Beet necrotic yellow vein virus ( benyvirus )
2006; Wiley; Volume: 36; Issue: 3 Linguagem: Norueguês
10.1111/j.1365-2338.2006.01037.x
ISSN1365-2338
Tópico(s)Plant Disease Resistance and Genetics
ResumoEPPO BulletinVolume 36, Issue 3 p. 429-440 Free Access Beet necrotic yellow vein virus (benyvirus) First published: 15 December 2006 https://doi.org/10.1111/j.1365-2338.2006.01037.xCitations: 7 European and Mediterranean Plant Protection Organization Organisation Européenne et Méditerranéenne pour la Protection des Plantes AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat PM 7/30 (2) Specific scope This standard describes a diagnostic protocol for Beet necrotic yellow vein virus (benyvirus). Specific approval and amendment This Standard was developed under the EU DIAGPRO Project (SMT 4-CT98-2252) through a partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2003-09. Revision approved in 2006-09. Introduction Rhizomania disease of sugar beet was first reported in Italy (Canova, 1959) and has since been reported in more than 25 countries. The disease causes economic loss to sugar beet (Beta vulgaris var. saccharifera) by reducing yield. Rhizomania is caused by Beet necrotic yellow vein virus (BNYVV), which is transmitted by the soil protozoan, Polymyxa betae (family Plasmodiophoraceae). The virus can survive in P. betae cystosori for more than 15 years. The symptoms of rhizomania, also known as ‘root madness’, include root bearding, stunting, chlorosis of leaves, yellow veining and necrosis of leaf veins. The virus is spread by movement of soil, primarily on machinery, sugar beet roots, stecklings, other root crops, such as potato, and in composts and soil. Water is important in the spread of the fungal vector; drainage water, ditches and irrigation with water from infected crops can favour the disease. In addition to high water content, high temperature can stimulate development of P. betae. Control measures include; cleaning soil from agricultural machinery after harvesting beets, avoiding the re-introduction to the farm of factory by-products, careful disposal of waste from seed processing and importing seed potatoes from rhizomania-free areas. Disease-tolerant sugar beet cultivars are widely used in affected regions. BNYVV is regulated within the European Union in protected zones (EU, 2000), currently Brittany (FR), Finland, Ireland, the Azores (PT), and Northern Ireland (GB). Identity Name: Beet necrotic yellow vein virus Acronym: BNYVV Taxonomic position: Viruses, Benyvirus EPPO computer code: BNYVV0 Phytosanitary categorization: EPPO A2 list no. 160; EU Annex designation I/B. Detection The disease affects all subspecies of Beta vulgaris, including sugar beet (Beta vulgaris subsp. maritime), fodder beet (Beta vulgaris subsp. vulgaris), red beet (Beta vulgaris subsp. cicla), mangolds (Beta vulgaris subsp. vulgaris), sea kale (Beta vulgaris subsp. vulgaris), Swiss chard (Beta vulgaris subsp. cicla), and also spinach (Spinacea oleracea). Symptoms of rhizomania Leaves Symptoms can often be seen very clearly from aerial photographs, as well on the ground, and consist of distinct yellow patches (Web Fig. 1). On inspection the following may be noted: • translucent, pale lettuce-green to lemon-yellow foliage • yellow veining following the midrib of the leaf (Web Fig. 2) • upright foliage with elongated petioles and narrowed leaf laminae (Web Fig. 3) • plants stunted and/or wilted (possibly without leaf symptoms). Roots • dark brown bearded roots (this may be slight and/or a single lateral root with bearding near the tip) (Web Fig. 4) • root constriction • pale yellow to dark brown vascular discoloration in transverse section • nodules (small tumorous growths along the taproot). The above symptoms are rarely found together in a single plant. Rhizomania-tolerant cultivars may only show typical symptoms at high virus infection levels. Identification Sampling Samples should be taken from identified yellow patches in beet crops (identified by aerial photography, etc.). A fork or spade should be used to dig up the roots (especially in dry hard baked soils). Care should be taken to lift the beet whole as the root tip and laterals with ‘rat tails’ can easily break off and be left behind in the ground. Each sample should consist of the lower third of the taproot of 5 or 6 plants showing symptoms. Each sample should be separately identified and placed in a labelled plastic bag2. Sample preparation For laboratory-based tests, the sugar beet samples should be thoroughly washed in cold water to remove loose soil from the roots and dried on absorbent paper. Samples should then be placed in labelled plastic bags for processing. Samples for soil-bait testing Soil samples from the field can be tested for rhizomania by growing susceptible beet in the soil (bait testing) in a glasshouse or in growing chambers. A total of 2.5 kg of field soil should be taken by walking in a W shape across each of the sampling areas. Each sample should be separately identified and placed in a labelled plastic bag. Sample preparation for lateral flow test kit See Appendix 6. Screening tests ELISA is the best and most cost-effective general screen (Appendix 1). The lateral flow test is appropriate if symptoms are seen in accordance with the pathogen key card provided with the kit (Appendix 6). Isolation Mechanical inoculation of virus to test plants Beta vulgaris (sugar beet): inoculated leaves usually develop chlorotic lesions after 6–8 days. Occasional bright yellow chlorotic lesions can be seen in leaf veins. Infection is rarely systemic. Chenopodium quinoa, Chenopodium amaranticolor, Tetragonia expansa: chlorotic or necrotic lesions develop after 5–7 days. Nicotiana tabacum, Lycopersicon esculentum and Phaseolus vulgaris are non-susceptible and can help to distinguish BNYVV from other rod–shaped viruses, e.g. Tobacco rattle virus, Tobacco mosaic virus, Pea early browning virus. For full description of host range, susceptible and insusceptible plants, see Tamada & Baba (1973). For new hosts, see Horváth (1994). At least two indicator plants and two non-susceptible hosts should be used. Sugar beet lateral roots are washed and ground in a mortar with a small spatula-full of celite and enough distilled water to make a thin paste. The indicator plant, e.g. Chenopodium quinoa, is inoculated at the stage of six or more fully expanded leaves by gently covering the leaves in root/celite suspension, using a finger covered in a disposable glove. After 5 min, plants are rinsed in tap water to remove debris and left covered overnight to exclude light. On the following day, the cover is removed and the plants are grown for 6–10 days at 18–20°C, with watering daily as required. Test plants infected with BNYVV produce characteristic chlorotic lesions (Web Fig. 5). There should be no symptoms on non-susceptible hosts. As mechanical inoculation is not always successful, false negative results may occur. Polymyxa betae The presence in roots of Polymyxa beta, the protozoan vector of BNYVV, can aid presumptive diagnosis of BNYVV, but its absence does not indicate that the roots are not infected. Suspect rootlets are washed in cold water and dried on absorbent paper. A selected sample, mounted gently flattened in water on a simple microscope slide with cover slip, is examined at × 10 under a light microscope for characteristic cystosori in the root cells. A magnification of × 40 may be used for closer examination (Web Fig. 6). Other stages in the life cycle of P. betae may also be seen, such as sporangia and plasmodia. Confirmation test(s) ELISA test A sample of 0.5–1 g of washed lateral or tap roots is processed following the procedure of Appendix 1. The ELISA value of the sample should be more than 2–3 times greater than the negative control. See specific instructions enclosed with the antisera. RT-PCR test A sample of washed lateral or tap roots (which may have been stored frozen) is processed following the