PHYTOSANITARY PROCEDURES

Transcription

1 EPPO Standards PHYTOSANITARY PROCEDURES PSEUDOMONAS SYRINGAE PV. PERSICAE SAMPLING AND TEST METHODS PM 3/44(1) English oepp eppo Organisation Européenne et Méditerranéenne pour la Protection des Plantes 1, rue Le Nôtre, Paris, France

2 APPROVAL EPPO Standards are approved by EPPO Council. The date of approval appears in each individual standard. REVIEW 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. AMENDMENT RECORD Amendments will be issued as necessary, numbered and dated. The dates of amendment appear in each individual standard (as appropriate). DISTRIBUTION 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. SCOPE 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. REFERENCES OEPP/EPPO (1996) Glossary of Phytosanitary Terms. EPPO Technical Documents no 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. DEFINITIONS 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. 2

3 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), , which corresponds approximately to the chronological order of appearance of the Phytosanitary Procedures. 3

4 EUROPEAN AND MEDITERRANEAN PLANT PROTECTION ORGANIZATION ORGANISATION EUROPEENNE ET MEDITERRANEENNE POUR LA PROTECTION DES PLANTES PM 3/44(1) English Phytosanitary procedure PSEUDOMONAS SYRINGAE PV. PERSICAE SAMPLING AND TEST METHODS Specific scope This standard describes the sampling and test methods for Pseudomonas syringae pv. persicae, to satisfy the requirements of EPP Standard PM 2/145(1). Specific approval and amendment First approved in September Edited as EPPO Standard in Introduction Pseudomonas syringae pv. persicae is an EPPO A2 quarantine organism and details about its biology, distribution and economic importance can be found in Data sheet no. 145 (OEPP/EPPO, 1986). The specific quarantine requirement for P.s. persicae (OEPP/EPPO, 1990a) requires a growing season inspection of the place of production, by a recommended procedure. Methods Visual inspection of peach orchards in spring will show up characteristic symptoms of the disease. However, the bacterium has a latent phase, persisting epiphytically on leaf surfaces through the summer to the autumn. It is accordingly necessary to test for epiphytic bacteria on the leaves, and a procedure bas been worked out for this specific purpose. This involves screening of leaf samples by immunofluorescence (IF), followed by isolation if IF is positive. The samples should be taken in autumn and, preferably, also in spring. See Appendix I for details of the methods. APPENDIX I Visual inspection P.s. persicae infects peach trees through the winter months, entering leaf scars in autumn and causing necrosis of shoots, branches and trunks. A characteristic symptom is olive-green discoloration, rapidly browning round dormant buds on young shoots. In spring, dieback of buds or shoots can be observed, affected tissues appearing brownish red. Symptoms on leaves (necrotic spots with chlorotic halo) are produced in wet conditions and are not very characteristic. Though symptoms may be seen on fruits, it is not recommended to inspect during the fruiting season, when more characteristic symptoms on shoots cannot so readily be seen. 4

5 Collecting leaf samples Leaf samples for detection of epiphytic populations of P.s. persicae should be collected in autumn, and possibly also in spring. Take one leaf at random from every fourth tree, to produce a sample of 500 leaves per unit of 2,000 trees. If the plot contains less than 2,000 trees, the sample will be correspondingly reduced, but not below 200 leaves. Divide into 5 subsamples, which will be used as follows: two for IF screening; two for isolation (if IF is positive); one for storage. Samples should preferably be processed within 2 days. IF screening Process the two subsamples separately. Shake in 500 ml (spring) or 1,000 ml (autumn) sterile distilled water for 2 h at room temperature. Dilute 1:10 and 1:100 with 0.01 M phosphatebuffered saline (PBS) ph 7.2. Dry 10 µl drops of the suspension and its dilutions on a 10-well multitest slide and fix in alcohol. Cover wells with 20 µl drops of anti-p.s. persicae antiserum, suitably diluted in 0.01 M PBS ph 7.2. The best working dilution is the last dilution which gives bright fluorescent cells at the predetermined titre of the antiserum. (The titre is defined as the highest dilution of antiserum at which coloration occurs, before the intensity of this coloration becomes noticeably reduced.) Include a PBS control for non-specific binding of conjugate. Set up positive controls with authentic P.s. persicae. See OEPP/EPPO (1990b) for example of layout of IF multitest slides. Table 1. Behaviour of Pseudomonas syringae pv. persicae in nutritional and physiological tests in comparison with pv. mors-prunorum and pv. syringae. pv. syringae pv. mors-prunorum pv. persicae Fluorescence on King's B or Fluorescence on CSGM 1 Levan Gelatin hydrolysis Esculin hydrolysis Acid produced from: inositol sorbitol erythritol Utilization of: DL lactate D( ) tartrate L( ) tartrate or or or 1 Casamino - sucrose - gelatin medium (Lelliott & Stead, 1987). or 5

6 Isolation Process the two subsamples separately. Triturate in 500 ml (spring) or 1,000 ml (autumn) sterile distilled water with a Waring blender. Decant. Dilute the supernatant by four serial tenfold steps in 0.01 M PBS ph 7.2. Plate 0.05 ml drops of supernatant and its dilutions on YGPA (Lelliott & Stead, 1987) in triplicate. Incubate 3-4 days at C. Colonies of P.s. persicae are usually grey, slightly bluish at the margin, translucent, or mucoid, 2 mm in diameter, with undulate margin. Testing Test suspect colonies for nutritional and physiological characters (Table 1). 6

7 References Lelliott, R.A., Stead, D.E. (1987) Methods or the Diagnosis of Bacterial Diseases of Plants. Blackwell, Oxford (GB). OEPP/EPPO (1986) Data sheets on quarantine organisms. No Pseudomonas syringae pv. persicae. Bulletin OEPP/EPPO Bulletin 16, OEPP/EPPO (1990a) Specific quarantine requirements. EPPO Technical Documents no OEPP/EPPO (1990b) Quarantine procedures. No. 25. Clavibacter michiganensis ssp. sepedonicus - inspection and test methods. Bulletin OEPP/EPPO Bulletin 20, Enquiries May be addressed to: A. Luisetti & J. L. Gaignard, INRA, Station de Pathologie Végétale, Route de St Clément, Beaucouzé, Angers (France). 7