Tomato yellow leaf curl and Tomato mottle begomoviruses
2005; Wiley; Volume: 35; Issue: 2 Linguagem: Inglês
10.1111/j.1365-2338.2005.00837.x
ISSN1365-2338
Tópico(s)Plant and Fungal Interactions Research
ResumoEPPO BulletinVolume 35, Issue 2 p. 319-325 Free Access Tomato yellow leaf curl and Tomato mottle begomoviruses First published: 03 October 2005 https://doi.org/10.1111/j.1365-2338.2005.00837.xCitations: 5 European and Mediterranean Plant Protection Organization PM 7/50(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 Tomato yellow leaf curl begomovirus (TYLCV) and Tomato mottle begomovirus (ToMoV). 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 2004-09. Introduction EU countries regulate a number of viruses infecting tomato and other Solanaceous crops, transmitted by the whitefly Bemisia tabaci. Most of these are members of the genus Begomovirus. Two groups in particular are concerned: (1) Tomato yellow leaf curl begomovirus (TYLCV), present in certain European countries, together with various closely related viruses which were originally considered as strains but are now distinguished as species, and may or may not be present in European countries; (2) non-European viruses transmitted by B. tabaci (Brown & Bird, 1992), of which the named example in EU regulations is Tomato mottle begomovirus (still referred to in the phytosanitary texts as ‘Florida tomato virus’); in recent years, a whole series of other non-European begomoviruses of Solanaceae has been described (Fauquet & Stanley, 2003; Fauquet et al., 2003). This Standard concentrates on the two named examples. Tomato yellow leaf curl virus Tomato yellow leaf curl virus (TYLCV) was first recorded as a whitefly-transmitted virus in tomato crops in Israel (Cohen & Harpaz, 1964) and later shown to be a member of the family Geminiviridae, genus Begomovirus. Together with related species which have recently been distinguished, it causes the most devastating virus disease complex of tomato in tropical and warm temperate regions of the world, where losses up to 100% are incurred (Moriones, 2000). Members of the TYLCV complex have been recorded in tomato crops in Spain, Italy, Portugal and France, and occur in most eastern Mediterranean countries (Idriss et al., 1997) and parts of sub-Saharan Africa, Asia, Australia, the Caribbean, and USA (Florida). In the EPPO region, two species are present (Moriones et al., 2000) according to the most recent nomenclature (Fauquet et al., 2000): Tomato yellow leaf curl begomovirus (TYLCV) (the first described species, originally known as the Israel strain (TYLCV-IL)), and Tomato yellow leaf curl Sardinia begomovirus (TYLCSV), originally known as the Sardinia strain (TYLCV-S). In this Standard, the two are mostly treated together, except at specific points where it is necessary to refer to them separately. The main whitefly vector Bemisia tabaci transmits TYLC viruses in a persistent, circulative manner (Cohen et al., 1966). There are no reports of seed transmission in tomato and mechanical transmission does not occur in nature (Moriones et al., 2000). Four biotypes of B. tabaci are currently present in Europe, of which biotypes B and Q efficiently transmit TYLCV and TYLCSV. Global expansion of the B biotype of B. tabaci, also termed the silverleaf whitefly, and proposed as a separate species, B. argentifolii by Bellows et al. (1994) is associated with the emergence of TYLCV in Europe and the Western Hemisphere (Polston & Anderson, 1997). The B biotype of B. tabaci is highly fecund and has the ability to adapt to new host crops unlike established regional populations of B. tabaci. In Spain, TYLCV has been found to be the causal agent of a novel disease of phaseolus beans (Navas-Castillo et al., 1999; Sánchez-Campos et al., 1999) and also occurs in capsicums (Reina et al., 1999). TYLCV isolates are monopartite and consist of geminate, quasi-isometric particles, 20 nm in diameter and 30 nm in length (Brunt et al., 1990). Tomato mottle begomovirus The whitefly transmitted geminivirus Tomato mottle begomovirus (ToMoV) was first observed in 1989, and recorded by Simone et al. (1990) and Abouzid & Hiebert (1991), on tomato crops in Florida (USA). A serious outbreak occurred in the 1990/1991, associated