dominantly present in Western Europe. Results Here we generated a multilevel pangenome for Pectobacterium, comprising 197 strains across 19 species, including type strains, with a focus on P. brasiliense. The extensive phylogenetic analysis of the Pectobacterium genus showed robust distinct clades, with most detail provided by 452,388 parsimony-informative single-nucleotide polymorphisms identified in single-copy orthologs. The average Pectobacterium genome consists of 47% core genes, 1% unique genes, and 52% accessory genes. Using the pangenome, we zoomed in on differences between virulent and avirulent P. brasiliense strains and identified 86 genes associated to virulent strains. We found that the organization of genes is highly structured and linked with gene conservation, function, and transcriptional orientation. Conclusion The pangenome analysis demonstrates that evolution in Pectobacteria is a highly dynamic process, including gene acquisitions partly in clusters, genome rearrangements, and loss of genes. Pectobacterium species are typically not characterized by a set of species-specific genes, but instead present themselves using new gene combinations from the shared gene pool. A multilevel pangenomic approach, fusing DNA, protein, biological function, taxonomic group, and phenotypes, facilitates studies in a flexible taxonomic context.

The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors. Phytophthora hybrids therefore pose a serious threat to agriculture as well as to natural ecosystems. Early and correct identification of hybrids is therefore essential for adequate plant protection but this is hampered by the limitations of morphological and traditional molecular methods. Identification of hybrids is also important in evolutionary studies as the positioning of hybrids in a phylogenetic tree can lead to suboptimal topologies. To improve the identification of hybrids we have combined genotyping-by-sequencing (GBS) and genome size estimation on a genus-wide collection of 614 Phytophthora isolates. Analyses based on locus- and allele counts and especially on the combination of species- specific loci and genome size estimations allowed us to confirm and characterize 27 previously described hybrid species and discover 16 new hybrid species. Our method was also valuable for species identification at an unprecedented resolution and further allowed correct naming of misidentified isolates. We used both a concatenation- and a coalescent-based phylogenomic method to construct a reliable phylogeny using the GBS data of 140 non-hybrid Phytophthora isolates. Hybrid species were subsequently connected to their progenitors in this phylogenetic tree. In this study we demonstrate the application of two validated techniques (GBS and flow cytometry) for relatively low cost but high resolution identification of hybrids and their phylogenetic relations This work was supported by the ERA-NET BiodivERsA project “Resipath” (Re sponse of European Forests and Society to Invasive Pathogens) co-financed by national funds: in Belgium through the Belgian Science Policy Office (grant number BR/132/A1/RESIPATH-BE) and in Portugal by the Portuguese Science and Technology Foundation (grant number BIODIVERSA/0002/2012).

There are more than 100 species that belong to the fungus-like genus Phytophthora, many of which can cause severe damage to plants in both natural and agricultural ecosystems. The availability of techniques for detection and identification are crucial for monitoring and control of these pathogens. In recent years, new methods using molecular approaches have been developed. However, the majority of them are designed to detect single Phytophthora species. Techniques that are able to target multiple species in one sample would offer advantages, especially for the assessment of Phytophthora diversity in the environment. This paper describes a multiplex assay for simultaneous detection and identification of 26 members of Phytophthora down to species level and another 22 to clade or subclade level through target-specific primer extension (TSPE) and the Luminex xTAG array detection system. The assay starts with PCR amplification of two genomic regions, ITS and coxI, followed by a multiplex TSPE reaction with clade-, subclade- and species-specific probes. As a result, biotin-dCTP labelled products are generated and subsequently detected through hybridization with a set of anti-TAG coupled, colour-coded paramagnetic beads. The specificity of the method has been tested using DNA extracts from over 400 isolates representing 110 Phytophthora species and subspecies. The sensitivity and robustness have been determined by the use of DNA mixtures, dilution series and environmental samples. Thus the developed technique allows simultaneous identification of multiple Phytophthora species, particularly useful for the detection of these pathogens in environmental samples such as soil, water and plant tissue.

Live plants, particularly when accompanied by soil or potting substrates, are considered the main pathway for international spread of plant pathogens. Modern, rapid shipping technologies for international plant trade increase the probability of plant pathogen survival during transport and the subsequent chances of disease outbreaks in new locations. The survival of two model pathogens, an Oomycete, Phytophthora cinnamomi, and a filamentous fungus, Fusarium verticillioides, was studied in two different commercial potting substrates (peat and peat-free) under glasshouse conditions in the absence of a plant host. Survival rates were analysed at 2, 7, 12 and 17 months after substrate inoculation. Fusarium verticillioides had the longest survival rate, and was still present at 17 months. In contrast, P. cinnamomi survived up to 7 months but was not recovered after 12 or 17 months. There was no significant difference in the number of colony-forming units (CFUs) of either pathogen in the two substrates, except at 2 months, when higher numbers were recovered from peat substrates.

