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Certified reference materials to promote quality assurance in the seed production chain and support Community policies for controlling quarantine organisms

Contract nr: FAIR-CT95-0517
Project nr: 517
Project type: SC
Starting date: 01/04/1996
Duration: 47 months
Total cost: 1,017,950 EUR
EC Contribution: 538,000 EUR
Scientific Officer: Richard HARDWICK
Research topic: Plant health

Disease-free planting material is essential for economically profitable and environmentally safe crop production. Production and selection of healthy seed can be ensured by testing seed for the presence of unwanted plant pathogens. In order to harmonise seed health testing in the EU, standardisation and validation of test methods is needed. To achieve standardised and validated seed health test methods, tools to determine the value and to verify the performance of the test methods are required. Reference materials (RMs), defined as encapsulated, preserved bacterial preparations containing a specified quantity of viable bacterial cells of a well described pathogenic strain, are an example of such tools (Van den Bulk and Taylor, 1997). The use of RMs of known contamination level is also recommended for quality control in seed health testing. In addition, reference materials are useful tools for laboratory accreditation bodies to guarantee that seed health tests are performed correctly.

The general objective of this project is the development and implementation of reference materials to be used in seed health testing for the presence of harmful bacteria.

Candidate reference materials will be developed for four seedborne bacteria which fall under plant health regulations of the EU, i.e. Clavibacter michiganensis subsp. michiganensis (causing bacterial canker of tomato), Erwinia stewartii (causing Stewart's bacterial wilt of maize), Pseudomonas syringae pv. phaseolicola (causing halo blight of beans) and Xanthomonas campestris pv. campestris (causing black rot of cabbage). The chosen bacteria are all agriculturally important organisms in terms of legislation by DG VI.

Candidate reference materials produced will be tested in intercomparative studies with 12 European seed test stations involved, to determine their value and usability as standards in seed health testing.

Current situation/results:
Various preservation techniques were tested. Preservation by freeze drying was the most stable over a range of storage temperatures. Overall it was concluded that the freeze dried paper disk system was the best method from the point of view of stability and practicality. The freeze dried paper disk procedure was optimised by adding a pre-drying step, omitting the pre-freezing step, and improving the composition of the preservation medium. For recovery of viable cells, the best resuscitation fluid (0.1% peptone broth) and most optimal disk soaking time (2 hours) were established. The physical representation of the developed reference materials is a 2-ml vial, 4 cm high and 14 mm in diameter, containing a 6-mm diameter paper disk inoculated with 20 µl of bacterial suspension. After a pre-drying step and over-night freeze drying, the vials are stoppered under vacuum with a rubber stopper, capped with an aluminium seal and stored at 4 °C.

Test batches of reference materials were shown to be rather stable in recovery up to 12 months of storage at 4°C. Homogeneity analyses of test batches showed the variance between reference materials to be acceptable.

An interlaboratory test was organised to determine whether the materials developed are usable as effective reference materials. Reference materials of each of the four pathogens were sent to and tested in 13 laboratories, with each laboratory following a precisely defined protocol for recovery, diluting, plating and recording. Statistical analysis of the results obtained for each pathogen/medium combination indicated that the overall reproducibility, including both within and between laboratory variance, was at an acceptable level. Results for X. campestris and P. syringae were particularly good, with similar levels of reproducibility for both selective and non-selective media. Results for E. stewartii and C. michiganensis were not as good, due mainly to poorer reproducibility on the selective media.

A second interlaboratory test established that the reference materials developed were useful as standards in actual seed test protocols. The results showed adequate performance of the reference materials in an environment with `live background', i.e. seeds with their normal microflora.


Plant Research International B.V.
P.O. BOX 16
NL-6700 AA Wageningen
Tel.: +31 317 47 69 80
Fax: +31 317 41 80 94


  • John Douglas TAYLOR
    Horticulture Research International
    UK-CV35 9EF Warwick
    Tel.: +44 1789 47 03 82
    Fax: +44 1789 47 05 52


  • Madalena SOUSA SANTOS
    DGPC - Direcção de Serviços de Fitossanidade
    Tapada da Ajuda
    Edificio 1
    P-1300 Lisboa
    Tel.: +351 21 363 40 54
    Fax: +351 21 363 50 16

  • Frank NIEPOLD
    Federal Biological Research
    Centre for Agriculture and Forestry
    Messeweg 11-12
    D-38104 Braunschweig
    Tel.: +49 531 299 45 15
    Fax: +49 531 299 30 08

    Istituto Sperimentale per la Patologia vegetale
    Via Carlo Giuseppe Bertero 22
    I-00156 Roma
    Tel.: +39 06 86 89 02 51
    Fax: +39 06 86 80 22 96

  • Ib Geltzer DINESEN
    Danish Plant Directorate
    Skovbrynet 20
    DK-2800 Lyngby
    Tel.: +45 45 96 66 00
    Fax: +45 45 96 66 10

  • Athanassios ALIVIZATOS
    Benaki Phytopathological Institute
    S. Delta Str. 8
    GR-145 61 Athens-Kifissia
    Tel.: +30 1 807 88 32
    Fax: +30 1 807 75 06

  • James REEVES
    National Institute of Agricultural Botany
    Huntingdon Road
    UK-CB3 0LE Cambridge
    Tel.: +44 1223 34 22 72
    Fax: +44-1223 27 76 02

  • Chiara DELOGU
    Ente Nazionale delle Sementi Elette
    Via Emilia 19
    I-20080 Tavazzano
    Tel.: +39 03 71 76 19 19
    Fax: +39 03 71 76 08 12

  • Harrie KOENRAADT
    Sotaweg 22
    P.O. BOX 27
    NL-2370 AA Roelofarendsveen
    Tel.: +31 171 31 91 02
    Fax: +31 171 31 62 56

  • Valerie OLIVIER
    Groupe d'étude et de controle des variétés et des semences -GEVES
    Rue Georges Morel
    P.O. BOX 24
    F-49071 Beaucouze CEDEX
    Tel.: +33 2 41 22 58 54
    Fax: +33 2 41 22 58 01

  • Paula PERSSON
    Swedish University of Agricultural Sciences
    P.O. BOX 7044
    S-750 07 Uppsala
    Tel.: +46 18 67 23 58
    Fax: +46 18 67 28 90

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