Plum pox potyvirus
2004; Wiley; Volume: 34; Issue: 2 Linguagem: Lituano
10.1111/j.1365-2338.2004.00726.x
ISSN1365-2338
Tópico(s)Plant Pathogenic Bacteria Studies
ResumoEPPO BulletinVolume 34, Issue 2 p. 247-256 Diagnostic protocols for regulated pests†Free Access Plum pox potyvirus First published: 10 September 2004 https://doi.org/10.1111/j.1365-2338.2004.00726.xCitations: 48 European and Mediterranean Plant Protection Organization PM 7/32(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 Plum pox potyvirus. 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 Sharka (plum pox) is considered one of the most devastating diseases of stone fruits in terms of agronomic impact and economic importance (Dunez & Sutic, 1988; Németh, 1994). The disease is very detrimental in apricot, peach and plum trees because it produces reduced quality and premature dropping of fruits. It is caused by Plum pox virus (PPV), a member of the genus Potyvirus in family Potyviridae (López-Moya & García, 1999). The PPV epidemic originated in eastern Europe. The disease was described for the first time around 1917 on plums and in 1933 on apricots in Bulgaria (Atanasoff, 1932, 1935). Since then, the virus has progressively spread to a large part of the European continent, around the Mediterranean basin and Near and Middle East. It has been found also in India and in America (Chile, USA and Canada). The introduction of infected plant propagation material is considered the most important means of long-distance spread of PPV. In addition, the virus is non-persistently transmitted by a number of aphid species existing in each region (Kunze & Krczal, 1971; Labonne et al., 1995). Non-aphid transmissible isolates have been described (Maiss et al., 1989; López-Moya et al., 1995). PPV particles are flexuous rods about 700 × 11 nm composed of a single mono-stranded RNA molecule close to 10 000 nucleotides coated by up to 2000 subunits of a single coat protein (CP). The expression strategy of PPV, as for other potyviruses, includes translation of a unique long open-reading frame (ORF) further processed to yield viral products. In recent years, knowledge of the molecular biology of PPV in particular, and of potyviruses in general (Riechmann et al., 1992; Shukla et al., 1994; Revers et al., 1999), has experienced a huge increase. Different biotechnological aspects related to PPV, including novel diagnostic techniques that facilitate detection and typing of virus isolates, strategies to implement pathogen-derived resistance through plant transformation, potential use of genetic elements derived from the virus, and PPV-based expression vectors (López-Moya et al., 2000). The numerous PPV isolates differ in biological and epidemiological properties such as aggressiveness, aphid transmissibility and symptomatology. Two main groups Dideron (D) and Marcus (M) have been established serologically (Kerlan & Dunez, 1976). PPV isolates belonging to D or M groups show different epidemiological behaviour. The M isolates are spread more readily by aphids than D isolates and cause more severe symptoms in peach. The D isolates are able naturally to infect apricot and plum, and rarely spread from these hosts to peach. The existence of these two groups is also based on: (1) different serological pattern or reaction with D- or M-specific monoclonal antibodies (Cambra et al., 1994; Boscia et al., 1997); (2) electrophoretic mobility of the viral CP (Ravelonandro et al., 1988; Bousalem et al., 1994; Pasquini & Barba, 1994); (3) nucleotide sequence information, either