with large outbreaks of Bemisia tabaci biotype B. It has since been recorded also in tomato in S. Carolina, Tennessee and Virginia, and on the weed Solanum viarum. ToMoV is mechanically transmissible. Symptoms on tomato are mild when compared with TYLCV or Tomato golden mosaic begomovirus (TGMV) (Abouzid et al., 1992). Experimental hosts include members of three solanaceous genera (Lycopersicon, Nicotiana, and Physalis) and Phaseolus vulgaris. The genome of ToMoV has been sequenced (Abouzid et al., 1992) and is bipartite. Approach to detection and diagnosis Serological methods have met limited success when used for whitefly-transmitted Geminiviridae (Harrison et al. 1991; Muniyappa et al., 1991) as antisera have proved difficult to produce. Relatively non-specific monoclonal antibodies have been produced, which detect TYLCV and ToMoV, but cross-react with a range of other B. tabaci-transmitted Geminiviridae. In the EPPO region, only TYLCV and TYLCSV currently occur, so field samples submitted for diagnosis are most likely to be these species. Serological tests on material from outside the EPPO region could also detect, but not distinguish, other species of the TYLC complex, and various other Begomovirus spp. including ToMoV. Molecular methods are then needed for diagnosis. Identity Name: Tomato yellow leaf curl begomovirus Synonyms: Tomato yellow leaf curl bigeminivirus, Tomato yellow leaf curl geminivirus Acronym: TYLCV Taxonomic position: Viruses: Geminiviridae: Begomovirus EPPO computer code: TYLCV0 Phytosanitary categorization: EPPO A2 list no. 182, EU Annex designation II/A2 Name: Tomato mottle begomovirus Synonyms: Tomato mottle virus, Florida tomato virus Acronym: ToMoV Taxonomic position: Viruses: Geminiviridae: Begomovirus EPPO computer code: TOMOV0 Phytosanitary categorization: EPPO A1 list no. 225, EU Annexes: listed as Florida tomato virus under non-European viruses transmitted by Bemisia tabacci in Annex I/A1 Detection Symptoms In tomato, symptoms caused by TYLC viruses vary depending on the growth stage at the time of initial infection, environmental conditions and the tomato cultivar; they include severe stunting, marked reduction in leaf size, upward cupping, chlorosis of leaf margins, mottling, flower abscission and significant yield reduction CSL (Morris, 2000). In phaseolus beans, TYLC symptoms include leaf thickening, leaf crumpling, upward curling of leaves, abnormal lateral shoot proliferation, deformation of pods and reduction in pod number. In capsicum, TYLC symptoms include interveinal and marginal leaf chlorosis, upward curling of leaf margins (or symptomless). In Lisianthus, TYLC symptoms include distortion of the growing tips, cup-shaped leaves, swelling of veins on the lower surface of the leaves, significant reduction in flower quality and stunting. Symptoms of ToMoV in tomato include chlorotic mottling, leaf distortion and curling and stunting. Sampling For TAS-ELISA, appropriate sample selection is critical for serological detection of TYLC viruses and ToMoV. There is more detectable virus in freshly expanded young leaves present in the uppermost regions of the plant than in older plant parts. Younger leaf material should be selected for TAS-ELISA sample preparation as described in Appendix 1. For PCR, detection of TYLC viruses and ToMoV is achieved using fresh or lyophilized leaf material. Leaf material should be derived from freshly expanded young leaves present in the uppermost regions of the plant and should be collected carefully to avoid cross contamination (by changing disposable gloves between samples, or preferably by punching a piece of leaf directly into an Eppendorf tube using the lid as a cutting implement). Recommended DNA extraction methods are described in Appendix 2. Identification The procedure for identification of TYLCV and ToMoV is summarized in the flow-diagram in Fig. 1. Figure 1Open in figure viewerPowerPoint Flow diagram for the identification of TYLCV and ToMoV. Screening Tests TAS-ELISA In cases where TYLC viruses or ToMoV are suspected, a TAS-ELISA test (Appendix 1) can be performed to aid in presumptive diagnosis. However, final diagnosis cannot be achieved using ELISA alone, since currently available antisera may miss