procedure of Appendix 23. Immunocapture PCR A sample of washed lateral or tap roots (which may have been stored frozen) is processed following the procedure of Appendix 3. TaqMan® RT-PCR A sample of washed lateral or tap roots (which may have been stored frozen or freeze dried) is processed following the procedure of Appendix 4. Electron microscopy tests A washed sample of lateral or tap roots is examined following EM, IEM or gold labelling procedures (Appendix 5). Soil tests Soil from fields suspected of being infested by rhizomania can be tested by baiting with seedling sugar beet (Appendix 7), which are then tested by ELISA (Appendix 1). The optimum time for bait testing is 6 weeks for the English test (Henry et al., 1992; Tuitert & Bochen, 1993) or 3–4 weeks for French test (LNPV Fleury method). If a more rapid method is needed, RT-PCR (Appendix 2) or TaqMan® RT-PCR (Appendix 4) can be done after 3 weeks using the English test (Henry et al., 1995). Beet soil-borne virus (pomovirus) is a different rod-shaped beet-infecting virus, also transmitted by Polymyxa betae. Beet soil-borne mosaic virus (benyvirus) is also closely related but serologically distinct. The ring-tested diagnostic tests recommended in this protocol are specific for BNYVV, and will not detect any other viruses. The procedures for detection and identification described in this protocol, and the decision scheme in 1, 2, should have been followed. Positive identification of BNYVV (in the original plant or in an indicator plant) should be made using ELISA and/or PCR methods (see Appendices). A confirmation of the presence of the virus may be required, using a method distinct from that originally used (e.g. if a serological method was used first, a molecular method is used for confirmation). See 1, 2 and Appendices. Any first finding should be confirmed by other tests. Figure 1Open in figure viewerPowerPoint Flow-diagram for detection and identification of Beet necrotic yellow vein benyvirus in sugar beet.(1) The most appropriate and rapid screening test would be ELISA (Appendix 1), which should be repeated if the result is unclear. Lateral flow devices (Appendix 6) can be used in the field and for small numbers of lab samples.(2) ELISA testing is normally sufficient for diagnosis but the following alternative tests can be performed as required: PCR (Appendix 2) and TaqMan® PCR (Appendix 4) Additional tests include mechanical inoculation of indicator plants and electron microscopy (Appendix 5). Figure 2Open in figure viewerPowerPoint Flow diagram for detection and identification of Beet necrotic yellow vein benyvirus in soil samples.(1) The standard size of soil sample is 0.5–2.5 kg. However, the procedure can be used for smaller samples.(2) Soil bait tests: (Appendix 7).(3) ELISA testing (Appendix 1) is the normal way of screening many samples for BNYVV and can be used as the sole screening test provided the antisera is of high specificity. The test can detect virus concentrations down to 10−3) and is cheap and fast. If an additional confirmatory test is required it should be preferably based on different biological principles.PCR (Appendix 2) being approximately 800 times more sensitive than the standard TAS ELISA, the detection limit is 10−6 for root extracts. TaqMan® PCR (Appendix 4) is approximately 10 000 times more sensitive than conventional PCR. Skilled operators are needed however, as extreme care is required during preparation to prevent contamination and false positives. Other tests used for BNYVV mainly for research purposes Protein profiling Whole cell extracts can be denatured and separated by SDS–PAGE. The coat protein of BNYVV is Mr 21 kDa. Western blotting can also be carried out (Torrance et al., 1988). Quantification of Polymyxa betae in rhizomania soil samples It is possible to estimate the number of infectious units of viruliferous P. betae in an infested soil by a series of soil dilutions, by the most probable number (MPN method) and bait