DNA barcoding protocols for selected EU-regulated arthropods, bacteria, fungi, nematodes and phytoplasmas were developed within the Quarantine organisms Barcoding of Life (QBOL) project financed by 7th framework program of the European Union. DNA barcodes generated with the developed protocols were stored in the Q-bank database. An test performance study (TPS) was set up involving 14 participating laboratories to validate the use of the developed protocols as a diagnostic tool and to identify possible difficulties in the use of the protocols and Q-bank. This paper describes the steps that were used to set up the TPS, to validate the protocols and to identify difficulties. TPS data shows that the developed tests are very robust and produce highly reproducible results. Participants managed to define good consensus sequences which allowed them to correctly identify their samples using Q-bank in 78% of all cases. Q-bank outperformed NCBI and BOLD in terms of diagnostic sensitivity and diagnostic specificity for all organism groups. Using general qualifiers, performance criteria and feedback from TPS participants, difficulties in the set-up of the TPS, the use of the protocols and databases, and the proficiency of participants were identified/ evaluated and recommendations for future work were made. The developed DNA barcoding protocols and Q-bank have proven to be useful tools in support of the identification of selected EU-regulated plant pests and pathogens on the desired taxonomical level.

Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan probe, designed from the internal transcribed spacer region of the multi-copy rDNA gene were tested in extracts from artificially and naturally infested soil. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable by guarding against false negative results. A calibrations curve was created by spiking zonal centrifuge fractions of clean soil samples with a dilution series of winter spores. The Taqman assay was also performed on infected potato plant material (stolons) along with the detection of the cytochrome oxidase gene as a potato endogenous control. Sensitivity of the TaqMan assay was improved at least 100-fold and proved to be reliable for accurate diagnosis of the disease.

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5′ and 3′ ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10 4 . A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

A zonal centrifugation method, known as the Hendrickx centrifuge technique, was tested for routine detection of winter sporangia of Synchytrium endobioticum in soil. In four experiments the ability of the Hendrickx centrifuge to extract the sporangia from soil was compared with a method used by the Dutch Plant Protection Service, which is a modification of the recommended EPPO method. Naturally and artificially contaminated soil samples were used to study the recovery percentage of and variation in numbers of winter sporangia. The effects of soil type and inoculum density were studied. The Hendrickx centrifuge method, developed originally for extraction of free living nematodes from soil, performed better than the method used by the Dutch Plant Protection Service. This was due to a better extraction recovery (60% higher), a lower measurement error (50% lower) and a lower detection level (down to 0.02 sporangia g(-16) soil). The Hendrickx centrifuge method is much less labour-intensive than the method used by the Dutch Plant Protection Service. It can be used to extract many different organisms from soil, and DNA can be subsequently extracted from the supernatant for further PCR analysis. Inclusion of the Hendrickx centrifuge method in the official EPPO diagnostic protocol for regulated pests is recommended as an alternative method for detection of sporangia in soil

Analysis of 12 polymorphic simple sequence repeats identified in the genome sequence of Phytophthora ramorum , causal agent of ‘sudden oak death’, revealed genotypic diversity to be significantly higher in nurseries (91% of total) than in forests (18% of total). Our analysis identified only two closely related genotypes in US forests, while the genetic structure of populations from European nurseries was of intermediate complexity, including multiple, closely related genotypes. Multilocus analysis determined populations in US forests reproduce clonally and are likely descendants of a single introduced individual. The 151 isolates analysed clustered in three clades. US forest and European nursery isolates clustered into two distinct clades, while one isolate from a US nursery belonged to a third novel clade. The combined microsatellite, sequencing and morphological analyses suggest the three clades represent distinct evolutionary lineages. All three clades were identified in some US nurseries, emphasizing the role of commercial plant trade in the movement of this pathogen.

Phytophthora fragariae, the cause of strawberry red stele disease, is a quarantine pathogen in Europe. Detecting low levels of infection requires sensitive and specific methods. In the past, Dutch and English inspection services have used bait plants to test strawberry propagation stocks destined for export. Increasingly though, PCR is being incorporated into these testing procedures in an effort to increase sensitivity and speed. Various combinations of baiting and PCR assays were compared with existing testing procedures. Water and root samples from the bait test were screened by nested PCR and the PCR amplicon was detected by several methods, including fluorescent labelled probes (TaqMan™ and Molecular Beacon™). PCR amplification was monitored in real-time and semi-quantitative detection was possible. Because PCR reactions are sensitive to inhibitors present in extracted DNA samples, an internal control containing the primer sequences specific for P. fragariae was developed to avoid false negatives.