samples at early stages of infection, or cross-react with other ssDNA viruses. The antiserum supplied by Adgen (http://www.adgen.co.uk/) is effective as a universal screen, but cannot differentiate between TYLC viruses and ToMoV. The antisera supplied for TYLCV detection by DSMZ (DSMZ AS-0421/DSMZ AS-0546/2/DSMZ 0546/4 –http://www.dsmz.de/plvirus) can be used for the detection and differentiation of TYLCV and TYLCSV (present in Europe), but cannot effectively resolve mixed species infections. Known infected plants should be used as positive controls in ELISA tests, in addition to healthy leaf material of the host species as a negative control. A buffer-only control should also be included. PCR For PCR, DNA is extracted from samples of plant material infected with TYLC viruses or other Begomovirus spp. according to the procedures described in Appendix 2. PCR for the detection of TYLCV/TYLCSV is carried out as described in Accotto et al. (2000) (Appendix 3). This confirms the diagnosis of TYLCV/TYLCSV, but also be a useful indicator of possible presence of other ssDNA virus. If a negative result is obtained with symptomatic material which gave a positive result in the screening text, or an atypical restriction digest pattern occurs using this method, the presence of another ssDNA virus such as ToMoV may be achieved by carrying out the PCR procedure of Deng et al. (1994) (Appendix 3), extended to ToMoV detection by Morris. Various other methods have been experimented but are not included in this Standard. An alternative screening method for TYLCV/TYLCSV is dot-blot hybridization, but the method is limited in that it cannot reliably distinguish between the members of the TYLC complex because of some probe cross reactivity, and can be influenced by virus concentration in the sample. Probes for all TYLC viruses have not yet been documented (Accotto et al., 2000). Thus this method could be used as a general screen for TYLCV/TYLCSV within the EPPO region but, as discussed for ELISA, would have to be backed up by one of the PCR methods given in this protocol to prevent false negative or false positive results. The degenerate primers of Polston et al. (1995) may be used for the detection of ToMoV followed by RFLP. Southern blotting for the detection of ToMoV (Murphy et al., 2000) is an alternative but of limited use since confirmation of ToMoV in Europe screened by any of the above methods currently also requires nucleotide sequence analysis. PCR extraction, PCR set-up, and post-PCR analysis should be performed in separate laboratory areas to avoid contamination. Filter tips and gloves should be used. A positive control should be included, and more than one negative control of the same species as the test plant. A master-mix control (no template in the reaction) blank is also required. Confirmation Confirmation of TYLCV or TYLCSV is achieved using RFLP (Accotto et al., 2000) as described in Appendix 3 or the PCR method of Martínez-Culebras et al. (2001) for the rapid discrimination of TYLCV and TYLCSV. PCR as described in Appendix 3 (Deng et al., 1994) is recommended to identify ToMoV, and all TYLC viruses, to be confirmed by sequencing. Within the EPPO region, confirmation of positive PCR tests for ToMoV would at present require sequence analysis (using standard methodology) to confirm a first record. However, sequence analysis is not recommended as a method for routine diagnosis since it is time-consuming and costly. Where species-specific tools (primers, antisera) for molecular or serological identification are not available, it is the only method. The results of TAS-ELISA and PCR screening tests for initial identification require confirmation. Where RFLP results positive for TYLCV or TYLCSV are obtained using the method of Accotto et al. (2000), the sample can be confirmed as positive. When negative results are achieved using the PCR method of Accotto et al. (2000), or if RFLP using this method produces a digestion pattern which is not indicative of TYLCSV or TYLCV, confirmation of the negative result or diagnosis of another ssDNA virus such as ToMoV should be achieved by the PCR procedure of Deng et al. (1994), followed by sequencing. Where the PCR result thus obtained is negative, the sample