complete or partial including the 3′ terminal region of the genome of several PPV isolates (Ravelonandro et al., 1988; Laín et al., 1989; Maiss et al., 1989; Teycheney et al., 1989; Wetzel et al., 1991a; Cervera et al., 1993; Palkovics et al., 1993); (4) sequence analysis of polymerase chain reaction (PCR) fragments corresponding to the C-terminal region of the PPV CP gene and RsaI restriction fragment length polymorphism (RFLP) (Wetzel et al., 1991b; Bousalem et al., 1994; Candresse et al., 1994); (5) different variants of PCR, hemi-nested PCR, nested PCR and Co-operational PCR (Co-PCR) using specific primers (Candresse et al., 1994; Olmos et al., 1997, 1999, 2002) including colorimetric detection of the amplicons with D- or M-specific probes. Two additional groups of PPV isolates are: El Amar (EA) and Cherry (C). PPV-EA isolates are different in nucleotide sequence (Wetzel et al., 1991a) and contain specific epitopes ( Myrta et al., 1998). Group C was described more recently, after the discovery that some PPV isolates, able to infect cherry, were molecularly and serologically different from the other groups (Nemchinov & Hadidi, 1996; Nemchinov et al., 1996) and gave specific reactions with monoclonal antibodies ( Myrta et al., 2000). Detection of any PPV isolate (universal) can be achieved by using monoclonal antibody 5B-IVIA (Cambra et al., 1994) or polyclonal antibodies. Selective and specific detection of PPV-D isolates (Cambra et al., 1994), PPV-M (Boscia et al., 1997), PPV-C ( Myrta et al., 2000) and PPV-EA ( Myrta et al., 1998) is possible using available ELISA kits. Molecular hybridization techniques (Varveri et al., 1988) and different PCR-based assays have been developed for the detection (Korschineck et al., 1991; Wetzel et al., 1991b, 1992; Candresse et al., 1994, 1995; Levy et al., 1994; Olmos et al., 1996) and for the simultaneous detection and typing of PPV isolates (Olmos et al., 1997). Different systems of viral target preparation prior to PCR have been developed based on immunocapture (Wetzel et al., 1992) or on print and squash capture (Olmos et al., 1996), which removes the need for extract preparation. Use of immobilized targets on paper (Cambra et al., 1997) has allowed detection of PPV in single aphids (Olmos et al., 1997) by squash-capture PCR. Nested PCR in a single closed tube (Olmos et al., 1999) has been applied for sensitive detection of PPV targets in plant material and in single aphids. A Co-PCR system using a universal probe for hybridization (Olmos et al., 2002) has been described, giving a sensitivity similar to that of nested PCR. Serological and molecular characterization of PPV isolates correlates very well (Candresse et al., 1998). Identity Name: Plum pox potyvirus Synonyms: Sharka virus Acronym: PPV Taxonomic position: Potyviridae, Potyvirus Bayer computer code: PPV000 Phytosanitary categorization: EPPO A2 list no. 96, EU Annex designation II/A2. Detection Under natural conditions, PPV readily infects fruit trees of the genus Prunus used as commercial varieties or rootstocks: apricot (Prunus armeniaca), European plum (Prunus domestica), Japanese plum (Prunus salicina), peach (Prunus persica), Prunus cerasifera and Prunus cerasifera x munsoniana cv. Marianna. Sour (Prunus cerasus) and sweet (Prunus avium) cherries and almond (Prunus dulcis) may be infected occasionally. The virus also infects many wild or ornamental Prunus species such as Prunus besseyi, Prunus insititia, Prunus tomentosa, Prunus triloba and Prunus spinosa. PPV can be artificially transmitted