testing (Ciafardini, 1991). Tissue print-immunoblotting of roots It is possible to show, by immunoblotting of a longitudinal section of a sugar beet root, where the virus is concentrated in a given plant. This has been used more as a research and development technique than in general diagnosis (Kaufmann et al., 1992). A, B & P pathotypes Different pathotypes of BNYVV, designated A and B, were first identified by Koenig et al. (1994). They have been classified into groups based on a number of molecular characteristics. The sequence differences between A & B types are subtle, a high percentage of the sequence being the same. Sequencing is now reliably used to detect strain differences. Koenig & Lennefors (2000) have used sequencing to provide a more reliable method of differentiating the European A, B and P types of BNYVV than using RFLP and SSCP analyses (Koenig et al., 1995), the genomes of the BNYVV isolates having been found to be very stable. The common isolates of BNYVV contain RNAs 1–4. The A type is widespread in most European countries (Kruse et al., 1994), the USA, China and Japan. The B type is more restricted, generally to Germany, France and the UK. Mixtures of these strains can occur. A and B isolates can be also differentiated using PCR (Ratti et al., 2005). BNYVV isolates containing additional genomic RNA (RNA 5) are found in Japan and China (Tamada et al., 1989; Miyanishi et al., 1999). Such isolates, which have been described as P pathotype have also been reported in Europe, near Pithiviers (FR) (Koenig et al., 1997), and near Norwich (GB) (Harju et al., 2002). Similar BNYVV strains with RNA 5 have been found in Kazakhstan (Koenig & Lennefors, 2000). There is some evidence to suggest that isolates containing RNA 5 are more virulent than those of the other pathotypes (Tamada et al., 1996), and that sugar beet cultivars with different degrees of resistance vary in their response to various pathotypes of BNYVV. B types appeared to be less damaging than A or P types. P types appear to give a higher virus content than A or B types (Heijbroek et al., 1999). A Japanese PCR test for RNA 5 was published by Kiguchi et al. (1996) and TaqMan® PCR has been used for detection of RNA 5 in the UK (Harju et al., 2002) and (Harju et al., 2005) (see Appendix 4). Future diagnostic developments Recently published papers described the use of a new type of antibody production from BNYVV-specific single-chain antibody variable fragments (scFvs) (Griep et al., 1999; Uhde et al., 2000). The latter authors achieved good results when testing stored sugar-beet roots with antibodies produced from scFvs. The specificity of these new antibodies in ELISA may in future have potential for their use as reagents in sensitive diagnostic assays for testing. A recombinant antibody ELISA test has been developed for Polymyxa betae (Kingsnorth et al. 2003). A close correlation was found between the numbers of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. In the United States remote detection using hyper-spectral leaf reflectance and multi spectral canopy reflectance to study rhizomania has been tested (Steddom et al., 2003). They found the total leaf nitrogen was significantly lower in symptomatic beets than in healthy beets. Chlorophyll and carotenoid levels were also reduced in symptomatic beets. Classification was best in August gradually decreasing in accuracy until harvest. These results indicate that remote sensing technologies can be used to facilitate detection of rhizomania. Reporting and documentation Guidelines on reporting and documentation are given in EPPO Standard PM 7/77 (1) Documentation and reporting on a diagnosis. Further information Further information on Beet necrotic yellow vein virus can be obtained from the Pest and Disease Identification Team (PLHB) and Immunological and Molecular Methods Team (PLHC), Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom, e-mail: v.harju@csl.gov.uk. Information can also be obtained from other