Antonio Tiberini

Jenny Tomlinson

United Fera

Kingdom

Alain Roques

CBGP Astrid Cruaud

Antoon Loomans

Jose Carlos

Rodrigues Univ

Peter Bonants
email: peter.bonants@wur.nl

Bonants Pjm

Françoise Petter

Suffert Muriel

Roy Anne Sophie

Griessinger Damien

Madeleine Mcmullen

Maja Ravnikar
email: maja.ravnikar@nib.si

Nina Prezelj

Petra Nikolič

Nataša Mehle

Rok Lenarčič

Tanja Dreo

Dany Morisset

David Dobnik

Jennifer Hodgetts
email: jennifer.hodgetts@fera.gsi.gov.uk

Neil Boonham

Matt Dickinson

Marina Dermastia

Bertaccini Assunta
email: assunta.bertaccini@unibo.it

Paltrinieri Samanta

Contaldo Nicoletta
email: nicoletta.contaldo2@unibo.it

Nicolaisen Mogens

Duduk Bojan

Hodgetts Jennifer

Jayne Hall

Boonham Neil

Irene González-Martín

Ravnikar Maja

Nikolić Petra

Nicoletta Contaldo

Assunta Bertaccini

Berkvens Nick
email: nick.berkvens@ilvo.vlaanderen.be

Braeken Kristien
email: kristien.braeken@gezondheid.belgie.be

Casteels Hans
email: hans.casteels@ilvo.vlaanderen.be

Cottyn Bart
email: bart.cottyn@ilvo.vlaanderen.be

Nicole Damme
email: nicole.damme@ilvo.vlaanderen.be

Mr Mathias De Backer
email: mathias.debacker@ilvo.vlaanderen.be

Jonghe De

Kris

Vos De

Paul

Mr Sven Inghelbrecht
email: sven.inghelbrecht@ilvo.vlaanderen.be

Leonard Sandrine
email: sandrine.leonard@afsca.bebelgium

Martine Maes
email: martine.maes@ilvo.vlaanderen.be

Multiplex pathogen detection using microarrays Neil Boonham, Fera, United Kingdom

Development and validation of a real-time RT-PCR assay for generic detection of pospiviroids

 30-18.30Drinks and close

André Lévesque

Using DNA Barcoding for diagnostics and monitoring of plant pathogens and pests, a Canadian perspective C. André Lévesque Agriculture and Agri-Food Canada, Central Experimental Farm, Ottawa, Canada DNA sequencing has been used to identify fungi pretty much since PCR became available. The need to develop internationally accepted identification standards supported by NCBI as well as databases housing information not available in GenBank became apparent. In 2003, the concept of DNA Barcoding proposed by Paul Hebert, Guelph University, Canada, addressed this issue. The success of DNA barcoding depends largely on the breadth of the reference databases which means that it must be an international endeavour. Shortly after the term DNA barcoding was coined, the Alfred P. Sloan Foundation supported the creation of the Consortium for the Barcode of Life to foster this activity internationally and more recently Genome Canada has supported the creation of the International Barcode of Life Initiative (iBOL).

The access to comprehensive DNA barcode databases is essential for accurate identification by Sanger sequencing and there has been significant progress made recently in DNA barcoding of both true Fungi and oomycetes. These sequence databases open up many technological possibilities that can be rapidly developed from analyses of this reference data. Quantitative PCR, DNA array hybridization and pyrosequencing are just a few examples of technologies for molecular detection based on DNA barcode data. The Genomics Research and Development Initiative (GRDI) is the counterpart to Genome Canada for Canadian Federal government scientists. In January 2012, GRDI approved a project on Quarantine and Invasive Species that is involving almost all science-based departments of the Canadian government. This project has a strong component on plant quarantine species of relevance to Canada. Further databases are planned for plant quarantine insects, nematodes, fungi, phytoplasmas and viruses as well as invasive plants and apply some of the next generation sequencing tools for direct detection. This new Canadian project will complement the seminal work done by QBOL Pedro Crous

Culture collections in relation to DNA Barcoding

Crous Pedro W. CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584CT Utrecht, the Netherlands p.crous@cbs.knaw.nl

The CBS Culture Collection (1904) is in the process of generating DNA barcodes of its entire holdings. Faced with a great diversity of undescribed and cryptic taxa, taxonomic types have the prominent function of anchoring existing species names. Taxonomic type sequence information is rare, because few types have to date been subjected to DNA sequencing. The barcode sequences of the more than 8000 ex-type cultures will be of tremendous value for fungal taxonomy and its user community. Furthermore, the CBS yeast collection holds almost 9000 strains including the 2240 ex-type strains of all described species. The resulting barcode data set will be an invaluable reference source for yeast research. For more than 100 years, strains entering the CBS collections have been identified based on state-ofthe-art techniques at the time of accession. DNA barcoding the holdings will allow identifications to be updated and the recognition of strains in need of further research, which will not only increase the value of the collection, but will aid the user community. The DNA barcoding approach and online database identification tool will also prove invaluable to confirm the identity of strains selected for whole genome analysis. For some years work has been ongoing to compile information to facilitate identification of non-native plants that pose a (potential) threat to the biodiversity of the ecozone comprising Northern Germany, the Netherlands, Belgium and North-Western France or are regulated by third countries and are likely to be present as contaminant in commercial exports originating from the Netherlands. This information is now available at http://www.qbank.eu/Plants/.

This information system also comprises a set of interactive image-driven identification keys for invasive plants at various growth stages in a range of "habitats": seed identification of contaminants in birdfeed, weeds in bonsai plants, seedling identification, invasive terrestrial plants and invasive aquatic plants. The idea of image-driven identification, using a multiple entry system is to avoid misunderstandings in terminology and failure in identification in the absence of certain characters as is sometimes the case in dichotomous keys. The interactive keys are linked to the species information in the database, including datasheets, distribution maps, specimen level information, barcodes of selected species etc. In addition to the above mentioned species information, the results of the EUPHRESCO DeCLAIM (Decision support systems for the control of alien invasive macrophytes) project are also incorporated in the database and available at http://www.q-bank.eu/Plants/ under the header "control".

Mogens Nicolaisen

QBOL -WP 7: DNA barcoding of phytoplasmas

Nicolaisen Mogens (1), Makarova Olga (1), Contaldo Nicoletta [START_REF] Piškur | Reference plant pathogenic bacteria in support of the European plant health policy: the Q-Bacco-net initiative Aspin A.(4)[END_REF], Paltrinieri Samanta [START_REF] Piškur | Reference plant pathogenic bacteria in support of the European plant health policy: the Q-Bacco-net initiative Aspin A.(4)[END_REF], Bertaccini Assunta (2) 1) Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark, mogens.nicolaisen@agrsci.dk 2) Alma Mater Studiorum, University of Bologna, Plant Pathology, Viale Fanin, 42, 40127 -Bologna, Italy, assunta.bertaccini@unibo.it Phytoplasmas infect a high number of plant species. Based on the 16S ribosomal gene sequence, over 20 groups have been established within the "Candidatus Phytoplasma" taxon, some of which are of quarantine status. To date, no protocol for in vitro cultivation of phytoplasmas has been developed, making classical microbiological identification procedures impossible. Phytoplasma identification is currently based on RFLP analysis of a 1.2 kbp region of the 16S rDNA, however, this method is not easy to set up and it only considers a minor part of the available phytoplasma sequence information. It has been suggested that universally amplified, short, and highly variable DNA barcodes may help to rapidly identify organisms. As part of the QBOL project, a universal DNA barcoding based tool for phytoplasma identification was developed. Two sets of primers amplifying a fragment of the Tuf gene and a fragment of the 16S rDNA gene, respectively, were designed and the potential of these fragments as DNA barcodes was verified. Successful amplification and sequencing of more than 150 phytoplasma strains, and ability to separate various phytoplasma strains to "Candidatus species" level, 16S ribosomal group and sub-group level confirm that these barcodes are efficient phytoplasma identification tools.

Session 2 Viruses

Abstracts of Presentations

Ian Adams

QBOL -WP 6: Next Generation Sequencing for Plant Virus Discovery and Diagnosis

Adams Ian(1), Glover Rachel(1), Hany Ummey(1), Vlugt Rene van der [START_REF] Piškur | Reference plant pathogenic bacteria in support of the European plant health policy: the Q-Bacco-net initiative Aspin A.(4)[END_REF], Dullemans Annette [START_REF] Piškur | Reference plant pathogenic bacteria in support of the European plant health policy: the Q-Bacco-net initiative Aspin A.(4)[END_REF], Kreuze Jan(3), Willmer Cuellar(3), Boonham Neil (1). 1) Food and Environment Research Agency, York, UK ian.adams@fera.gsi.gov.uk 2) Plant Research International, Wageningen, NL 3) International Potato Centre, Peru

The advent of next generation sequencing has revolutionised the sequencing of plant viruses. When previously it could take months or even years to sequence the genome of a novel virus it is now possible to carry this out in a matter of days. As part of the EU funded QBOL project a range of different methods have been optimised using Roche 454 FLX and Illumina HS2000 sequencers to allow the rapid and cheap sequencing of virus genomes. As part of the project, these methods have then been used to sequence over 50 plant virus genomes. The methods have also been used to identify novel viruses in diseased material. New viruses discovered include Piper DNA virus 1 & 2 found on pepper plants in India and Watercress white vein virus, a virus identified on diseased watercress plants.

Using the newly developed sequencing techniques it is now possible to go from a diseased plant to a genome sequence of a novel virus and finally to a high throughput real-time PCR assay to detect the novel virus in a matter of weeks.

Ellis Meekes

Detection of Potato spindle tuber viroid and Tomato chlorotic dwarf viroid in seeds of tomato

Koenraadt H.M.S. (1), Van Vliet A. (1), Jodlowska A. (1), Meekes E.T.M. (1), Verhoeven J.Th.J. [START_REF] Piškur | Reference plant pathogenic bacteria in support of the European plant health policy: the Q-Bacco-net initiative Aspin A.(4)[END_REF] and Bakker D.

(1). 1) Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, the Netherlands E.Meekes@Naktuinbouw.nl 2) National Plant Protection Organization, P.O. Box 9102, 6700 HC Wageningen, the Netherlands Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) are contagious pathogens of tomato and several other hosts. Phytosanitary regulations for seeds of tomato have led to the development of a PSTVd/TCDVd seed assay, although the significance of seed transmission of both viroids is still unclear. In the assay sub samples of 1000 tomato seeds are incubated in an extraction buffer overnight to soften the seed coat. Then the seeds are extracted in a mini bagmixer. After subsequent RNA extraction a real-time RT-PCR based on TaqMan® technology is carried out for detection of PSTVd/TCDVd. For each sub sample two independent PCR reactions are carried out. In one tube PSTVd/TCDVd is detected in a singleplex reaction. In another tube detection of PSTVd/TCDVd is combined with the detection of endogenous Nad5 RNA amplicons as an internal amplification control to monitor RNA extraction and PCR inhibition. For PSTVd/TCDVd positive seed lots additional RT-PCRs are performed in order to sequence the viroid genome for definite identification to the species level. A validation study was performed to determine several performance characteristics of the assay. It was shown that one PSTVdcontaminated seed could be detected in sub samples of 1000 tomato seeds. Results will be presented of > 3000 commercials seed lots that were tested in the routine laboratory.

Jan Kreuze

Small RNA sequencing and assembly as a DNA barcoding method for plant viruses Kreuze Jan International Potato Center (CIP), Aveninida La Molina, Lima 12, Peru, j.kreuze@cgiar.org Novel and emerging plant viruses, arising from pathogen evolution, global trade, crop intensification and potentially climate change, pose a key threat to agriculture worldwide. Even apparently symptomless virus infections can cause considerable yield losses, which can be further exacerbated by synergistic interactions with other viruses. RNA silencing constitutes a fundamental antiviral defence mechanism in plants in which host enzymes cut viral RNA into pieces of 20-24 nt. When isolated, sequenced en mass and properly aligned these virus-derived small RNA (sRNA) sequences can reconstitute genomic sequence information of the viruses being targeted in the plant. This approach is independent of the ability to culture or purify the virus and does not require any specific amplification or enrichment of viral nucleic acids as it automatically enriches for small RNAs of viral origin by tapping into a natural antiviral defence mechanism. To date the method has been used to identify numerous new viruses including single and double stranded RNA, DNA and reverse transcribing viruses and viroids from hosts as divergent as plants and invertebrate animals. In several cases unexpected apparently symptomless viruses were also identified, providing an important reminder that there may be many more viruses infecting our crop plants than we have previously been aware of, and their potential impacts on agricultural productivity remain to be understood.

Neil Boonham

Multiplex pathogen detection using microarrays

Neil Boonham (1), Jenny Tomlinson (1), Ian Adams (1), Sarah Goodchild (2) and Simon Weller (2) 1) Food and Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK neil.boonham@fera.gsi.gov.uk 2) DSTL, Porton Down, Salisbury, Wiltshire, SP4 OJQ, UK Screening for the presence of multiple pathogens in samples using generic microarray techniques has been in development for some time, yet using genomics based platforms (e.g. high density glass slide arrays) has failed to make any impact in the diagnostics arena. In addition development of probes that give the desired level of specificity and sensitivity has been a challenge, since predictions based on bioinformatics are often not borne out by empirical testing. Working with panels of pathogens efforts have been invested in understanding in greater detail factors affecting probe binding of pathogens and the impact this will have on testing real samples in terms of sensitivity and specificity. In addition by performing the work to a diagnostics focused platform several screening tests for EU quarantine pathogens are now close being transferable to the routine testing laboratory and making these tests more widely available. The paper will discuss the approaches used to design primers and probes, the lessons learned as well as specific examples of the tests becoming available.

Antonio Tiberini

Microarray-based methods for a multiple and simultaneous detection of viruses highlighted the need for a collection database

Tiberini Antonio (1) and Barba Marina (1) 1) CRA-PAV Centro di ricerca per la patologia vegetale -Plant Pathology Research Centre -Via C.G. Bertero 22 00156 Rome Italy antonio.tiberini@entecra.it

Since the first reports in 2002 on the use of this tool to detect viruses, the microarray-based approaches offer an alternative to the routine methods, as microarray virus-specific probes are potentially capable of detecting an almost unlimited number of virus species in one assay. Among these approaches, developing different microarray platforms, the microarray-based diagnosis methods, aimed to identify crop specific viruses or to develop a "universal" microarray to identify all the known viruses reported in plants.

The Combimatrix platform, has recently shown its potential to detect a large number of viruses in different plant matrices as tomato (Solanum lycopersicum L.) and globe artichoke (Cynara scolymus L.), allowing the identification of 37 viruses and 1 viroid on tomato and 14 viruses on globe artichoke. The repeatability and specificity of the Combimatrix platform showed how the identification of plant viruses is limited only by the spectrum of the viruses probes present in microarray. The increasing number of plant viruses" nucleotide sequences may allow a great number of probes to be designed which must be evaluated to recognize the greatest number of strains and isolates belonging to the species target, highlighting the need of the creation and maintaining of a wide microorganism collection databases.

Jenny Tomlinson

RT-LAMP for detection of plant pathogenic viruses including Cassava brown streak virus

Tomlinson Jenny (1), Ostoja-Starzewska Sioban (1), Boonham Neil (1), Smith Julian (1), Dickinson Matthew (2) 1) The Food and Environment Research Agency, Sand Hutton, York, UK, YO41 1LZ jenny.tomlinson@fera.gsi.gov.uk 2) University of Nottingham School of Biosciences, Sutton Bonington Campus, Loughborough, UK, LE12 5RD

Loop-mediated isothermal amplification (LAMP) assays have been developed for detection of a range of plant pathogens, including fungal, bacterial and viral pathogens. Primers have been developed for the specific amplification of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (genus Ipomovirus, family Potyviridae), by reverse transcriptase-LAMP (RT-LAMP). Performance of the RT-LAMP assays compared favourably with that of conventional RT-PCR and TaqMan real-time RT-PCR. LAMP-based detection is simple, rapid and flexible, and methods using this technology have the potential to be used in situations where PCR-based testing is too complex, time-consuming or expensive. Potential workflows can be compared in terms of equipment costs, cost per sample and suitability for use in the field, and include real-time fluorescence detection using a specialised instrument such as the OptiGene Genie II, as well as non-instrumented detection using modified primers and lateral flow devices for detection of incorporated labels. The development and characterisation of RT-LAMP assays for CBSV and UCBSV detection, in addition to assays for other plant pathogen targets, will be discussed. et al., 2004).

Marleen Botermans

Development and validation of a real-time RT-PCR assay for generic detection of pospiviroids

The method used at the plant health laboratory at Anses aims to detect PSTVd and TCDVd using RT-PCR developed by [START_REF] Shamloul | Sensitive detection of potato spindle tuber viroid using RT-PCR and identification of a viroid variant naturally infecting pepino plants[END_REF]. To estimate the presence of the other viroids on Solanaceae , different methods were evaluated based on 2 techniques of end-point RT-PCR (Verhoeven et al., 2004;Spieker et al., 1996) Hydroponic systems and intensive irrigation in horticulture, are widely used, but can potentially lead to rapid and efficient spread of water-transmissible plant pathogens throughout the whole crop. Although numerous plant viruses have been detected in aqueous environments, for many of them, the survival in water and the potential for direct transmission through irrigation water are still unknown. Therefore it was decided to explore whether water can be a source of infection with PepMV, PVY and PSTVd, that are relatively stable and contagious viruses/viroid, and a serious threat to tomato and/or potato production.

