Tomato spotted wilt tospovirus , Impatiens necrotic spot tospovirus and Watermelon silver mottle tospovirus
2004; Wiley; Volume: 34; Issue: 2 Linguagem: Inglês
10.1111/j.1365-2338.2004.00728.x
ISSN1365-2338
Tópico(s)Plant and Fungal Interactions Research
ResumoEPPO BulletinVolume 34, Issue 2 p. 271-279 Diagnostic protocols for regulated pests†Free Access Tomato spotted wilt tospovirus, Impatiens necrotic spot tospovirus and Watermelon silver mottle tospovirus First published: 10 September 2004 https://doi.org/10.1111/j.1365-2338.2004.00728.xCitations: 10 European and Mediterranean Plant Protection Organization PM 7/34(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 onFacebookTwitterLinked InRedditWechat Specific scope This standard describes a diagnostic protocol for Tomato spotted wilt tospovirus, Impatiens necrotic spot tospovirus and Watermelon silver mottle tospovirus. 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 The Tospovirus Tomato spotted wilt virus (TSWV) was first described in Australia (Brittlebank, 1919) and its viral etiology presented (Samuel et al., 1930). TSWV is now classified as the type member of its genus (family Bunyaviridae), which to date includes 13 species (van de Wetering, 1999), including the Tospoviruses Impatiens necrotic spot virus (INSV) and Watermelon silver mottle virus (WSMoV). Tospovirus isolates were originally identified based on serological differences and a serogroup classification system was established (de Avila et al., 1990) in which TSWV is the sole member of serogroup I, INSV is the sole member of serogroup III, and WSMoV is a member of serogroup IV. Tospovirus spp. are further classified based on molecular data (de Avila et al., 1993). They are all transmitted and spread in nature by thrips (Thysanoptera: Thripidae) which acquire virus during larval stages and transmit virus via the adults. However, less is known of the vector-virus relationship for WSMoV than for TSWV and INSV (OEPP/EPPO, 1999). These viruses have quasi-spherical enveloped particles 70–110 nm in diameter (OEPP/EPPO, 1999). Identity Name: Tomato spotted wilt tospovirus Acronym: TSWV Taxonomic position: Bunyaviridae, Tospovirus Bayer computer code: TSWV00 Phytosanitary categorization: EPPO A2 list: No. 290, EU Annexes: I/B and II/A2 Name: Impatiens necrotic spot tospovirus Acronym: INSV Taxonomic position: Bunyaviridae, Tospovirus Bayer computer code: INSV00 Phytosanitary categorization: EPPO A2 list: No. 291, EU Annexes: not specifically listed, but not distinguished at the time TSWV was included in Annexes I/B and II/A2. Name: Watermelon silver mottle tospovirus Acronym: WSMOV Taxonomic position: Bunyaviridae, Tospovirus Phytosanitary categorization: WSMoV EPPO A1 list: No. 294, EU Annexes: not specifically listed, but not distinguished at the time TSWV was included in Annexes I/B and II/A2) Biology Tomato spotted wilt tospovirus (TSWV) TSWV is one of the most widespread and economically important plant viruses (Goldbach & Peters, 1994). It occurs in countries within the EPPO region, Asia, Africa, N. America, C. America and the Caribbean, S. America and Oceania (OEPP/EPPO, 1999). Of the TSWV insect vectors cited, the most important is Frankliniella occidentalis Pergande, which transmits TSWV in a persistent propagative fashion (Gera et al., 2000). Other vector(s) of TSWV are also important in some countries both within and outside Europe (Mumford et al., 1996a; OEPP/EPPO, 1999; Chatzivassiliou et al., 2000). TSWV is considered not to be seed-transmitted. Impatiens necrotic spot tospovirus (INSV) INSV occurs where F. occidentalis is present as a vector (Naidu et al., 2001) and causes damage and losses akin to those of TSWV, largely on ornamental hosts, but also some vegetable crops (Vicchi et al., 1999). INSV has not often been reported on outdoor crops, but the detection of INSV in tomato in Italy represents a further step in what seems to be a progressive adaptation of INSV to outdoor vegetable crops (Finetti & Gallitelli, 2000). INSV has a more restricted geographic