A new devastating disease in the United States, commonly known as Sudden Oak Death, is caused by Phytophthora ramorum. This pathogen, which previously was described attacking species of Rhododendron and Viburnum in Germany and the Netherlands, has established itself in forests on the central coast of California and is killing scores of native oak trees (Lithocarpus densiflora, Quercus agrifolia, Q. kelloggii, and Q. parvula var. shrevei). The phytosanitary authorities in the European Union consider non-European isolates of P. ramorum as a threat to forest trees in Europe. To date, almost all European isolates are mating type A1 while those from California and Oregon are type A2. The occurrence of both mating types in the same region could lead to a population capable of sexual recombination, which could generate a new source of diversity. To prevent contact between these two populations, a rapid, reliable, and discriminating diagnostic test was developed to easily distinguish the two populations. Based on a DNA sequence difference in the mitochondrial Cytochrome c oxidase subunit 1 (Cox1) gene, we developed a single-nucleotide polymorphism (SNP) protocol to distinguish between isolates of P. ramorum originating in Europe and those originating in the United States. A total of 83 isolates of P. ramorum from Europe and 51 isolates from the United States were screened and all isolates could be consistently and correctly allocated to either the European or the U.S. populations using the SNP protocol.

The genetic structure within and between USA and European populations of the emerging phytopathogen Phytophthora ramorum was examined. Four primer combinations were used for amplified fragment length polymorphism (AFLP) fingerprinting of 67 USA isolates from California and Oregon, and 18 European isolates from Belgium, Germany, The Netherlands, Spain and the UK. In addition, three DNA regions (ITS, cox II, and nad 5) of additional Phytophthora species were amplified by polymerase chain reaction, sequenced, and analysed to provide better phylogenetic understanding of P. ramorum within the genus Phytophthora. AFLP banding patterns indicate that the 85 isolates form two distinct lineages within a monophyletic group, distinct from the closely related outgroup species P. lateralis. With the exception of two isolates from an Oregon nursery, European and USA isolates clustered separately within individual clades. The AFLP profiles also indicate that a single clonal lineage dominates the North American population, while the European population consists of an array of mainly unique, closely related AFLP types. Sequences from the three DNA regions were identical among all P. ramorum isolates, and phylogenetic analysis indicates that P. ramorum is closely related to P. lateralis and P. hibernalis.

Based on the ITS regions of the ribosomal DNA, specific primer sets were developed for the citrus pathogen Guignardia citricarpa and the common citrus endophyte, G. mangiferae, and tested for their specificity against 37 isolates of G. citricarpa, 29 isolates of G. mangiferae, 10 isolates of related species and other fungi found on citrus. The efficacy of the PCR-detection method for G. citricarpa was approximately 60–70% for lesions without pycnidia, and approximately 90% for lesions with pycnidia. A reliability of 99% can be reached by analysing multiple lesions per sample. An internal control was developed to monitor DNA samples for PCR inhibition; samples with PCR inhibition should be re-examined. Detection by PCR is more rapid than the current five-day incubation method prescribed by the European Union for diagnosis of black spot lesions lacking the diagnostic pycnidia. The latter method had an efficacy of 40–50%, while culturing of suspected lesions had an efficacy of 10%. Species-specific primers and ITS sequence data showed that G. citricarpa can occur as a symptomless endophyte in leaves. This shows that wild and cultivated plants occurring in citrus groves are potential carriers of this quarantine fungus. Application of the presently developed PCR method for the detection of G. citricarpa will enable citrus producing as well as importing countries to prevent further spread of this harmful organism.

Red core is a serious root rot of strawberry caused by Phytophthora fragariae. Despite having a‘nil tolerance’in propagation schemes and being a quarantine pest, P. fragariae has spread steadily throughout Europe such that only a few Mediterranean countries are now apparently free from it. Field inspections of nurseries are inadequate for detection of red core and a bait test, although sensitive, takes too long to be useful commercially. The European Union, through its Standards, Measurements and Testing Programme has funded the further development of prototype molecular and immunological tests for red core to routine tests that meet ISO 9002 standards. The principal thrust has been the development of a nested polymerase chain reaction (PCR), supported by immunological tests for rapid screening of large numbers of routine samples. Nested PCR has suitable speed, sensitivity and specificity for testing and is now being used routinely in some laboratories. The finished tests will be freely available to all parties after the final stage of the project, a demonstration phase in which the technique will be tested widely in Europe.