to numerous Prunus spp., Sorbus domestica and several herbaceous plants. Nicotiana benthamiana, Nicotiana glutinosa, Pisum sativum and Chenopodium foetidum are frequently used as experimental host plants for different purposes. Symptoms Symptoms may appear on leaves, petals, fruits and stones (Web Figs 1, 2). They are particularly clear on leaves in spring: mild light green discoloration, chlorotic spots, bands or rings, vein clearing or yellowing, or even leaf deformation. Flower symptoms can occur on petals (discoloration) of some peach varieties. Infected fruits show chlorotic spots or lightly pigmented yellow rings or line patterns. Fruits may become deformed or irregular in shape and develop brown or necrotic small areas. Diseased fruits may show internal browning of the flesh and reduced quality. In some cases the diseased fruits drops prematurely from the tree. In general early varieties are much more sensitive for symptom expression on fruits than late varieties. Stones from diseased apricot fruits show pale rings or spots. Identification Sampling Appropriate sample selection is critical for serological or molecular detection. If typical symptoms are present, symptomatic flowers, leaves or fruits should be collected. In symptomless plants, a standard sample should be taken of 5 shoots or 10 fully expanded leaves collected around the canopy of each individual tree from the middle of each scaffold branch, until the appearance of high temperatures at the beginning of summer. Sampling from July to the beginning of September should be avoided in Mediterranean climates. Plant material should preferably be selected from the internal structure of the tree. Samples in spring can be flowers, young shoots or small fruits. Mature leaves can be collected for analysis in autumn. Samples can be stored at 4 °C for not more than 7 days before processing. Fruits can be stored for 1 month at 4 °C. Dormant buds or bark from shoots or branches can be selected in winter. Preparation of the sample for testing Approximately 1 g of plant material is weighed, cut into small pieces and placed in a suitable tube or plastic bag for processing. Approximately 20 volumes of extraction buffer are added (Cambra et al., 1994) and the sample is homogenized in tubes using a Polytron (Kinematica) or similar apparatus. Alternatively, the sample can be homogenized in plastic bags using the Homex 6 machine (Bioreba) or a manual roller, hammer, or similar implement. The composition of the extraction buffer is: phosphate-buffered saline (PBS) pH 7.2–7.4 (Appendix 1), supplemented with 2% Polyvinylpyrrolidone (PVP-10) and 0.2% sodium diethyl dithiocarbamate (DIECA). Samples for serological testing can be prepared in tubes or in plastic bags. Samples for molecular testing should be prepared in individual plastic bags. Biological testing The object of a biological test is to detect the presence of PPV in plant accessions or selections, or in samples whose sanitary status is to be assessed. The main indicator plants used for PPV testing are: seedlings of Prunus persica GF 305 or Nemaguard, or Prunus tomentosa. The indicators should be graft-inoculated according conventional methods (Desvignes, 1999), with 4–6 replicates, and held under standard conditions. Symptom onset should be compared with positive and negative control plants. Serological tests DASI-ELISA (Double Antibody Sandwich Indirect ELISA), or Triple Antibody Sandwich (TAS), is performed according to Cambra et al. (1994) using the detailed protocol described in Appendix 3 