; EPPO Standards ;
SAMPLING AND TEST METHODS
PM 3/40(1) English
Organisation Européenne et Méditerranéenne pour la Protection des Plantes
1, rue Le Nôtre, 75016 Paris, France
EPPO Standards are approved by EPPO Council. The date of approval appears in each individual standard.
EPPO Standards are subject to periodic review and amendment. The next review date for this set of EPPO Standards is decided by the EPPO Working Party on Phytosanitary Regulations.
Amendments will be issued as necessary, numbered and dated. The dates of amendment appear in each individual standard (as appropriate).
EPPO Standards are distributed by the EPPO Secretariat to all EPPO member governments. Copies are available to any interested person under particular conditions upon request to the EPPO Secretariat.
EPPO Phytosanitary Procedures are intended to be used by National Plant Protection Organizations, in their capacity as bodies responsible for the inspection, testing and treatment of plants and plant products moving in trade, or for the implementation of surveys against quarantine pests.
OEPP/EPPO (1996) Glossary of Phytosanitary Terms. EPPO Technical Documents no. 1026.
CABI/EPPO (1997) Quarantine Pests for Europe, 2nd edition (Ed. by Smith, I.M.; McNamara, D.G.; Scott, P.R.; Holderness, M.), CAB International, Wallingford, UK.
OEPP/EPPO (in preparation) Specific Quarantine Requirements. Available as electronic documents from the EPPO Web Site.
Phytosanitary procedure: Any officially prescribed method for performing inspections, tests, surveys or treatments in connection with plant quarantine.
Inspection: Official visual examination of plants, plant products or other regulated articles to determine if pests are present and/or to determine compliance with phytosanitary regulations.
Survey: An official procedure conducted over a defined period of time to determine the characteristics of a pest population or to determine which species occur in an area.
Test: Official examination, other than visual, to determine if pests are present or to identify pests. Treatment: An officially authorized procedure for the killing, removal or rendering infertile of pests.
OUTLINE OF REQUIREMENTS
EPPO Phytosanitary Procedures describe the methods to be followed for performing inspections, tests, or treatments of commodities moving in trade, or surveys against quarantine pests. For many quarantine pests, a reference to the relevant EPPO Phytosanitary Procedure is made in the corresponding EPPO Specific Quarantine Requirements. The development of EPPO phytosanitary procedures started many years ago, and these methods have been published in the Bulletin OEPP/EPPO Bulletin under several titles: ‘Fumigation standards’, ‘Quarantine Inspection Procedures’ and ‘Quarantine Procedures’. All of them are now appearing under the title ‘EPPO Phytosanitary Procedures’ and are being edited into EPPO Standard format. The numbering of these procedures will continue to follow the sequence described in the Bulletin OEPP/EPPO Bulletin 20(2), 229-233, which corresponds approximately to the chronological order of appearance of the Phytosanitary Procedures.
EUROPEAN AND MEDITERRANEAN PLANT PROTECTION ORGANIZATION
ORGANISATION EUROPEENNE ET MEDITERRANEENNE POUR LA PROTECTION DES PLANTES
PM 3/40(1) English
SAMPLING AND TEST METHODS
Specific scope Specific approval and amendment
This standard describes the sampling and test methods First approved in September 1991.
for Erwinia amylovora, to satisfy the requirements of Edited as EPPO Standard in 1998.
the EPPO Standard PM 2/52(1).
Erwinia amylovora is an EPPO A2 quarantine organism and details about its biology, distribution and economic importance can be found in Data sheet no. 52 (OEPP/EPPO, 1983). The EPPO specific quarantine requirements for E. amylovora (OEPP/EPPO, 1990a) include requirements that host plants should have
been grown in areas where E. amylovora does not occur or else in a place of production found free from E.
amylovora during the last growing season. In the latter case, countries which consider themselves to be at high risk can specify that the field, as well as the surrounding zone of radius of at least 250 m, must be inspected at least once in July/August and once in September/October and that spot checks should be carried out in the surrounding zone of radius of at least 1 km in places where host plants are grown, at least once in July/October. For the southern hemisphere, equivalent periods would apply. While these inspections are essentially visual, for symptoms of the disease, any suspect material will need to be tested for the presence of E. amylovora. In addition, exporting countries may need to perform
random or systematic surveys of areas from which host plants are exported and in which the absence of E.
amylovora has to be confirmed. Such surveys may involve sampling of apparently healthy host material in or on which E. amylovora may occur latently or epiphytically.