There are approximately 60 described species in the genus Phytophthora. Despite the economic and ecological damage they cause worldwide, the genus is poorly understood. A major difficulty, with implications for scientific communication as well as phytosanitary regulations, is species identification. With a paucity of stable morphological characters, even specialists can have difficulty in identification beyond the genus level. Appropriate molecular methods offer an objective alternative. In this paper, the utility of the internal transcribed spacer (ITS) regions of the subunit which encodes ribosomal RNA will be discussed. At the Scottish Crop Research Institute, an examination of the ITS restriction digest patterns of over 450 Phytophthora isolates representing almost all known taxa has allowed an evaluation of the method. Sophisticated band-matching software has been used to compare new isolates rapidly with those in the database. Limitations of such methods are also discussed. In some cases (e.g. closely related taxa with identical ITS regions, or where discrimination at the intraspecific level is needed), amplified fragment length polymorphism (AFLP) analysis is necessary. The database of AFLP fingerprints developed in Plant Research International is discussed in this context.

Phytophthora fragariae var. fragariae is pathogenic to strawberry only and is an EPPO A2 quarantine pest. Seventy-five isolates of P. fragariae from all over the world have been characterized by amplified fragment length polymorphism (AFLP) analysis. However, a sensitive, specific and fast method of detection is now needed, and polymerase chain reaction (PCR)-based methods have now been set up. Based upon sequences of the ITS (internal transcribed spacer) regions of rDNA of several Phytophthora species, PCR primers specific for P. fragariae have been developed together with SCRI (Dundee, GB). These primers were tested on DNA extracted from water, soil and plant material. A nested PCR procedure has been included in the currently used bait test to improve sensitivity and reliability. Also several methods have been studied to detect the amplicon of the PCR reaction: gel electrophoresis, DIAPOPS (detection of immobilized amplified products in a one-phase system), PCR-ELIS A, TaqMan and molecular beacon. With the last two methods, using the ABI 7700 detection system, quantification of target DNA is possible.

The monophyletic origin of host-specific taxa in the plant-pathogenic Fusarium oxysporum complex was tested by constructing nuclear and mitochondrial gene genealogies and amplified fragment length polymorphism (AFLP)-based phylogenies for 89 strains representing the known genetic and pathogenic diversity in 8 formae speciales associated with wilt diseases and root and bulb rot. We included strains from clonal lineages of F. oxysporum f. spp. asparagi, dianthi, gladioli, lilii, lini, opuntiarum, spinaciae, and tulipae. Putatively nonpathogenic strains from carnation and lily were included and a reference strain from each of the three main clades identified previously in the F. oxysporum complex; sequences from related species were used as outgroups. DNA sequences from the nuclear translation elongation factor 1α and the mitochondrial small subunit (mtSSU) ribosomal RNA genes were combined for phylogenetic analysis. Strains in vegetative compatibility groups (VCGs) shared identical sequences and AFLP profiles, supporting the monophyly of the two single-VCG formae speciales, lilii and tulipae. Identical genotypes were also found for the three VCGs in F. oxysporum f. sp. spinaciae. In contrast, multiple evolutionary origins were apparent for F. oxysporum f. spp. asparagi, dianthi, gladioli, lini, and opuntiarum, although different VCGs within each of these formae speciales often clustered close together or shared identical EF-1α and mtSSU rDNA haplotypes. Kishino-Hasegawa analyses of constraints forcing the monophyly of these formae speciales supported the exclusive origin of F. oxysporum f. sp. opuntiarum but not the monophyly of F. oxysporum f. spp. asparagi, dianthi, gladioli, and lini. Most of the putatively nonpathogenic strains from carnation and lily, representing unique VCGs, were unrelated to F. oxysporum f. spp. dianthi and lilii, respectively. Putatively nonpathogenic or rot-inducing strains did not form exclusive groups within the molecular phylogeny. Parsimony analyses of AFLP fingerprint data supported the gene genealogy-based phylogram; however, AFLP-based phylogenies were considerably more homoplasious than the gene genealogies. The predictive value of the forma specialis naming system within the F. oxysporum complex is questioned.

