PHYTOSANITARY PROCEDURES

Transcription

1 EPPO Standards PHYTOSANITARY PROCEDURES PHOMA EXIGUA VAR. FOVEATA INSPECTION AND TEST METHODS PM 3/23(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. 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. 2

3 EUROPEAN AND MEDITERRANEAN PLANT PROTECTION ORGANIZATION ORGANISATION EUROPEENNE ET MEDITERRANEENNE POUR LA PROTECTION DES PLANTES PM 3/23(1) English Phytosanitary procedure PHOMA EXIGUA VAR. FOVEATA INSPECTION AND TEST METHODS Specific scope This standard describes the inspection and test methods for Phoma exigua var. foveata, to satisfy the requirements of EPPO Standard PM 2/78(1). Specific approval and amendment First approved in September Edited as EPPO Standard in Introduction Phoma exigua var. foveata is an A2 quarantine organism and details about its biology, distribution and economic importance can be found in EPPO Data Sheet no. 78. (Bulletin OEPP/EPPO Bulletin 12 (1)). According to the EPPO Specific Quarantine Requirement (SQR), importing countries may require countries exporting seed or ware potato tubers to inspect them as follows before issuing a Phytosanitary Certificate: seed potatoes should have been found free from the pathogen and ware potatoes should have been found practically free; in addition, countries require that seed potatoes entering nuclear stock schemes in disease-free areas should be tested by an EPPO-recommended method. Methods Under certain circumstances a single visual inspection of potato tubers is sufficient to detect the gangrene symptoms caused by P. exigua var. foveata and importing countries may require no more than this. However, tubers may carry a latent infection in the periderm which cannot be detected by visual symptoms alone. If tubers are bruised and then incubated at a low temperature (5 C), any latent infection should develop to give symptoms. Other fungal rots may also develop and it is, therefore, advisable to check the identity of the pathogen. Two methods are available: (1) isolation and identification in agar culture (relatively simple to perform but rather slow, requiring several days); (2) chromatography test on rotted tissue (a faster technique, but requiring special equipment and expertise). These methods may be used in conjunction with visual inspection or after the incubation procedure. See Appendix I for details of the methods and Fig.1 for a sequential scheme for their use. 3

4 Visual inspection for symptoms negative positive does importing country require testing? reject or yes no: accept bruise and incubate for symptoms symptoms present no symptoms: accept check identity of P. exigua var. foveata Fig.1. Inspection scheme (pre-export). present: reject absent: accept Appendix I Visual inspection Examine the surface of 100 tubers for depressed gangrene patches of irregular outline. If suspicious blemishes are seen, cut tubers and look for internal cavities lined with grey or dark brown to purple mycelium (see EPPO Data Sheet no. 78). If desired, check for Phoma exigua var. foveata as below. Low-temperature incubation of bruised tubers In order to detect latent infection of lots of potato tubers with Phoma exigua var. foveata, the following procedure is recommended: (1) take a sample of 100 tubers from each lot; (2) bruise the tubers (approx. 4 wounds per tuber) and store them at 5 C for 2 weeks 1 ; disinfect the bruising equipment between each sample with alcohol (30%); (3) after 4 weeks check the tubers for the presence of rotting tissue near the wounds (as above). If suspect symptoms are seen, check for Phoma exigua var. foveata as below. 1 French method proposes instead 8 weeks at 4-5 C 4

5 Identification of Phoma exigua var. foveata (1) Isolation and culture Isolations can be made from the rots which develop, and the fungus identified by its cultural characteristics, on 2% malt agar (MA) (EPPO Data Sheet no. 78). When the two varieties of Phoma exigua (foveata and exigua) are grown together in the same Petri dish, on MA, a violet line occurs in the agar where the colonies meet. This criterion can be used systematically to identify P. exigua var. foveata if fragments of potato tuber material for testing are laid on MA around a central inoculum of P. exigua var. exigua. (2) Chromatography P. exigua var. foveata induces the formation of anthraquinone derivatives (such as chrysophanol), which can be detected on silica gel chromatograms, as a proof of rotting by P. exigua var. foveata. (a) Place rotted tissue in a glass bottle and add chloroform. Close the bottle. (b) Remove the rotted tissue after 16 h. (c) Evaporate the chloroform. During this process, the temperature should not exceed 55 C. (d) Add a few drops of chloroform to redissolve the residue. (e) Spot the extract on a silica gel sheet (for example, DC-Alufolien Kieselgel 60), with a distance between the spots of 1.5 cm. Include a positive control. (f) Develop the silica gel chromatogram with a mixture of toluene and acetone (95:5 v/v) as solvent. (g) When the front of the solvent has nearly reached the top of the sheet, remove the silica gel sheet and dry it. (h) The various components can be seen under UV-light with a wave length of 366 nm. If P. exigua var. foveata is present in the rotted tissue, an orange-yellow spot is visible with an Rf value of References Jacob, U., Zott, A. (1980) Thin-layer chromatography as a method for identifying Phoma exigua var. foveata. Archiv für Phytopathologie und Pflanzenschutz Berlin 16, Langerfeld, E. (1974) [Identification of Phoma exigua var.foveata in rotted potato tubers]. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes (Braunschweig) 26, Mosch, W.H.M., Mool, J.C. (1975) A chemical method to identify tuber rot in potato caused by Phoma exigua var. foveata. Netherlands Journal of Plant Pathology 81, Enquiries For identification by symptoms: E. Langerfeld, BBA, Messeweg 11/12, 3300 Braunschweig (DE). For chromatography: W.H.M. Mosch, IPO, P.O. Box 9060, 6700 GW Wageningen (NL). 5