can be confirmed as negative. Where the PCR result is positive, preliminary identification of ToMoV could be determined by PCR amplicon size, followed by sequencing. Confirmation of ToMoV requires sequence analysis both in samples derived from imports to the EPPO region and in field samples from EPPO countries to confirm a first record of a ‘new’Begomovirus in the region. Reporting and documentation Guidelines on reporting and documentation are given in EPPO Standard PM7/– (in preparation). Further information Further information on these organisms can be obtained from Virology teams PLHC and PLHB, Central Science Laboratory, Sand Hutton, York Y041 1LZ (UK). E-mail: jane.morris@csl.gov.uk. Footnotes 1 The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. Acknowledgements This protocol was originally drafted by J. Morris, Central Science Laboratory, York (GB). Appendices Appendix 1 ELISA test Preparation of the sample for ELISA testing Weigh approximately 1 g of the infected plant material. Place each sample in a suitable polythene bag for processing. Add the correct volume of extraction buffer (Macintosh et al., 1992) and homogenize the sample using a Homex 6 machine (Bioreba) or using a wallpaper seam roller, or similar. Pipette 100 µL of homogeneous sample into a pair of wells on the microtitre plate for testing. Conserve the remainder of the extract at 4°C until testing is completed. TAS-ELISA test This ELISA, based on the method of Thomas et al. (1986), employs African cassava mosaic virus (ACMV) polyclonal antiserum as the trapping antibody, and mouse monoclonal antisera for detection. Microtitre plates (Nunc Maxisorp Immunoplate) are used. Known infected plants are used as positive controls, together with healthy plants of the same species as the test plants as a negative control. Add the crude ACMV polyclonal antibody at the recommended dilution (10−3) to the coating buffer. Pipette the solution into the microtitre plates, 100 µL per well. Incubate for 3 h at 33°C. Flick out the contents of the wells. Wash the 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. Wash the wells four times with PBS-Tween with 3 min soaks between washes. Blot dry on absorbent paper. Add TYLCV/ToMoV monoclonal antibody SCR 23 at the recommended dilution in dried milk buffer at 100 µL per well. Incubate for 2 h at 33°C. Flick out the contents of wells. Wash the wells four times with PBS-Tween with 3-min soaks between washes. Blot dry on absorbent paper. Prepare 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. Wash the wells three times with PBS-Tween with 3-min soaks between washes. Blot dry on absorbent paper. Prepare alkaline phosphatase substrate solution. Add 100 µL to each well. Incubate at ambient temperature for 1 h. Read absorbance at 405 nm. Interpretation of the ELISA test The ELISA test is negative if the absorbance of the sample is less than 2 times the absorbance of the healthy control. The ELISA test is positive if the absorbance of the sample is equal or greater than 2 times the absorbance of the healthy control. Materials used for TAS-ELISA • Extraction buffer for tissue maceration (Macintosh et al., 1992): 0.05 m Tris-HCl pH 8.5, 6.05 g; 0.06 m Na2SO3, 7.56 g. Make up to just below 1 L with deionized distilled water, adjust pH to 8.5 with HCl and make up to 1 L with additional water • Carbonate coating buffer pH 9.6: Na2CO3, 1.59 g: NaHCO3, 2.93 g; distilled water, 1 L. Dissolve the ingredients and check pH. Store solution at 4°C • 10 × Phosphate-buffered saline (PBS) 1 × = pH 7.2: NaCl, 80 g; KH2PO4, 2 g; Na2HPO4·12H2O, 11.5 g; KCl, 2 g; distilled water, 1 L. Dissolve all ingredients and check pH. Dilute to 1 × for use • Phosphate-buffered saline Tween (PBS-T): 10 × PBS, 100 mL; 10% Tween 20, 5 mL; distilled water, 895 mL. Mix ingredients well • Antibody buffer: PBS-T, 100 mL; 5% dried milk powder, 5 g. Prepare on day of use • Substrate buffer (diethanolamine buffer 1 m): diethanolamine, 95 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. Store solution at 4°C • 2 (p-nitrophenyl phosphate) phosphatase substrate: dissolve two 5 mg tablets (Sigma 104) per 10 mL of substrate solution • TYLCV-detection kits incorporating the detecting monoclonal antisera SCR23 within