Two Fusarium species, F. oxysporum f.sp. asparagi and F. proliferatum, are known to be involved in the root and crown rot complex of asparagus. We have investigated reports on the involvement of F. redolens, a third species, which until recently was considered conspecific with F. oxysporum because of morphological similarities. RFLP analysis of the rDNA internal transcribed spacer region and AFLP fingerprinting identified eight strains from asparagus unambiguously as F. redolens. Four of these were tested and found to be pathogenic to asparagus either in this study (two strains) or in a previous one in which they were classified as F. oxysporum (three strains). Disease symptoms and disease development were the same as with F. oxysporum f.sp. asparagi and F. proliferatum. Present data and literature reports identify F. redolens as a host-specific pathogen involved in root, crown and spear rot of asparagus. The pathogen is formally classified as F. redolens Wollenw. f.sp. asparagi Baayen.

Three similar isolates of Phytophthora (Phytophthora sp-h) were obtained from diseased Spathiphyllum and Primula plants. Cultural characteristics did not fit any known description of Phytophthora species. The Phytophthora sp-h isolates are papillate, are homothallic, possess 80 to 86% amphigynous antheridia, and have a maximum temperature for growth of 36.5°C. Isozyme analysis of the Phytophthora sp-h isolates revealed a three-banded pattern with malic enzyme and a three-banded pattern with malate dehydrogenase on the second putative locus. The fastest band at both enzyme loci comigrated with the single P. nicotianae band, the slowest band comigrated with the single P. cactorum (and also P. pseudotsugae) band, and one band in between was concluded to represent the heterodimeric isozyme. The random amplified polymorphic DNA patterns of the Phytophthora sp-h isolates almost exclusively consisted of bands that were also present in either P. nicotianae or P. cactorum. Southern hybridization showed that bands specific for P. nicotianae were present as comigrating bands in the Phytophthora sp-h isolates. The same was found for species-specific bands of P. cactorum. It is concluded that the three Phytophthora sp-h isolates represent interspecific hybrids, P. nicotianae being the one parent and P. cactorum the other. Analysis of mito-chondrial DNA with restriction enzymes revealed banding patterns in all the Phytophthora sp-h isolates identical with those of P. nicotianae, confirming that indeed P. nicotianae was one of the parents.

The rate of introduction and establishment of damaging plant pests and diseases has increased steadily over the last century as a result of expanding globalisation of trade in plant material, climate change, EU expansion, and by a recognised decline in the resources supporting plant health activities. Furthermore there is a constant decline in the number of taxonomic specialists in the different disciplines (mycology, bacteriology, etc.), capable of identifying plant pathogens, and funds to support this kind of work are very hard to obtain. Also the number of other specialists in phytopathology and other fields, which are vital for sustaining sound public policy on phytosanitary issues, are diminishing. These problems affect all countries. In this context QBOL (www.qbol.org), an EU project on DNA barcoding, started in 2009 to generate DNA barcoding data of quarantine organisms and their taxonomically relatives to support plant health diagnostics. The data are included in a database, called Q-bank (www.Q-bank.eu), which now consists of a dynamic open-access database of quarantine plant pests and look-alikes, linked to curated and publicly accessible reference collections. It contains sequence and morphological data including photographs, nomenclatural and diagnostic data of specimens available in reference collections. Within Q-bank curators from many countries with expertise on taxonomy, phytosanitary and collection issues for the different groups have been appointed and links with other databases have been made; this in order to provide Q-bank an international role in supporting plant health agencies.

This book is part of the Plant Pathology in the 21st Century Series, started in the occasion of the IX International Congress of Plant Pathology, Torino, 2008. In conjunction with the Xth International Congress of Plant Pathology, held in Beijing in August 2013. Although deriving from a Congress, the book will not have the format of traditional Proceedings, but will be organized as a resource book. It will be based on invited lectures presented at the Congress as well as by other chapters selected by the editors among offered papers. This book will cover a topic very important in the field of plant pathology, dealing with detection and diagnostics. This field of research is continuously moving forwards, due to innovation in techniques. The application of new detection and diagnostic technologies are relevant to many applied fields in agriculture. The different chapters will provide a very complete figure of the topic, from general and basic aspects to practical aspects. Contents: 1. New developments in identification and quantification of airborne inoculum 2. siRNA deep sequencing and assembly: piecing together viral infections 3. Use of airborne inoculum detection for disease management decisions 4. Proximal sensing of plant diseases Case studies and special applications 5. Diagnostic Challenges for the Detection of Emerging Pathogens: A Case Study Involving the Incursion of Pseudomonas syringae pv. actinidiae in New Zealand 6. Detection of Human Pathogens on Plants - 7. Plant Disease Diagnostics For Forensic Applications III. Role of diagnostics in plant disease management 8. Results of the EU project QBOL, focusing on DNA barcoding of quarantine organisms, added to an international database (Q-bank) on identification of plant quarantine pathogens and relatives 9. On-site testing - moving decision making from the lab to the field 10. Virtual Diagnostic Networks: A platform for collaborative diagnostics 11. Development and implementation of rapid and specific detection techniques for seed-borne pathogens of leafy vegetable crops. 12. Diagnosis of plant pathogens and implications for plant health regulation: the European Food Safety Authority perspective