institutes including the Institute for Plant Virology, Microbiology and Biosafety, Messeweg 11/12, D-38104, Braunschweig, Germany, e-mail: Biosearch@bba.de and Institut International de Recherches Betteravièrs 195, Avenue de Tervuren B-1150 Bruxelles, Belgium, e-mail: mail@iirb.org. Footnotes 1 The figures in this standard marked ‘Web Fig.’ are published on the EPPO Website http://www.eppo.org. 2 In Poland (Jezewska & Piszczek, 2001) sugar beet leaves are routinely sampled for ELISA testing for BNYVV. The suitability of this method has not generally been assessed in the EPPO region and may depend on factors such as virus concentration and local environmental conditions. 3 A multiplex PCR that can detect BNYVV, BSBV, Beet virus Q and Polymyxa betae has been developed (Meunier et al., 2003). This has been tested by the authors (Université Catholique de Louvain-la Neuve) (UCL) in comparison with the DIAGPRO PCR protocol. 4 L. Potyondi (Beta Kutato Kft, Sopronhorpacs, HU); C. Bragarde (UCL Unit of Phytopathology, Louvain-la Neuve, BE); S. L. Neilsen (Danish Institute of Agricultural Sciences, Flakkebjerg, DK); M. Jezewska (Institute of Plant Protection, Poznan, PL); C. Ratti (DISTA, University of Bologna, IT); C. H. B. Olsson (SLU, Plant Pathology and Biocontrol Unit, Göteborg, SE); G. W. van den Bovenkamp (Laboratory Methods & Diagnostics, NAK, Emmeloord, NL); S. Steyer (Research Station of the Ministry of Agriculture, Gembloux, BE); D. Vilsan (Central Laboratory for Phytosanitary Quarantine, Bucharest, RO); E. Pocsai (Fejér Megyei Növény-és Talajvédelmi Szolgálat, Virológiai Laboratórium, Velence, HU). Acknowledgements This protocol was originally drafted by: V. Harju, Central Science Laboratory, York (GB). The French soil bait test (LNPV Fleury method) was drafted by F. Vey, LNPV Fleury les Aubrais. The main diagnostic tests recommended in this protocol were ring-tested in different European laboratories4. Individual samples were tested in all cases. Bulked samples sometimes used in large surveys were not tested. References Canova A (1959) [On the pathology of sugar beet]. Informatore Fitopatologico 9, 390– 396 (in Italian). Chang S, Puryear J & Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Molecular Bioliology Reporter 11, 113– 116. Ciafardini G (1991) Evaluation of Polymyxa betae Keskin contaminated by beet necrotic yellow vein virus in soil. Applied and Environmental Microbiology 57, 1817– 1821. EU (2000) Council Directive 2000/29 of 8 May 2000 on protective measures against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community. Official Journal of the European Communities L169, 1– 112. Grassi G, Cerato C, Benso P & Borgatti S (1988) Monoclonal and conventional antibodies for the detection of beet necrotic yellow vein virus (BNYVV) in sugar beet. Phytopathologia Mediterranea 27, 125– 132. Griep RA, Twisk C & Van Schots A (1999) Selection of beet necrotic yellow vein virus specific single-chain Fv antibodies from a semi-synthetic combinatorial antibody library. European Journal of Plant Pathology 105, 147– 156. Harju VA, Mumford RA, Blockley A, Boonham N, Clover GRG, Weekes R & Henry CM (2002) The identification of isolates of Beet necrotic yellow vein virus from the United Kingdom which contain RNA 5. Plant Pathology 51, 811. Harju VA, Skelton A, Clover GRG, Ratti C, Boonham N, Henry CM & Mumford RA (2005) The use of real time RT- PCR (TaqMan®) and post ELISA virus release for the detection of Beet necrotic yellow vein virus types containing RNA 5 and its comparison with conventional PCR. Journal of Virological Methods 123, 73– 80. Harness AM, Johnson JC, Kulemeka BP, Sutula CL & Badla MD (2003) Immunocapture RT-PCR as a confirmation tool for ELISA. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 110, 101. (Abstract) [ http://www.agdia.com/references/immunocaptureposter.pdf]. Heijbroek W, Musters PMS & Schoone AHL (1999) Variation in pathenogenicity and multiplication of Beet necrotic yellow vein virus (BNYVV) in relation to the resistance of sugar beet cultivars. European Journal of Plant Pathology 105, 397– 405. Henry CM, Barker I, Morris J & Hugo SA (1995) Detection of beet necrotic yellow vein virus using reverse transcription and polymerase chain reaction. Journal of Virological Methods 54, 15– 28. Henry CM, Harju V, Brewer G & Barker I (1992) Methods for the detection of Rhizomania in soil. Aspects of Applied Biology 32, 129– 133. Horváth J (1994) Beet necrotic yellow vein furovirus 1 new host.Acta Phytopathologica et Entomologica Hungarica 29, 109– 118. Hughes DW & Galau G (1988) Preparation of RNA from cotton leaves and pollen. Plant Molecular Biology Reporter 6, 253– 257. Jezewska M & Piszczek J (2001) Surprisingly high frequency of detection of Beet necrotic yellow vein virus in sugar beet leaves by ELISA. Phytopathologic Polonica 21, 165– 170. Kaufmann A, Koenig R & Lesemann DE (1992) Tissue print-immunoblotting reveals an uneven distribution of beet necrotic yellow vein and beet soil-borne viruses in sugar beets. Archives of Virology 126, 329– 335. Kiguchi T, Saito M & Tamada T (1996) Nucleotide sequence analysis of RNA-5 of five isolates of beet necrotic yellow vein virus and the identity of a deletion mutant. Journal of General Virology 77, 575– 580. Kingsnorth CS, Asher MJC, Keane GJP, Chwarszczynska DM, Luterbacher MC & Mutasa-Göttgens ES (2003) Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening. Plant Pathology 52, 673– 680. Koenig R, Haeberle AM & Commandeur U (1997) Detection and characterization of a distinct type of beet necrotic yellow vein virus RNA 5 in a sugar beet growing area in Europe. Archives of Virology 142, 1499– 1504. Koenig R, Kruse M, Hoffmann H, Heijbroek W, Buttner G, Lindsten K & Paul (1994) The existence of possible pathotypes of Beet necrotic yellow vein virus (BNYVV) and their possible impact on partially resistant sugar beet varieties. In Proceedings of the 57th IIRB Winter Congress. IIRB, Bruxelles (BE). Koenig R & Lennefors BL (2000) Molecular analyses of European A, B and P type sources of beet necrotic yellow vein virus and detection of the rare P type in Kazakhstan. Archives of Virology 145, 1561– 1570. Koenig R, Lesemann DE & Burgermeister W (1984) Beet necrotic yellow vein virus: purification, preparation of antisera and detection by means of ELISA, immunosorbent electron microscopy and electro-blot immunoassay. Phytopathologische Zeitschrift 111, 244– 252. Koenig R, Luddecke P & Haeberle AM (1995) Detection of beet necrotic yellow vein virus strain, varieties and mixed infections by examining single-strand confirmation polymorphism of immunocapture RT-PCR products. Journal of General Virology 76, 2051– 2055. Kruse M, Koenig R, Hoffmann A, Kaufmann A, Commandeur U, Solovyev AG, Savenkov I & Burgmeister W (1994) Restriction fragment length polymorphism analysis of reverse transcription-PCR products reveals the existence of two major strain groups of beet necrotic yellow vein virus. Journal of General Virology 75, 1835– 1842. Meunier A, Schmit J-F, Stas A, Kutluk N & Bragard C (2003) Multiplex RTPCR for the simultaneous detection of Beet necrotic yellow vein virus, Beet soil borne virus, Beet virus Q and their vector Polymyxa betae Keskin on sugar beet. Applied and Environmental Microbiology 69, 2356– 2360. Miyanishi M, Kusume T & Tamada T (1999) Evidence for three groups of sequence variants of Beet necrotic yellow vein virus RNA 5. Archives of Virology 144, 879– 892. Morris J, Clover GRG, Harju VA, Hugo SA & Henry CM (2001) Development of a highly sensitive nested RT-PCR method for Beet necrotic yellow vein virus detection. Journal of Virological Methods 95, 163– 169. Mumford RA, Walsh K, Barker I & Boonham N (2000) Detection of Potato mop-top and Tobacco rattle virus using a multiplex real-time fluorescent reverse transcription polymerase chain reaction assay. Phytopathology 90, 448– 453. Ratti C, Clover GRG, Rubies-Autonell C, Harju VA & Henry CM (2005) A Multiplex RT- PCR assay