EPPO BulletinVolume 34, Issue 2 p. 229-237 Diagnostic protocols for regulated pests†Free Access Beet necrotic yellow vein benyvirus First published: 10 September 2004 https://doi.org/10.1111/j.1365-2338.2004.00724.x European and Mediterranean Plant Protection Organization PM 7/30(1) 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 Specific scope This standard describes a diagnostic protocol for Beet necrotic yellow vein benyvirus. Specific approval and amendment This Standard was developed under the EU DIAGPRO Project (SMT 4-CT98-2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2003-09. Introduction Rhizomania disease of sugarbeet was first reported in Italy (Canova, 1959) and has since been reported in more than 25 countries. The disease causes economic loss to sugarbeet (Beta vulgaris var. saccharifera) by reducing yield. Rhizomania is caused by Beet necrotic yellow vein benyvirus (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 disease is spread by movement of soil, primarily on machinery, sugarbeet 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 sugarbeet cultivars are widely used in affected regions. BNYVV is regulated within the European Union in protected zones (EU, 2000), currently Denmark, Brittany (FR), Finland, Ireland, Azores (PT), Sweden and Northern Ireland (GB). Identity Name: Beet necrotic yellow vein benyvirusvirus Acronym: BNYVV Taxonomic position: Viruses, Benyvirus Bayer 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 sugarbeet, fodder beet, red beet, mangolds, sea kale, Swiss chard, and also Spinacea oleracea (spinach). Symptoms of rhizomania Above-ground parts 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 the glasshouse. A total of 0.5–1 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 5. 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 5). Isolation Mechanical inoculation of virus to host plants Beta vulgaris (sugarbeet): inoculated leaves 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 tobravirus, Tobacco mosaic tobravirus, Pea early browning tobravirus. 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. Sugarbeet 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. Plants infected with BNYVV should produce characteristic chlorotic lesions in susceptible host plants (Web Fig. 5). There should be no symptoms on non-susceptible hosts. 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 coverslip, 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 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. 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. Electron microscopy tests A washed sample of lateral or tap roots is examined following EM, IEM or gold labelling procedures (Appendix 4). Soil tests Soil from fields suspected of being infested by rhizomania can be tested by baiting with seedling sugarbeet (Appendix 6), which is then tested by ELISA (Appendix 1). The optimum time for bait testing is 6 weeks (Henry et al., 1992; Tuitert & Bochen, 1993). If a more rapid method is needed, RT-PCR (Appendix 2) can be done after 3 weeks (Henry et al., 1995). 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 sugarbeet 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), 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 (C.Ratti, pers.com.). BNYVV isolates containing additional genomic RNA (RNA 5) are found in Japan and China (Tamada et al., 1989; Miyanishi et al., 1999). Such isolates have also been reported in Europe, near Pithiviers (FR), the P pathotype (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 pathogenic than those of the other pathotypes (Tamada et al., 1996), and that sugarbeet 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). 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 sugarbeet 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. Possible confusion with similar species Beet soil-borne pomovirus is a different rod-shaped beet-infecting virus, also transmitted by Polymyxa betae. Beet soil-borne mosaic 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. Requirements for a positive diagnosis The procedures for detection and identification described in this protocol, and the decision scheme in 11, 12, 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 11, 12 and Appendices. Any first find should be confirmed by other tests. Figure 11Open in figure viewerPowerPoint Decision scheme for detection and identification of Beet necrotic yellow-vein benyvirus in sugarbeet. Figure 12Open in figure viewerPowerPoint Decision scheme for detection and identification of Beet necrotic yellow-vein benyvirus in soil samples. Report on the diagnosis The report on the execution of the protocol should include: • results obtained by the recommended procedures • information and documentation on the origin of the infected material • a description of the disease symptoms • an indication of the magnitude of the infection • comments as appropriate on the certainty or uncertainty of the identification. Further information Further information on this organism can be obtained from: Virology Diagnostic Team, Plant Health 2 and Research Team, Plant Health 6, Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom. (GB). E-mail: v.harju@csl.gov.uk Footnotes 1 The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. 