In irrigation waters, the viruses are usually present in concentrations lower than the detection limit of classical methods, but that may be sufficient to infect plants; therefore the development of highly sensitive diagnostic methods is necessary. Small sample volumes may lead to non-representative testing, therefore, a concentration step allowing the handling of larger water volumes may improve water monitoring diagnostic scheme. A fast and efficient way to concentrate highly diluted viruses consists on using CIM monolithic chromatographic supports. The application of such a strategy to the above mentioned plant viruses/viroid and the results of survival in water at room temperature and transmission efficiency through nutrient solutions in hydroponic cultivation of tomatoes and potatoes will be presented. Application of molecular diagnostic methods has greatly accelerated identification of arthropod pests. At the same time, DNA barcoding based on the mtDNA COI gene as well as nuclear markers, has shown great potential to improve the detection of pests. Through a series of examples the potential of DNA barcoding to detect quarantine arthropods efficiently will be illustrated, the potential drawbacks will also be discussed. From the author"s experience, DNA barcoding may provide an efficient new tool in the biosurveillance armoury for detection of pests but may sometimes be misleading. Nevertheless, developing a worldwide DNA library of barcodes of quarantine species, possibly including their main natural enemies that could be used in biological control projects, is of strategic importance to enhance our ability to detect and manage future invasive pests for agriculture and forests.

Cornelis Schoen

LAMP as a tool for

Marja van der Straten

Morphological and molecular identification of EU quarantine species of Spodoptera (Lepidoptera, Noctuidae) Noyes of the British Natural History Museum have been unable to establish taxonomical differences among individuals from these three populations. Nevertheless, minor morphological differences suggest the possibility of taxonomical divergence, which could lead to distinctive biological control potential. Genomic sequences of individuals from these populations were compared to determine their identity and their degree of genetic difference. Haplotypes of the three populations were different and the population from Puerto Rico was not genetically diverse, in contrast with those of Barbados and Florida. This study was able to provide evidence of differentiation of Leptomastidea spp. in Puerto Rico from other Caribbean populations in order to support the classical biological control of HCM.

Michel Dollet

Characterization of two haplotypes of Haplaxius (Myndus) crudus, vector of different phytoplasmas associated with palm diseases, using cytochrome oxidase I gene.

Michel Dollet (1), Jean-Luc Dzido(2), Jean-François Julia (1), Thierry Bourgoin (3), Paula Ceotto (3), Carlos Oropeza (4), Brown Judith (5). In the 1980"s Haplaxius (Myndus) crudus (Cixiidae) was reported to be the vector of the phytoplasma disease "lethal yellowing" (LY) of coconut palm in Florida. Most recently, the LY phytoplasmas have been shown to belong to the group 16S rIV-A. Transmission of the 16Sr IV-D phytoplasma ("Texas Phoenix palm decline") by H. crudus to Pritchardia pacifica was obtained in Yucatan, however transmission to coconut was not demonstrable. The taxonomy of the Cixiidae is confounding owing to the often cryptic nature of closely related variants and species, particularly of the tribe Oeclini Muir, 1922 to which Haplaxius belongs. Recently several Myndus species were transferred to other genera. Haplaxius itself was moved to the genus Myndus and re-classified as Haplaxius. In 2008 it was shown that phylogenetic trees reconstructed using the cytochrome oxidase I gene (mtCOI) and the 18S rDNA sequences were congruent, and that the trees were consistent with morphological classification for Oeclini. The highly evolving mitochondrial mtCOI were used to ascertain if differences occurred between H. crudus populations from Mexico with respect to the transmission of group 16S rIV-A and 16S rIV-D. Results indicated that two haplotypes of H. crudus exist in Mexico, possibly involved in the transmission of one of the two phytoplasma groups.

Session 4 Nematodes

Abstracts of Presentations

Sebastian Kiewnick Identification of quarantine plant pests needs to be fast and accurate to enable timely plant protection measures.

QBOL

Incorrect diagnosis could cause serious financial losses for trade and producers. Genetically based diagnostics is a reliable alternative to the classical identification generally based on morphological features requiring expert taxonomic skills. Genetic diagnostics through the use of DNA-barcodes, stretches of DNA that contain taxon-specific information, can be performed by any skilled laboratory-worker.

The European Union 7th Framework project QBOL: "Development of a new diagnostic tool using DNA barcoding to identify quarantine organisms in support of plant health" aims to establish DNA-barcodes for all European quarantine organisms and their close relatives, including plant parasitic nematodes. For quarantine nematodes, several gene regions such as COI, COII, SSU, LSU and RNA polymerase subunit II have been evaluated for their barcoding potential. The results and protocols for successful use of DNA-barcoding of quarantine nematodes and their close relatives are available through an online database, www.Q-bank.eu, freely accessible to all interested users, such as national plant protection organizations. A group of curators will ensure that data incorporated into the Q-bank database are validated and linked to specimen or populations in reference collections across Europe.

Johannes (Hans) Helder

Use of a phylum-wide SSU rDNA-based molecular framework for the detection of plant parasitic nematode species in complex DNA backgrounds Helder, Johannes (1), Landeweert, Renske (2), Sven van den Elsen (1), Paul Mooyman (1), Hanny van Megen (1) Winfried Mulder ( 2), Gerrit Karssen (3), and Jaap Bakker (1) 1) Wageningen University, Laboratory of Nematology, Droevendaalsesteeg 1, 6708 PB Wageningen Hans.Helder@wur.nl 2) ClearDetections, Binnenhaven 5, 6709 PD Wageningen, the Netherlands 3) Plant Protection Service, Nematology Section, Geertjesweg 15, 6706 EA Wageningen, the Netherlands Although the overwhelming majority of nematodes play essential roles in the soil food web, some nematode species are serious parasites of major crops such as potato, sugar beet and soybean. Among the most notorious ones are cyst (e.g. Globodera rostochiensis and G. pallida), root knot (e.g. Meloidogyne chitwoodi, M. fallax and M. minor), stem and bulb (e.g Ditylenchus dipsaci and D. destructor), and foliar nematodes (various Aphelenchoides species). A number of these plant parasites have a quarantine status, and as a consequence substrates (often soil) and plant materials should be checked for the presence of certain plant parasitic nematode species. So far, most molecular tests offer "local solutions"; they enable for instance the detection of certain Meloidogyne species in a pre-selected pool of root knot nematodes. In an attempt to reconstruct the evolution of the phylum Nematoda, a phylum-wide molecular framework has been constructed, consisting of appr. 2, 500 taxa. This framework was used to define unique DNA sequence signatures that enable "blind" identification of individual nematode species in highly complex DNA backgrounds. Over 20 relatively simple, (Q-)PCR-based diagnostic tests for species identification, and a number of examples will be presented during this conference.

Geraldine Anthoine

Validation of morphological identification method -the example of a morphological key for Bursaphelenchus xylophilus species

Anthoine Geraldine(1), Buisson Alain ( 2) & Sarniguet Corinne (2) 1) Plant Health Laboratory, 7 rue Jean Dixméras, 49044 ANGERS cedex 01, FR, geraldine.anthoine@anses.fr 2) Plant Health Laboratory, Domaine de la Motte au Vicomte BP35327, 35653 LE RHEU cedex, FR Many diagnostic laboratories have now developed quality management systems in order to ensure the quality of their official analysis. One of the requirements of such a quality management system, especially for accredited laboratories, is the validation of internal or non-standard tests.

The validation process is often discussed either at national or regional level, e.g. within EPPO panels. This validation process is often considered as impossible for morphological tests. This presentation will describe, beyond the results of the validation, the procedure applied for the validation of a morphological key for the identification of a regulated nematode, Bursaphelenchus xylophilus. The different performance criteria defined in the EPPO diagnostic protocol (PM 7/98), such as sensitivity, specificity and reproducibility, have been evaluated by using confusing Bursaphelenchus species, different stages of B. xylophilus and several operators. This evaluation led to the description of the most accurate, reliable and simple morphological identification key.

Martijn Holterman

Evaluation of barcoding and phylogenetic potential of four genes in the genus Meloidogyne

Holterman Martijn H.M.(1), Frey Juerg E.(1), Helder Hans(2), Mooyman Paul J.W.( 2), Kiewnick Sebastian(1) 1) Agroscope Changins-Waedenswil, Schloss, 8820 Waedenswil, Switzerland martijn.holterman@acw.admin.ch 2) Laboratory of Nematology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands Identification of plant pests, in particular quarantine species, needs to be fast and accurate to enable timely plant protection measures. It is now well established that genetically based diagnosis is a reliable alternative to the classical identification procedures which are generally based on morphological features, requiring expert taxonomic skills. On the other hand, genetic diagnosis through the use of so-called DNA-barcodes, stretches of DNA that contain taxonspecific information, can be performed by any skilled laboratory-worker. Root-knot nematodes of the genus Meloidogyne are some of the most important plant parasitic nematodes world-wide. As part of the QBOL project, four potential barcoding regions were tested and evaluated in the genus Meloidogyne. These were the small subunit (SSU) ribosomal RNA gene, the large subunit (LSU) ribosomal RNA gene as well as the mitochondrial cytochome oxidase c subunit 1 (COI) and subunit 2 (COII) genes. As well as evaluating their barcoding potential, results were compared with earlier phylogenetic studies of the genus Meloidogyne.

Renske Landeweert

Diagnostic kits for the detection of plant parasitic nematodes.

Landeweert Renske (1), Mulder Winfried (1), Van den Elsen Sven (2), Mooyman Paul (2), Helder Johannes (2) 1) ClearDetections, Binnenhaven 5, 6709 PD Wageningen, The Netherlands, renske.landeweert@cleardetections.com or info@cleardetections.com 2) Wageningen University, Laboratory of Nematology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

The highly conservative morphology of nematodes means that identification requires expert knowledge. Current nematode analyses are mostly based on microscopic examinations with limited throughput, sensitivity and specificity. This hampers standardized control for quarantine nematode species in importing and exporting countries. A solution would be a radical switch to DNA barcode based identification. Previous studies demonstrate that the small subunit ribosomal DNA (SSU rDNA) gene harbours enough phylogenetic resolution to distinguish between nematode families, genera and often even species (Holterman et al. 2009, Van Megen et al. 2009). On the basis of a framework of ~ 2,500 full length nematode SSU rDNA sequences, diagnostic Q-PCR tests for nematodes have been developed and offered to agricultural laboratories either as a detection kit or as dedicated system.

Recently, many agricultural laboratories have invested in the set-up of a molecular laboratory, indicating that molecular identification and detection of plant pathogens will be the golden standard in the near future. The planned product range introduced here enables inspection laboratories to implement standardized and validated molecular tests and allows them to work according to the latest taxonomic insights. These tests enable high throughput, specific and sensitive detection of quarantine nematodes and provide a major means for durable disease control and risk management.

Sylvie Gamel

Direct detection of plant parasitic nematodes by real time PCR: experience gained from development and validation of different tests

Sylvie Gamel (1), Anne-Marie Chappe (1), Fabrice Ollivier (1) and Géraldine Anthoine (2) 1) French Agency for Food, Environmental and Occupational Health and Safety, Plant Health Laboratory, Nematology unit, Domaine de la Motte au Vicomte, BP35627, 35653 Le Rheu, France. sylvie.gamel@anses.fr 2) French Agency for Food, Environmental and Occupational Health and Safety, Plant Health Laboratory, 7 rue Jean Dixméras, 49044 Angers, France.

Plant parasitic nematodes cause important damage on many crops and this occurrence is expected to increase over the next few years due to pesticides bans, increasing trade etc.. Some nematodes are regulated in Europe to limit their dispersal over non-infested areas. As a result, the development of reliable screening methods to detect quarantine nematodes in different matrices is important in order to detect and manage new infested areas. Morphological analysis is commonly performed to identify nematode species. However, this technique is time-consuming and not suitable to analyse large series of samples. Over the last few years, several real time PCR tests were adapted for direct detection, after isolation of nematodes from matrix. The example of the Bursaphelenchus xylophilus and Meloidogyne chitwoodi and M. fallax detection in nematodes suspensions respectively from wood and soil will be presented. Assessment procedure for validation according to the EPPO Standard (PM 7/98) is presented; the key points of its evaluation for detection purpose are highlighted.

Session 5 Posters

Abstracts of Posters

Françoise Petter POSTER PRESENTED BY THE AUTHOR

EPPO databases

Françoise Petter 1 , Roy Anne Sophie 1 , Griessinger Damien An extensive die-back of European hop hornbeam (Ostrya carpinifolia) has been observed in Slovenia since 1997, which coincided with unusually hot and dry weather conditions. Numerous fungal isolates, belonging to Botryosphaeriaceae were isolated from the diseased trees. Closely related Botryosphaeriaceae species cannot be easily resolved due to overlapping morphological characteristics. In order to unravel the obtained Botryosphaeriaceae complex from the diseased O. carpinifolia trees, the analyses of sequence data for the ITS rDNA and translation elongation factor 1-α were performed. The sequence data revealed four groups; the majority of isolates were classified as Botryosphaeria dothidea, but a small group of isolates was identified as Dothiorella spp., with three subgroupings, of which at least one most likely represents an undescribed species. The implementation of molecular data proved to be an indispensable step in the identification of the Botryosphaeriaceae found in diseased tissues of O. carpinifolia and enabled further characterization of the pathogenicity and significance of resolved species in the disease development. Yükselbaba Utku, İkten Cengiz, Göçmen Hüseyin, Akdeniz Üniversitesi, Ziraat Fakültesi, Bitki Koruma Bölümü, Antalya,TURKEY uyukselbaba@akdeniz.edu.tr

Cotton whitefly Bemisia tabaci (Gennadius) is a polyphagous pest that is widely distributed in tropical and subtropical regions. Many biotypes of this pest have been identified and reported from different regions of the world. In this study, the biotypes of B. tabaci populations collected from 7 different regions of Antalya province were identified by sequence analysis of the mitochondrial Cytochrome Oxidase I (mtCOI) region. DNA extracted from each individual whitefly was amplified using primers specific to mtCOI region. An amplicon of approximately 800 bp of which 770 were used in sequence analysis were obtained. The sequences were subject to aligment analysis.

Results of the analysis revealed that all the populations of B.tabaci studied here were biotype B. These results also showed that some populations from some regions previously known to be biotype Q had changed to biotype B, leading to the assumption that biotype B could take over and become the dominant biotype in a short period when introduced into new areas.

Béatrice Courtial POSTER

Use of the Nuclear Region ITS2 for Arthropods Species Identification

Courtial Béatrice (1), Auger-Rozenberg Marie-Anne (1), Roques Alain (1) 1) INRA, Unité de Recherche de Zoologie Forestière 2163 Avenue de la Pomme de Pin CS 40001 Ardon 45075 ORLEANS Cedex 2, France, beatrice.courtial@orleans.inra.fr

During the last decade, many barcode projects were launched for species identification by DNA sequencing in order to improve taxonomic knowledge of biodiversity and to try to assign sample individuals to species. In the case of Arthropods the most often used region is a part of the mitochondrial gene Cytochrome Oxydase I, which is often insufficient to reliably delineate and name the species. To confirm the identification of specimens it is necessary to use another DNA sequence. This sequence is very often the nuclear ribosomal internal transcribed spacer 2 (ITS2).

The data presented here show the preliminary results of ITS2 sequencing on 40 arthropods species belonging to different orders (Coleoptera, Lepidoptera, Hymenoptera). The results presented show the difficulty of defining universal primers for this target ITS2 and the large variability of obtained sequences (presence of deletion, repeats, etc.

). An example of sequences obtained for the genus Dendroctonus (Coleoptera, Curculionidae) is also supplied. To detect and identify phytophthora species several molecular methods have been developed for single species only. A uniform method for detection of all Phytophthora species would be very useful for research and regulatory communities. Therefore a diagnostic method was developed to detect a range of Phytophthora species, including P. ramorum. The method includes a generic TaqMan PCR amplification method for all Phytophthora species combined with species specific padlock probe (PLP) detection on a dedicated universal micro-array. Twenty-three padlock probes for 22 Phytophthora species relevant for the Netherlands were developed based on sequence differences in the ITS-1 region. After point mutation specific ligation of a mixture of the 23 PLPs on the generic amplicon, exonuclease treatment to degrade the unreacted probes, amplification of the ligated probes and hybridization on a micro-array, a unique signature on the micro-array can be obtained for each Phytophthora species included in the test. Specificity and sensitivity of this padlock based diagnostic tool has been combined with a cost effective microtiter plate array detection device and has been evaluated using reference Phytophthora cultures as well as mixed infected material collected from field surveys, including air-, root-, water-and plant tissue samples.

Martijn Holterman POSTER Barcoding in the Ralstonia solanacearum complex

Holterman Martijn H.M.(1), Frey Jürg E.(1), Maes Martine(2), Duffy Brion(1) 1) Agroscope Changins-Wädenswil, Schloss, 8820 Wädenswil, Switzerland martijn.holterman@acw.admin.ch 2) Institute for Agricultural and Fisheries Research, Plant-Crop Protection, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium

Ralstonia solanacearum is an important plant pathogen worldwide with a wide host range. It causes wilting diseases in, amongst others, solanaceous crops. Furthermore, races 2 and 3 of R. solanacearum have quarantine status in Europe. As part of the QBOL project (barcoding of European quarantine organisms) 11 genes were examined for their potential use as barcoding regions in the genus Ralstonia. Three genes, the mutS, egl and rpoB genes, were selected as the most promising barcoding regions. R. solanacearum is a complex species, consisting of four phylotypes, one of which also includes R. syzygii and the Banana Blood Disease bacterium. Recently it was proposed to divide this complex into three new species. All three selected genes allowed the phylotype to be determined and supported the division of the R. solanacearum complex into these three new species. was detected in the Netherlands, and in the following years it was also found in several other countries. It is reported that the bacterium is more aggressive than both P. atrosepticum and D. dianthicola, can be found in a wider range of climatic conditions, needs lower bacterial concentrations to cause disease and spreads faster and more efficiently. The name "Dickeya solani' has been proposed for this pest.