distribution than TSWV within the EPPO and EU region, Asia, N. America, Central America and Caribbean (OEPP/EPPO, 1999). INSV is not reported to be seed-transmitted. Watermelon silver mottle tospovirus (WSMoV) WSMoV naturally infects Cucurbitaceae, and experimentally infects other hosts including tomato (Yeh et al., 1992). It is spread naturally with its vector Thrips palmi (OEPP/EPPO, 1999). The virus currently has a restricted geographic distribution occurring in India, Japan and Taiwan. It causes significant losses in watermelon (Yeh & Chang, 1995). WSMoV is not present in the EU, but eradicated outbreaks of its vector T. palmi (Fang-Hua Chu et al., 2001) have been reported in the Netherlands (EPPO/CABI 1997) and the UK (OEPP/EPPO, 2001). WSMoV is not reported to be seed-transmitted. Vectors Vectors cited for the Tospoviruses in this protocol (OEPP/EPPO, 1999a–c) are as follows TSWV – besides F. occidentalis (already mentioned), Thrips tabaci Lindeman, Thrips setosus Moulton, Frankliniella fusca Hinds, Frankliniella intonsa Trybom, Frankliniella schultzei Trybom and Scirtothrips dorsalis Hood. INSV –F. occidentalis, Frankliniella fusca. WSMoV –T. palmi. Principal hosts TSWV TSWV infects at least 900 plant species, with the number of natural host species recorded steadily increasing (Peters, 1998). It occurs in ornamental, vegetable and weed hosts (OEPP/EPPO, 1999). The principal vegetable and industrial host crops in the EPPO region (EPPO/CABI, 1997; OEPP/EPPO, 1999b) are tomato, tobacco, lettuce, faba bean, capsicum, chicory, potato, aubergine and artichoke. The principal ornamental plants (OEPP/EPPO, 1999) are: Alstroemeria, Anemone, Antirrhinum, Araceae, Aster, Begonia, Bouvardia, Calceolaria, Callistephus, Celosia, Cestrum, Columnea, Cyclamen, Dahlia, Dendranthema× grandiflorum, Eustoma, Fatsia japonica, Gazania, Gerbera, Gladiolus, Hydrangea, Impatiens, Iris, Kalanchoe, Leucanthemum, Limonium, Pelargonium, Ranunculus, Saintpaulia, Senecio cruentus, Sinningia, Tagetes, Verbena, Vinca and Zinnia. 104 plant species were found to be infected with TSWV in the Netherlands (Verhoeven & Roenhorst, 1998). TSWV and INSV often occur together (OEPP/EPPO, 1999a). INSV INSV currently has a much more restricted host range than TSWV, but the number of natural host species recorded is steadily increasing (Roggero et al., 1999). It is much more frequently found on ornamental crops than on vegetables (OEPP/EPPO, 1999a) which include: Impatiens, Aconitum, Alstroemeria, Anemone, Antirrhinum, Begonia, Bouvardia, Callistephus, Columnea, Cyclamen persicum, Dahlia, Dendranthema × grandiflorum, Eustoma grandiflorum, Exacum affine, Fatsia japonica, Gerbera, Gladiolus, Limonium, Lobelia, Pittosporum, Primula, Ranunculus, Senecio cruentus, Sinningia speciosa, and Zantedeschia aethiopica. Vegetable hosts (OEPP/EPPO, 1999; Finetti et al., 2000) include: Capsicum annuum, Cichorium endivia, Cucumis sativus, Lactuca sativa, Ocimum basilicum, Valerianella olitoria and Lycopersicon esculentum. 41 plant species were identified to be infected with INSV in the Netherlands (Verhoeven & Roenhorst, 1998). WSMoV The principal hosts of WSMoV are watermelon (Citrullus lanatus) and melon (Cucumis melo) (OEPP/EPPO, 1999c). These crops are widely grown in Mediterranean countries and in northern countries in glasshouses. WSMoV presents a clear risk to these crops (OEPP/EPPO, 1999c). Detection Symptoms TSWV can induce a wide variety of symptoms on economically important ornamental plants, vegetables and industrial crops (EPPO/CABI, 1997) including: necrotic or chlorotic local lesions, ring spots, ring spots in concentric rings, green island mosaic, stem discoloration, line patterns, wilting, stunting, mottling, bronzing, distortion, chlorosis, necrosis which may vary on the same host species (Web Figs 1, 2 and 3). Variables affecting symptom expression include the cultivar, age, and nutritional and environmental conditions of the plant, and differences between different isolates of TSWV on the same hosts (OEPP/EPPO, 1999; Mumford