test kit 1072-05 were obtained from Adgen (Auchincruive, Scotland). Appendix 2 DNA extraction methods DNA extraction method 1 is recommended for PCR, with extraction method 2 supplied as an alternative. The leaf squash method of Atzmon et al. (1998) is also an alternative, but details are not provided. DNA extraction method 1 The method of Accotto et al. (2000) can be used for TYLCV and ToMoV DNA extraction. Leaf material can easily be collected using an Eppendorf tube without touching the leaf with fingers, thus avoiding cross contamination. The method can be carried out using either 10 mmβ-mercaptoethanol in the extraction buffer or 10 mm citric acid. Grind 0.15 g of leaf material to a fine powder in liquid nitrogen using either a pestle and mortar, or a plastic grinding bag and wallpaper roller. Ensure the tissue does not thaw once frozen. Add 500 µL of extraction buffer (100 mm Tris-HCl pH 8, 50 mm EDTA, 500 mm NaCl, 1% SDS and 10 mmβ-mercaptoethanol or citric acid 10 mm). Mix tubes by vigorous shaking and then incubate at 65°C for 5 min. Add 150 µL of 5 m potassium acetate and shake tubes vigorously to mix, then incubate at 0°C for 10 min. This step removes most proteins and polysaccharides as a complex with the insoluble potassium dodecyl sulphate precipitate. Tubes are then spun at 13 000 rev min−1 for 10 min and the supernatant (about 500 µL) withdrawn to a tube containing 350 µL of cold isopropanol, then mixed well and centrifuged at 13 000 rev min−1 for 10 min. Wash precipitate by addition of 500 µL of 70% ethanol and centrifuge for 5 min at 13 000 rev min−1. Carefully discard ethanol and desiccate for 15 min in a vacuum chamber or thoroughly air-dry. Resuspend pellets in 100 µL of 10 mm Tris, 1 mm EDTA pH 8.0. DNA extraction method 2 For the method of Lohdi et al. (1994), add 200 mg of plant material to liquid nitrogen. Add 2–3 mL of CTAB extraction buffer (Appendix 4). Homogenize the sample using a wooden/plastic wallpaper roller or similar. Decant ground sap into a 1.5 mL microfuge tube. Incubate for 10–15 min at 65°C. Centrifuge for 5 min at 13 000 rev min−1. Remove 700 µL of supernatant into a new microfuge tube. Extract with an equal volume (700 µL) of chloroform: IAA (24 : 1). Vortex at low speed for 2 s. Centrifuge at 13 000 rev min−1 at ambient temperature for 10 min. Collect the upper aqueous layer and transfer the contents to another Eppendorf. Repeat. Precipitate with 0.5 volume of 5 m sodium chloride and an equal volume of ice-cold isopropanol. Incubate at −20°C overnight. Pellet the DNA by centrifugation at 13 000 rev min−1 for 10 min. Wash precipitate by addition of 500 µL of 70% ethanol and centrifuge for 5 min at 13 000 rev min−1. Carefully discard ethanol. Desiccate for 15 min to dry the pellet. Resuspend pellet in 100 µL of 1 × TE (Appendix 4). Alternatively the EZNA extraction kit Omega Biotek is recommended. Appendix 3 PCR tests PCR method 1 For the TYLCV/TYLCSV PCR test in plants (Accotto et al., 2000), prepare the PCR reaction mix in a 1.5 mL microfuge tube. Final concentrations for PCR reaction components (kept on ice) are: 2 mm MgCl2, 0.2 mm dNTPs, 0.4 µm (final concentration) primer TY1, 0.4 µm (final concentration) primer TY2, 0.4 U µL−1 Taq DNA polymerase for a total of 25 µL. For further reactions, calculate the quantity of each component for the required number of reactions (25 µL per sample aliquot). Make up 1 more than needed for easier pipetting. For PCR after mixing the components, transfer 25 µL of PCR reaction mix, with 1 µL of nucleic acid DNA to a 0.2 mL Eppendorf tube. Place the tubes in the thermal cycler heating block. Run the following programme on the thermal cycler: 1 cycle (4 min at 95°C), 35 cycles (30 s at 95°C, 30 s at 60°C, 30 s at 72°C), 1 cycle (7 min at 72°C). Analyse 5 µL of PCR product on a 1% agarose gel or store tubes at −20°C until analysis is performed. Interpretation of the PCR test result If an amplicon of the expected size of 580 bp is present, precipitate with ethanol the remaining 20 µL of PCR product and carry out RFLP to confirm TYLCV identity as described. If no amplicon is present or amplicon of unexpected size, PCR method 2 should be performed to determine whether the sample is negative or other ssDNA virus