In Norway, D. solani has not yet been detected. Bioforsk Plantehelse has received potato samples with unusual blackleg symptoms in the past two years, some of which were found to be infected by virulent strains of Pectobacterium wasabiae. In 2011 a small, initial Dickeya-survey was carried out. More than hundred isolates have been collected from 20 arbitrarily gathered samples of symptomatic potato plants and tubers. These have been analyzed with realtime PCR assays using primers and probes for Pectobacteria and Dickeya. Dickeya solani was not detected.

Johannes Hallmann POSTER Q-bank -The nematological approach of offering ecological, morphological and molecular information for the identification of regulated and other relevant plant-parasitic nematodes

Hallmann Johannes (1), van Heese Evelyn (2), Kiewnick Sebastian (3) 1)Julius Kühn-Institut, Federal Research Center for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Toppheideweg 88, D-48161, Münster, Germany, johannes.hallmann@jki.bund.de 2)Plant Protection Service/National Reference Centre (PPS), The Netherlands, e.y.j.van.heese@minlnv.nl 3)Agroscope Changins-Wädenswil, Schloss 1, 8820 Wädenswil, Switzerland, sebastian.kiewnick@acw.admin.ch Taxonomic expertise is dramatically declining in the scientific community. In an attempt to conserve existing knowledge but also provide modern tools for nematode identification, Q-bank was started. This database comprises ecological, morphological, physiological, and sequence data of properly documented strains of plant pests and diseases allowing plant protection organizations, inspection bodies and private laboratories an accurate identification.

The main focus of Q-bank is on regulated plant pests and pathogens, however, for accurate distinction of regulated from non-regulated pests and diseases, descriptions of the latter are equally important. Different groups of pests and diseases require different information. For the field of nematology, the concept and structure of Q-bank is presented and discussed. The available information will be listed. One of the main questions raised during the pest risk analysis (PRA) for M. enterolobii was the possible source(s) and route(s) of introduction into Europe. To ensure that appropriate phytosanitary measures are at hand, reliable detection and identification tools are needed. This EUPHRESCO project will deliver new and validated protocols for sensitive detection and reliable identification of M. enterolobii and consequently improve diagnostic expertise in Europe. It will provide a test performance study and a workshop establishing DNA-barcoding and the use of the Q-bank database as a useful identification tool. It will also support the decision making process for eradication or integrated control strategies once the nematode has been detected and correctly identified.

Anne-Laure Boutigny POSTER

An optimised duplex real-time PCR tool for highly sensitive detection of the quarantine oomycete Plasmopara halstedii in sunflower seeds

Ioos Renaud (1), Fourrier Céline (1), Wilson Véronique (1), Webb Kathryn (2), and Tourvieille de Labrouhe Denis(3) 1) ANSES, Laboratoire de la Santé des Végétaux, Unité de mycologie, IFR 110, Domaine de Pixérécourt, F-54220 Malzéville, France.

2) FERA, 04GA08/09, Sand Hutton, York, YO41 1LZ, United Kingdom.

3) INRA, Plate-Forme IPM, UMR 1095, Domaine de la Crouelle, 234 avenue du Brézet, F-63100 Clermont-Ferrand, France.

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an obligate parasite that is subjected to phytosanitary regulation. This pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported by international commercial trade of these seeds. In order to prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. hasltedii in sunflower seed samples is required. This work reports the optimisation of a novel real-time detection tool that targets the oomycete within sunflower seed lots, and provides statistically supported validation data for that tool. The test is shown to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as little as 45 copies of target DNA. A fully optimised DNA extraction protocol was also developed starting from a sample of 500 sunflower seeds, and enabled the final detection of less than one infected seed / 500. In order to ensure reliability of the results, a set of controls was used systematically during the assays, including a plant specific probe used in a duplex qPCR fashion that enabled the assessment of the quality of each DNA extract. As part of the Great Lakes Cassava Initiative (GLCI), which is providing disease free planting material to African farmers, a diagnostic method was developed. Using next generation sequencing, isolates of CBSV and UCBSV were genome sequenced and real-time PCR assays were designed to detect both viruses. The performance of these assays was validated using 493 cassava samples from Kenya, Uganda and Tanzania and on extracts of pools of diseased and healthy cassava leaves. Using this data, the appropriate sampling and pooling strategy was (300 leaves in 30 sub-samples) was developed to give a 95% confidence of detecting infected plants that make up 1% of the plants in a field. Next generation sequencing was also used to confirm the absence of any virus in samples which appeared to have visual symptoms but tested negative by the real-time assay.

The diagnostic method has now been used to screen 150000 leaf samples from GLCI multiplication sites.

Mathieu Rolland POSTER

Clavibacter michiganensis subsp michiganensis : evaluation of efficiency of different detection methods

Rene Mathis(1), Celine Fricot(1), Magali Larenaudie(1), Thomas Baldwin(2), Rodolphe Germain (2), Valerie Olivier(3), Anne Quillevere(1), Mathieu Rolland(1), and Valerie Grimault(1) 1) GEVES, rue G Morel, BP 90024, 49071 Beaucouzé Cedex. mathieu.rolland@geves.fr 2) Union Française des Semenciers, 17 rue du Louvre, 75001 Paris 3) ANSES, 7 rue Jean Dixmeras, 49044 Angers Cedex 01

Clavibacter michiganensis subsp. michiganensis (Cmm) is one of the most important tomato seed transmitted pathogens.

In recent years the number of outbreaks has increased. Due to absence of chemical control on tomato plants, the only way of controlling the spread of this disease is seed testing. The efficiency of the existing detection/identification methods were compared on a collection of 213 bacterial isolates including 141 Cmm strains, saprophytes, other Clavibacter and other genera pathogenic on tomato. Methods compared were immunofluorescence, isolation on media, PCR and pathogenicity. Sensitivity and specificity of each method were determined. Comparison of growth of bacteria on two different semi selective media showed that slow and fast growing isolates must be taken into consideration for seed analysis. Extraction methods and efficiency of detection on treated/untreated seeds were studied on a naturally contaminated seed lot obtained by artificial contamination of a tomato culture and extraction of seeds produced and on commercial seed lots. A promising confirmation method, direct PCR on seed macerate used for immunofluorescence, was set up and is under evaluation. This study contributed to the validation of a detection method on media of the International Seed Federation and to the EPPO Standard.

Session 6 Bacteria

Abstracts of Presentations

Thursday 2012-05-24

Bart Cottyn

QBOL -WP 4: Barcode identification of quarantine bacteria, the QBOL strategy and results

Cottyn Bart (1), De Vos Paul ( 2), Holterman Martijn (3), Zaluga Joanna (2), Gavrila Elena (1), and Maes Martine (1) 1) Institute for Agricultural and Fisheries Research -ILVO, Merelbeke, Belgium, martine.maes@ilvo.vlaanderen.be 2)Laboratory of Microbiology, University Ghent, Belgium 3)Agroscope Changins-Wädenswil -ACW, Zwitserland Within QBOL, barcode sequences have been defined for a group of quarantine bacteria from the combined EU/EPPO lists, comprising the three Clavibacter michiganensis subspecies, Ralstonia solanacearum, Xylella fastidiosa and a selection of Xanthomonas pathogens. The challenges were to compose a relevant collection of quarantine and closely related bacteria, to unravel their taxonomic and pathogenic identity, and to define representative barcode sequence regions.

A barcoding strategy had to be developed. For this it was not possible to use other barcoding of life (BOL) programs, which study existing diversity of eukaryotes, but not of prokaryotes. Moreover, several quarantine bacteria are named as pathovars, an infra-species division that refers to a pathological specialization and does not necessarily correlate with a taxonomic unit. Barcode identification of the quarantine bacteria uses several gene sequences in a step-wise approach following different flows in a decision scheme. The barcoding principle and process will be illustrated for a set of Xanthomonas pathovars.

Isabelle Robène-Soustrade PCR-based assays for detecting Xanthomonas axonopodis pv. allii in onion seed.

Isabelle Robène-Soustrade(1), Marion Perret(1), Emmanuel Jouen(1), Aude Chabirand(2) and Olivier Pruvost(1) 1) CIRAD, UMR Peuplements végétaux et Bioagresseurs en Milieu Tropical CIRAD-Université de la Réunion, Pôle de protection des plantes, 7, chemin de l"IRAT, 97410 Saint Pierre, La Réunion, France 2) Laboratoire de la Santé des Végétaux , Anses, Pôle de protection des plantes, 7, chemin de l"IRAT, 97410 Saint Pierre, La Réunion, France. isabelle.robene@cirad.fr Bacterial blight of onion is an emerging disease threatening world onion production, and causing damage to other Allium crops. The causal agent, Xanthomonas axonopodis pv. allii (Xaa) has been listed on the EPPO A1 list of pests recommended for regulation as quarantine pests, since 2009. A duplex nested-PCR assay, targeting two markers specific to Xaa has been recently developed (Robène-Soustrade et al., 2010). A triplex quantitative real-time PCR assay (Taqman® technology) was developed targeting the same Xaa-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Xaa strains were detected by the amplification of one or both of the two specific markers. The internal control signal validates both the extraction process and the reaction itself. Several successive steps have to be performed before detection from seed: seed maceration for 48h at 4°C, followed by homogenization of the seed macerate with a stomacher® and DNA extraction using DNeasy® Plant mini kit (Qiagen). This assay is currently being validated following the European standard EN ISO 16140: 2003 and the EPPO standard PM7/98 (1). The performance of Nested-PCR and real-time PCR assays are discussed. These PCR-based tools could be useful for the international sanitary surveillance of seed exchanges.

Maria Bergsma-Vlami

Invasion of Xanthomonas arboricola pv pruni (Xap) in the ornamental P. lauroceracus (cherry laurel) in the Netherlands

Bergsma-Vlami Maria (1), Koenraadt Harrie (2), Teunissen Hedwich ( 2 The aim of the study was characterization of strains recently from infected potato plants in Poland. Phenotypic features such as the ability to chelate iron, biosurfactant production, motility and activity of cellwall degrading enzymes have been analyzed. Tested strains produce a vast amount of pectolytic enzymes, which facilitate the degradation of plant cell walls and maceration of the plant tissue. Significant differences between tested strains, in the ability to produce pectolytic enzymes in anaerobic conditions and at different temperatures, were observed. All tested strains indicated both identical sequences for the housekeeping genes (dnaX, gyrA, recA, rpoS) and identical REP-PCR and PFGE patterns.

Reference: Toth I, van der Wolf J, Saddler G, Lojkowska E, Hellas V, Pirhonen M, Tsror L, Elphinstone J. 2011 Plant Pathology, 60: 385-399.

René Mathis

Clavibacter michiganensis strains characterized as non-pathogenic by phylogenetic and polyphasic analyses are transmitted by tomato seeds and interfere with C. michiganensis subsp. michiganensis detection. Clavibacter michiganensis subsp. michiganensis (Cmm), the agent responsible for bacterial wilt and canker of tomato, causes severe losses worldwide. This quarantine phytopathogen is under regulation in order to control its spread. The best way to fight this seed borne pathogen is based on seed control. Improving pathogen detection tools requires a precise knowledge of the pathogen diversity. Therefore the diversity of a worldwide Cmm strain collection was investigated to explore the genetic diversity and to establish the phylogenetic relationship between strains using a MLSA-MLST type analysis based on the partial sequences from six housekeeping genes. Working on a collection of more than 200 strains isolated from tomato seeds, it was possible to show differences in aggressiveness between Cmm strains and to identify non-pathogenic strains. Sequence polymorphism observed in a subset of this collection shows that Cmm is monophyletic and is distinct from its closest taxonomic neighbours. Few Cmm clonal complexes were identified in MLST. The taxonomic identity of the C. michiganensis saprophytic strains was confirmed by 16S rDNA sequencing. Based on MLSA, we show that these strains are phyllogenetically distinct from the pathogenic strains. However, these strains cross-react with the detection tools we tested. Our data provide a large data set to study this pathogen and to develop new detection tools.

Marie-Agnès

Paul De Vos

Is identification by bar-coding of Q-clavibacters in conflict with the existing bacterial taxonomy of this group of bacteria?

De Vos P. (1,2) and Maes M.( 3) 1) Department of Biochemistry and Microbiology, Gent University, Gent, Belgium; Paul.DeVos@Ugent.be 2) BCCM/LMG Bacteria Collection, Gent, Belgium 3) Institute for Agricultural and Fisheries Research-ILVO, Merelbeke, Belgium,

The genus Clavibacter contains mainly plant pathogenic bacteria elaborating a host specificity that makes them of great economic importance and most of them are regarded as quarantine and/or q-alert organisms. Taxonomically they are known to form a well delineated clade in the Microbacteriaceae. Bacterial taxonomy concerns grouping, naming and identification of bacteria at the species or sub-species level. The molecular backbone of the taxonomic system is the 16S rRNA gene sequence that allows delineating groups of higher taxonomic ranks. Species and subspecies delineation is based on comparative analysis of overall genome relatedness coupled with phenotypic discriminatory characteristics. Recent developments in bacterial taxonomy reveal that species delineation can be achieved by the MLSA (Multilocus Sequence Analysis) based on a number of well-selected house-keeping genes.

The number of genes to be sequenced for obtaining a reliable identification is probably depending on the group of organisms under study. Within the frame of QBOL (Quarantine Barcoding of Life) European Project it is aimed to develop a reliable identification based on DNA barcoding to significantly improve the current diagnostics. The question may be raised whether the bar-coding approach would conflict the existing Clavibacter taxonomy. Two hundred named clavibacter strains were subjected to sequence analysis of 16S rRNA gene and gyrB. A 500bp sequence of the housekeeping gene -gyrB was found to be a discriminative barcode at the subspecies level for the included clavibacters. Including phylogenetically related species confirmed the robustness of the method at this taxonomic level. However, it is known that bacteria exchange part of their genomes horizontally and this may affect the long term validity of existing bar-coding schemes for identification.

In order to back up the results of the bar-coding, MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) patterning was introduced. It turned out that the MALDI-TOF profiles allowed selecting for subspecies specific biomarkers, herewith supporting the sequencing data. In 2007 there were several worldwide outbreaks of Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato. Amplified Fragment Length Polymorphism (AFLP) was initiated to investigate the genetic diversity within Cmm strains and closely related strains. It revealed extensive genetic variation and showed that two contaminated seed lots were responsible for some of the outbreaks of Cmm. At present, for Cmm and related strains there is a database with more than 750 entries. The outbreaks have intensified international research to improve the reliability of the seed assay. Extraction of Cmm from contaminated seeds and concentration of the bacteria were identified as critical steps to improve the sensitivity of the assay. New media were developed that allow good growth of Cmm while the growth of many saprophytes is inhibited. Spiking, a technique in which a marked Cmm strain is added to samples, showed that antagonistic micro-organisms could obscure Cmm presence. Spiked and non-spiked versions of seed extracts are now routinely plated. A valid test result is only obtained when the Cmm spike is recovered. The molecular identification procedure was also improved. The adaptations in the Cmm protocol resulted in the new ISHI 4.0 protocol. The outbreaks initiated cooperation between seed companies and transplant producers leading to the foundation of "Good Seed and Plant Practices" (GSPP). The impact of GSPP and the new validated ISHI 4.0 protocol will be discussed.

Françoise Poliakoff

Emergence of Pseudomonas syringae pv. actinidiae (Takikawa, 1989) in France: methods of characterization

Poliakoff Françoise (1), Amandine Cunty ( 2), Carène Rivoal (1) 1) French Agency for Food, environmental and occupational health and safety -Anses -Plant Health Laboratory, 7 rue jean Dixméras 49044 ANGERS Cedex 01 (FR), lsv.angers@anses.fr francoise.poliakoff@anses.fr 2) SCAAP Kiwifruits de France, 2398, Boulevard de l"Océan, RN 117, 40 300 LABATUT (FR).

Pseudomonas syringae pv actinidiae (Psa), causal agent of Kiwi canker, has been known since recent outbreaks in Italy in 2007 (Balestra, 2009) as an aggressive pathogen on Kiwi fruit, spreading rapidly in orchards and the environment. The first recordings of Psa were in 1984 in Japan (Takikawa et al., 1989, Serizawa et al. 1989), then in 1992in Korea (Lee & Koh, 1994) and Italy (Scortichini, 1994) where outbreaks had been eradicated.

This bacterium was first detected in France in July 2010 by isolation on nutrient agar media and identification. Its spread has been rapid since then, to Portugal (Balestra, 2010), New Zealand (2010, MAF Biosecurity Website) it was then reported in 2011 in Chile, Australia, Switzerland and Spain. The bacterial canker poses a serious threat to the cultivation of the yellow-fleshed kiwifruit in Italy (Actinidia chinensis), but also to green-fleshed kiwifruit (Actinidia deliciosa cv. Hayward). In France, this variety comprises about 95% of orchards.

The French strains isolated in 2010 were shown to belong to the same population as the strains from recent outbreaks in Italy. These genetics profiles where confirmed for most of the strains isolated during 2011 by using different biomolecular tools. The genetic structure of the population of strains isolated from French Kiwifruit is under investigation.