et al., 1996a). The range of symptoms for INSV is similar to that of TSWV and these viruses can occur together (OEPP/EPPO, 1999). Some symptoms are given below for TSWV (OEPP/EPPO, 1999), INSV and principal WSMoV hosts. Given the wide host range, and variables affecting symptom expression discussed, it is not feasible to give a comprehensive symptom list in this protocol for all TSWV and INSV host plants. Further symptom descriptions are given by Daughtrey (1996), Chatzivassiliou et al. (2000), Lisa et al. (1990), Yeh et al. (1992). TSWV On tomato, leaf symptoms include bronzing, curling, necrotic spots, necrotic streaks, stunting. Fruit symptoms are usually either irregular yellow/orange flecks and occasionally rings on red fruits, or necrotic lesions or rings. Ripe fruits show paler red or yellow areas on the skin. Sometimes affected plants are killed by severe necrosis On capsicum, symptoms include stunting and yellowing of the plant, chlorotic line patterns or mosaic with necrotic spots on leaves, necrotic streaks on stems extending to terminal shoots and yellow target spots or necrotic streaks may be observed on ripe fruits. On lettuce, symptoms include leaf discoloration and one-sided growth. On tobacco, symptoms include necrotic lesions, necrotic rings, chlorotic rings. On aubergine, symptoms include necrotic lesions on leaves. On faba bean, symptoms include necrotic lesions on leaves. INSV On New Guinea Impatiens hybrids, symptoms include stunting, leaf spots and black discoloration at leaf bases. WSMoV On watermelon, symptoms include foliar mottling, crinkling, yellow spotting and narrowed leaf laminae, small malformed fruits with necrotic spots or silver mottling, reduced fruit set and upright growth of branches and tip necrosis. On melon, symptoms include foliar mottling, stunting, upright growth of branches and tip blight. Systemic symptoms are induced in experimental hosts including tomato and cucumber (Yeh et al., 1992). Sampling Indicator Plants The sampling strategy recommended for TAS-ELISA is also recommended for selection of material for inoculation of indicator plants. The use of indicator plants is an essential aid to reliable tospovirus diagnosis due to uneven virus distribution in plant hosts. It is thus recommended that indicator plants as recommended in this protocol (Appendix 2) as diagnostic hosts for TSWV, INSV and WSMoV are inoculated from the original host plant with symptoms or suspect, at an early stage, before deterioration of the sample occurs. Lateral Flow Tests Whole or parts of leaves can be used with the extraction bottle. It is recommended that fresh, young expanded leaf material is used and not senescent material. The tests are designed to detect TSWV or INSV in symptomatic material (see pathogen key card provided with the lateral flow device test kits). When sampling a plant it is advisable to take tissue from several locations on the plant, in case of uneven distribution. ELISA TSWV, INSV and WSMoV tend to be unevenly distributed in natural hosts. Symptomatic plant material (leaves, stems) should be sampled where possible, but not senescent material. Expanded young leaves tend to have more detectable virus than older plant parts. RT-PCR/MPCR The sampling strategy recommended for ELISA is also recommended for selection of plant material for nucleic acid extraction. Identification Sample preparation See Appendix I. For RT-PCR and MRT-PCR, it is recommended to include internal control primers to confirm RNA extraction (Appendix 3). The CTAB RNA extraction method recommended by Boonham et al. (2001) is best suited to the broad range of tospovirus hosts detailed in this protocol. Alternatives given in this protocol (Appendix 1), on the basis of good RNA yield and ease of use, comprise the methods of (Logemann et al., 1987) and a commercial kit for RNA extraction. Screening tests Detailed protocols as well as material for testing and indicator plants are described in Appendices 1–6. A decision scheme is presented in Fig. 4. Figure 4Open in figure viewerPowerPoint