capable of distinguishing beet necrotic yellow vein virus types A and B. Journal of Virological Methods 124, 41– 47. Spiegel S & Martin RR (1993) Improved detection of potato leafroll virus in dormant potato tubers and microtubers by the polymerase chain reaction and ELISA. Annals of Applied Biology 122, 493– 500. Steddom K, Heidel G, Jones D & Rush CM (2003) Remote detection of rhizomania in sugar beets. Phytopathology 93, 720– 726. Tamada T & Baba T (1973) Beet necrotic yellow vein virus from rhizomania-affected sugar beet in Japan. Annals of Phytopathological Society of Japan 39, 325– 331. Tamada T, Kusume T, Uchino H, Kiguchi T & Saito M (1996) Evidence that Beet necrotic yellow vein virus RNA 5 is involved in symptom development of sugar beet roots. In Proceedings of the Third Symposium of the International Working Group on Plant Viruses with Fungal Vectors (Ed. JL Sherwood & CM Rush), pp. 49– 52. American Society of Sugar beet Technologists, Denver (US). Tamada T, Shirako Y, Abe H, Saito M & Kiguchi T (1989) Production and pathogenicity of isolates of beet necrotic yellow vein virus with different numbers of RNA components. Journal of General Virology 70, 399– 409. Torrance L, Pead MT & Buxton G (1988) Production and some characteristics of monoclonal antibodies against beet necrotic yellow vein virus. Annals of Applied Biology 113, 519– 530. Tuitert G (1990) Assessment of the inoculum potential of Polymyxa betae and beet necrotic yellow vein virus (BNYVV) in soil using the most probable number method. Netherlands Journal of Plant Pathology 96, 331– 341. Tuitert G & Bochen GJ (1993) Recovery of resting spores of Polymyxa betae from soil and the influence of duration of the bioassay on the detection level of beet necrotic yellow vein virus in soil. Netherlands Journal of Plant Pathology 99 (Suppl. 3), 219– 230. Uhde K, Kerschbaumer RJ, Koenig R, Hirschl S, Lemaire O, Boonham N, Roake W & Himmler G (2000) Improved detection of Beet necrotic yellow vein virus in a DAS ELISA by means of antibody single chain fragments (scFv) which were selected to protease-stable epitopes from phage display libraries. Archives of Virology 145, 179– 185. Appendix 1. ELISA test Materials recommended for the ELISA test Homogenization buffer This buffer is used for tissue maceration: polyvinylpyrrolidone (PVP) 20.0 g; phosphate-buffered saline (PBST) 1 L (see below). Add 500 mL of PBST to 20 g of PVP. Dissolve by machine stirring rapidly. Make up to 1 L, stirring thoroughly. This buffer should be made up freshly as required. Carbonate coating buffer pH 9.6: Na2CO3 1.59 g; NaHCO3 2.93 g; distilled water l L. Dissolve the ingredients and check pH. Store at 5°C. 10 × Phosphate Buffered Saline (PBS), 1× = pH 7.2: NaCl 80 g; KH2PO4 2 g; Na2HPO4·12H2O 29 g; KCl 2 g; distilled water 1 L. Dissolve all ingredients and check pH. Dilute to 1 × for use. Phosphate-Buffered Saline-Tween (PBST) 10 × PBS 100 mL; 10% Tween-20 5 mL; distilled water 895 mL. Stir ingredients briefly. Antibody buffer (prepare fresh) PBST 100 mL; 5% dried milk powder 5 g or 0.2%; bovine serum albumin 0.2 g. Stir ingredients briefly. Alkaline phosphatase substrate solution pH 9.8: Diethanolamine 97 mL; distilled water 800 mL. Mix and adjust to pH 9.8 with concentrated HCl. Make up to 1 L with distilled water. Add 0.203 g of MgCl2 and store at 5°C. Dissolve two phosphatase substrate 5 mg tablets (Sigma) per 15 mL of substrate solution. Antibodies Suitable antibodies (including Anti-species AP-conjugated antibody) for use in the ELISA test for BNYVV are those of Koenig et al. (1984), Grassi et al. (1988) and Torrance et al. (1988). Commercial detection kits • Bio-Rad, Phyto-Diagnostics, 3 bd. Raymond Poincaré, 92430 Marnes-la-Coquette (FR) [previously Sanofi Cie (Libourne, FR] . Tel. +33 (0)1 47 60 00, Fax +33 (0)1 47 41 91 33, http://www.bio-rad.com; • Bioreba AG (Switzerland), Chr. Merian-Ring 7, CH 4153 Reinach BL1 (CH) http://www.bior
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