2 In Poland (Jeżewska & Piszczek, 2001), sugarbeet 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 new multiplex PCR that can detect BNYVV, BSBV, Beet virus Q and Polymyxa betae has just been published (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, Poznań, 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 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. Appendices Appendix 1. ELISA test Materials used for the ELISA test Homogenization buffer: This buffer is used for tissue maceration: polyvinylpyrrolidone (PVP) 20.0 g; phosphate-buffered saline (PBST) L (see Appendix 2). 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. 10x Phosphate Buffered Saline (PBS), (1x = 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; 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 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 95 62, Fax 33(0)1 47 95 64 http://www.bio-rad.com; • Bioreba AG (Switzerland), Chr. Merian-Ring 7, CH 4153 Reinach BL1 (CH) http://www.bioreba.ch; • Adgen Ltd, Nellies Gate, Auchincruive, Ayr KA6 5HW, Scotland (GB). Tel ++44(0)1292525275, Fax ++44(0)1292525477 http://www.adgen.co.uk; ELISA test – Triple Antibody Sandwich (TAS) This test, based on Henry et al. (1992), has been ring–tested. The use of Nunc-Maxisorp microtitre plates or those of a similar quality is recommended. Include negative sugar beet controls, homogenizer buffer control and positive control (either known positive sugarbeet material or positive leaf material from BNYVV inoculated onto Chenopodium quinoa). Add BNYVV antibody at the recommended dilution to the coating buffer. Pipette the solution onto the microtitre plates, 100 µL per well. Incubate for 3 h at 33 °C. Flick out the contents of the wells, and wash wells three times with PBS-Tween with 3-min soaks between washes. Blot dry on absorbent paper. Add sample homogenate at 100 µL per well, using two wells per test sample. Incubate at 4 °C overnight. Flick out the contents of the wells as before but wash 4 times. Add specific BNYVV monoclonal antibody diluted as appropriate in dried milk buffer at 100 µL per well. Incubate for 2 h at 33 °C. Flick out contents of wells and wash 4 times. Prepare anti-species alkaline phosphatase conjugate at appropriate dilution in dried milk buffer. Add 100 µL to each well. Incubate for 2 h at 33 °C. Flick out the contents of the wells and wash 4 times. Prepare alkaline phosphatase substrate solution. Add 100 µL to each well. Incubate at ambient temperature for 1 h. Read absorbance at 405 nm. Some commercial kits for BNYVV detection use the double antibody sandwich (DAS) ELISA. If this method is being used add antibody conjugate instead of monoclonal, and omit the following step of adding conjugate. The ELISA test is negative if the absorbance of the sample is less than 3 times the absorbance of the healthy control, or positive if equal or greater than 3 times that value (Web Fig. 7). Appendix 2. PCR test Materials for the PCR test Oligonucleotide primer sequence: Downstream primer: (BNYVV 017 (R) 5′-ACT-CGG-CAT-ACT-ATT-CAC-T(T) 3′ Upstream primer: (BNYVV 016 (F) 5′-CGA-TTG-GTA-TGA-GTG-ATT-T (A) 3′ Expected size of amplicon 500 bp. Nested PCR primers: Downstream primer: (Rhzn 17 (R) 5′-GAC-GAA-AGA-GCA-GCC-ATA-GC) 3′ Upstream primer: (Rhzn 15 (F) 5′-ATA-GAG-CTG–TTA-GAG-TCA-CC) 3′ Expected size of amplicon 326 bp. RNA extraction method: RNA is extracted from sugarbeet roots by a method adapted from Hughes & Galau (1988) with modifications from Spiegel & Martin (1993). Extraction buffer: Tris base-HCl 200 mM pH 8.5; 1.5% lithium dodecyl sulphate; EDTA 10 mM; NaCl 300 mM; 1% Sodium deoxycholate. Autoclave (121 °C at 1.2 bar for 30 min) and store. Prepare fresh extraction buffer in required amount for the number of samples extracted (3 mL per sample) by adding 1% Ipegal CA-630 (Sigma-Aldrich); DTT (DL-Dithiothreitol) 10 mM; Thiourea (CH4N2S) 5 mM. The resulting buffer will keep for 2 weeks at room temperature. Potassium acetate: potassium acetate 6 M; molecular grade water. Adjust pH to 6.5 using acetic acid and make up to 1 L. Autoclave prior to use. Lithium chloride: LiCl 4 M; molecular grade water. Make up to 1 L. TE Buffer: Tris-HCl 10 mM; EDTA 1 mM; molecular grade water. Adjust pH to 8.0 and make up to 1 L. Sodium chloride: NaCl 5 M; molecular grade water. Make up to 1 L. Autoclave prior to use. Ethidium bromide: TBE 600 mL; ethidium bromide 60 µL. Keep in covered plastic box covered to prevent photodegradation. 