Decision scheme for the detection and identification of Tospoviruses (Tomato spotted wilt virus, Impatiens necrotic spot virus, Watermelon silver mottle virus). Indicator plants Mechanical inoculation of appropriate indicator plants selected from those listed in Appendix 3 is carried out according to the method described in Appendix 1. Symptoms develop within 7 days on indicator plants inoculated with TSWV or INSV grown at 20 °C, and with WSMoV at 20–25 °C. Serological Tests TAS-ELISA and Lateral Flow Devices (LFD) are rapid methods for screening tospovirus samples. The TAS-ELISA methods employed in this protocol allow the specific detection of TSWV, INSV or WSMoV. For a serological comparison of tospovirus isolates using other commercially available antisera, refer to Adam et al. (1996). LFDs allow rapid detection (within minutes) of TSWV or INSV. Positive LFD results should be confirmed by ELISA or PCR-based methods. The sensitivity of detection of TAS-ELISA and LFD serological tests is not as great as that of PCR-based methods, so RT-PCR/MPCR should also be used where tospoviruses are suspected to be present but are not detected by serological tests in the original host (at low concentration in plant hosts, or at an early stage of infection). Positive controls (infected plant material, preferably hosts of the same species as the test plants where available) and negative controls (healthy plant material and buffer) should be included if possible. The use of healthy controls is important as certain plant extracts, e.g. Fuchsia may give false positive results (Louro, 1996). A buffer-only control should also be included. The ELISA value of the sample should be twice or more than that of the negative control. RT-PCR/MRT-PCR RT-PCR/MRT-PCR methods for the detection of TSWV, INSV or WSMoV are carried out as described in Appendix 1. PCR-based methods are more sensitive than TAS-ELISA and are suitable for detection of tospoviruses present at low concentration in plant hosts. Methods given in this protocol enable both specific RT-PCR/MRT-PCR identification of TSWV, INSV or WSMoV or MRT-PCR using universal degenerate primers which provides a broad screening test for tospovirus species in serogroups I (TSWV), II (GRSV,TCSV), III (INSV) and (IV) WSMoV. Confirmation Where ELISA tests (TAS-ELISA-Appendix 1) for TSWV, INSV or WSMoV are positive, specific virus presence is confirmed. Where RT-PCR/MRT-PCR tests are positive, specific virus presence is confirmed. Confirmation of WSMoV where a positive test is achieved currently also requires sequencing to confirm a first record in Europe. Confirmation of TSWV, INSV and WSMoV species where serological screening test(s) (Appendix 1), and RT-PCR/MRT-PCR tests of original plant host material are negative, is achieved by ELISA testing of indicator plants using TAS-ELISA as described in this protocol. The use of indicator plants is an essential aid to reliable tospovirus diagnosis due to uneven virus distribution in plant hosts. Possible confusion with similar species None. Requirements for a positive diagnosis The procedures for detection and identification described in this protocol, and the decision scheme in Fig. 4, should have been followed. Where positive results are achieved using ELISA or molecular tests described in this protocol, the sample is positive. Positive LFD results should be confirmed by ELISA or PCR-based methods. Where positive results are achieved using ELISA tests for indicator plants, the sample is positive (but infection of indicator plants from the original host material is not always successful). Where negative results are obtained using serological tests for samples from the original host, appropriate PCR-based tests should be applied to the original host. This procedure aids positive identification where virus is at low titre in the original host. Where original host material is screened by RT-PCR/MRT-PCR methods, there is a possibility of false negative results due to virus distribution in the original host. The material should be inoculated to indicator plants, and virus presence or absence confirmed by ELISA testing of the indicator plants. 