Gilles Cellier

Story of an array based technology designed to grasp and detect the complex plant pathogen Ralstonia solanacearum

Gilles Cellier (1), Sandrine Arribat (2), Isabelle Robène (2), and Philippe Prior (2,3) 1) ANSES -LSV, unité Ravageurs et Agents Pathogènes Tropicaux (RAPT), 7 chemin de l'Irat, Pole de Protection des Plantes, 97410 SAINT PIERRE, gilles.cellier@anses.fr 2) CIRAD, UMR Peuplement Végétaux et Bioagresseurs en Milieu Tropical (PVBMT), 7 chemin de l'Irat, Pole de Protection des Plantes, 97410 SAINT PIERRE 3) INRA, 75000 PARIS gilles.cellier@anses.fr

The ancient soil borne plant vascular pathogen Ralstonia solanacearum evolved and adapted to cause severe damage on an unusually wide range of plants. Based on the phylotype classification, it was questioned how genetically and phenotypically diverse strains of Ralstonia solanacearum may be, in an attempt to produce adapted diagnostic tools.

A pangenomic microarray was first used to better describe and understand the phylogeny of R. solanacearum, especially three particular ecotypes in the phylotype II: (i) Brown rot strains from sequevars IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from sequevar IIB-4NPB that lack pathogenicity to Cavendish banana but can infect many other plant species; and (iii) Moko disease-causing strains from sequevars IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia. Results revealed a phylogeographic structure within Brown rot strains, allowing European outbreak strains of Andean and African origins to be distinguished, and also showed a close but distinct relationship between Moko ecotype IIB-4 and the emerging IIB-4NPB strains. Based on those results, it is proposed to develop new DNA-based diagnostic tools, including array and routine PCR, to ensure better detection of this complex plant pathogen.

Xiang (Sean) Li

Graft-transmission of 'Candidatus Liberibacter solanacearum', the causal agent of potato zebra chip disease, from greenhouse-grown latently-infected tomato.

Li Xiang, Nie Jingbai, Hammill Desmond L., Arsenault Heidi, and De Boer H. Solke. Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mt Edward Road, Charlottetown, PE, C1A 5T1, Canada, sean.li@inspection.gc.ca.

In contrast to potato plants propagated in growth chambers that developed severe symptoms of zebra chip (ZC) when infected by "Candidatus Liberibacter solanacearum" (CLS), ZC-infected tomato plants served as a latent carrier of CLS. Tomato plants (varieties Money Maker and Roma) graft-inoculated with scions from latently infected tomato plants remained symptomless, but tested positive in a ZC-specific PCR assay while potato plants (cultivars Jemsam, Atlantic, Shepody, Frontier Russet, and Russet Norkotah) showed typical symptoms of purple top and leaf scorch four weeks after being grafted with scions from the same tomato plants. Tubers from the graft-inoculated potato plants also showed typical symptoms of brown discoloration in the vascular ring and medullary rays. CLS could not be detected in the aerial tubers of graft-inoculated greenhouse-grown plants, but the bacterium was readily detected in the stems and progeny tubers of the same plants. It was also considered important to differentiate CLS from the potato witches" broom phytoplasma because they cause similar foliage symptoms in potato. Therefore, a culture-independent approach was employed to clone and sequence the rrn operon of the two phytopathogens. Sequence analysis provided ample targets to differentiate the two pathogens as would be expected for phylogenetically different microorganisms.

Session 7 Fungi

Abstracts of Presentations

Johannes Zacharias Groenewald Fungal organisms were selected from the EU Council Directive and EPPO A1/A2 lists based on the availability of cultures and/or taxonomic expertise and were sequenced for (potentially) informative barcode regions. The following fungi and their related organisms were selected for the QBOL Work Package 2 (Fungi): Ceratocystis fagacearum, C. fimbriata f. sp. platani, C. virescens, Pseudocercospora angolensis, Ps. pini-densiflorae, Melampsora farlowii, Me. medusae, Monilinia fructicola, Mycosphaerella dearnessii, My. larici-leptolepis, My. populorum, Puccinia pittieriana, Septoria malagutii and Thecaphora solani. The internally transcribed spacer (ITS) regions of the nrDNA operon were initially used to confirm the taxonomic identity of all strains and to evaluate the resolution for species resolution of this commonly used region. One or more additional house-keeping genes, e.g. actin, beta-tubulin and translation elongation factor 1-alpha, were screened for almost all species to supplement data for species with poor ITS resolution. A molecular decision scheme was developed to support decision making of which loci to use for which species. This presentation will give an overview of the obtained results and highlight the more important findings.

Wellcome Ho

DNA barcoding of the fungal genus Phoma from New Zealand

Wellcome, Ho(1), Kun Pan(1), Gerald Bills(2), Brett Alexander(1) 1) Plant Health and Environment Laboratory, Ministry of Agriculture and Forestry, PO Box 2095, Auckland 1140, New Zealand, wellcome.ho@maf.govt.nz 2) Fundación MEDINA, Avenida de Conocimiento 3, Parque Tecnológico Ciencias de la Salud, Armilla, Granada 18100, Spain

The New Zealand Ministry of Agriculture and Forestry, Plant Health & Environment Laboratory (PHEL) is responsible for the identification of new and exotic plant pests and diseases that may be damaging to the country"s primary industries and environment. The PHEL has developed a DNA barcoding system using the fungal group Phoma as a model for the identification of pests and diseases of biosecurity concern. The barcode database comprises DNA sequences from the ITS regions, the actin and beta-tubulin genes. Around 800 sequences were generated from 320 Phoma isolates that were collected from culture collections in New Zealand. In addition, over 800 authentic sequences from type or representative specimens have been selected from around 2,000 published sequences.

Using the barcode database, 80% of the 320 isolates were identified to species level. Among these, 32 isolates were identified as belonging to 14 Phoma or closely-related species that had not been reported in New Zealand previously.

The barcode database complements the morphological identification technique used for plant pathogenic fungi and has been demonstrated to accurately identify surveillance samples. The barcoding process has been extended to other groups of plant pests and diseases for rapid and accurate identification.

William Quaedvlieg

Barcoding Mycosphaerella species of quarantine importance to Europe Quaedvlieg W., Groenewald J.Z. and Crous P.W. CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands w.quaedvlieg@cbs.knaw.nl

The EU 7 th Framework Program funded by the Quarantine Barcoding of Life (QBOL) project, aims to develop a quick, reliable and accurate DNA barcode-based diagnostic tool for Mycosphaerella species on the European and Mediterranean Plant Protection Organization (EPPO) A1/A2 lists of pests recommended for regulation as quarantine pests. In order to determine which barcoding loci would be best suited for identifying Mycosphaerella and/or its anamorph species on these A1/A2 lists, seven nuclear genomic loci (β-tubulin (Btub), internal transcribed spacer( ITS), 28S nrDNA (LSU), Actin (ACT), Calmodulin (CAL), Translation elongation factor 1-alpha (EF1) and RNA polymerase II second largest subunit (RPB2)) were tested on their Kimura-2-parameter-based inter-and intraspecific variation, their PCR amplification success rate and their ability to distinguish between quarantine species and their closest known neighbours. The research showed that none of the test loci was ideally suited individually to serve as a reliable barcoding locus; however, a combination of a primary and secondary barcoding loci can compensate for individual weaknesses and give reliable identification results. A combination of either ITS and EF1α or Btub would be an ideal combination to serve as barcoding loci for EPPO A1/A2-listed Mycosphaerella species of quarantine importance.

Mathias De Backer

New SNP markers for the genetic characterization of the quarantine pathogen Puccinia horiana and their application in pathogen migration analysis

Mathias De Backer (1,2), Martine Maes (1), Erik Van Bockstaele (1,2), Theo van der Lee (3) and Kurt Heungens (1) 1) Plant Sciences Unit -Crop Protection; Institute for Agriculture and Fisheries Research (ILVO); Burg. Van Gansberghelaan 96 bus 2; 9820 Merelbeke, Belgium mathias.debacker@ilvo.vlaanderen.be 2) Ghent University, Faculty of Bioscience Engineering, Department of Plant Production, Coupure Links 653, 9000 Ghent 3) Plant Research International, Biointeractions and Plant Health, Droevendaalsesteeg 1, 6708 PB Wageningen

The quarantine pathogen Puccinia horiana, causal agent of chrysanthemum white rust, is one of the most important diseases in chrysanthemum-producing regions worldwide. During this study the genetic variability of forty five isolates originating from North and South America, Asia, and Europe was determined based on neutral Single nucleotide polymorphism-markers. Together with information on collection year and geographical origin this allowed questions of migration, survival, and recombination to be addressed. A high level of genotypic diversity was observed and in most cases phylogenetic clustering was related to the geographic origin. Local survival was demonstrated based on multi-year findings of specific clones in a given geographic area. Recent long distance migration events were demonstrated but were limited in number. Exclusive clonal propagation of this microcyclic rust was disproved given clear indications of recombination between specific genotypes. The genotype data were also combined with the pathotype data of specific isolates but pathotype-specific markers were only observed in one of the clades. In combination with a fast SNP detection system, this technique could allow rapid characterization of intercepted or local isolates, which may help in questions relating to the need for quarantine action. A multiplex diagnostic method, based on macroarray, has been set up for the detection in soil of the following fungi: Verticillium dahliae, V. albo-atrum, Armillariella mellea, Nectria galligena, Phytophthora cactorum and Chondrostereum purpureum. The method has been set up to provide a diagnostic tool for the production of certified pome fruit propagation materials as regulated by the Italian Ministry of Agriculture following the European Directives. The macroarray method is an open system which enables a high level of multiplexing and allows new oligonucleotides detectors to be added for new target identification and/or to change sensitivity/specificity of detection. With this technology, oligonucleotides detectors are immobilized on a nylon membrane and used as target for microorganism detection. The DNA to be tested is amplified, labelled and then hybridized to the membrane. In this system, DNA is directly labelled with Alkaline Phosphatase which reacts with CDP-Star giving a chemiluminescent signal. Oligonucleotides detectors have been designed on the ITS regions of the rDNA, because of its high sequence variability and availability in gene bank databases. Species and genus-specific oligonucleotide detectors have been designed. The system has been validated using DNA extracted from pure fungal cultures and from infested soil. The characteristics of the macroarray system are discussed.

Hans de Gruyter

Molecular phylogeny of

James Woodhall

Using real-time PCR to detect plant pathogens in soil James W Woodhall, Ian P Adams, Jeff C Peters & Neil Boonham The Food and Environment Research Agency, Sand Hutton, York YO41 1LZ, UK james.woodhall@fera.gsi.gov.uk Soil borne diseases are a major constraint to crop production worldwide. Detection of a soil-borne plant pathogen prior to planting could therefore inform grower decisions concerning planting and soil treatment. Real-time PCR is a robust detection method and combined with a suitable DNA extraction method, could be used to quantify a specific pathogen in a particular soil sample. However, the detection of a pathogen in soil presents several challenges. These include the ability to extract DNA from biologically relevant sample sizes, effective sampling strategies and techniques to cope with inhibition due to the presence of humic compounds within a sample. Measures to overcome these challenges are discussed and several examples of successful diagnostic assays for important soil-borne plant pathogens are presented. DNA barcoding is a generic diagnostic method that uses a short standardised genetic marker in an organism's DNA to aid species identification. DNA barcoding protocols for 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. The use of a single barcode region was found to be insufficient for the identification of the majority of regulated plant pests. Therefore, several short standardised genetic markers have been appointed as "barcodes" for the identification of plant pest groups. To determine the usability of the developed tests for diagnostics, an international collaborative test performance study among 14 laboratories was organised. Participants were provided with the protocols (presented as a draft EPPO standard), interactive forms, primers, DNA extraction kits, test material and positive amplification controls in order to perform the tests and a USB-key to report the data. USB-keys were collected by PPS and the data was analysed to determine the diagnostic sensitivity, diagnostic specificity, reproducibility, repeatability and robustness of the tests.

Vincent Robert

QBOL and Q-bank data management and analysis system Robert V., Szoke S., Jabas B., Vu D., Chouchen O. and Amor, A. Bioinformatics, CBS-KNAW fungal biodiversity centre, Utrecht, Netherlands, v.robert@cbs.knaw.nl

For the Quarantine Barcoding of Life (QBOL, EU project) as well as the Q-bank (international consortium mainly supported by the Dutch government) projects a complete scientific data management system based on the BioloMICS software has been developed. Morphological, physiological, chemistry, ecological, molecular, geographical, administration, bibliography and taxonomy data etc. can each be stored, handled and analyzed in the most appropriate ways. The system includes all relevant biological data related to Bacteria, Fungi, Insects, Nematodes, Phytoplasmas, Plants and Viruses associated with the quarantine problematic. The system not only allows data to be saved but also to be analysed (polyphasic identification, classification, statistics, automated curation, etc.) but also to be published online. The system is completely dynamic in the sense that new characters or fields, tables and records can be added to the system within minutes without the intervention of software developers.

Rachel Glover

Biodiversity sequencing -next generation DNA barcoding

Glover Rachel (1), Adams Ian (1), Hany Ummey (1), Chisholm James (1), Boonham Neil (1) 1) Food and Environment Research Agency, Sand Hutton, York, YO41 1LZ, United Kingdom rachel.glover@fera.gsi.gov.uk

The application of next-generation sequencing technologies to environmental samples is not new, with 16S bacterial metagenomic studies being commonplace. However, recent improvements in the 454 pyrosequencing chemistry have resulted in sequence lengths of 600-700bp which enables the expansion of metagenomic amplicon sequencing to other loci, in particular those currently being used for DNA barcoding (COI, ITS, rbcL, 18S). This presentation will discuss these recent developments and their application to biodiversity studies and large-scale diagnostics in the near future.

Ming-Fu Li

Research on and applications of DNA Barcodes for species identification

Ming-fu Li, Zhu Zhao, Jing xu, Yun Song, Shuifang Zhu Chinese Academy of Inspection and Quarantine, Beijing, China limf9@caiq.gov.cn

Along with the development of China's economy and trade, Import and export of goods and staff exchanges have become increasingly frequent in 2011. More than 500 thousand batches of goods were imported and exported, and thousands of harmful biological species intercepted. Facing more and more biological safety issues, the department of inspection and quarantine is looking for more effective methods to identify the species, and deal with the risks such as invasive alien species, genetic resources losses, economic fraud etc. With the birth of DNA barcode technology, a lot of research have been carried out in China , its application has been explored actively and some scientists have been involved in relevant international cooperation, So far, more than 20 different flora and 15 kinds of fauna were investigated and identified with DNA barcode technology and some important gene barcodes were acquired and verified; A few of specific barcode databases were built, and applied preliminary for inspection and quarantine, the preservation of Chinese medicine resources and the conservation of biodiversity. Meanwhile, several nationally important DNA barcode research projects have been funded in the twelfth five-year plan, that will be expected to further improve the technology system of inspection and quarantine in China, and play an important role in performing the related international treaty, realize the objectives of the Convention on Biological Diversity (CBD) by promoting conservation, sustainability, and the equitable sharing of benefits arising from use of genetic resources.

Bénédicte Moignot

How to prioritize the development of diagnostic tests for plant pests based on a risk ranking tool?

Moignot Bénédicte (1), Ouvrard Florian (1), Mouttet Raphaëlle (1), Suffert Frédéric (2), Reynaud Philippe (1) 1) Anses, Plant Health Laboratory, 7 rue Jean Dixméras, 49044 Angers cedex 01, France benedicte.moignot@anses.fr 2) INRA, UMR BIOGER-CPP, Campus AgroParisTech, 78850 Thiverval-Grignon, France.

Many regulated and exotic plant pests threaten crops in the European Union. While inspections and surveys using diagnostic tests are useful to mitigate the risk of their introduction, prevent their permanent establishment and limit their subsequent spread, resources are dramatically limited to cover all regulated pests. Therefore detection efforts should be performed in accordance to the assessed pest risks. A pragmatic way to address this objective is to prioritize the pests according to their potential phytosanitary impact. Thus, a ranking system was developed using explicit datadriven criteria. It allows a rapid risk assessment which result in a pest prioritization in the French context (climate, crops, trade…). This tool has been applied for 100 selected pests. We compared the pests ranking first and the pests for which validated diagnostic protocols are available. Diagnostic protocols already exist for top priority pests, which confirmed that this tool gives similar results to expert judgment used so far to set priorities within the French NPPO. As a consequence, this system is an opportunity to prioritize needs to develop new diagnostic tests for emerging pests compared to already regulated pests ranked with a medium or low phytosanitary risk profile.

Tanja Dreo

qPCR, low level detection and validation data

Dreo Tanja (1), Nataša Mehle(1), Petra Nikolić (1), Manca Pirc(1), Marina Dermastia(1), Kristina Gruden (1) and Maja Ravnikar(1) 1) National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia Generation of validation data has become more systematic with the adoption of EPPO standards on assuring quality in detection and diagnosis of plant pathogens (PM 7/76, PM 7/84, PM 7/98) In the case of qPCR its use and validation data has led to the realization that due to its high sensitivity there is usually a range of low target concentrations that can only be detected as positive using q PCR and not by other less sensitive methods or methods based on different biological principles. This can give rise to difficulties in reporting results and their interpretation; particularly in cases of zero-tolerance quarantine pathogens when usually at least two methods with positive results are required to report a sample as suspicious and a pure culture is needed for final confirmation (e.g. for Ralstonia solanacearum). Several approaches to addressing this issue will be presented and discussed including use of several qPCR targets and determination of a so called 'cut-off value'.