and materials (specific monoclonal antibody 5B-IVIA) described in Appendix 1, for universal detection of any PPV isolate. Characterization or typing of PPV-D or PPV-M can be performed following the protocol described in Appendix 3 and materials (specific D or M monoclonal antibodies) described in Appendix 1. Conventional (biotin/streptavidin system) DAS-ELISA is performed according to Clark & Adams (1977) using the detailed protocol described in Appendix 3 and materials (specific monoclonal antibody 5B-IVIA or polyclonal antibodies) described in Appendix 1, for universal detection of any PPV isolate. Molecular tests Immunocapture RT-PCR (IC-RT-PCR) The immunocapture phase and the RT-PCR are performed according to Wetzel et al. (1991b, 1992), Olmos et al. (1997) and Rosner et al. (1998), using the detailed protocol described in Appendix 4 and materials described in Appendix 2 (oligonucleotide primer sequences and buffers), for universal detection of any PPV isolate. Characterization or typing of PPV-D or PPV-M can be performed following the protocol described in Appendix 4 and materials (PD and PM specific primers) described in Appendix 2. Characterization can also be done by molecular hybridization of IC-RT-PCR amplification products (P1/P2 primers) using specific D and M probes as described in Appendix 4 and Appendix 2. Co-operational amplification (Co-PCR) Co-PCR using purified RNA is performed according to Olmos et al. (2002) using the detailed protocol described in Appendix 4 and materials described in Appendix 2 (oligonucleotide primer sequences, buffers and probe), for universal detection of any PPV isolate. Characterization or typing of PPV-D or PPV-M can be performed following the protocol described in Appendix 4 and materials (specific D and M probes) described in Appendix 2. Possible confusion with similar species None. Requirements for a positive diagnosis If PPV is diagnosed for the first time, or in critical cases (import/export), the following should be performed and provided (see also Fig. 3): Figure 3Open in figure viewerPowerPoint Decision scheme for detection and identification of Plum pox potyvirus. • the original sample (with labels, if applicable) should be kept under proper conditions as long as possible. Sample extract and PCR amplification product should be kept at −80 °C for 3 months (or longer for legal purposes) • the combination of two different screening methods, based on biological tests (inoculation of woody indicator plants), on serological tests or on molecular detection (with the validated protocols and reagents) is required to support a positive PPV detection • to type a PPV isolate from a PPV-infected host, a characterization method based on DASI-ELISA, IC RT-PCR or molecular hybridization, using specific reagents, is required. For first findings of PPV, at least PPV coat protein (CP) should be sequenced for comparison with previously described D, M, EA and C PPV types. 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 plant 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: Instituto Valenciano de Investigaciones Agrarias (IVIA), Department Protección Vegetal y Biotecnología, Carretera de Moncada-Náquera km 5, 46113 Moncada (Valencia) Spain. E-mail: mcambra@ivia.es Footnotes 1 The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. 