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 (with photographs if possible) • 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: J. Morris, Central Science Laboratory, Virology team PLH6, 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, Virology team PLH6 Sand Hutton, York, Y041 1LZ, UK. Appendices Appendix 1 Materials Materials for tests TAS-ELISA 10× Phosphate buffered saline solution (PBS): NaCl 80 g (136 mm); KH2PO4 2 g (1.5 mm); Na2HPO4·12H2O 29 g (9 mm); KCl 2 g (2.7 mm); distilled water to 1 L. This concentrate gives pH 7.4 at × 1. Extraction buffer (PBS-T): used for tissue maceration, and microtitre plate washes (Clark & Adams, 1977). 10× PBS 100 mL; 10% Tween 20 5 mL (0.05%); distilled water 895 mL. Mix well. Carbonate coating buffer pH 9.6: Na2CO3 1.59 g; NaHCO3 2.93 g; distilled water to 1 L. Check pH. Store at 4 °C. Antibody buffer: PBS-T 100 mL; 5% dried milk powder or 0.2% bovine serum albumin. Prepare on day of usage. Substrate buffer: 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 0.02% MgCl2. Store at 4 °C. Add substrate (disodium p-nitrophenyl phosphate) to microtitre plates at 1 mg/mL dissolved in substrate buffer. TSWV antisera: trapping antiserum (rabbit polyclonal) – use at 1 : 1000 (ADGEN-1071-01); detecting antiserum (TSWV monoclonal) – use at 1 : 100 (ADGEN-1071-03); rabbit antirat AP conjugate (SIGMA) – use at 1 : 5000. INSV antisera: trapping antiserum (rabbit polyclonal) – use at 1 : 1000 (ADGEN-1028-01); detecting antiserum (INSV monoclonal) – use at 1 : 100 (ADGEN-1028-03); rabbit antimouse AP conjugate (SIGMA) – use at 1 : 5000. WSMoV antisera: trapping antiserum (rabbit polyclonal) – use at 1 : 1000 (DSMZ kit T-0118) or prefix D for DAS-ELISA kit; detecting antiserum (WSMoV monoclonal) – use at 1 : 500 (DSMZ kit T-0118-MAb 1B4); rabbit antimouse AP conjugate (SIGMA) – use at 1 : 5000. Lateral Flow Devices: INSV 14-768 (10 tests) or 14-769 (100 tests) (ADGEN); TSWV 14-766 (10 tests) or 14-767 (100 tests) (ADGEN). Indicator plants TSWV Selected susceptible host species and symptoms (Plant Viruses Online http://image.fs.uidaho.edu/vide/, van de Wetering, 1999; Gera et al., 2000): Cucumis sativus– chlorotic spots with necrotic centres in cotyledons, not systemic; Petunia × hybrida– necrotic local lesions, not systemic; Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana tabacum (Web Fig. 6), Nicotiana rustica– necrotic local lesions, systemic necrotic patterns and leaf deformation; Impatiens– chlorotic to necrotic spots or rings on inoculated leaves, systemic chlorotic to necrotic spots; Datura stramonium– chlorotic and necrotic spots and rings on inoculated leaves, systemic mosaic and mottle; Nicotiana benthamiana– chlorotic to necrotic ring spots on inoculated leaves, systemic chlorosis, stunting; Lycopersicon esculentum– chlorotic to necrotic spots and rings on inoculated leaves, systemic mosaic, systemic chlorosis and necrotic spotting. INSV Selected susceptible host species and symptoms (Brunt et al., 1996): Impatiens (as for TSWV); N. benthamiana (as for TSWV; Web Fig. 7); D. stramonium – local lesions (some isolates); P. hybrida: small necrotic spots on inoculated leaves (not systemic); L. esculentum – variable between isolates; lesions on inoculated leaves only. WSMoV Systemic hosts (Yeh et al., 1992): D. stramonium, N. benthamiana (Web Fig. 8). Local lesion host, not systemic: P. hybrida. Other experimentally susceptible hosts (Yeh et al., 1992; all systemic except C. amaranticolor and C. quinoa): Citrullus lanatus, Cucumis metuliferus, Cucumis sativus, D. stramonium, L. esculentum, N. glutinosa, N. rustica, Gomphrena globosa, Chenopodium amaranticolor, Chenopodium quinoa. RT-PCR/MRT-PCR Oligonucleotide Primer Sequences (Mumford et al., 1996b): Degenerate primers for the detection of TSWV, INSV and WSMoV S1 UNIVF 5′-TGT A (G/A) TG (T/G)TCCAT(T/A)GCA-3′ S2 UNIVR 5′-AGA GCA AT (T/C) GTG TCA-3′ Specific primers for