EPPO Standards PHYTOSANITARY PROCEDURES TOMATO RINGSPOT NEPOVIRUS IN FRUIT TREES AND GRAPEVINE INSPECTION AND TEST METHODS. PM 3/32(1) English

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1 EPPO Standards PHYTOSANITARY PROCEDURES TOMATO RINGSPOT NEPOVIRUS IN FRUIT TREES AND GRAPEVINE INSPECTION AND TEST METHODS PM 3/32(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 3/32(1) English Phytosanitary procedure TOMATO RINGSPOT NEPOVIRUS IN FRUIT TREES AND GRAPEVINE INSPECTION AND TEST METHODS Specific scope This standard describes the inspection and test methods for tomato ringspot nepovirus in fruit trees and grapevine, to satisfy the requirements of EPPO Standard PM 2/102(2). Specific approval and amendment First approved in September Edited as EPPO Standard in Introduction Tomato ringspot nepovirus (TomRSV) is an A2 quarantine organism and details about its biology, distribution and economic importance can be found in Data sheet no. 102 (OEPP/EPPO, 1982) and Stace-Smith (1984). It also affects pelargonium, Ribes and Rubus and separate Quarantine procedures exist for TomRSV on these hosts (OEPP/EPPO, 1990a, 1991). It should be noted that in the EPPO region TomRSV has not been found on any fruit crop but only on pelargonium. So for fruits it practically has an A1 status. According to the EPPO Specific quarantine requirements (SQR) (OEPP/EPPO, 1990b) for TomRSV, plants for planting (except seeds) of apple, cherry, peach and apricot have to come from a field inspected for TomRSV and found free from the virus. If they come from a country where TomRSV occurs, they have to be derived (not further than the second generation) from mother plants tested for TomRSV, and maintained under conditions designed to prevent reinfection. Methods are thus needed for visual inspection in the field and for testing mother plants. Currently, the SQR does not cover grapevine but similar requirements seem appropriate in this case. Methods Fruit trees and grapevine infected by TomRSV can show very striking symptoms, so visual inspection is important in practice. However, it will not be sufficient as a method for detecting TomRSV in mother plants, which have to be tested. As a nepovirus, TomRSV has classically been detected by sap inoculation to herbaceous indicators and this method is still recommended by ISHS (ISHS, 1983). It is a simple technique but requires 1-2 weeks for symptoms to appear. Indexing by grafting onto woody indicators will detect TomRSV, and ISHS recommends suitable indicators for detecting the virus in fruit trees. However, this method is extremely slow and would only be appropriate if such testing was in any case being done for other viruses. Nepoviruses are readily detected by serological techniques such as ELISA, which is in practice the most reliable and rapid method, requiring relatively simple equipment and only 4

5 short training. This method is equally suitable for fruit trees and grapevine (and indeed also Ribes and Rubus). Finally, maintenance under conditions designed to prevent reinfection implies absence of the nematode vector Xiphinema americanum sensu lato. This requires soil extraction, a simple task requiring little equipment, and nematode identification, a task for the specialist. See Appendix I for details of the methods. APPENDIX I Visual inspection Symptoms of TomRSV infection depend on the fruit-tree host: all Prunus spp. show stem pitting, associated with graft-union abnormalities; with the yellow bud mosaic strain, almond and peach show symptoms of yellow bud mosaic (pale green to pale yellow, oblong, featheredged blotches along the main vein or large lateral veins of the leaves; buds produce rosettes of small and often distorted leaves, with or without mottling, or are pale yellow and later die; fruits may be dwarfed or malformed). Apple shows union necrosis (necrosis of the graft union and symptoms on the tree similar to those following trunk girdling). Grapevine shows general decline, ringspot and mottling on the leaves, dwarfing and rosetting of the leaves, berry abortion, spongy phloem tissue with numerous necrotic pits. Mechanical transmission ISHS (1983) recommends to use Chenopodium quinoa or Cucumis sativus for nepoviruses generally. However, it only specifically recommends the latter for TomRSV. ISHS recommends 5 replicates, at 20 C for 20 days. For further details on the method of inoculation and on symptoms produced, refer to the Quarantine procedure no. 28 for tomato ringspot nepovirus on pelargonium (OEPP/EPPO, 1990a), which also mentions Chenopodium amaranticolor, Nicotiana tabacum, Petunia hybrida, Phaseolus vulgaris and Vigna unguiculata as herbaceous indicators for TomRSV. Note also that ISHS considers apricot a very poor inoculum source for this test. Woody indicators ISHS recommends the following woody indicators to detect TomRSV in almond, cherry, peach or plum: Prunus persica cvs Elberta or GF305, in the glasshouse, with 5 replicates, at 20 C, for 3 months; Prunus tomentosa IR 473/1 or IR 474/1, in the glasshouse, with 3 replicates, at 22 C, for 3 months. For field indexing, ISHS recommends that P. persica cvs Elberta or GF305 can be used to detect TomRSV in peach only (3 replicates, 4 years). ISHS does not recommend the use of woody indicators for apple or apricot, nor is it recommended for grapevine. ELISA TomRSV can be detected in cambial and inner bark and/or bud tissues of scions and rootstocks of all hosts by direct and indirect forms of ELISA. Knives or cork borers (15 mm 5