Phytophthora fragariae var. fragariae Hickman, which causes red core disease of strawberry, is a quarantine organism on which a nil tolerance is placed. Detection of the fungus is by a root tip bait test which is highly specific and sensitive but time-consuming (5–6 weeks), has to be done at 12°C and can require some taxonomic experience. Even with the test, detecting red core can still be difficult, especially with early primary infection of propagation beds or infected symptomless resistant cultivars. Moreover, P. cactorum, a closely related fungus, causes crown rot of strawberry. It is not a quarantine organism, typical tolerances of 1␒nfection are allowed, and can confuse diagnosis. Techniques based to the Polymerase Chain Reaction (PCR) are promising alternatives. Various parts of the ribosomal RNA gene repeat (rDNA) have been used to design primers with different specificities: specific PCR primers that can differentiate between P. fragariae and P. cactorum and others designed to detect a wider range of Peronosporales, to which Phytophthora belongs. In nested PCR, combinations of these primers can detect both pathogens with high specificity and sensitivity. Competitive PCR has also been developed to allow quantification of P. cactorum infection in strawberry crowns. With these techniques, P. fragariae was detected in strawberry plants one day after inoculation whereas the symptoms were only visible after one week. Moreover, very low numbers of zoospores could be detected in water samples. The test has been evaluated on samples provided by the inspection services of Scotland and The Netherlands.

Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.

Aims To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs). Methods and Results Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans. Pyrosequencing resulted in 16 965 reads, detecting all species in the template DNA mixture. Reducing the ITS1 sequence identity threshold resulted in a decrease in numbers of unmatched reads but a concomitant increase in the numbers of false MOTUs. The total error rate was 0·63% and comprised mainly mismatches (0·25%) Conclusions Pyrosequencing of ITS1 region is an efficient and accurate technique for the detection and identification of Phytophthora spp. in environmental samples. However, the risk of allocating false MOTUs, even when demonstrated to be low, may require additional validation with alternative detection methods. Significance and Impact of the Study Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthora complexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments.

Stagonosporopsis andigena and S. crystalliniformis are serious foliage pathogens on potato (Solanum tuberosum) and tomato (Solanum lycopersicum). As both species have been recorded only in the Andes area, S. andigena is listed as an A1 quarantine organism in Europe. The actin region of isolates of Stagonosporopsis and allied species of Boeremia, Didymella, Peyronellaea and Phoma was amplified using generic primers. DNA sequence differences of the actin gene were utilised to develop species-specific real-time (TaqMan) PCR assays for the detection of S. andigena and S. crystalliniformis in leaves of potato or tomato. The specificity of the TaqMan PCR assays was determined on genomic DNA extracted from two S. andigena and two S. crystalliniformis isolates and 16 selected isolates of Stagonosporopsis, Phoma and Boeremia, which are the closest relatives. The validation of the methods developed included the DNA extraction and the TaqMan PCR assays. The performance criteria specificity, analytical sensitivity, reproducibility, repeatability and robustness of the TaqMan PCR assays demonstrated the reliability of both methods for the detection of S. andigena and S. crystalliniformis in leaf material. The TaqMan PCR assays were tested on symptomatic leaves of potato and tomato that were obtained after artificial inoculation of detached leaves with both pathogens under quarantine conditions. In the artificial inoculation experiments both S. andigena and S. crystalliniformis caused leaf infections on potato and tomato.

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5′ and 3′ ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes. [KEYWORDS: cytochrome c oxidase subunit I; DNA barcoding; internal transcribed spacer; oomycete; species identification]

Co-existence of both mating types A1 and A2 within the EU1 lineage of Phytophthora ramorum has only been observed in Belgium, which begs the question whether sexual reproduction is occurring. A collection of 411 Belgian P. ramorum isolates was established during a 7-year survey. Our main objectives were genetic characterization of this population to test for sexual reproduction, determination of population structure, evolution and spread, and evaluation of the effectiveness and impact of control measures. Novel, polymorphic simple sequence repeat (SSR) markers were developed after screening 149 candidate loci. Eighty isolates of P. ramorum, broadly representing the Belgian population, were analyzed using four previously described and three newly identified polymorphic microsatellite loci as well as amplified fragment length polymorphisms. SSR analysis was most informative and was used to screen the entire Belgian population. Thirty multilocus genotypes were identified, but 68% of the isolates belonged to the main genotype EU1MG1. Although accumulated mutation events were detected, the overall level of genetic diversity within the Belgian isolates of P. ramorum appears to be limited, indicating a relatively recent clonal expansion. Based on our SSR analysis there is no evidence of sexual recombination in the Belgian population of P. ramorum. Metalaxyl use decreased the genetic diversity of P. ramorum until 2005, when the majority of the isolates had become resistant. Most genotypes were site-specific and despite systematic removal of symptomatic and neighbouring plants, some genotypes were detected over a period of several years at a single site, sometimes discontinuously, indicating (latent) survival of the pathogen at those sites.