Visual observation is the main method used for detecting fireblight in and around places of production of host plants. The symptoms have been very fully described in various publications (OEPP/EPPO, 1983). Testing for E. amylovora can be performed using the following techniques: (1) IFAS (indirect
immunofluorescence antibody stain); (2) agglutination test; (3) API-20E test strips; (4) pathogenicity. The first test can be applied to suspensions in sterile phosphate-buffered saline (PBS) of plant material, as a screening technique, and also to pure cultures. The other tests first require dilution plating of suspect material on 5 % sucrose agar and/or CCT medium. Typical colonies of E. amylovora are levan-positive and
non-fluorescent under UV light. Such colonies can be tested by agglutination with a drop of diluted E.
amylovora antiserum on a slide, by IFAS, by use of standard-test strips or by routine biochemical tests (Dye, 1983) allowing a + or – response for some 20 biochemical characters. Finally they can be tested for hypersensitive reaction of tobacco plants, and for pathogenicity on immature pear fruits. For material showing fireblight symptoms, the normal procedure would be dilution plating, followed by agglutination or IFAS. A positive agglutination or IFAS test is generally considered a definite proof of the presence of E. amylovora. For apparently healthy material, possibly carrying latent or epiphytic E.
amylovora, IFAS is used first as a screening method. If a positive result is obtained, the sample is dilution-plated and tested as above. In all cases, it may be useful to confirm the serological identification by use of
biochemical tests and by pathogenicity tests. Such confirmation would certainly be needed for material coming from an area where E. amylovora does not occur.
The steps in testing of apparently healthy material are illustrated in Fig. 1. See Appendix I for details of methods.
IFAS test on concentratedSample considered free suspension from twig tipsfrom E. amylovoranegative
Dilution-plate and select typical colonies
none found Try again oncefound
Sample considered free Test them by agglutination test and/or IFASfrom E. amylovora negative
Sample considered positive for E. amylovora
If desired, confirm by biochemical
and pathogenicity tests
Fig. 1. Testing scheme for latent or epiphytic infections by Erwinia amylovora.
Sampling procedure for detection of latent Erwinia amylovora in woody material before export
Take samples from each nursery from which host plants will be exported (or other unit under investigation). A sample consists of 100 twigs about 10 cm in length, from 100 plants of different species and/or cultivars. If there are several genera in the lot, these should be equally represented in the sample,
with a maximum of three genera per sample. Each sample is placed in a plastic bag and labelled.
From each sample, randomly take 30 cut twigs to be tested. Store the remaining twigs of the sample, appropriately labelled, at 5?C in a cold chamber, in order to be able to test genera separately if an infection is found. From each of the 30 twigs, cut 4 pieces (= 120 stem pieces).
Place the 4 stem pieces for 1.5 h at room temperature in sterile 0.01 M PBS Tween, pH 7.2 (containing -1in g litre distilled water: NaHPO•12 HO, 2.7; NaHPO•2HO, 0.4; NaCl, 8; Tween 20, 1 ml) sufficient to 242242
cover the sample and shake in a rotary shaker in Erlenmeyer flasks.
Filter the decanted supernatant through filter paper (Whatman no. l) held in a sintered glass filter (no. 2 = 40-100 µm) using a water vacuum pump, and collect the filtrate in a centrifuge tube. Centrifuge for 20 min at 10000 g. Suspend pellet in 1 ml sterile 0.01 M PBS (as above without Tween).
To 0.6 ml of resuspended pellet, add one drop of Difco Bacto glycerol and retain for reference at -20?C. The other part is used for the immunofluorescence test and isolation on different media.
Indirect immunofluorescence antibody stain (IFAS)
Use an antiserum to E. amylovora of which the titre (last dilution of the antiserum giving still a bright
fluorescence) has been determined, using a homologous antigen control. General indications of the IFAS procedure can be found in the EPPO Quarantine procedure for Clavibacter michiganensis ssp. sepedonicus
Pipette 20 µl of test suspension to windows (8 mm diameter) of a 10-well multitest slide (Flow Laboratories). This suspension may derive from the resuspended pellet from the sampling procedure -1(above), or from a 48-h colony on a dilution plate (diluted to about 10 cells ml). Air-dry slides at room
temperature and gently heat-fix by flaming (if the antiserum was made using heat-fixed cells, but not otherwise). Cover all (except the first) windows with 20 µl two-fold dilutions of E. amylovora antiserum up
to one step above the titre. The first window is covered with 0.01 M PBS as a control for non-specific binding of the conjugate. Antiserum dilutions are made in 0.01 M PBS. -1A control slide is prepared with a suspension of about 10 cells ml of a 48-h colony of E. amylovora
grown on Difco nutrient agar (NA), using the same antiserum dilutions on 20 µl drops of the heat-fixed bacterial suspension.