2 E Grabensteiner and B Suárez (Osterreichische Agentur für Gesundheit und Ernährungssicherheit Gmbh, Wien, AT); J Kummert, S Steyer and E Demonty (Centre de Recherches Agronomiques, Gembloux, BE); P Gentit, N Grasseau & F Chappoux (CTIFL, Prigonrieux, FR); VMJ Boeglin, ENSA-INRA, Montpellier, FR); T Candresse, MJ Delucq & L Svanella-Dumas, INRA, Villenave d’Ornon, (FR); W Jarausch & G Krczal, Centrum Grüne Gentechnik, SLFA Neustadt, DE; C Varveri, Benaki. Phytopathological Institute, Kifissia, (GR); L Krizbai, D Sebestyén, I Ember & M Kölber, Central Laboratory for Pest Diagnosis, Gödollo, (HU); A Myrta & N Abou-Ghanem, Istituto Agronomico Mediterráneo, Valenzano, (IT); O Potree, D Boscia, A Minafra, L Barbarossa & M Al Rwahnih, Istituto di Virologia Vegetale del CNR, Bari, (IT); G Pasquinic & M Barba, Istituto Sperimentale per la Patología Vegetale, Roma, (IT); P Martínez-Gómez, M Rubio & F Dicenta. Campus Universitario de Espinardo, Murcia, (ES); RF González & C Muñoz Noguera, Laboratorio de Sanidad Vegetal de Sevilla, Montequinto, (ES); MÁ Cambra & ML Palazón, Centro de Protección Vegetal, Zaragoza, (ES); E Bertolini, MC Martínez & MT Gorris, IVIA, Laboratorio de Serología, Moncada, Valencia, (ES); R Mumford, A Blockley, B Jarvis & V Harju, Central Science Laboratory, York, (GB). Acknowledgements This protocol was originally drafted by: M. Cambra, A. Olmos and M. T. Gorris, Instituto Valenciano de Investigaciones Agrarias (IVIA), Department Protección Vegetal y Biotecnología, Carretera de Moncada-Náquera km 5, 46113 Moncada (Valencia), Spain. This protocol was ring-tested in different European laboratories22 E Grabensteiner and B Suárez (Osterreichische Agentur für Gesundheit und Ernährungssicherheit Gmbh, Wien, AT); J Kummert, S Steyer and E Demonty (Centre de Recherches Agronomiques, Gembloux, BE); P Gentit, N Grasseau & F Chappoux (CTIFL, Prigonrieux, FR); VMJ Boeglin, ENSA-INRA, Montpellier, FR); T Candresse, MJ Delucq & L Svanella-Dumas, INRA, Villenave d’Ornon, (FR); W Jarausch & G Krczal, Centrum Grüne Gentechnik, SLFA Neustadt, DE; C Varveri, Benaki. Phytopathological Institute, Kifissia, (GR); L Krizbai, D Sebestyén, I Ember & M Kölber, Central Laboratory for Pest Diagnosis, Gödollo, (HU); A Myrta & N Abou-Ghanem, Istituto Agronomico Mediterráneo, Valenzano, (IT); O Potree, D Boscia, A Minafra, L Barbarossa & M Al Rwahnih, Istituto di Virologia Vegetale del CNR, Bari, (IT); G Pasquinic & M Barba, Istituto Sperimentale per la Patología Vegetale, Roma, (IT); P Martínez-Gómez, M Rubio & F Dicenta. Campus Universitario de Espinardo, Murcia, (ES); RF González & C Muñoz Noguera, Laboratorio de Sanidad Vegetal de Sevilla, Montequinto, (ES); MÁ Cambra & ML Palazón, Centro de Protección Vegetal, Zaragoza, (ES); E Bertolini, MC Martínez & MT Gorris, IVIA, Laboratorio de Serología, Moncada, Valencia, (ES); R Mumford, A Blockley, B Jarvis & V Harju, Central Science Laboratory, York, (GB). . Appendices Appendix 1. Materials Materials for detection and characterization of PPV in plant tissues by serological tests Standard PPV-infected and healthy controls and PPV-specific monoclonal antibodies are commercially available, or can alternatively be obtained by non-profit institutions at Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera km 5. 