Mariette Edema

Validation as a project: 2 laboratories, 24 organisms, 3 years

Harry Arijs

Role of diagnostics in the EU plant health management

Brent Larson

IPPC diagnostic protocols: underpinning global phytosanitary systems

Brent Larson, IPPC Secretariat, viale delle Terme di Caracalla, Roma, Italy, brent.larson@fao.org National plant protection organizations worldwide rely on diagnostic protocols to detect and identify pests. Diagnosis serves various purposes, in relation to helping ensure safe trade and prevent pest introductions and spread, or provides support for such basic phytosanitary activities such as surveillance or determining pest status. This presentation will cover the aims and content of IPPC diagnostic protocols, the process for their development, and challenges in their implementation. The Commission on Phytosanitary Measures has established the Technical Panel on Diagnostic Protocols (TPDP) to facilitate the development of IPPC diagnostic protocols. The TPDP work programme focuses on pests for which there is a specific need for harmonized protocols. IPPC diagnostic protocols aim at providing a range of options for detection and identification (including morphological and molecular methods and methods based on biological properties). The methods are selected on the basis of their sensitivity, specificity and reproducibility, as well as practicability (for example ease of use, speed and cost). IPPC diagnostic protocols need to also be suitable for various levels of capabilities in IPPC contracting parties, in terms available expertise and equipment. Three IPPC diagnostic protocols were adopted so far, and 29 are currently under development. Additional expertise is needed to contribute to the development of these protocols, as well to suitable ways of integrating the new validated methods as they become available, such as barcoding. Finally, in order to implement diagnosis, contracting parties need access to laboratories, reference material and taxonomists, and capacity development programmes will be necessary.

Hans Smolders

Diagnostic tools in perspective; Netherlands policy and the need for robust phytosanitary chains

Hans Smolders, Ministry of Economic Affairs, Agriculture and Innovation, the Netherlands Agricultural trade, both import and export, is a highly relevant segment of the Netherlands agro-economy. Annual export on planting materials alone was valued at 1.6 billion Euros in 2009, representing a 75% increase since 2001; growth figures on imports are similar. Approximately 40% of total world trade for seeds, 60% for seed potatoes, and 80% for ornamental bulbs originates from the Netherlands. Such a high contribution to worldwide trade of agriculture products requires a high responsibility to both the Dutch government and private stakeholders to manage phytosanitary risk and to avoid the spread of harmful organism. Moreover, any interception or outbreak can be very damaging for the sector(s) involved.

In view of future growth of global trade, there is a need to strengthen the phytosanitary aspects of the international trade. Besides a strong role of governments, a higher involvement of primary stakeholders in the supply chain is required, which implies closer collaboration in the production chain with producers and traders upstream, including those in third countries. To build robust international phytosanitary chains, availability of new rapid and cost-effective diagnostic tools for traders and producers as well as the NPPO are vital. However, to keep risk control in the chain manageable, there is a need to combine new diagnostic tools with smart knowledge management, other technical innovation and non-regulatory measures to promote stakeholders willingness to take effective measures during growth, sorting, packing and transport. Some examples are provided of research and measures initiated by the Netherlands government to strengthen these phytosanitary aspects of the agriculture production and supply chain.

Wim van Eck

Q-bank: more than a database

Wim van Eck Netherlands Food and Consumer Product Safety Authority, Chairman Steering Committee Q-bank Q-bank is a tool for the identification of quarantine plant pests and invasive plant species. It is a web-based database. It contains data, amongst others on DNA sequences, linked to reliable reference collections. Q-bank is complementary to other databases, e.g. those of EPPO. A network of internationally recognized experts, the curators, ensures that only validated data are stored in the database. When QBOL was launched, it was agreed that data on DNA sequences generated through QBOL project would be stored in Q-bank. The link with reference collections and the validation of data by curators are key features of Q-bank. Q-bank therefore is not only a useful diagnostic tool but it contributes to strengthen national and international plant health systems. Q-bank is originally a Dutch initiative. The enhanced rate of the introduction of plant pests, mainly as a result of the globalization of the trade of plant products, called for mechanisms to rapidly disclose and share information on quarantine pests. This is the more important as resources in the area of plant health are limited, including the number of specialists able to identify this type of organisms. Q-bank is funded by the Dutch government until the end of 2013. Q-bank is governed by a steering committee. Recently senior persons from amongst the member states of EPPO (Germany, France, United Kingdom and the Russian Federation) have joined this committee. This transformation into an international steering committee is with the view to strengthen the governance of Q-bank and to enhance the international profile of the database to make it a really international diagnostic data base.

Challenges for the future are to ensure that Q-bank will remain a living and attractive database, encouraging the phytosanitary community to forward new data to the database whenever these data become available. To ensure the high quality of the database an active international network of curators is indispensable as is the link with reference collections. Sustainable funding is a prerequisite for the future of Q-bank. The ultimate objective is to find a international home for Q-bank.

WP 5 :

 5 Barcoding as a new tool for identification of quarantine nematodes and their close relatives Kiewnick Sebastian (1) Holterman Martijn (1) Helder Hans (2) Frey Jürg E. (1) 1) Agroscope Changins-Wädenswil, Schloss, 8820 Wädenswil, Switzerland 2) Laboratory of Nematology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

  WP2 of QBOL: Barcoding fungi of Quarantine importance Groenewald Johannes Z.(1), Quaedvlieg William(1), Crous Pedro W.(1), Woodhall James(2), Boonham Neil(2), Verstappen Els C.P.(3), Dullemans Annette M.(3), Bonants Peter J.M.(3) 1) CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands e.groenewald@cbs.knaw.nl 2) The Food and Environment Research Agency, Sand Hutton, York, YO41 1LZ, United Kingdom 3) Plant Research International BV, Business Unit Biointeractions & Plant Health, PO BOX 69, 6700 AB Wageningen, the Netherlands

QBOL-EPPO Conference on DNA barcoding and diagnostic methods for plant pests Haarlem, NL, 2012-05-21/25 Programme QBOL final Conference combined with EPPO Conference on diagnostic methods for plant pests

  Monday 21 May 2012-Friday 25 May 2012

		Session 4 Nematodes
	Monday Evening 2012-05-21
	18.00	Reception
	19.30	Welcome	Wim van Eck
			VWA, the Netherlands
	19.35	QBOL, an EU project delivering Barcodes to support
		Plant Health

Session leaders: Jeurg Frey (ACWm, Switzerland) and Géraldine Anthoine (Anses, France)

	10.50-11.10 Friday 2012-	Clavibacter michiganensis strains characterized as	René Mathis,
		non-pathogenic by phylogenetic and polyphasic	Variety and seed study and control
		analyses are transmitted by tomato seeds and interfere	group, France
	11.10-11.40	QBOL-WP 5: Barcoding as a new tool for with C. michiganensis subsp. michiganensis detection
		identification of quarantine nematodes and their close
		relatives

05-25 Session 8 Validation & Databases and other Diagnostic Tools Session leaders: Peter Bonants (PRI, the Netherlands) and Françoise Petter (EPPO)

	15.20-15.40	Diagnostic tools in perspective; Netherlands policy	Hans Smolders, Ministry EL&I, the
		and the need for robust phytosanitary chains	Netherlands
	15.40-16.00	Q-bank: more than a database	Wim van Eck, Q-bank steering
			committee, the Netherlands
	16.00-16.20	International trade of high graded plant material : the	John van Ruiten, Inspection Service
	08.30-09.00	QBOL-WP 10: DNA barcoding as identification tool need for harmonized and validated detection	Bart van de Vossenberg, Naktuinbouw, the Netherlands
		for regulated plant pests: an international techniques	NRC-NPPO, the Netherlands
	16.20-16.40	collaborative test performance study among 14 The view of horticultural producers on diagnostic	George Franke, AIPH, the
		laboratories tools	Netherlands
	09.00-09.30 16.40-17.00	QBOL and Q-bank data management and analysis TESTA -Seed health: development of seed treatment	Christine Henry, coordinator TESTA,
		system methods, evidence for seed transmission and	Fera, United Kingdom
		assessment of seed health
	17.00-17.30	Discussion / Concluding remarks	Eric Regouin (Euphresco, the
			Netherlands) and Alan Inman
			(Euphresco, the United Kingdom)
	17.

Session 1 Invasive Plants & Phytoplasmas Abstracts of Presentations Tuesday 2012-05-22 van Valkenburg Johan Interactive image-driven identification keys for invasive plants van

  Valkenburg Johan National Reference Centre, National Plant Protection Organization P.O. Box 9102 6700 AA Wageningen, the Netherlands j.l.c.h.van.valkenburg@minlnv.nl

of methods for detection of pospiviroids infecting Solanaceae Michèle

  Potato spindle tuber viroid, because it can cause serious damage in potato and tomato crops. Because other pospiviroids can cause similar damage and appear to be widely spread in ornamental crops, there was a need for a reliable and cost-effective generic testing method. An assay was developed and validated that detects all known species of the genus Pospiviroid, using real-time RT-PCR based on TaqMan technology. This GenPospi assay consists of two reactions running in parallel, the first targeting all pospiviroids, except Columnea latent viroid, the second specifically targeting the latter viroid. To monitor RNA extraction Nad5primers were included with a newly designed probe in each assay. Method validation on tomato leaves showed that the GenPospi assay detects all pospiviroids up to a relative infection rate of 0.13% (equal to a 770 times dilution). No cross reactivity was observed, either with other viroids and viruses, or with leaf material. Repeatability and reproducibility were 100% and the assay appeared robust in an interlaboratory comparison. The new GenPospi assay has been shown to be a suitable tool for large-scale screening for all known pospiviroids. The assay has been validated for tomato leaves but can potentially be used for any crop.

	Dekter (2), E.T.M. Meekes (2)
	1) National Plant Protection Organization, P.O. Box 9102, 6700 HC Wageningen, the Netherlands
	m.botermans@minlnv.nl
	2) Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands
	In many countries phytosanitary regulations apply to Pascal Gentit
	Comparison

M. Botermans (1), B.T.L.H. van de Vossenberg (1), J.Th.J. Verhoeven (1), J.W. Roenhorst (1), M. Hooftman (2), R. Visage, Xavier Tassus, Françoise Poliakoff, Pascal Gentit Anses, Laboratoire de la Santé des Végétaux, Unité Bactériologie Virologie OGM 7 rue Jean Dixméras, 49044 Angers Cedex Corresponding author: xavier.tassus@anses.fr Since the end of the 1980s, new Pospiviroids such as Tomato Chlorotic Dwarf Viroid (TCDVd) and Columnea Latent Viroid have emerged in the European territory. Often they are latent on ornamental Solanaceae which become reservoirs of these pathogens and which facilitate their spread (Verhoeven

State of the art multiplex Luminex xMAP and xTAG -detection of plant viruses

  and one technique of real-time RT-PCR(Monger et al., 2010) towards a panel of target samples (Pospiviroids) and non-target samples (Solanaceae and viruses). The criteria of relative sensitivity, relative specificity, repeatability and reproducibility allowed a scheme to be elaborated for detection of pospiviroids including confirmation by genetic characterization. Department of Plant Virology, Institute of Plant Molecular Biology, Biology Centre of Academy of Sciences of the Czech Republic, v.v.i., Branišovská 31, 370 05, České Budějovice, CZ Multiplex detection of viruses in a single sample would be an elegant way to improve DAS-ELISA efficiency and reduce costs. The Luminex xMAP technology allows such multiplex serological virus detection while retaining the standard DAS-ELISA 96-well format and workflow. Different Luminex colour-coded xMAP beads, each specifically coated, capture virus particles. Virus-specific conjugated antibodies, in combination with the unique bead colour-code identify a unique combination, allowing specific detection of each individual virus. Sensitivity is comparable to DAS-ELISA, while detection is completed in less than 3 hours with higher specificity.

	René van der Vlugt
	van der Vlugt René (1). van Raaij Henry (1) de Weerdt Marjanne (1) van Brunschot Sharon (2) Koloniuk Igor (3) van
	Beckhoven José (1) Bergervoet Jan (1)
	1) Plant Research International, Droevendaalsesteeg 1, 6708 PB, Wageningen NL rene.vandervlugt@wur.nl
	2) CRC for National Plant Biosecurity, LPO Box 5012, Bruce, ACT, 2617, Australia.
	3) Natasa Mehle
	Plant

When specific antibodies are not available, viral or viroid nucleic acid is detected on the same platform using Luminex xTAG beads. Similar colour-coded bead sets, labelled with virus-specific oligonucleotides probes, capture specific (RT)-PCR fragments. Similar to TaqMan probes, Template Specific Primer Extension (TSPE) adds an extra level of specificity to the xTAG test format while allowing a much higher level of multiplexing than Taqman. Luminex xMAP and xTAG tests were developed for a variety of plant viruses and viroids that allow the multiplex detection of more than 10 analytes in a single sample. They offer a state of the art, true multiplex and cost-effective alternative to the current DAS-ELISA and TaqMan formats.

viruses and viroids in an aqueous environment -survival, water mediated transmission and detection

	Mehle Natasa (1,4), Gutierrez-Aguirre Ion (1,4), Prezelj Nina (1), Delić Duska (2), Vidic Urska (1), Rupar Matevž
	(1), Rušić Jelena (3), Krajačić Mladen (3), Ravnikar Maja (1,4)
	1) National Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia,
	natasa.mehle@nib.si
	2) University of Banjaluka, Faculty of Agriculture, Banjaluka, Bosnia and Herzegovina
	3) University of Zagreb, Faculty of Science, Department of Biology, Zagreb, Croatia
	4) Center odlicnosti za biosenzoriko, instrumentacijo in procesno kontrolo, CoBIK, Solkan, Slovenia

On-Site Confirmation and Monitoring of plant pathogens

	Schoen Cor (2),Boonham Neil (1),Tomlinson Jenny (1), Michael Andreou (5), Zorovic Maja (3), Spinelli Franscesco
	(5),Morisset Dany (3),Lenarčič Rok (3),Ravnikar Maja (3),Blasioli Sonia (5), Biondi Enrico (5), Simonato Mauro
	(5),Goransson Jenny (6),Buehlmann Andreas (4)and Li Ming-fu (7).
	1) Food and Environment Research Agency (Fera), United Kingdom
	2) Plant Research International (PRI), Netherlands; E-mail: cor.schoen@wur.nl
	3) National Institute of Biology (NIB), Slovenia
	4) Agroscope Changins-Waedenswil Research Station ACW (ACW), Switzerland
	5) University of Bologna (UniBO), Italy
	6) Q-Linea (Q-Linea), Sweden 15) OptiSense (OptiSense), United Kingdom
	7) Chinese Academy of Inspection and Quarantine (CAIQ), China
	Effective detection methods are critical for the efficient functioning of inspection services and National Plant
	Protection Organisations (NPPOs). Yet in many cases, inspection services in particular, lack techniques that will
	enable efficient detection of quarantine pests. In WP7 of Q-detect different detection methods are developed. LAMP
	primer-sets for the detection of the whitefly transmitted viruses, bacteria and spindle tuber viroid (PSTVd) were
	developed.
	Applicability of different simple and fast extraction methods for isolation of both DNA and RNA were tested. The
	Quick extract RNA Extraction procedure from Epicentre, suitable for both ssRNA and ssDNA virus extractions, meet
	the criteria of being sensitive, fast, simple and portable. For bacterial extractions two other on-site nucleic acid
	extraction methods were tested. One method is dedicated to the extraction of DNA from bacteria and based on a
	simple boiling procedure. Another procedure that has been designed for the nucleic acid extraction relies on the lateral
	flow device (LFD) extraction approach published by Tomlinson et al. (Phytopathology, 2010, 100: 143-149). Both
	procedures have been shown to allow nucleic acid extraction from plant tissue in just a few minutes. Preliminary
	experiments with the Genie II as a LAMP stand-alone detection device, "point-of-care" diagnostics could be
	demonstrated.
	Annelien Roenhorst

Quality control in bioassays used for broad screening for plant viruses

	Roenhorst JW, Botermans M & Verhoeven JThJ
	National Plant Protection Organization, PO Box 9102, 6700 HC Wageningen, the Netherlands,
	j.w.roenhorst@minlnv.nl
	Test plants are often used for broad screening for plant viruses. Mechanical inoculation of a series of test plants
	enables generic detection of mechanically transmitted viruses in only one assay. Moreover, such an assay is suitable
	for known as well as unknown viruses and variants. In comparison to serological and molecular methods, however,
	quality control in bioassays has hardly been addressed. The system of positive and negative controls (1 st line), blind
	samples (2 nd line) and proficiency tests (3 rd line) is applicable, provided that a broader interpretation of positive and
	negative controls is used. For validation, performance criteria can only be determined for individual viruses.
	However, results often can be extrapolated. Sensitivity is addressed by dilution and comparison with other methods.
	Specificity is considered of minor importance for screening. Selectivity depends on the plant species tested, because
	some hosts contain components that inhibit transmission. Repeatability, reproducibility and robustness are determined
	by general features, such as environmental conditions and expertise of the virologist. Therefore, these performance
	criteria should be considered more generally. This presentation demonstrates that quality control can be implemented
	in bioassays, although it is more complicated than in serological and molecular assays.

Session 3 Insects & Mites Abstracts of Presentations Wednesday 2012-05-23 Jean-Yves Rasplus QBOL-WP 3: Barcoding Quarantine Arthropods

	Rasplus J-Y
	Inra, CBGP (Centre de Biologie pour la Gestion des Populations), UMR 1062, Montferrier-sur-Lez, France

Combining DNA barcoding and morphological analysis for the diagnosis of alien pests. The case study of Epitrix potato flea beetles (Coleoptera: Chrysomelidae)

  These quarantine species need to be distinguished reliably and quickly from other Spodoptera species of which S.exigua is most frequently intercepted. Morphological identification is straightforward when the adult life stage is concerned. However, in trade it is more common that the immature stages are intercepted, and identification cannot wait until the specimen is reared to the adult stage. Morphological characters for identification of larval stages of the four EU quarantine species are presented here as well as real-time PCR tests based on TaqMan technology developed for rapid molecular identification. These tests were found to be reliable, reproducible and repeatable tools, applicable for the identification of all developmental stages of the four Spodoptera quarantine species.