species present. RFLP Resuspend DNA (ethanol-precipitated 20 µL volume of PCR product) and digest with Ava 11 in 10 µL volume. Load all the reaction mix on a 3% NuSieve agarose gel (or 2% agarose). Restriction products of 68 bp, 360 bp, and 150 bp are expected for TYLCSV. Restriction products of 277 bp and 302 bp are expected for TYLCV. PCR Method 2 For the MPCR/PCR test for geminiviruses in plants (TYLC viruses and ToMoV) (Deng et al., 1994), prepare the PCR reaction mix in a 1.5 mL microfuge tube. A typical PCR reaction mixes is: 10 × PCR buffer 5 µL, 1.75 mm MgCl2 3.5 µL, 0.2 mm dNTPs 1 µL (of 100 µL stock solution), 0.2 µm (final conc) primer 540 2 µL, 0.2 µm (final conc) primer 541 2 µL, 2 U Taq DNA polymerase 0.4 µL, sterile ultra-pure water 34.6 µL, total 48.5 µL. For further reactions, calculate the quantity of each component for the required number of reactions (48.5 µL per sample aliquot). Make up 1 more than needed for easier pipetting. For PCR after mixing the components, transfer 48.5 µL of PCR reaction mix, with 1.5 µL of DNA to a 0.5 mL Eppendorf tube. Place the tubes in the thermal cycler heating block. Run the following programme on the thermal cycler: 1 cycle (2 min at 94°C), 1 cycle (1 min at 55°C), 1 cycle (2 min at 72°C), 32 cycles (45 s at 94°C, 1 min at 55°C, 2 min at 72°C), 1 cycle (45 s at 94°C, 1 min at 55°C, 5 min at 72°C). Analyse PCR product or store tubes at −20°C until analysis is performed. Analysis of PCR product The PCR fragments are detected by agarose gel electrophoresis and stained with ethidium bromide. Prepare a 1–2% agarose gel by gently bringing to the boil agarose in 1 × TBE (Appendix 4). Cool the molten agarose to 50–60°C, pour into the mould and insert the comb. Allow the gel to set. Remove the comb; submerge the gel in 1 × TBE. Add 10 µL of loading buffer to tubes containing 50 µL of sample, flick to homogenize the solution. Load the wells carefully (12 µL of sample + buffer). Include appropriate markers and positive control, amplified DNA. Run gel at 100 V/40 mA for 1 h until the gel dye front is within 1 cm of the end of the gel. Remove gel and stain in ethidium bromide solution (0.5 µg mL−1) for 45 min. Rinse the gel in distilled water. Visualize the amplified DNA fragments by UV trans-illumination. The PCR product for TYLCV with degenerate primers (Appendix 4) is 540 bp in length. Verify results against DNA marker and positive control. Photograph the gel to provide a permanent record. Interpretation of the PCR/MPCR test result Where more than one PCR product is present per lane, the test is termed MPCR. The test is negative if the characterized 540 bp fragment (TYLC or other geminivirus) or the 377 bp fragment (ToMoV) is not detected and the fragments for the respective positive control isolate(s) are detected. The test is positive if the 540 bp fragment (TYLC or other geminivirus) or the 377 bp fragment (ToMoV) are detected and the fragment is identical with the positive control isolate(s). RFLP utilising PCR products derived from PCR method 2 Sequence analysis is required to confirm a first record of ToMoV. Definitive RFLP patterns are not available for all TYLCV species, so sequencing is recommended to confirm virus identity. Appendix 4 Materials for detection of TYLC viruses or ToMoV in plants by PCR Oligonucleotide primer sequences TY1 (+): 5′-GCC CAT GTA (T/C) C G (A/G) AAG CC-3′ TY2 (–): 5′-GG (A/G) TTA GA (A/G) GCA TG (A/C) GTA C-3′ Deng 541: 5′-TAA TAT TAC CKG WKG VCC SC-3′ Deng 540: 5′-TGG ACY TTR CAW GGB CCT TCA CA-3′ (where K = G or T, R = A or G, S = C or G, W = A or T, Y = C or T, B = C, G or T, and V = A, C or G) Extraction buffer for DNA extraction method 1: 100 mm Tris-HCl pH 8, 50 mm EDTA, 500 mm NaCl, 1% SDS, 10 mmβ-mercaptoethanol or 10 mm citric acid. Extraction buffer for DNA extraction method 2: 2% CTAB, 100 mm Tris-HCl pH 8.0, 20 mm EDTA, 1.4 m NaCl, 1% Na sulphite, 2.0% PVP-40. Mix first 4 reagents. Make up to 1 L with distilled water. Store solution at ambient temperature. Add PVP and sodium sulphite fresh to aliquot of stock buffer (this will keep for about 2 weeks). TE Buffer for DNA extraction methods 1 and 2: 10 mm Tris-HCl, 1 mm EDTA, molecular grade water. 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