the detection of TSWV L1 TSWVR 5′-AAT TGC CTT GCA ACC AAT TC-3′ L2 TSWVF 5′-ATC AGT CGA AAT GGT CGG CA-3′ Specific primers for the detection of INSV S1 INSVF 5′-AAA TCA ATA GTA GCA TTA-3′ S2 INSVR 5′-CTT CCT CAA GAA TAG GCA-3′ Specific primers for the detection of WSMoV (Chu et al., 2001) H1R 5′-ACA GAA AGG TTA GCA CTG AA-3′ H2F 5′-ACA GAG GAC TCC ACT CCC GG-3′ Internal control primers (Universal plant 5SrRNA primers) PLANT-UNI F TTT AGT GCT GGT ATG ATC GC PLANT-UNI R TGG GAA GTC CTC GTG TTG CA Universal plant 5SrRNA primers, from Kolchinsky et al. (1991). RNA extraction method This follows Logemann et al. (1987). Extraction buffer: 4 m guanidine thiocyanate; 20 mm MES (2(N-morpholino) ethanesulphonic acid) pH 7.0; 20 mm EDTA; 50 mm 2-mercaptoethanol; 1% polyvinylpyrrolidone (w/v). This buffer does not need to be autoclaved. Alternatively the Promega SV RNA kit used as per kit instructions (Promega) is also recommended. Detection of TSWV from plant samples and thrips (Boonham et al., 2001) TSWV oligonucleotide primers and probe designed within the conserved regions of the TSWV nucleoprotein gene: TSWV-CP-17F TSWV 5′-CTC TTG ATG ATG CAA AGT CTG TGA-3′ TSWV-CP-100R TSWV 5′-TCT CAA AGC TAT CAA CTG AAG CAA TAA-3′ TSWV-CP-73T 5′-AGG TAA GCT ACC TCC CAG CAT TAT GGC AAG-3′ Thrips primers and probe (Boonham et al., 2001) – designed within the actin gene: WFT-RNA-25F WFT-actin 5′-GGT ATC GTC CTG GAC TCT GGT G-3′ WFT-RNA-93R WFT-actin-C 5′-GGG AAG GGC GTA ACC TTC A-3′ WFT-RNA-48T WFT-actin 5′-CGG TGT CTC CCA CAC TGT CCC CA-3′ Appendix 2 Detection and identification methods Triple Antibody Sandwich TAS-ELISA Extract saps in grinding buffer (PBS-T) 1 : 10 (w/v). Pipette 100 µL of homogeneous sample into a pair of wells on the microtitre plate for testing after step 2.2 of the ELISA test methods below. Conserve the remainder of the extract at 4 °C until testing is completed. This ELISA, based on the method of Clark & Adams (1977), employs polyclonal antisera to trap the virus and monoclonal antisera to detect the virus (Appendix 3). 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, where practicable, as negative control. Coat microtitre plate(s), 100 µL per well, with appropriate polyclonal antisera diluted at a predetermined rate (Appendix 3) in coating buffer. Cover plates and incubate at 33 °C for 3 h. Flick out the contents of the wells. Wash the wells three times with PBS – Tween (Appendix 3) 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 and wash four times as before. Dilute specific monoclonal antibody at a predetermined rate (Appendix 1) in conjugate buffer. Add 100 µL per well. Cover plates and incubate at 33 °C for 2 h. Wash 3 times as before. Add appropriate conjugate, 100 µL per well (Appendix 3). Cover plates and incubate at 33 °C for 2 h. Wash 3 times as before. Add substrate (Appendix 3) at 1 mg mL−1 in substrate buffer. Incubate at room temperature for 1 h. Read at 405 nm. An alternative to the above method for TSWV or INSV is a ‘cocktail’ ELISA using the antisera and the method as above, but adding monoclonal antisera and conjugate together to conjugate buffer at the predetermined rate (Appendix 3), then adding to plates at 100 µL per well. Mechanical inoculation of indicator plants The method follows Mumford (1995). Place selected indicator plants (Appendix 4) in the dark for 24 h prior to inoculation, to enhance susceptibility. Grind infected material with chilled inoculation buffer (0.01 m phosphate buffer, pH 7.0, containing 1% sodium sulphite), using a chilled pestle and mortar. Apply sap extract to the leaves of young plants with a small amount of celite (mixed with sap) or carborundum powder (applied as a light application to leaves). Use a gloved finger, dipped into the sap and gently rubbed down the top surface of the lamina, away from the plant stem. Wash plants carefully to remove any residual abrasive powder. Lateral flow tests Remove a leaf or a portion of leaf (about 3 × 4 cm) with suspicious symptoms. Unscrew lid from extraction bottle and place leaf inside (bottle