The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment. Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (

Hybrid isolates of Phytophthora nicotianae × P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns. The hybrid nature of these isolates was tested by restriction fragment length polymorphism analysis of the internal transcribed spacer (ITS) region of rDNA, generating fragments typical for both P. nicotianae and P. cactorum. In hybrid isolates, polymerase chain reactions (PCR) with primers derived from unique parts of the ITS region (ITS-PCR) of both species yielded a combination of unique amplicons typical of both parental species. Eleven hybrid isolates, three isolates of each parental species and two atypical isolates from Rhododendron and Idesia spp. close to P. cactorum, were analyzed for amplified fragment length polymorphisms (AFLP). Consistent differences in AFLP patterns existed among the hybrid isolates, strongly indicating that these hybrids have arisen from independent hybridization events between P. nicotianae and P. cactorum. The two atypical isolates morphologically resembling P. cactorum were identical to the latter species in ITS-restriction fragment length polymorphism and response to the specific PCR primers but were intermediate between P. nicotianae × P. cactorum and P. cactorum in isozyme profiles and AFLP patterns. Since the introduction of hydroponic systems in greenhouses in the Netherlands, outbreaks of Phytophthora diseases are occurring in previously unaffected host species. This may be due to interspecific hybridization events resulting in novel pathogenic behavior.

In this study, six methods for the detection of Phytophthora ramorum in planta were compared using naturally infested rhododendron plant material. The methods included two immunological methods, one an enzyme-linked immunosorbent assay (ELISA) and the other using a lateral flow format (LFD). Three molecular tests based on the polymerase chain reaction (PCR) using TaqMan chemistry also were assessed, including two assays designed for specific detection of P. ramorum and one designed for genus-level detection of Phytophthora. Isolation followed by morphological identification also was assessed. The diagnostic values of each of the methods, evaluated based on diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value, were calculated based upon the test results from 148 field samples. The “gold standard” used for the calculations was the final diagnosis, which was based on either a positive PCR result or successful isolation of P. ramorum. The Phytophthora spp. TaqMan PCR, ELISA, and LFD had higher sensitivities than the P. ramorum-specific methods, which make them useful as prescreening methods, where positive results must be confirmed by PCR or isolation. The article discusses practical advantages and disadvantages of each of the methods and how they are valuable in the diagnostic process, according to the circumstances of use (that is, diagnosis or surveillance) and in relation to the prevalence of P. ramorum infestation in the population to be tested.

With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real-time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real-time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi-copy rDNA gene. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real-time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real-time PCR methods showed that the TaqMan method was more sensitive.

Analysis of 44 isolates of Phytophthora cactorum, isolated from strawberry and other hosts, by AFLP showed that the crown rot pathotype is different from leather rot isolates and from P. cactorum isolated from other hosts. 16 of 23 crown rot isolates, including isolates from Europe, Japan, Australia, and New Zealand, were identical in an analysis based on 96 polymorphic bands from seven primer combinations. Leather rot isolates of strawberry could not be distinguished from isolates from other hosts. The pathogenicity test of all 44 isolates on strawberry plants mostly gave unambiguous results, except for three American isolates, which seemed to have reduced aggressiveness compared to the crown rot isolates. These isolates also differed in the AFLP analysis. Comparing information on the origin of the isolates with results from the pathogenicity test, showed that isolates from strawberry fruits or petioles could be either leather rot or crown rot pathotypes. None of the isolates from hosts other than strawberry caused crown rot symptoms in strawberry.

An F1 mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.

A Phytophthora species was found on blighted foliage of Ipomoea longipedunculata, a morning glory native to the highlands of central Mexico. Based on host range, morphology, allozymes, mitochondrial DNA haplotype and rDNA sequences it is concluded that a new Phytophthora species, P. ipomoeae sp. nov., is the causal agent of leaf blight disease on I. longipedunculata.