	Marja van der Straten & Bart van de Vossenberg National Reference Centre, National Plant Protection Organization, P.O.Box 9102, 6700 HC Wageningen, NL, m.j.van.der.straten@minlnv.nl The genus Spodoptera comprises 30 species of which four are enlisted on the EU quarantine list: S. littoralis, S. litura, S. frugiperda and S. eridania. Of the EU listed Spodoptera, S. littoralis is intercepted the most frequently, followed Jean-François Germain (1), Isabelle Meusnier (2), Jean-Yves Rasplus (2) and Astrid Cruaud (2) 1) Anses-Laboratoire de la Santé des Végétaux, unité entomologie et plantes invasives. CBGP campus international de Baillarguet CS30016 FR-34988 Montferrier-sur-Lez cedex Jean-francois.germain@anses.fr 2) INRA.-CBGP campus international de Baillarguet CS30016 FR-34988 Montferrier-sur-Lez cedex meusnier@supagro.inra.fr, rasplus@supagro.inra.fr, cruaud@supagro.inra.fr Fast and reliable identification of newly introduced pest species is essential to allow NPPOs to implement appropriate control measures. Identification can be performed using morphological characters and/or molecular data. Based on the example of two American species of potato flea beetles recently introduced in Europe, Epitrix cucumeris (Harris) and E. similaris Gentner (Coleoptera: Chrysomelidae) the advantages and disadvantages of both methods for quarantine entomology will be presented. by S. litura. Astrid Cruaud, CBGP, France, presented on behalf of Jean-François Germain, Anses, France Antoon Loomans

Morphological identification of Thrips palmi in the framework of ISO/IEC 17025

	Jose Carlos Rodrigues
	Vierbergen Bert, Koch Jeanette C., Loomans Antoon J.M.
	Ministry of Economic Affairs, Agriculture and Innovation, Netherlands Food and Consumer Product Safety
	Authority, National Plant Protection Organisation, National Reference Centre, PO Box 9102, 6706 EA Wageningen,
	the Netherlands. a.j.m.loomans@minlnv.nl
	Morphological identification of larvae and adults of the EU quarantine pest Thrips palmi is one of the routine
	procedures of the National Reference Centre (NRC) of the Dutch NPPO. Every year many samples are analysed from
	national import inspections. The quality of a morphological identification depends on several technical
	requirements, the most important of which are laboratory facilities, the literature, the reference collection, the quality
	of the available specimen(s) and the skills of the specialist. To be able to consistently produce valid identification
	results, the NRC has implemented a documented quality management system within the laboratory since 2007. Since
	March 1 st 2012 the laboratory has been accredited by the Dutch National Accreditation Body. For the accreditation
	process, ISO/IEC 17025 was used to demonstrate competence for Thrips palmi identification under a fixed scope,
	"Morphological identification of Thrips palmi adults". The EPPO Standard PM 7/98 Specific requirements for
	laboratories preparing accreditation for a plant pest diagnostic activity guidance for entomology sets specific
	requirements for this process to enable the validation of morphological identification of Thrips palmi adults. The
	performance characteristics validated were analytical sensitivity, specificity, repeatability and reproducibility,
	however selectivity was not considered as being relevant as a performance characteristic for morphological
	identifications in entomology. In this presentation the results and value of these performance characteristics are
	discussed in detail.

Molecular Characterization of Leptomastidea spp. (Encyrtidae, Hymenoptera) Populations from Puerto Rico, Barbados and Florida

  Leptomastidea are parasitoids of mealybugs, and at least one species, L. abnormis (Girault) is commonly used in biological control programs against mealybugs worldwide. Parasitoid wasps of the genus Leptomastidea (Hymenoptera: Encyrtidae) have been identified as important natural enemies of the Harrisia cactus mealybug (HCM), Hypogeococcus pungens (Granara de Willink) (Hemiptera: Pseudococcidae) in the Caribbean region. Three populations of Leptomastidea spp. have been identified attacking HCM in the region, and individuals were collected from Barbados, Puerto Rico and Florida. Morphological examinations by Dr. J. S.

	Rodrigues Jose Carlos V (1), Galindo-Cardona Alberto (1), Ciompelink Matthew (2), Segarra Alejandro (3) and Amy
	Roda (4)
	1) University of Puerto Rico, Agricultural Experimental Station, Jardín Botánico Sur, 1193 Calle Guayacán, San Juan, Puerto
	Rico 00926 jose_carlos@mac.com
	2) USDA-APHIS-PPQ-CPHST 22675 N. Moorefield Road, Bldg. 6414 Edinburg, TX 78541-5033, USA
	3) University of Puerto Rico, Crops and Agro-Environmental Sciences Department, College of Agricultural Sciences, Mayagüez,
	Puerto Rico 00681
	4) USDA-APHIS-PPQ-CPHST . 13601 Old Cuttler Rd., Miami, FL 33158, USA
	All known

BY THE AUTHOR Molecular data to unravel the Botryosphaeriaceae complex found in diseased European hop-hornbeams: a case study Piškur

  1 , Suffert Muriel 1 , McMullen Madeleine 1 EPPO, 21 Boulevard Richard Lenoir, 75011 Paris, France hq@eppo.int PQR (Plant Quarantine data Retrieval system) PQR (Plant Quarantine data Retrieval system) is the EPPO database on quarantine pests. It provides detailed information on the geographical distribution and host plants of quarantine pests. Its search tools also allow the users to identify commodities which are able to act as pathways in international trade for the movement of pests and diseases. In recent years, the database has been extended to cover invasive alien plants. The new PQR version also includes world maps, pictures and active links to the EPPO Reporting Service. PQR can be downloaded free of charge from this web site.http://www.eppo.int/DATABASES/pqr/pqr.htm EPPO database on Diagnostic expertise This database provides an inventory of the diagnostic expertise available in the EPPO region. Its aim is to cover the expertise on regulated pests (i.e. pests of EPPO A1 and A2 Lists, pests mentioned in EPPO Standards PM4: Production of Healthy Plants for Planting), pests possibly presenting a risk to EPPO member countries (EPPO Alert List) and plants of the EPPO List of invasive alien plants. This database does not include common pests which are widely distributed in the EPPO region. The EPPO Secretariat is maintaining the database but note that all information included in the database is based on individual expert's own declarations of their expertise.

http://dc.eppo.int/ Barbara Piskur POSTER PRESENTED Barbara(1), Pavlic Draginja(2), Slippers Bernard(2), Jurc Dušan(1) 1) Slovenian Forestry Institute, Department of Forest Protection, Večna pot 2, SI-1000 Ljubljana, Slovenia (barbara.piskur@gozdis.si) 2) Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, 0002, South Africa

THE AUTHOR Diagnostics for quarantine plant pathogens: DNA barcoding and MALDI-TOF MS revealed a new group of non-pathogenic to tomato Clavibacter isolated from tomato seeds

  Clavibacter contains mainly plant pathogenic bacteria. They have specific hosts and are mostly quarantine organisms. The genus Clavibacter contains pathogens of economically important crops. The QBOL project aims to develop reliable identification using DNA-barcoding. The work presented here focused on C. michiganensis subsp. michiganensis (Cmm) which is considered one of the most destructive diseases on tomato. Recently, many outbreaks of bacterial canker have been recorded. In the first part of this work 200 strains were subjected to sequencing of gyrB and 16S rRNA genes and MALDI-TOF MS. A 500bp gyrB sequence was found to be a discriminative barcode for Clavibacter. MALDI-TOF MS with specific biomarkers can be used as an alternative for a sequence-based identification. Data also revealed the existence of a new group of non-virulent bacteria isolated from tomato seeds. Because comparative physiological and phylogenetic characterization of this group versus genuine Cmm is generally unknown, tests were performed in planta as well as DNA-DNA hybridization. Their lack of pathogenicity was investigated by checking the presence of known molecular virulence determinants.

	Joanna Zaluga POSTER PRESENTED BY Zaluga Joanna (1), Heylen Kim (1), Maes Martine (3) De Vos Paul (1,2)
	1) Department of Biochemistry and Microbiology, Laboratory of Microbiology, Gent University, Gent, Belgium
	joanna.zaluga@ugent.be
	2) BCCM/LMG Bacteria Collection, Gent, Belgium (3)Institute for Agricultural and Fisheries Research-ILVO,
	Merelbeke, Belgium
	The genus May Bente Brurberg	POSTER PRESENTED BY

Furthermore, ALFP and ISSR-PCR are being tested to optimize a reliable typing method which helps to better understand the epidemiology of Clavibacter.

THE AUTHOR Outbreak of apple proliferation phytoplasma in Norway Blystad

  Candidatus Phytoplasma mali, is one of the most serious plant diseases in apple production areas all over Europe, and losses are estimated to be very high. The phytoplasma induces a range of symptoms, such as witches" brooms, enlarged stipules, yellowing, growth suppression, and undersized fruits. Candidatus Phytoplasma mali spread by vegetative propagation (grafting) or they are naturally transmitted by phloem-feeding insects. AP was first reported in Italy in 1950 and has since then been detected in several European countries where apple is grown commercially. Candidatus Phytoplasma mali was detected in Norway for the first time in 1996, but the situation was monitored carefully and the few infected trees detected in the following decade were eradicated. In September 2010, however, a new more serious out-break of AP occurred in one of the major apple production areas in western Norway. This led to the start-up of a survey with a particular focus on nurseries and their surroundings in four apple producing counties. Symptomatic and non-symptomatic samples were analyzed using real-time PCR. No infected trees were detected in the nurseries, but infected trees were detected in close surroundings of 70 % of the nurseries indicating the seriousness of the situation.

	Stefan Wagner	POSTER PRESENTED BY

, Toppe Brita

[START_REF] Piškur | Reference plant pathogenic bacteria in support of the European plant health policy: the Q-Bacco-net initiative Aspin A.(4)[END_REF]

, Holst Maren Anna Holst (2), Brurberg May Bente (1) 1) Bioforsk -Norwegian Institute for Agricultural and Environmental Research, Ås, Norway, may.brurberg@bioforsk.no 2) Mattilsynet (Norwegian food safety authority, Norway) Apple proliferation (AP), caused by

THE AUTHOR Lab-on-a-chip detection of Phytophthora species

  Phytophthora species are a worldwide threat to natural ecosystems and the nursery industry. To prevent the spread of these pathogens with infected plants, easy to handle diagnostic systems with high sensitivity and high throughput are demanded. Furthermore such methods should give results within a short time on site of inspection. In a three year project a chip-formatted PCR system combined with a chip-based electrical microarray will be adapted for the detection of important Phytophthora species in plant tissue (EPPO A2 / Alert list). A stationary PCR chip with integrated microstructured heaters and temperature sensors has been developed for the amplification of specific Phytophthora DNA fragments. A microfluidic system connects the PCR chip with a microarray where the labelled DNA fragments are detected. The miniaturization of the chip formatted PCR and array system enables high portability, low input of energy and expensive analytic chemicals and very fast reaction times due to low reaction volumes.

	Gian Luca Bianchi	POSTER
	Wagner Stefan(1), König Stephan(1), Weber Karina(2), Kirsch Konstantin(2), Schwenkbier Lydia(2), Weber Jörg(3),
	Hentschel Martin(3), Werres Sabine(1)
	1) Julius Kühn Institut -Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Horticulture
	and Forests, Messeweg 11/12, D -38104 Braunschweig, Germany email: Stefan.Wagner@jki.bund.de;
	sabine.werres@jki.bund.de
	2) Institute of Photonic Technology, Albert-Einstein-Straße 9, 07745 Jena, Germany
	3) Analytik Jena AG, Konrad-Zuse-Strasse 1, 07745 Jena, Germany

Multiplex real time RT-PCR in the diagnosis of grapevine viruses

  Bianchi Gian Luca, De Amicis Francesca, De Sabbata Luca, Di Bernardo Nicoletta, Nonino Fabio, Marrazzo Teresa ERSA -Servizio Fitosanitario e chimico, Via Sabbatini 5, 33050 Pozzuolo del Friuli (Udine), Italia gianluca.bianchi@ersa.fvg.it Diagnosis of grapevine viruses is very important in certification of grapevine propagation materials. The aim of this work was to develop two multiplex real time RT-PCR assays for the detection of some viruses of grapevine (ArMV, GFLV, GLRaV-1, GLRaV-3 and GVA). 6790 grapevine samples of woody mature canes were collected from 299 stock nurseries of propagating material and assayed in ELISA and multiplex real time RT-PCR using group analysis procedure. ELISA tests were done on samples of 5 canes according to official procedures adopted in Italy while multiplex real time RT PCRs were done on samples of 20 canes. The health of 280 stock nurseries was evaluated using ELISA and was confirmed by multiplex real time RT PCR. 10 stock nurseries had positive results using the molecular test and negative results to serological tests while 9 stock nurseries with positive ELISA results did not have these results confirmed by the multiplex real time RT PCR. Relative accuracy of the multiplex real time RT-PCR in comparison to the ELISA test was 93.7 %. The multiplex real time RT-PCR developed in this work was shown to be a useful tool for the diagnosis of grapevine viruses in comparison to the ELISA test, being faster and cheaper than the official serological method.

	Utku Yükselbaba	POSTER

Determination of the biotypes of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations from Antalya Province of Turkey by sequence analysis of mitochondrial Cytochrome Oxidase I ( mtCOI) gene region

Using DNA Sequencing for Diagnosis of Certain Pests of Quarantine Concern for the Russian Federation

  The possibility of Ypt-1 gene sequencing for diagnosis of Phytophthora fragariae var. ruby and Phytophthora fragariae var. fragariae was investigated. Sequencing the PPV coat protein gene enabled strains to be determined for 133 PPV isolates from various regions of the Russian Federation. PPV-W strain (Winona) was found to be widely spread in Russia. A new PPV strain was identified and preliminarily called PPV-CR (Cherry Russian). Sequencing the 12S rRNA gene DNA in 12 samples of bark beetles allowed identification of the variability between genera and species of beetles sufficient to develop methods for their diagnosis. Sequencing amplification products allowed confirmation of detection of previously absent pathogens (Ralstonia solanacearum, Beet necrotic yellow vein virus, Impatiens necrotic spot virus) or poorly identified pathogens (Tobacco ringspot virus, Phytophthora fragariae). A PCR-analysis for Andean potato viruses was developed. It was tested on the DSMZ virus isolates.

	Françoise Poliakoff	POSTER
	Natalia Sherokolava	POSTER
	Prihodko Juri, Mazurin Evgeniy FGBU
	All-Russian Plant Quarantine Center Pogranichnaya str., 32, Bykovo, Ramensky region, Moscow oblast, 140150,
	Russian Federation prihodko_juri59@mail.ru

Methods comparison for the detection of Pepino Mosaic Virus (PepMV) on tomato seeds

  Anne Saison, Xavier Tassus, Françoise Poliakoff Anses, Laboratoire de la Santé des Végétaux, Unité Bactériologie Virologie OGM 7 rue Jean Dixméras, 49044 Angers Cedex Corresponding author :To review the French official detection method for PepMV, three detection methods were assessed. The reference method which is a serological detection test (DAS ELISA) was compared to a conventional RT-PCR test(Ling et al, 2008) and a real time RT-PCR test(Ling et al, 2007). Results obtained allowed specificity, sensitivity and repeatability criteria to be calculated. In this study, the real-time RT-PCR test had the best performance criteria with 100% for the three performance criteria assessed.DAS ELISA test will remain the initial test used because it has good sensitivity, repeatability, reproducibility and low cost. Because of its higher sensitivity, the real-time RT-PCR test will be used for the confirmation of undetermined results obtained with the serological test.

References: Ling, K.

S. et al., (2008)

Genetic composition of Pepino mosaic virus population in North American greenhouse tomatoes. Plant disease, 92 (12), 1683-1688 Ling, K.S., et al. (2007). Development of a one-step immunocapture real-time TaqMan RT-PCR assay for the broad spectrum detection of Pepino mosaic virus. J. Virol. Methods, 144, 65-72 Peter Bonants POSTER Multiplex detection of Phytophthora: Padlock probe based Universal detection Multiplex Array (PUMA) Katarzyna Gaszczyk, Odette Mendes, Els Verstappen, Cor Schoen and Peter Bonants Plant Research International, Wageningen UR, Droevendaalsesteeg 1, 6708 PB Wageningen, NL, peter.bonants@wur.nl Phytophthora spp. are responsible for many diseases worldwide and can occur on a wide range of different crops and other plants.

Development and validation of molecular diagnostic tools for detection and identification of the root-knot nematode Meloidogyne enterolobii, a new EPPO A2 pest recommended for regulation.

  Q-bank is supported by the Dutch Ministry of Economic Affairs, Agriculture and Innovation and by QBOL, an EU-project with partners from 20 countries. Several interceptions of ornamental plants infested with M. enterolobii by the Dutch and German authorities in 2008, lead EPPO (European and Mediterranean Plant Protection Organization) to add it to its list of A2 list of pests recommended for regulation in 2010.

	Sebastian Kiewnick	POSTER

S. Kiewnick, A. Braun-Kiewnick, J. Frey Agroscope Changins-Wädenswil, Schloss 1, 8820 Wädenswil, Switzerland sebastian.kiewnick@acw.admin.ch Root-knot nematodes (Meloidogyne spp.) pose a significant risk to agricultural production systems all over Europe. Meloidogyne enterolobii is a polyphagous species and has been found on many host plants. It is considered to be a very aggressive root-knot nematode species that is able to reproduce on root-knot nematode-resistant plants.