Incubate slides with antiserum for 30 min at room temperature in a humid chamber. Wash carefully (control slides separate) in three changes of 0.01 M PBS. Remove excess moisture by carefully blotting with filter paper.
Cover each window with 20 µl of fluorescein isothiocyanate (FITC) conjugate in a 100-fold dilution (for each batch of conjugate the appropriate dilution should be determined) and incubate at room temperature for 30 min in a humid chamber. Wash and remove moisture as above. When slides are dry, add one droplet of 0.1 M phosphate-buffered glycerine, pH 7.6 (containing NaHPO•12HO, 3.2g; 242
NaHPO•2HO, 0.15 g; glycerol 50 ml and distilled water to 100 ml) to each window and cover slide with 242
a long coverglass.
Examine slides with a microscope with an epifluorescent light source and filters suitable for working with FITC, using a 40 x or 100 x oil immersion objective and a 10 x eyepiece. The titre is first checked in the control slide and thereafter sample windows are scanned across two diameters at right angles. Count morphologically typical fluorescent cells (at or near the titre) in at least 10 microscope fields per window. If no typical fluorescent cells are found, IFAS is negative and the sample is regarded as negative. If morphologically typical fluorescent cells at the titre are found IFAS is positive. However, this is not sufficient in itself to identify E. amylovora.
Plate 10 µl of pellet suspension (see above) with a Drigalsky stick on two subsequent plates of NA -1containing 50 g litre sucrose and 200 mg cycloheximide (added after autoclaving) and/or on two 1subsequent plates of semi-selective CCT medium. After 48 h, place plates under an ultraviolet lamp (366
nm) and mark all typical levan-positive, fluorescence-negative colonies with a pencil.
Typical levan-positive, fluorescence-negative colonies are agglutinated in a drop of 0.01 M PBS with a drop of E. amylovora antiserum in a 20-fold dilution on a slide.
In certain cases it may be necessary to identify agglutination-positive, fluorescence-negative, levan-positive colonies. The easiest way to do so is by using IF and the API-20E test strip (API System, La Balme-les-Grandes, 38390 Montalieu-Vercieu, France, or local distributor) where tests should have the following results:
ONPG – MAN –
ADH – INO –
LDC – SOR –
ODC – RHA –
CIT – SAC +
HS – MEL – 2
URE – AMY –
TDA – ARA +
IND – Oxidas –
If API strips are not available, confirmatory tests as described by Dye (1983) should be performed.
8-1Pathogenicity can be checked by the hypersensitivity test on tobacco. A suspension of 10 cells ml of a
48-h NA culture is infiltrated into the tobacco leaf. After 10-12 h the infiltrated area becomes glassy, later thin, white necrotic. If an IF-positive, API-20E positive culture is hypersensitivity-reaction negative, a pathogenicity test on immature pear fruits should be performed, using the method of Billing et al. (1960).
The bacterium should be reisolated from infected tissue and checked by agglutination.
Billing, E., Crosse, J.E., Garrett, C.M.E. (1960) Laboratory diagnosis of fireblight and bacterial blossom
1 Add to 970 ml distilled water, 100 g sucrose, 10 g sorbitol, 30 ml 1% aqueous tergitol anionic, 2 ml 0.1% crystal violet in absolute ethanol, 23 g nutrient agar. Autoclave 10 min at 115?C. Add after autoclaving 2 ml 1% aqueous thallium nitrate and 0.05 g cycloheximide.
blight of pear. Plant Pathology 9, 19-25.
Dye, D.W. (1983) The Amylovora and Herbicola group. In Plant Bacterial Diseases, A Diagnostic Guide (eds
Fahy, P.C., Persley, G.J.), pp. 67-86. Academic Press, London (GB). Ishimura, E.S., Klos, E.J. (1984) New medium for detection of Erwinia amylovora and its use in
epidemiological studies. Phytopathology 74, 1342-1345.
OEPP/EPPO (1983) Data sheets on quarantine organisms. No. 52. Erwinia amylovora. Bulletin OEPP/ EPPO Bulletin 13 (1).
OEPP/EPPO (1990a) Specific quarantine requirements. EPPO Technical Documents no. 1008.
OEPP/EPPO (1990b) Quarantine procedures. No. 25. Clavibacter michiganensis ssp. sepedonicus -
inspection and test methods. Bulletin OEPP/EPPO Bulletin 20, 235-254.
May be addressed to: Ir. J. D. Janse, Plantenziektenkundige Dienst, Geertjesweg 15, P.O. Box 9102, 6700
HC Wageningen (Netherlands).