contains buffer and sodium azide). Replace lid tightly, ensuring dropper cap is on. Shake firmly for about 20 s until a green extract is visible. See also instruction leaflet for lateral flow kit. Remove a test device from its foil pack, avoiding to touch the viewing window. Remove cap from lid of bottle and discard 2–3 droplets by inverting and gently squeezing bottle. Hold device horizontally and gently squeeze 2 drops onto the sample well of the device. Keep device horizontal until extract is absorbed (about 30 s) and a blue dye appears in the viewing window. Wait until the control line appears (labelled C on the device). The control line should be clearly visible in the viewing window of the device after 3 m, and the test (labelled T on the device) result visible in 1–3 min). Two blue lines (C & T) indicate a positive result, test satisfactory; one blue line (C only) indicates a negative result, test satisfactory; faint blue T line, strong C line indicates a possible positive, test satisfactory. A faint or absent line may indicate a low concentration of the pathogen, uneven distribution within the plant or recent infection. See also instruction leaflet for lateral flow kit and Danks & Barker (2000). RNA extraction method of Boonham et al. (2001) Macerate 100–200 mg leaf tissue to a fine powder in liquid nitrogen. Mix ground tissue with 1 mL buffer (2% CTAB, 100 mm Tris-HCl pH 8.0, 20 mm EDTA, 1.4 m NaCl, 1% Na2SO3, 2% PVP-40). Incubate in microfuge tube at 65 °C for 10–15 min. Extract twice with chloroform: isoamyl alcohol (24 : 1). Precipitate RNA from the aqueous layer overnight with an equal volume of 4 m lithium chloride at 4 °C. Centrifuge for 30 min at 13 000 g at 4 °C. Resuspend pellet in 200 µL of TE buffer containing 1% sodium dodecyl sulphate (SDS). Incubate at −20 °C for 30 min with 100 µL of 5 m NaCl and 300 µL of ice-cold isopropanol. Centrifuge for 10 min at 13 000 g at 4 °C, wash with 70% ethanol, re-pellet, and dry pellet. Re-suspend pellet in 50 µL DEPC-treated water and store at −20 °C. RNA extraction method of Logemann et al. (1987) Freeze 200 mg of plant tissue in liquid nitrogen in a polythene bag and homogenize to a fine powder using a hand roller. Add two volumes (400 µL) of Logemann buffer (Appendix 3) and homogenize the tissue further. Decant the homogenate into a 1.5-mL microfuge tube. In a safety cabinet, add an equal volume (400 µL) of phenol:chloroform 5 : 1 (e.g. Amresco), vortex and then centrifuge at 13 000 g at 4 °C for 15 min. Without disturbing the interphase, pipette the upper aqueous phase into a new 1.5-mL microfuge tube. To this add 0.1 volumes of 3M sodium acetate and 2.5 volumes of ice-cold absolute ethanol. Incubate at −20 °C for 1 h. Recover the RNA by centrifugation at 13 000 g at 4 °C for 15 min. Discard the ethanol phase. Resuspend the pellet in 200 µL of 3M sodium acetate. Centrifuge for 5 min at 13 000 g at room temperature. Discard the supernatant. Wash the RNA by resuspending in 500 µL of ice cold 70% ethanol. Centrifuge at 4 °C for 15 min as previously. Remove the ethanol wash. Dry the pellets in a vacuum desiccator for 10–15 min, or on the bench for 30 min or until pellets are dry. Resuspend in 100 µL of DEPC-treated sterile distilled water and store at −20 °C. Centrifuge for 30 s prior to use. Alternatively, the Promega SV RNA kit used as per kit instructions (Promega – See Appendix 3) is also recommended. RT-PCR/MRT-PCR Tests The methods are adapted from Weekes et al. (1996) and Mumford et al. (1996b). Prepare the RT reaction mix in a microfuge tube: 3.5 mm dNTPs 3 µL (1 mm); Primer 2 µL; 5xM-MLV Buffer 2 µL; distilled H2O 1.1 µL; 200 U µL−1 M-MLV 0.5 µL; 120 U µL−1 RNase inhibitor 0.4 µL, for each 10 µL final volume reaction. Add 9 µL of master mix to 1 µL of RNA sample in each microfuge tube. Reaction conditions: 42 °C for 15 min, 99 °C for 5 min, 5 °C for 5 min. Prepare the PCR reaction mix (kept on ice): Primer 2 µL; 50 mm MgCl2 1.5 µL; 10× Buffer 5 µL; distilled H2O 31.25 µL
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