POSTER From information to diagnosis: management of an alien pest: a case study of Drosophila suzukii (Diptera: Drosophilidae) Valérie

  At the time of emergence of a new pest, it is important that information flows quickly to the plant protection services. This information must be complemented by identification tools. The identification should be accessible at all levels, for field workers and for researchers. The emergence of Drosophila suzukii (Diptera: Drosophilidae) in Europe in 2009 illustrated the management of this new pest.

	Ian Adams	POSTER

Astrid Cruaud, CBGP, France, presented on behalf of Jean-François Germain, Anses, France Balmès* and Jean-François Germain* Anses-Laboratoire de la Santé des Végétaux. Unité entomologie et plantes invasives. CBGP Campus International de Baillarguet. CS 30016. FR-34988 Montferrier-sur-Lez Cedex. valerie.balmes@anses.fr jeanfrancois.germain@anses.fr

Development of a System for High Throughput Screening of Planting Material for Cassava Brown Steak Disease Causing Viruses

  College of Agricultural and Environmental Sciences, Makerere University, Kampala, Uganda Cassava provides more than half the dietary calories for half of the population of sub-Saharan Africa. Cassava brown streak disease caused by the viruses Cassava brown streak virus and Ugandan cassava brown streak virus is currently spreading throughout sub-Saharan Africa and can cause up to 70% yield losses.

	Adams Ian(1), Abidrabo Phillip (2,6), Miano Douglas(3), Alicai Titus(2), Kinyua Zachary (3), J Clarke Jasper(5)
	Macarthur Roy(1), Weekes Rebecca(1), Laurenson Lynn(1), U Hany Ummey(1), Peters Dai(4), Potts Michael(4),
	Glover Rachel(1), Boonham Neil(1), Smith Julian(1)
	1) The Food and Environment Research Agency, York, UK,
	2) National Crops Resources Research Institute, Kampala, Uganda,
	3) Kenya Agricultural Research Institute, Nairobi, Kenya
	4) Catholic Relief Services, Nairobi, Kenya
	5) Centre for Novel Agricultural Products, University of York, York, UK
	6)

of real-time PCR for detection and identification of Xanthomonas fragariae

  Xanthomonas arboricola pv pruni (Xap) is a serious disease of Prunus. In other European countries Xap infects stone fruits, however, in the Netherlands it has only been detected in ornamental cherry laurel. So far, Dutch Xap strains were isolated from symptomatic leaves. The identity of the Xap isolates was confirmed by Pagani-and Pothier-PCR. For typing, gyrB and BOX/ERIC were applied. Both demonstrated a high degree of homology among Xap isolates. The genotypic diversity of 25 Xap isolates was assessed by MLVA. These isolates could be divided into two groups, however, no correlation with their geographical origin was established. These results indicated the high discriminatory potential of the MLVA typing. Additionally, 5 Xanthomonas spp. isolates reacted in the Pagani-but not in the Pothier-PCR. Their characterisation with BOX/ERIC differed significantly with Xap. AFLP data and gyrB typing confirmed this observation. Their sequence analysis on gyrB did not match any published Xap gyrB sequences but demonstrated high homology Xanthomonas fragariae is a quarantine organism (EPPO A2 List of pests recommended for regulation/EU A2) causing bacterial angular leaf spot of Fragaria spp. (strawberry). The National Plant Protection Organization has recently validated a real-time PCR based on the gyrB gene (Weller et al. 2007, J.Microbiol.Methods 70:379-3) for detection of X. fragariae in leaves and rhizomes of Fragaria spp. and for identification of pure cultures, according to the Dutch validation guideline for plant pathogens and pests. Limit of detection (LOD), measuring range, trueness, analytical specificity, selectivity, robustness, repeatability and reproducibility of the assay has been assessed. The LOD for detection, as determined by spiking rhizoom-and leaf-extracts of two Fragaria spp. varieties with five X. fragariae isolates, is 8.0x10 4 cfu/mL and for identification, determined with the same isolates, is 7.8x10 4 cfu/mL. These LODs are approximately 100 x lower than those for a conventional PCR, making it more suitable for detection of lower population densities on infected plants. The assay identified all X. fragariae reference isolates used and did not give any cross reactivity with either plant DNA or DNA of other Xanthomonads, including X. arboricola pv. fragariae. The repeatability and reproducibility were both 100%. The real-time PCR has been shown to be a valid tool for the detection and identification for X. fragariae.

	), Pothier Jöel (3), Duffy Brion (3) and van 1) National Plant Protection Organization, P.O. Box. 9102, 6700 HC Wageningen, the Netherlands, Doorn Joop (4) m2.bergsma@minlnv.nl 2) Naktuinbouw, P.O. Box. 40, 2370 AA Roelofarendsveen, the Netherlands, 3)Agroscope Changins-Wädenswil ACW,Plant Protection Division, Schloss 1, CH-8820 Wädenswil, Switzerland, 4) Wageningen University and Research Centre, P.O. Box. 85, 2160 AB Lisse, the Netherlands Bacterial spot disease, caused by Marcel Westenberg Validation Ewa Lojkowska Phenotypic

with Xanthomonas dyei, X. arboricola pv. celebensis and Xanthomonas arboricola. In conclusion, MLVA is a promising technique to assess variation within Xap populations. Additionally, use of complementary methods for identification such as BOX/ERIC, gyrB, MLVA, AFLP and the Pothier-PCR, are strongly recommended in order to guarantee a reliable diagnosis of Xap.

M. Westenberg (1), M. Bergsma-Vlami (1), B.T.L.H. van de Vossenberg (1) 1) National Plant Protection Organization, P.O. Box 9102, 6700 HC Wageningen, The Netherlands m.westenberg@minlnv.nl

and genotypic characteristics of Dickeya strains isolated from potato in Poland Lojkowska

  Ewa, Potrykus Marta, Golanowska Malgorzata, Jafra Sylwia Intercollegiate Faculty of Biotechnology UG and MUG, ul. Kladki 24, Poland, lojkowska@biotech.ug.gda.pl Pectolytic bacteria from Pectobacterium and Dickeya genera, that cause blackleg and soft rot, cause substantial crop reduction. The genus Dickeya is a highly diverse group, which at the moment contains six species. However, Dickeya strains, with significantly distinct genetic and biochemical profiles, have recently been isolated from symptomatic potato plants in Poland and other European countries. The new species Dickeya 'solani' has been postulated(Toth et al. 2011).

of Clavibacter michiganensis subsp. michiganensis in seeds of tomato Koenraadt

	Harrie Koenraadt
	Detection

The existence within the Clavibacter group of a new group of seed-borne non-pathogenic bacteria is discussed elsewhere (see Zaluga et al.). H.M.S., Van Vliet A., Bakker D., Teunissen H.A.S. Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands H.Koenraadt@Naktuinbouw.nl

Phoma: tool for development of validated real-time (TaqMan) PCR assays for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of tomato and potato

  TaqMan) PCR assays are presented for the detection of Stagonospora andigena and S. crystalliniformis in leaves of potato or tomato cultivars. Both species, originally described in the anamorph genus Phoma, are serious foliage pathogens and have been recorded only in the Andes area so far. Stagonosporopsis andigena is listed as an A1 quarantine organism in Europe. The specificity of the TaqMan PCR assays was determined on genomic DNA extracted from strains of S. andigena, S. crystalliniformis and closely related species of Stagonosporopsis, Phoma and Boeremia. The validation of the methods developed included the DNA extraction and the TaqMan PCR assays. The specificity, analytical sensitivity, reproducibility, repeatability and robustness of the TaqMan PCR assays are demonstrated. Both TaqMan PCR assays were tested on symptomatic leaves of potato or tomato obtained after artificial inoculation with both pathogens under quarantine conditions.

	de Gruyter Hans (1) van Rijswick Patricia (1) Bonants Peter (2) van Gent-Pelzer Marga (2) Crous Pedro (3)
	Woudenberg Joyce (3)
	1) National Reference Centre, National Plant Protection Organization, P.O. Box 9102, 6700 HC Wageningen, the
	Netherlands, e-mail: j.de.gruyter@minlnv.nl
	2) Plant Research International BV, P.O. Box 16, 6700 AA Wageningen, The Netherlands Meekes Ellis
	Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands
	3) CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands
	Species-specific real-time (Anne-Laure Boutigny
	Real-

time PCR detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

  Anne-Laure Boutigny, Cécile Guinet and Renaud Ioos ANSES, Laboratoire de la Santé des Végétaux, Unité de Mycologie, Domaine de Pixérécourt, 54220 Malzéville, France anne-laure.boutigny@anses.fr Melampsora medusae Thümen is one of the causal agents of leaf rust of poplars and is an EPPO A2 listed pest recommended for regulation as a quarantine pest. Two formae speciales have been described within M. medusae: M. medusae f. sp. deltoidae and M. medusae f. sp. Tremuloidae, on the basis of their preference for cultivated poplars (e.g. Populus deltoides) or wild poplars (e.g. Populus tremuloides), respectively. Given the host preference, it is assumed that only M. medusae f. sp. deltoidae was reported to be present in Europe so far. In this context, a M. medusae f. sp. deltoidae specific real-time PCR assay was developed. A set of primers and hydrolysis probe was designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and sensitivity, specificity, repeatability, reproducibility and robustness of the detection method were assessed. This assay was very sensitive as it allowed the consistent detection of one single urediniospore of M. medusae f. sp. deltoidae in a mixture of 2 mg (corresponding approximately to 800000 urediniospores) of other Melampsora species. This test can be used for specific surveys in nurseries and phytosanitory controls, in order to avoid introduction and spread of this pathogen in Europe

	Anita Rose Haegi
	A DNA-

Based Macroarray for Multiplex Detection of Soil-Borne Fungi

	Haegi Anita, Belisario Alessandra, Infantino Alessandro, Pucci Nicoletta, Tizzani Lorenza, Vitale Salvatore, Riccioni
	Luca
	Plant Pathology Research Center (CRA-PAV), Via C.G. Bertero 22, 00156 Roma, Italy, anita.haegi@entecra.it

Session 8 Validation & Databases and other Diagnostic Tools Abstracts of Presentations Friday 2012-05-25

	Bart van de Vossenberg
	DNA barcoding as identification tool for regulated plant pests: an international collaborative test performance
	study among 14 laboratories
	B.T.L.H. van de Vossenberg (1), Jean-Yves Rasplus (2), Martine Maes (3), Ewald Groenewald (4), Juerg Frey (5),
	Mogens Nicolaisen (6), Neil Boonham (7), Peter Bonants (8)
	1) Plant Protection Service (PPS), Geertjesweg 15, 6706 EA Wageningen, the Netherlands,
	b.t.l.h.van.de.vossenberg@minlnv.nl
	2). Institut National de la Recherche Agronomique (INRA), 2 Place Viala, 34060 Montpellier cedex 1, France 3).
	Instituut voor Landbouw-en Visserijonderzoek (ILVO), Burgemeester Van Gansberghelaan 96 , 9820 Merelbeke,
	Belgium
	4) Fungal Biodiversity Centre(KNAW-CBS), Uppsalalaan 8, 3584 CT Utrecht, the Netherlands
	5) Agroscope Changins-Wädenswil (ACW), Schloss 1, Labor 4, 8820 Waedenswil, Switzerland
	6) Aarhus University (UA), Nordre Ringgade 1, 8000 Aarhus C, Denmark
	7) Food and Environment Research Agency (FERA), FERA, Sand Hutton York YO41 1LZ, United Kingdom
	8) Plant Research International (PRI), Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands

  National Reference Centre, National Plant Protection Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands Tel: +31-317496248 -Fax: +31-317 421701 b.t.l.h.van.de.vossenberg@minlnv.nl VAN DOORN Joop (Mr) Applied Plant Research, Wageningen University and Research Institute, Prof. van Slogterenweg 2, 2161 DW Lisse, Netherlands Tel: +31-252462121 -Fax: +31-252462100 joop.vandoorn@wur.nl VAN ECK Wim H. (Mr) Food and Consumer Product Safety Authority, Division Plant Post Box 9102, 6700HC Wageningen Netherlands Tel: +31-317496655 -Fax: +31-317421701 wim.van.eck@vwa.nl VAN HOLST Ineke (Ms) Hilbrands Laboratory for Soil-borne Pest and Diseases (HLB BV),

	Canada Latvia Netherlands Netherlands Netherlands Netherlands Netherlands Slovenia	LÉVESQUE André (Mr) Agriculture and Agri-Food Canada, KW Neatby Building BRICE Elina (Ms) State Plant Protection Service , Lielvārdes iela 36/38, LV-1006 DE GRUYTER Hans (Mr) National Reference Centre, National Plant Protection FRANKE George (Mr) AIPH, c/o PO Box 1000, 1430 BA Aalsmeer, Netherlands LOOMANS Anton (Mr) National Reference Centre, National Plant Protection VAN DE VOSSENBERG WESTENBERG Marcel National Reference Centre, National Plant Protection RAVNIKAR Maja (Ms) National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana,
	(continued) (continued) (continued) (continued) (continued) (continued)	Bart (Mr) (Mr)	960 Carling Avenue, K2A 2P8 Ottawa (Ontario), Canada Rīga, Latvia Organization, P.O. Box 9102, 6706 EA Wageningen, Netherlands Tel: +31-297395007 Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands Organization, P.O. Box 9102, 6706 EA Wageningen, Netherlands Slovenia
			Tel: +1-6137591579 Tel: +371-67543138 Tel: +31-0653700550 gfranke@vbn.nl Tel: +31-622602540 -Fax: +31-317421701 Tel: +31-646200605 Tel: +386-59232801
			andre.levesque@agr.gc.ca elina.brice@vaad.gov.lv j.de.gruyter@minlnv.nl a.j.m.loomans@minlnv.nl m.westenberg@minlnv.nl maja.ravnikar@nib.si
		GROENEWALD Johannes	CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT
	Canada Netherlands Spain	LI Xiang (sean) (Mr) ASHRAFI Samad (Mr) DE HAAN Eisse (Mr) Zacharias (Mr) MEEKES Ellis (Ms) WOUDT Bert (Mr) CUBERO Jaime (Mr)	Canadian Food Inspection Agency, 93 Mount Edward Road, C1A National Reference Centre, Geertjesweg 15, 6706 EA NAK, Randweg 14, 8304 AS Emmeloord, Netherlands Utrecht, Netherlands Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, Syngenta Seeds B.V., P.O. Box 2, 1600 AA Enkhuizen, National Institute for Agriculture and Food Research, Ctra Coruña
	(continued)		5T1 Charlottetown, Canada Wageningen, Netherlands Tel: +31-527635400 -Fax: +31-527635411 Tel: +31-302122600 Netherlands Netherlands Km 7,5, 28040 Madrid, Spain
			Tel: +1-9023680950 Tel: +31-651014907 ehaan@nak.nl e.groenewald@cbs.knaw.nl Tel: +31-713326236 -Fax: +31-713326366 Tel: +31-228366539 -Fax: +31-228312818 Tel: +34-913474162
			sean.li@inspection.gc.ca samad.ashrafi@vwa.nl e.meekes@naktuinbouw.nl bert.woudt@syngenta.com cubero@inia.es
		DE HAAS Jan (Mr) HELDER Johannes (Mr) Wageningen University, Laboratory of Nematology, Department HZPC Holland B.V. Research & Development, Roptawei 4, 9123
	China	LI Ming-Fu (Mr) ASMA Margreet (Ms) MENDES Odette (Ms) ZIJLSTRA Carolien (Ms) WUR-Plant Research International, PO Box 69, 6700AB Animal & Plant Quarantine Institute, Chinese Academy of Bejo Zaden B.V., Trambaan 1c P.O. Box 50, 1749 ZH JB Metslawier, Netherlands of Plant Sciences, Wageningen University, Droevendaalsesteeg 1, Plant Research International (Wageningen UR), Building 107 PÉREZ-SIERRA Ana Mª Instituto Agroforestal Mediterráneo-Universitat Politècnica de
		(Ms)	Inspection and Quarantine, Huixin xije, Huixinli No.241 Chaoyang Warmenhuizen, Netherlands Tel: +31-622987583 6708 PB Wageningen, Netherlands (Radix), Droevendaalsesteeg 1, 6708 PB Wageningen, Netherlands Wageningen, Netherlands València, Escuela Técnica Superior de Ingeniería Agronómica y
			Dis., 100029 Beijing, China Tel: +31-226396162 jande.haas@hzpc.nl Tel: +31-317483136 -Fax: +31-317484254 Tel: +31-0317480754 Tel: +31-317480635 del Medio Natural, Edificio 3K-2ª Planta, Camino de Vera s/n,
			Tel: +86-01085380628 -Fax: +86-01085380628 m.asma@bejo.nl hans.helder@wur.nl odette.mendes@wur.nl carolien.zijlstra@wur.nl 46022 Valencia, Spain
		limf9@sina.com DE KOCK Maarten (Mr) Wageningen UR -Applied Plant Research, Prof. van Slogterenweg Tel: +34-963879254 -Fax: +34-963879269
	BAKKER Daniël (Mr) HIDDINK Gerbert (Mr) MIGLINO Roberto (Mr) Bloembollenkeuringsdienst (BKD), Postbus 300, 2161 AH Lisse, Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, 2, 2161DW Lisse, Netherlands Enza Zaden Seed Operations B.V., Haling 1E -P.O. Box 7, 1600 New Zealand HO Wellcome (Mr) Netherlands Ministry of Agriculture and Forestry, P.O. Box 2095, 1140 aperesi@eaf.upv.es
	(continued)	ZHAO Zhu (Ms)	Chinese Academy of Inspection and Quarantine, Gaobeidian Beilu Netherlands Tel: +31-252462148 AA Enkhuizen, Netherlands Netherlands Auckland, New Zealand
	Switzerland	FREY Jürg E. (Mr)	A3, Chaoyang District, 100121 Beijing, China Tel: +31-713326237 -Fax: +31-713326366 maarten.dekock@wur.nl Tel: +31-228350100 -Fax: +31-228315960 Tel: +31-252419101 -Fax: +31-252 417856 Kampsweg 27, 9418 PD Wijster, Netherlands Tel: +64-99095730 -Fax: +64-99095739 Agroscope Changins-Wädenswil Research Station ACW, Schloss
			Tel: +86-01064912743 -Fax: +86-01064912743 d.bakker@naktuinbouw.nl g.hiddink@enzazaden.nl roberto.miglino@bkd.eu Tel: +31-593582812 -Fax: +31-593582829 wellcome.ho@maf.govt.nz 1, Laboratory 4, P.O. Box 185, 8820 Wädenswil, Switzerland
		DEN NIJS Loes (Ms)	zhaozhu-100@163.com National Reference Centre, National Plant Protection vanholst@hlbbv.nl Tel: +41-447836332 -Fax: +41-447836434
	Norway	BERGSMA-VLAMI Maria JONGEDIJK Gerard (Mr) Propagation Nurseries the Netherlands, Tienrayseweg 9A, 5961 National Reference Centre, National Plant Protection Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands MOOYMAN Paul (Mr) Laboratory of Nematology, WUR, Radix, Building no. 107, BRURBERG May Bente Norwegian Institute for Agricultural and Environmental Research, juerg.frey@acw.admin.ch
	Czech Republic CERMAK Vaclav (Mr) (Ms) VAN LEEUWEN Gerard (Ms)	State Phytosanitary Administration, Division of Diagnostics, Organization, P.O. Box 9102, 6700 EA Wageningen, Netherlands Tel: +31-651255798 -Fax: +31-317421701 NK Horst, Netherlands Droevendaalsesteeg 1, 6708 PB Wageningen, Netherlands National Reference Centre, National Plant Protection Høgskoleveien 7, N-1430 Ås, Norway
		(Mr) HOLTERMAN Martijn	Slechtitelu 23/773, 779 00 Olomouc, Czech Republic Tel: +31-317496830 l.j.m.f.den.nijs@minlnv.nl Tel: +31-773986969 Tel: +31-317485085 Organization, P.O. Box 9102, 6706 EA Wageningen, Netherlands Tel: +47-92609364 Agroscope Changins-Wädenswil Research Station ACW, Schloss
		(Mr)	Tel: +420-585570171 -Fax: +420-585227790 m2.bergsma@minlnv.nl jongedijk@vermeerderingstuinen.nl paul.mooyman@wur.nl Tel: +31-629604595 -Fax: +31-317421701 may.brurberg@bioforsk.no 1, Laboratory 4, P.O. Box 185, 8820 Wädenswil, Switzerland
		DIJKSTRA Eddy (Mr)	vaclav.cermak@srs.cz National Reference Centre, National Plant Protection g.c.m.van.leeuwen@minlnv.nl Tel: +41-447836312
		BONANTS Peter (Mr) KARSSEN Gerrit (Mr) OOSTERHOF Jessica (Ms) Rijk Zwaan Breeding B.V., Postbus 40, 2678 ZG De Lier, Plant Research International, Droevendaalsesteeg 1, 6708 PB Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands National Reference Centre, National Plant Protection PERMINOW Juliana Irina Norwegian Institute for Agricultural and Environmental Research, martijn.holterman@acw.admin.ch
	Denmark	HANSEN Henrik Jorskov VAN RAAK Marcel (Mr) National Reference Centre, National Plant Protection Plant Diagnostic Laboratory, the Danish Veterinary and Food Wageningen, Netherlands Tel: +31-615946599 -Fax: +31-317421701 Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands Netherlands Spies (Ms) Hoyskoleveien 7, 1432 Aas, Norway
		(Mr) KIEWNICK Sebastian	Administration, Skovbrynet 20, 2800 Lyngby, Denmark Tel: +31-317480649 -Fax: +31-317418094 e.g.m.dijkstra@minlnv.nl Tel: +31-317496846 -Fax: +31-317421701 Tel: +31-0174532300 Organization, P.O. Box 9102, 6700HC Wageningen, Netherlands Tel: +47-92059165 Agroscope Changins-Wädenswil Research Station ACW, Schloss
		(Mr)	Tel: +45-45263849 -Fax: +45-45263613 peter.bonants@wur.nl g.karssen@minlnv.nl j.oosterhof@rijkzwaan.nl Tel: +31-317496834 -Fax: +31-317421701 juliana.perminow@bioforsk.no 1, Laboratory 4, P.O. Box 185, 8820 Wädenswil, Switzerland
		DULLEMANS Annette	hjh@pdir.dk Plant Research International, P.O. box 67, 6700 AB Wageningen, marcel.van.raak@minlnv.nl Tel: +41-447836336
	Pakistan	BOOMSMA Doretta (Ms) HZPC Holland B.V. Research & Development, Roptawei 4, 9123 (Ms) Netherlands KOCH Jeanette (Ms) National Reference Centre, National Plant Protection QUAEDVLIEG William CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT JANJUA Safia (Ms) Bioresource Research Center, 34-Bazar Road, G-6/4. sebastian.kiewnick@acw.admin.ch
		NICOLAISEN Mogens (Mr) VAN RUITEN John (Mr) Naktuinbouw, P.O.Box 40 Sotaweg 22, 2370 Aa Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark JB Metslawier, Netherlands Tel: +31-317480653 -Fax: +31-317418094 Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands Utrecht, Netherlands Islamabad, 44000 Islamabad, Pakistan
	Turkey	(Mr) YUKSELBABA Utku (Mr) Akdeniz University, Akdeniz University, Faculty of Agriculture, Tel: +45-87158137 Tel: +31-0519244381 annette.dullemans@wur.nl Tel: +31-615036257 Tel: +31-302122600 Roelofarendsveen, Netherlands Tel: +92-3325136610
			mogens.nicolaisen@agrsci.dk doretta.boomsma@hzpc.nl jeanette.koch@vwa.nl w.quaedvlieg@cbs.knaw.nl Tel: +31-713326166 -Fax: +31-713326364 safiajanjua@hotmail.com Plant Protection Department, 07070 Antalya, Turkey
		EBBING Ralph (Mr)	Bloembollenkeuringsdienst (BKD), Postbus 300, 2160 AH Lisse, j.v.ruiten@naktuinbouw.nl Tel: +90-5334110055
	Peru	NIELSEN Steen Lykke BOONEKAMP Piet (Mr) Plant Research International, Wageningen UR, The Netherlands, Aarhus University, Faculty of Agricultural Sciences, Dept. of Netherlands KOENRAADT Harrie (Mr) Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, REGOUIN Eric (Mr) Ministry of Economic Affairs, Agriculture and Innovation, KREUZE Jan (Mr) International Potato Center, Apartado 1558, Lima 12, Peru uyukselbaba@akdeniz.edu.tr
		(Mr) VAN SCHADEWIJK Ton	Integrated Pest Management, Forsogsvej 1, DK-4200 Slagelse, Droevendaalsesteeg 1, P.O. Box 69, Tel: +31-252419101 Netherlands Agroknowledge Department, Postbus 20401, 2500 EK Den Haag Bloembollenkeuringsdienst (BKD), Postbus 300, 2160 AH Lisse, Tel: +51-13496017
	United	(Mr) ADAMS Ian (Mr)	Denmark 6700 AB Wageningen, the Netherlands ralph.ebbing@bkd.eu Tel: +31-713326234 -Fax: +31-713326366 Netherlands Netherlands j.kreuze@cgiar.org The Food and Environment Research Agency, Sand Hutton, YO41
	Kingdom		Tel: +45-89993671 Tel: +31-317480626 h.koenraadt@naktuinbouw.nl Tel : +31-707573151 Tel: +31-252433059 1LZ York, United Kingdom
	Poland	steenl.nielsen@agrsci.dk piet.boonekamp@wur.nl EBSKAMP Michel (Mr) Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, e.j.m.regouin@minlnv.nl ton.van.schadewijk@bkd.eu LOJKOWSKA Ewa (Ms) Intercollegiate Facluty of Biotechnology University of Gdansk & Tel: +44-1904462553
		KOX Linda (Ms)	Netherlands National Reference Centre, National Plant Protection Medical University of Gdańs, Kladki 24, 80-822 Gdansk, Poland ian.adams@fera.gsi.gov.uk
	Finland	SOUKAINEN Mirkka (Ms) Finnish Food Safety Authority Evira, Mustialankatu 3, FI-00790 BUIMER Mark (Mr) Naktuinbouw, Sotaweg 22, 2371 GD Roefolareendsveen, Tel: +31-713326237 Organization, P.O. Box 9102, 6706 EA Wageningen, Netherlands RIBBENS Inge (Ms) Frugi Venta, Bezuidenhoutseweg 82, 2594 AX Den Haag, VAN VALKENBURG National Reference Centre, National Plant Protection Tel: +48-725991070
		Johan (Mr) BOONHAM Neil (Mr)	Helsinki, Finland Netherlands m.ebskamp@naktuinbouw.nl Tel: +31-317496754 Netherlands Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands lojkowska@biotech.ug.gda.pl The Food and Environment Research Agency, Sand Hutton, YO41
			Tel: +358-405834943 -Fax: +358-207725085 Tel: +31-713326158 l.f.f.kox@minlnv.nl Tel: +31-703355010 Tel: +31-621294830 -Fax: +31-317421701 1LZ York, United Kingdom
		EDEMA Mariette (Ms) MOSZCZYŃSKA Justyna	mirkka.soukainen@evira.fi m.buimer@naktuinbouw.nl National Reference Centre, National Plant Protection ribbens@frugiventa.nl j.l.c.h.van.valkenburg@minlnv.nl Main Inspectorate of Plant Health and Seed Protection, Central Tel: +44-1904462332
		Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands KOOMAN Miriam (Ms) Dutch General Inspection service (NAK), Randweg 14, 8304 AS (Ms) Laboratory, ul. Żwirki i Wigury 73, 87-100 Toruń, Poland neil.boonham@fera.gsi.gov.uk
		TEGEL Jukka (Mr) CROUS Pedro (Mr) ROBERT Vincent (Mr) VERSTAPPEN Els (Ms) Plant Research International, P.O. Box 69, 6700 AB Wageningen, Finnish Food Safety Authority Evira, Mustialankatu 3, FI-00790 CBS Fungal Biodiversity Institute, P.O. Box 85167, 3508AD Tel: +31-317496800 -Fax: +31-317421701 Emmeloord, Netherlands Fungal Biodiversity Center, Uppsalalaan 8, 3534CT Utrecht, Tel: +48-566391110 -Fax: +48-566391111
		Helsinki, Finland Utrecht, Netherlands m.j.edema@minlnv.nl Tel: +31-651191133 Netherlands Netherlands j.moszczynska@piorin.gov.pl ELPHINSTONE John (Mr) Food and Environment Research Agency, Sand Hutton, YO41 1LZ
			Tel: +358-405759174 -Fax: +358-207725034 Tel: +31-302122643 -Fax: +31-302122601 mkooman@nak.nl Tel: +32-478285764 Tel: +31-317480676 -Fax: +31-317418094 York, United Kingdom
		ELLENS Renate (Ms) SALDAT Marta (Ms)	jukka.tegel@evira.fi p.crous@cbs.knaw.nl Hilbrands Laboratory for Soil-borne Pest and Diseases (HLB BV), v.robert@cbs.knaw.nl els.verstappen@wur.nl Main Inspectorate of Plant Health and Seed Protection, Central Tel: +44-1904462334
		LANDEWEERT Renske	Kampsweg 27, 9418 PD Wijster, Netherlands Clear Detections, Binnenhaven 5, NL-6709 PD Wageningen, Laboratory, ul. Żwirki i Wigury 73 , 87-100 Toruń, Poland john.elphinstone@fera.gsi.gov.uk
	France	AMATO Marcos (Mr) DEES Robert (Mr) (Ms) ROENHORST Annelien WESSELS-BERK Brigitta	Agdia-Biofords, 5 Rue Henri Desbruères, 91030 Evry, France Applied Plant Research -WUR Flowerbulbs, Nurserystock & Tel: +31-593582812 Netherlands National Reference Centre, National Plant Protection National Reference Centre, National Plant Protection Tel: +48-566391110 -Fax: +48-566391111
		(j.w.) (Ms) (Ms)	Tel: +33-633060214 Fruits, Prof. van Slogterenweg 2, 2161 DW Lisse, Netherlands ellens@hlbbv.nl Tel: +31-652561829 Organization, P.O. Box 9102, 6700 HC Wageningen, Netherlands Organization, P.O. Box 9102, 6706 EA Wageningen, Netherlands m.saldat@piorin.gov.pl
			marcos.amato@biofords.com Tel: +31-252462121 renske.landeweert@cleardetections.com Tel: +31-317496911 -Fax: +31-317421701 Tel: +31-615946601 -Fax: +31-317421701
			robert.dees@wur.nl j.w.roenhorst@minlnv.nl b.f.wessels-berk@minlnv.nl

NOUWEN Ria

Martine Maes

Session 9 Stakeholder views and needs

Abstracts of Presentations

Results of all Work Packages of QBOL, from DNA barcodes to Database and Validation.

Peter Bonants Plant Research International, Wageningen UR, Droevendaalsesteeg 1, 6708 PB Wageningen, NL, peter.bonants@wur.nl

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. In addition the number of other specialists in phytopathology and other fields who are vital for sustaining sound public policy on phytosanitary issues are diminishing. These problems affect all members of the EU and other nations. 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). The six work packages (WP) to generate the barcodes on Fungi (WP2), Arthropods (WP3), Bacteria (WP4), Nematodes (WP5), Viruses (WP6) and Phytoplasmas (WP7) and also WP9 on the Database and WP 10 on Validation will be presented in an overview. The Plant Health Quads (PHQuads) is an international partnership in which senior regulators from the national plant protection organizations of Australia, Canada, New Zealand and the United States may engage in collaborative projects and discussions of plant health issues of mutual concern. In 2006, the PHQuads established the Quads Scientific Collaboration Working Group (SCWG). The purpose of the SCWG in support of the PHQuads is: to share and exchange technical information; to foster cooperation and collaboration in phytosanitary programs; and to advance plant health knowledge, science, and technology in areas of mutual interest. The missions of the respective PHQuads national plant quarantine/ biosecurity organizations are to safeguard plant resources from invasive plant pests/pathogens. When a pest is detected it is imperative that the identity of that pest or pathogen is established for appropriate action to be taken. Misidentification of a pest may result in unnecessary actions that hinder trade. The PHQuads Diagnostic Tools project team was formed to specifically, identify and coordinate the development of diagnostic tools for plant pests. To facilitate PHQuads understanding and application of DNA barcoding technology, a project on the topic was initiated in 2010. The PHQuads barcoding project has a number of aims that include: Identifying opportunities for cooperation on DNA barcoding projects between Quads members; Discussing and evaluating barcode libraries and protocols for pests of concern to Quads members; and Exploring the regulatory implications at the international level, especially the issue of species delimitation and its implications for regulators. Details of this project will be presented on behalf of the project members.

Wellcome Ho

Barcoding, Multilateral Initiatives for Biosecurity Outcomes

John van Ruiten

International trade of high graded plant material: the need for harmoniszed and validated detection techniques.

John van Ruiten, Director Naktuinbouw, Sotaweg 22, Postbus 40, NL-2370 AA Roelofarendsveen Nederland the Netherlands Growers of agricultural and horticultural products are faced with many challenges. There is a great necessity to increase production and productivity and at the same time decrease use of fossil fuels and crop protection compounds.

To achieve this goal it is required that high graded, absolutely disease free seeds and young plants/tubers are available for starting growing a crop. Companies, inspection agencies and phytosanitary services have to develop better (even more sensitive) techniques/protocols for producing, testing and monitoring healthy seeds and plants. As material is produced and marketed globally it is vital that sampling and testing techniques are much better harmonized than at present. An initiative such as Q-bank offers the conditions and connections amongst specialists to develop (DNA-based) diagnostic and detection methods that are validated (and are therefore proven to be effective against occurring strains of a wide range of pathogens).

George Franke

The view of horticultural producers on diagnostic tools

Christine Henry

TESTA -Seed health: development of seed treatment methods, evidence for seed transmission and assessment of seed health C. M. Henry, FERA; M-A. Jacques, INRA ; P. Bonants, DLO ; V. Cockerell, SASA; D. Spadaro, UNITO; M.J.M. Ebskamp, NAKT; T.A.S. Aveling, University of Pretoria; S. L. Nielsen, Aarhus University; J. E. Thomas, NIAB; E. Stefani, University of Modena and Reggio Emilia; V. Grimault, GEVES; F. Petter, EPPO ; J.M. Carstensen, Videometer.

The TESTA initiative plans to develop a range of novel methods to underpin the control of seed-borne diseases and pests, including faster, more accurate methods to assess the mode of seed transmission, economic and practical sampling approaches for the detection of low levels in large seed lots, novel and efficient generic detection methodologies, non-destructive testing methods and improved, effective and sustainable disinfection methods. Target crop and disease/pest combinations have been identified in consultation with EPPO, ISHI-ISF and ISTA. Outcomes from the project will include a comprehensive electronic database of seed transmitted diseases and pests, validated detection methods for target species, a validation protocol for assessing the efficacy of disinfection, as well as many key scientific publications. These will provide supporting methods and sources for the EU seed testing laboratories and plant health services.

Eric Regouin

Discussion /concluding remarks United Kingdom (continued) GLOVER Rachel (Ms) The Food and Environment Research Agency, Sand Hutton, YO41