6 diameter) are used to remove pieces of bark, from scion and rootstock portions. Then, phloem and cambial tissue is scraped from the inner surface of the bark pieces and from the exposed wood. These tissue samples and/or excised buds are triturated with pestle and mortar or homogenized with a Polytron homogenizer in grinding buffer consisting of phosphate (0.01 M)-buffered saline, 0.05%, Tween 20 and 2% (w/v) polyvinyl pyrrolidone (PVP). When samples of Prunus and Vitis spp. are to be tested, it is essential to supplement the grinding buffer with 2% nicotine. Either the double-antibody sandwich ELISA (Clark & Adams, 1977) is used or, when serologically deviant strains of TomRSV are likely to occur (Stace-Smith, 1984), an indirect form of ELISA is suggested (Clark & Bar-Joseph, 1984). The optimal dilutions of the individual reagents used in ELISA should be determined prior to routine testing. Either form of ELISA is conducted in polystyrene microtitre plates using volumes of 200 µl per well for each reagent. Following incubation of each reagent, plates are thoroughly washed at least three times using either PBS-Tween or simply tap water. Plates should be wrapped in plastic bags and should not be stacked during the incubation periods. Direct ELISA (1) Plates are coated by filling the wells with TomRSV-specific immunoglobulins (IgG) or serum diluted (e.g. 1: 500 to 1: 2000) in 0.05 M sodium carbonate buffer, ph 9.6, or in PBS, ph 7.4, and by incubating the plates for about 1 h at 37 C. (2) Phloem, cambial or bud tissue is homogenized in grinding buffer at a ratio of 1:5 to 1:20. Extracts are placed in plates (two wells per sample) and incubated for 2-4 h at 37 C or overnight at 4 C. (3) TomRSV-specific IgG conjugated to alkaline phosphatase is appropriately diluted (e.g. 1: 500 to 1: 2000) in PBS-Tween-PVP containing 0.2%, (w/v) ovalbumin, is placed into the wells and is incubated for about 4 h at 30 C. (4) A freshly prepared solution of 0.1%, (w/v) p-nitrophenyl phosphate in diethanolamine buffer, ph 9.8, is filled into the wells and incubated at room temperature for 0.5 to 4 h. The intensity of the colour reaction is determined visually or by photometric measurement at 405 nm. Indirect ELISA Although various forms of indirect ELISA are known (Clark & Bar-Joseph, 1984), the following procedure is suggested: (1) as for direct ELISA: use TomRSV-specific IgG produced in a rabbit; (2) as for direct ELISA; (3) fill wells with chicken or mouse antivirus IgG (or monoclonal antibody) appropriately diluted in PBS and incubate plates for 2-4 h at 37 C; (4) fill wells with rabbit anti-chicken or anti-mouse IgG conjugated to alkaline phosphatase and diluted as above, and incubate plates at 30 C for 2-4 h; (5) as for direct ELISA under 4. Barrat et al. (1984) and Hoy & Mircetich (1984) are also useful references. Nematode testing Extract nematodes (Flegg, 1967) from 200 ml samples of soil taken from the cm layer. The soil is soaked in 300 ml water for 1 h and then transferred to a bucket with approximately 10 litres of water. The suspension is stirred and after 25 s the supernatant is poured through a bank of 3 sieves of 150-µm pore; this is repeated after a 15-s settling period. The soil debris 6

7 retained on the sieves is transferred to a Baermann funnel with a 90-µm pore sieve and left for 24 h. Identification is performed at x 400 magnification of freshly killed specimens mounted in TAF or FAA fixative (Southey, 1986), with reference to the details in Lamberti & Bleve- Zacheo (1979). References Barrat, J.G., Scorza, R. & Otto, B.E. (1984) Detection of tomato ringspot virus in peach orchards. Plant Disease 68, Clark, M.F. & Adams, A.N. (1977) Characteristics of the microplate method of enzymelinked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34, Clark, M.F. & Bar-Joseph, M. (1984) Enzyme immunosorbent assays in plant virology. In Methods in Virology (eds Maramorosch, K. & Koprowski) vol. 8, pp Academic Press, New York (US). Flegg, J.J.M. (1967) Extraction of Xiphinema and Longidorus species from soil by a modification of Cobb's decanting and sieving technique. Annals of Applied Biology 60, Hoy, J.W. & Mircetich, S.M. (1984) Prune brownline disease: susceptibility of prune rootstocks and tomato ringspot detection. Phytopathology 74, ISHS (1983) Detection of virus and virus-like diseases of fruit trees. Acta Horticulturae no. 130, Lamberti, F. & Bleve-Zacheo, T. (1979) Studies on Xiphinema americanum sensu lato with descriptions of fifteen new species Nematologica Mediterranea 7, OEPP/EPPO (1982) Data sheet on quarantine organisms no. 102: tomato ringspot virus. Bulletin OEPP/EPPO Bulletin 12 (1). OEPP/EPPO (1990a) Quarantine procedure no. 28: tomato ringspot nepovirus in pelargonium. Bulletin OEPP/EPPO Bulletin 20, OEPP/EPPO (1990b) Specific quarantine requirements. EPPO Technical Documents no OEPP/EPPO (1991) Quarantine procedure no. 31: Rubus viruses. Bulletin OEPP/EPPO Bulletin 21, Southey, J.F. (1986) Laboratory Methods for Work with Plant and Soil Nematodes. Reference Book 402. HMSO, London (GB). Stace-Smith, R. (1984) Tomato ringspot virus. CMI/AAB Descriptions of Plant Viruses no AAB, Wellesbourne (GB). Enquiries May be directed to: H. L. Weidemann, BBA, Messeweg 11/12, Braunschweig (DE). 7

EPPO Standards SCHEMES FOR THE PRODUCTION OF HEALTHY PLANTS FOR PLANTING NURSERY REQUIREMENTS. PM 4/7(2) English. oepp eppo

EPPO Standards SCHEMES FOR THE PRODUCTION OF HEALTHY PLANTS FOR PLANTING NURSERY REQUIREMENTS PM 4/7(2) English oepp eppo European and Mediterranean Plant Protection Organization 1, rue Le Nôtre, 75016