46113 Moncada (Valencia), Spain and at Instituto di Virologia Vegetale del CNR, Sezione di Bari, via Amendola 165/A, I-70126 Bari, Italy. (Dr D. Boscia, E-mail: csvvdb08@area.ba.cnr.it) Complete DAS-ELISA or DASI-ELISA kits based on specific monoclonal antibodies 5B-IVIA (PPV universal), 4D (PPV-D specific) and AL (PPV-M specific), for PPV detection and characterization, are commercially available from REAL (validated in ring tests), CE Durviz S.L., Parque Tecnológico de Valencia, Leonardo Da Vinci 10, 46980 Paterna (Valencia), Spain http://www.durviz.co; Grittiest S.R. (validated in ring tests), Str. Prove. Per Casamassima Km. 3, I-70010 Valenzano, Italy http://www.agritest.it; Agdia Incorporated, 30380 County Road 6, 46514 Elkart, USA http://www.agdia.com. Complete DAS-ELISA kits (conventional) for universal PPV detection, based on polyclonal or monoclonal antibodies (different from 5B-IVIA) are commercially available from: Adgen Limited, Nellies Gate.Anchincruive, Ayr KA6 5HW (GB) http://www.adgen.co.uk; BIORAD Laboratories-SANOFI, Rue Raimond Poincaré 3-BD, 92430 Marnes La Coquette (FR) http://www.bio-rad.com; Bioreba, Chr. Merian-Ring 7, 4153 Reinach BL1 (CH) http://www.bioreba.ch; DSMZ, Deustche Sammlung von Mikroorganismen und Zellkulturen GmbH, Messenweg 11/12, 38104 Braunschweig (DE) http://www.dsmz.de/nf-plvirus; Hortitech, Hortitech Diagnostic & Crop Protection Services, Horticulture Research International, Stockbridge House, Cawood, YO8 3TZ Selby (GB) E-mail: plantclinic.sh@hri.ac.uk; LOEWE Biochemica GmbH, Mühiweg 2a, D-82054 Sauerlach (DE) http://www.loewe-info.com; Plant Research International B.V., PO Box 16, 6700 AA Wageningen (NL) http://www.plant.wageningen-ur.nl. For the biotin/streptavidin system, they are available from: INGENASA, Hermanos García Noblejas 41, 2a planta, 28037 Madrid (ES) http://www.ingenasa.es. Alkaline-phosphatase linked goat antimouse immunoglobulins Cat No A-3562 are available from Sigma (Steinhein), Germany, and streptavidin alkaline phosphatase linked. goat antimouse immunoglobulins Cat no. 1089 161 from Roche Diagnostics GmbH, Mannheim, Germany. Buffers Phosphate-Buffered Saline (PBS) pH 7.2–7.4: NaCl 8 g, KCl 0.2 g, Na2HPO4 × 12H2O 2.9 g, KH2PO4 0.2 g, distilled water 1 L. Carbonate buffer pH 9.6: Na2CO3 1.59 g, NaHCO3 2.93 g, distilled water 1 L. Washing buffer (PBS, pH 7.2–7.4 with 0.05% Tween 20): NaCl 8 g, KCl 0.2 g, Na2HPO4 × 12H2O 2.9 g, KH2PO4 0.2 g, Tween 20 500 µL, distilled water 1 L. Substrate buffer for alkaline phosphatase: diethanolamine 97 mL; dilute in 800 mL of distilled water; adjust pH 9.8 with concentrated HCl; adjust to 1000 mL with distilled water. Materials for detection and characterization of PPV in plant tissues by molecular tests Standard PPV-infected and healthy controls and PPV-specific oligonucleotide primer and probe sequences are available for non-profit institutions at Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera km 5. 46113 Moncada (Valencia), Spain and at Institute National de Recherche Agronomique (INRA), Centre de Bordeaux, Equipe de Virologie UMR GD2P, IBVM., BP 81, 33883 Villenave d’Ornon Cedex, France. (Dr Thierry Candresse, E-mail: tc@bordeaux.inra.fr). RNA purification kit (validated in ring tests) is available from Rneasy Plant Mini Kit – Cat no. 74904 – Qiagen GmbH (Hilden), Germany. The oligonucleotide primer sequences (validated in ring tests) are: • P10: 5′–3′ GAG AAA AGG ATG CTA ACA GGA • P20: 5′–3′ AAA GCA TAC ATG CCA AGG TA • P1: 5′–3′ ACC GAG ACC ACT ACA CTC CC • P2: 5′–3′ CAG ACT ACA GCC TCG CCA GA • PD: 5′–3′ CTT CAA CGA CAC CCG TAC GG • PM: 5′–3′ CTT CAA CAA CGC CTG TGC GT The oligonucleotide 3′DIG labelled probe sequences (validated in ring tests) are: • PPV Universal Probe: TCG TTT ATT TGG CTT GGA TGG AA-Digoxigenin • PPV-D Specific Probe: CTT CAA CGA CAC CCG TAC GGG CA-Digoxigenin • PPV-M Specific Probe: AAC GCC TGT GCG TGC ACG T-Digoxigenin Colorimetric detection of amplicons (validated in ring tests) is available from Roche Diagnostics GmbH, Cat no. 1585 762 (DIG Wash