1x Tris borate EDTA buffer (for gel electrophoresis): Tris BASE 107.8 g; EDTA 7.4 g; H3BO3 55.0 g; de-ionized water to 10 L. Adjust to pH 8.2. RNAse-free DEPC water: add 0.05% diethyl pyrocarbonate (DEPC) to water that needs treating. Decant into small bottles, or as required (coat all internal surfaces). Leave bottle tops loose. Leave overnight in fume hood. Autoclave at 121 °C at 1.2 bar for 30 min. This treated water cannot be used for Tris buffers. 6x loading buffer (for gel electrophoresis): 0.25% bromophenol blue; 0.25% xylene cyanol FF; 30 % glycerol in water. Store at 4 °C. PCR test This test, based on Morris et al. (2001), has been ring-tested. Sterile filter-plugged pipette tips, Eppendorfs etc. should be used, and gloves worn during all stages of sample preparation and other manipulations involving PCR. Nucleic acid extraction (Hughes & Gallau, 1998) Weigh 200 mg of fresh or frozen root material (or 100 mg dried sample). Place in suitable labelled, small, strong, polythene bags (such as Stomacher) and immerse in a flask of liquid nitrogen. Add 0.5–1 mL of RNA extraction buffer with DTT and thiourea freshly added. Homogenize. Decant 600 µL of the homogenate into a labelled 1.5 mL microfuge tube. Add an equal volume of 6 M potassium acetate to each tube and incubate on ice for 15 min. Spin the samples in a microfuge at 13 000 rev min−1 for 10 min. Transfer 600 µL of the supernatant to a fresh, labelled microfuge tube. Add an equal volume of 4 M LiCl. Incubate the samples at 4 °C overnight. Centrifuge samples at 13 000 rev min−1 at 4 °C for 30 min. Resuspend the pellet in 200 µL TE buffer containing 1% SDS. Add 100 µL of 5 M NaCl and 300 µL ice-cold isopropanol, vortex and incubate samples at −20 °C for 30 min. Centrifuge the samples at 13 000 rev min−1 at 4 °C for 10 min. Discard the supernatant and wash the pellet with 500 µL of 70% ethanol. Centrifuge at 13 000 rev min−1 at 4 °C for 5 min. Carefully pour off the ethanol and dry the pellet in a vacuum drying centrifuge for about 15 min. Resuspend the pellet in 50 µL of RNase-free water. Use for PCR or freeze at −20 °C until needed. One-step RT-PCR Keep defrosted extractions, chemicals and buffers on ice prior to use. For each sample: pipette 1 µL of RNA template into a labelled 0.5 mL Eppendorf tube. Include positive and negative controls and RNAse–free water control. Make up the following RT master mix in a 1.5-mL Eppendorf tube (amounts per sample, but adding 2 extra multiples to allow for pipetting errors): 10 × PCR buffer 5 µL (Promega) (100 mM Tris-HCl, 500 mM KCl, pH 9.0); dNTPs (10 mM) 1 µL: forward primer 016F (5 µM) 2 µL; reverse primer 017R (5 µM) 2 µL; MgCl2 (25 mM) 3 µL; DEPC water (RNAse free) 35.45 µL; MMLV 0.05 µL (10 units, Promega, Southampton, GB); Taq polymerase (add last) 0.5 µL (2.5 units, Promega). Add this 49 µL of the master mix to each Eppendorf already containing 1 µL of template RNA. Run the following programme in the thermal cycler: 30 min at 37 °C, 2 min at 94 °C; followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C. Finally 3 min at 72 °C. Analyse PCR product, or store vials at 5 °C (short term ) or −20 °C until analysis is performed. When normal PCR is complete, use 0.5 µL of PCR product as the template for the nested PCR as below or run products on a gel. Nested PCR PCR nested primers are used as an additional amplification step, if required. The method is 1000 times more sensitive than conventional PCR. Special care is needed to ensure the product does not become contaminated. An additional Eppendorf tube containing 0.5 µL of DEPC water that has not been through the initial PCR should be used as an additional control with the nested PCR. Master mix (per sample): 10 × PCR Buffer (Promega, 100 mM Tris-Hcl, 500 mM KCl, pH 9.0) 5 µL; dNTPs (10 mM) 1 µL; Primer rhzn15 (5 µM) 2 µL; Primer rhzn17 (5 µM) 2 µL; MgCl2 (25 mM) 3 µL; Taq polymerase (Promega 2.5 units) 0.5 µL; DEPC water (RNAse free) 36 µL; amplified product from normal BNYVV PCR 0.5 µL. Add the 49.5 µL of Master mix to each tube containing 0.5 µL of PCR product. Run the following programme in the thermal cycler: 94 °C for 2 min; 30 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min; finally, 