Alternaria alternata, A. radicina and A. dauci are important seed-borne fungi on carrot, with the first two species having a high toxigenic potential, for which a specific and sensitive detection method is required. Because both the traditional deep-freeze-blotter method and plating on selective medium are time consuming and laborious, a PCR-based assay was developed. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) from 45 different Alternaria isolates were determined, a restriction fragment length polymorphism analysis was performed and a phylogenetic tree was constructed. Based on the sequences, specific primers for detection and identification of the three Alternaria species on carrot seeds and roots were designed. The primers were highly sensitive and were shown to be able to differentiate between the three Alternaria species. A. alternata and A. radicina could be detected in DNA isolated from carrot material applying the specific primers, even at low infection levels. The PCR-assay was compared to the deep-freeze-blotter method (DFBM) and plating on Alternaria radicina Selective Agar (ARSA, for A. radicina) by testing naturally infected seed samples and root material. Results of the PCR-assay were similar to those of the blotter method and plating on ARSA for the detection of A. alternata and A. radicina. A positive correlation was found between the percentage of seed infection established by the blotter method and the intensity of the amplified, specific product. The PCR-assay based on the specific primers developed seems to be a good alternative for the deep-freeze-blotter method and plating on ARSA, especially when time is an important issue.

In the last decade, nucleic acid sequence based amplification methods have been developed for many important plant pathogenic bacteria. Although they possess excellent properties with respect to sensitivity and specificity, the introduction of these techniques for routine detection has been hampered by in particular the lack of robustness. In different ring tests, both for human and plant pathogens, the failures of PCR amplification to diagnose correctly infected and non-infected sample material have been demonstrated many times. Carry-over contamination of amplicons has been recognized as the main source of false-positive results, whereas the presence of PCR inhibiting components in sample extracts is known as the main reason for false-negative results. In this paper new technologies and approaches are described to improve the quality of DNA and RNA amplification methods for routine detection.

Since 1993, a hitherto unidentified Phytophthora species has been found associated with twig blight disease in Rhododendron and, sporadically, Viburnum. The morphology and growth characteristics of fourteen isolates from Germany and the Netherlands were investigated, together with their breeding system, the internal transcribed spacer (ITS) regions of the ribosomal DNA, amplified fragment length polymorphism (AFLP) fingerprints, and isozyme profiles, which were compared to those of a number of outgroup species. Morphologically the isolates are characterized by abundant production of chlamydospores and elongate, ellipsoid, deciduous sporangia with a short pedicel, in which they resemble P. palmivora. However, sporangia were semi-papillate, chlamydospores were much larger and cardinal temperatures much lower than those of P. palmivora. Oogonia with amphigynous antheridia and plerotic oospores were produced in dual cultures with an A2 mating type strain of P. cryptogea. ITS1 and ITS2 sequences of the unidentified species were closest to those of P. lateralis, but differed in three and eight nucleotides respectively from the latter species. AFLP fingerprints and isozyme patterns of malate dehydrogenase (MDH-2) and malic enzyme (MDHP) showed that the isolates formed a homogeneous group, distinct from all examined outgroup species, including P. lateralis. It was concluded that they represent a new Phytophthora species, described here as P. ramorum sp. nov. In pathogenicity tests all isolates of P. ramorum were pathogenic to Rhododendron.

Recently, several outbreaks of brown rot caused by Ralstonia solanacearum (Smith) Smith biovar 2, race 3 were reported in Western Europe. Knowledge of the variation in the population of the target pathogen forms the basis of epidemiological studies and control measures. In this study, the genetic diversity of race 3 strains from France, The Netherlands and the UK was determined with different molecular techniques. AFLP and pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction enzymes (RC-PFGE) were able to separate race 3 strains in different groups, indicating that several clonal lines are present in Europe. ERIC- and BOX-PCR were less discriminative. No correlation was found between geographical origin and profiles obtained.

The fungus Phytophthora fragariae Hickman is a quarantine organism and is as well pathogenic on strawberry as on raspberry [1]. It means that all plantmaterial for export has to be tested on the presence of the fungus. A sensitive and specific detection method is therefore necessary. At IPO-DLO in the Department of Detection we work on the development of such a detection method. In the past work was done to solve this problem by serological methods [2,3,4,5]. Unfortunately with limited succes. In this study a detection method is developed using nested PCR.

Scanning electron micrographs of the nematode-egg-parasitic fungus Paecilomyces lilacinus infecting eggs of the root-knot nematode Meloidogyne spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N- and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode Meloidogyne hapla induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33.5 kDa, a pH optimum of 10.3, a temperature optimum of 60 degrees C and an isoelectric point above pH 10.2. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes.