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Development of standardised molecular techniques for the identification of insect quarantine pests

Contract nr: FAIR-CT96-1972
Project nr: 1972
Project type: SC
Starting date: 01/02/1997
Duration: 36 months
Total cost: 1,107,000 EUR
EC Contribution: 809,000 EUR
Scientific Officer: Arnaud BORCHARD
Research topic: Plant health
Acronym: INSECT DIAGNOSTICS

Background:
The detection and identification of harmful insect pests are critical to the maintenance of EU plant health, both as regards the introduction of new pests into Europe and the spread of established pests within Europe. It is evident that procedures for pest diagnostics will be of growing importance as an increase in legal challenges to Member State plant health decisions can be expected in response to the EC Plant Health Directive and the General Agreement on Tariffs and Trade (GATT) rules. Molecular diagnostic techniques can facilitiate quarantine identifications as well as the study of the biology and epidemiology of existing pest species and their biotypes (e.g. Pesticide resistant biotypes, host specific biotypes) both in the support of current EU plant health policy and in the development of future EU policy. Currently, insufficient DNA sequence data exists to enable the design of molecular-based tests which will be specific to target species, yet be adaptable enough to cope with potentially high levels of intraspecific variation.

Objectives:
The objectives of this proposed project are:
1) to develop novel, standardised and robust molecular diagnostic procedures for the identification of Tephritid and other quarantine insect species;
2) to develop molecular markers for monitoring gene flow of Tephritid insects within the EU and to aid identification of the sources of new infestations;
3) to develop novel, standardised and robust molecular diagnostic procedures for the identification of high risk insect biotypes exhibiting pesticide resistance;
4) to establish a DNA sequence database for insects of quarantine importance as well as non-quarantine species which need to be distinguished from quarantine pests;
5) to disseminate identification protocols, diagnostic materials (e.g. PCR primers, `type' DNA) and DNA sequence data to EU laboratories with responsibility for quarantine pest identification.

Description:
The development of validated, user-friendly and robust diagnostic tests has been achieved by the production of standard PCR primer sets designed to detect diagnostic insect DNA sequences. A range of techniques were used to detect these sequences. These included random amplified polymorphic DNA (RAPD) PCR, Amplified Fragment Length Polymorphism (AFLP) analysis, sequence analysis of mitochondrial and ribosomal DNA, microsatellite marker analysis, multilocus enzyme electrophoresis (MLEE), and single copy nuclear DNA polymorphisms, subtraction hybridisation and conserved primer PCR. The approach has maximised the likelihood of identifying usable sequences and developing the robust diagnostic protocols required for quarantine work. The efficacy of the different approaches in isolating useful diagnostic sequences from quarantine insects was assessed using seven Tephritid species (Ceratitis capitata, Ceratitis rosa, Bactrocera oleae, Bactrocera dorsalis, Anastrepha suspensa, Anastrepha ludens, Rhagoletis cerasi) and the efficacy of diagnostic primers investigated using laboratory tests combined with optimised storage and DNA extraction procedures. Protocols, DNA sequence information, PCR primers and standard DNA samples will be archived and disseminated, as required, to quarantine services and laboratories.

Current situation/results:
Experimental work within the project has been completed resulting in good progress in a number of areas:
Experiments have identified optimised protocols for storage of Tephritid insects after collection from quarantine inspection. In addition, the project has tested a range of DNA extraction protocols and identified those procedures which optimise high yielding DNA extraction from all insect life stages.
RAPD-PCR, analysis of mitochondrial and ribosomal DNA, subtraction hybridisation, multilocus enzyme electrophoresis (MLEE) microsatellite DNA , AFLP and single copy nuclear DNA polymorphisms, have all produced specific DNA sequences for Tephritid diagnostics, allowing identification of Ceratitis capitata, Ceratitis rosa, Bactrocera oleae, Bactrocera dorsalis, Anastrepha suspensa, Anastrepha ludens, Rhagoletis cerasi and other Tephritid species. The DNA-based tests can be used on all life stages and can give positive identification from even small parts of adult flies including legs and wings.
The development of intraspecific markers for C. capitata populations (e.g. rDNA, mtDNA, RAPD primers and SCARs) has been productive resulting in genetic markers for assessing gene flow and geographic origins in the species.
Work on Frankliniella occidentalis has generated primers targeting kdr, super kdr and cyclodiene resistance genes. These markers for insecticide resistance should facilitate risk assessments for new insect populations and facilitate the management of established populations.
The DNA sequence database, comprising raw sequence, haplotype information, PCR primers and probe sequences represents a valuable resource for future studies. In addition, PCR primers, DNA probes and Tephritid DNA samples stored at the Central Science Laboratory are available for interested researchers. For access to this information please contact the coordinator, Dr Colin Fleming.

Website: http://www.qub.ac.uk/afs/aps/insect.htm


Coordinator
Colin FLEMING
Queen's University of Belfast
Applied Plant Science
Newforge Lane
UK-BT9 5PX Belfast
Tel.: +44 2890 25 52 76
Fax: +44 2890 66 83 75
E-mail: C.Fleming@qub.ac.uk


Partners

  • Anna Rodolfa MALACRIDA
    Università degli Studi di Pavia
    Dipartimento di Biologia Animale
    Laboratorio di Zoologia
    Piazza Botta 9
    I-27100 Pavia
    Tel.: +39 38 250 62 94
    Fax: +39 38-250 62 90
    E-mail: malacrid@ipv36.unipv.it

  • Ann BURNELL
    National University of Ireland
    Department of Biology
    IRL-Maynooth, Co. Kildare
    Tel.: +35 3 17 08 38 40
    Fax: +35 3 17 08 38 45
    E-mail: burnell@may.ie

  • Dominique Warren COLLINS
    Central Science Laboratory
    Hatching Green
    UK-AL5 2BD York
    Tel.: +44 1582 71 52 41
    Fax: +44 1582 76 21 78
    E-mail: d.collins@csl.gov.uk

  • Dolores OCHANDO
    Universidad Complutense de Madrid
    Genetics Department
    Biological Sciences Faculty
    Ciudad universitaria s/n
    E-28040 Madrid
    Tel.: +34 1 394 49 99
    Fax: +34 1 394 48 44
    E-mail: ochando@eucmax.sim.ucm.es

  • Juerg Ernst FREY
    Swiss Federal Research Station for Fruit-growing, Viticulture and Horticulture
    Schloss
    CH-8820 Wädenswil
    Tel.: +41 17 83 63 32
    Fax: +41 17 80 63 41
    E-mail: juerg.frey@faw.admin.ch
 
 
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