72 °C for 3 min. Analyse PCR products or store vials at −20 °C until analysis is performed. Analysis of PCR product The PCR products are detected by agarose gel electrophoresis and staining with ethidium bromide. Prepare a 1.2% agarose gel by gently bringing to the boil (molecular grade, general purpose) agarose in Tris borate EDTA buffer. Cool the molten agarose to 50–60 °C, pour into the gel tray and insert the comb. Allow the gel to set. Remove the comb, submerge the gel in Tris Borate EDTA buffer so that it is covered with 2–3 mm of liquid. On parafilm or in new Eppendorf tubes, take 2 µL of 6× loading buffer and mix with 10 µL of PCR product. Load 10 µL of loading buffer/PCR product mix into the wells carefully. Include appropriate 1 Kb marker(s) used at 5 µL per well or 100 base-pair ladder and positive control amplified DNA. Run gel at 100 V for 1–1.5 h (gel dye front about 15 cm. Remove and soak in ethidium bromide solution (0.5 µg mL−1) for 30–45 min. Destain by rinsing in distilled water. Visualize the amplified DNA products with a UV transilluminator. The PCR product of BNYVV with primer set complementary to the nucleotides 1301–1320 and 1781–1800 on RNA 2 is 500 bp in length. The nested PCR product is 326 bp. Check against DNA marker and against positive control. The water control should be negative in every case. If positive contamination has occurred, the test should be repeated. Photograph the gel to provide a permanent record. The PCR test is negative if the characterized 500 bp (PCR) fragment (or 326 bp for nested PCR) is not detected and the fragment for the positive control isolate of BNYVV is detected., and positive if the 500 bp (PCR) fragment (or 326 bp for nested PCR) is detected and it is identical in size with the fragment for the positive control isolate of BNYVV. Appendix 3. One-step Immunocapture RT-PCR Materials for Immunocapture PCR For ELISA grinding buffer and PBST, see Appendix 1. For PCR primers etc., see Appendix 2. The following one-step Immunocapture RT-PCR has been found to be slightly less sensitive than a one-step RT-PCR (Morris et al., 2001) using purified RNA and detects dilutions of sap extracted from infected sugarbeet and C. quinoa down to 1 × 10−2 and 1 × 10−3 respectively. Ring-testing was inconclusive, and it is only included here for completeness as it is used in place of normal PCR in some laboratories. Coat 500-µL microfuge tubes with polyclonal anti-BNYVV antiserum (e.g. Adgen) 3 h at 33 °C. Wash tube three times with PBST (Appendix 1). Grind sample roots in ELISA extraction buffer 1:9 w/v (as in ELISA Appendix 1). Add 100 µL of root homogenate to the coated microfuge tube and incubate overnight at 4 °C. Wash three times with PBST and twice with sterile distilled water. Add 50 µL of the following RT Master mix to each washed sample tube: Forward primer (016F, 5 µM) 2 µL; Reverse primer (017R, 5 µM) 2 µL; 10 × Taq reaction buffer (100 mM Tris-HCl, 500 mM KCl, pH 9.0) 5 µL; MgCl2 (25 mM) 3 µL; dNTPs (10 mM) 1 µL; MMLV reverse transcriptase (Promega, Southampton, GB) 10 units 0.05 µL; RNAse inhibitor (Promega, Southampton, GB) 20 units 0.5 µL; Taq DNA polymerase (Promega, Southampton, GB) 2.5 units 0.5 µL; molecular grade/DEPC water 35.95 µL. Place tubes in a thermocycler set to the following programme: 30 min at 37 °C, 2 min at 94 °C, followed by 30 cycles of 1 min at 94 °C for, 1 min at 55 °C and 1 min at 72 °C, followed by 3 min at 72 °C. Analyse PCR product (see previous section ) or store vials at −20 °C until analysis is performed. Appendix 4. Electron microscopy tests Materials for electron microscopy tests 2% Uranyl acetate (U/A) stain: uranyl acetate 2 g; distilled water to 100 mL. Dissolve and store in a brown glass dropper bottle at 4 °C. Phosphate buffer pH 6.5 (Sorenson's) (SPB): mix 3 mL of Na2HPO4 (9.469 g L−1) with 7 mL KH2PO4 (9.079 g L−1). PBS – see Appendix 1. Transmission Electron Microscopy (TEM) Wash roots free from soil and blot dry on absorbent paper. Grind 1 g of root using a mortar and pestle in about 1 mL of SPB pH 6.5 (choose lateral roots or thin slivers from lateral root). Take up the liquid with a pipette and place two 20-µL drops in a Petri dish lined with plastic laboratory film. Place two TEM carbon-coated grids, one on each of the drops, coated side down, using fine forceps. Incubate with lid on Petri dish for 10 min. Remove grids using forceps and wash off excess sap with 20 drops of distilled water from a Pasteur pipette. Stain sample with 3 drops of 2% U/A using a dropper bottle. Gently dry the grids by touching their edge on filter paper, and store. Examine in the TEM at × 46 000 fo