and Block Buffer Set), Cat no. 1093 274 (Anti-Digoxigenin-AP Fab fragments), Cat no. 1465 341 (Multicolor Detection Set) – Mannheim, Germany. Buffers and substrate solutions Carbonate buffer pH 9.6: Na2CO3 1.59 g, NaHCO3 2.93 g, distilled water 1 L Washing buffer (PBS, pH 7.2–7.4 with 0.05% Tween 20): NaCl 8 g, KCl 0.2 g, Na2HPO4 × 12H2O 2.9 g, KH2PO4 0.2 g, Tween 20 500 µL, distilled water 1 L. 50X TAE buffer: Tris 242 g, 0.5 m Na2EDTA pH 8.0100 mL, glacial acetic acid 57.1 mL, distilled water to 1 L. Loading buffer: 0.25% bromophenol blue, 30% glycerol in H2O Buffer 20X SSC: 3M NaCl, 300 mm sodium citrate, pH 7.0; dissolve 175.3 g NaCl and 88.2 g sodium citrate-2H2O in 800 mL H2O, 2 g KCl, Na2HPO4-7H2O and 2.4 g KH2PO4 in 800 mL H2O; adjust pH 7.4 with HCl; adjust volume to 1 L; sterilize by autoclaving. SDS 10% (w/v) in sterile water, filtered through a 0.2–0.45 µm membrane; dissolve 100 g sodium dodecyl sulphate crystals (SDS) in 900 mL H2O; heat to 68 °C to dissolve crystals; adjust pH to 7.2 with HCl (about 50 µL): adjust volume to 1 L with H2O. Maleic acid buffer: 100 mm maleic acid, 150 mm NaCl, pH 7.5; this buffer 10X is available in a ready-to-use form in the Roche DIG Wash and Block Buffer Set (Cat. no. 1585762); bottle (2). Washing buffer: 100 mm maleic acid, 150 mm NaCl, pH 7.5, 0.3% (v/v) Tween 20; this buffer 10X is available in a ready-to-use form in the Roche DIG Wash and Block Buffer Set (Cat. no. 1585762); bottle (1). Blocking reagent solutions: for stock solution 10% (w/v), dissolve 10 g blocking reagent (Roche, Cat. no. 1096 176) in 100 mL maleic acid buffer with several 30-s heat pulses in the microwave (3–4 min total); avoid boiling; alternatively dissolve 10 g blocking reagent in 100 mL maleic acid buffer; heat at 60 °C for approximately 1 h until completely dissolved; sterilize by autoclaving; dilute as appropriate with maleic acid buffer; the stock solution 10X (10% w/v) is available in a ready-to-use form in the Roche DIG Wash and Block Buffer Set (Cat. no. 1585762); bottle (3) TE buffer pH 8.0: 10 mm Tris-HCl, 1 mm EDTA. Washing solution 2X: 2X SSC with 0.1% SDS. Washing solution 0.5X: 0.5X SSC with 0.1% SDS. N-lauroylsarcosine: 10% (w/v) in sterile water filtered through 0.2–0.45 µm membrane Formamide: 500 mL formamide, 50 g ion exchange: AG 501-X8 Resin (Bio-Rad); stir 30 min slowly on a stirrer, then remove resin by filtration and store the deionized formamide at 20 °C. Standard hybridization buffer: 5X SSC, 0.1% lauroylsarcosine, 0.02% SDS, 1% w/v blocking solution. Standard hybridization buffer + 30% formamide: 5X SSC, 30% deionized formamide, 0.1% sodium lauroylsarcosine, 0.02% SDS, 2% w/v blocking solution. Standard hybridisation buffer + 50% formamide: 5X SSC, 50% deionized formamide, 0.1% sodium lauroylsarcosine, 0.02% SDS, 2% w/v blocking solution. Anti-digoxigenin-AP stock solution: 750 units/mL antidigoxigenin, Fab fragments (available in Roche Cat. no. 1093274) conjugated to alkaline phosphatase; working concentration 150 mU/mL; dilute antidigoxigenin-AP stock solution 1 : 5000. Detection buffer pH 9.5: 100 mm Tris-HCl, 100 mm NaCl. this buffer 10X is available in a ready-to-use form in the Roche DIG Wash and Block Buffer Set (Cat. no. 1585762; bottle 4). NBT solution: 75 mg/mL nitro blue tetrazolium salt in 70% (v/v) dimethylformamide. BCIP solution: 50 mg/mL 5-bromo-4-chloro-3indolyl phosphate (BCIP) toluidinium salt in 100% dimethylformamide; mix 45 µL NBT solution and 35 µL BCIP solution in 10 mL of detection buffer. Appendix 2. Detailed protocols for serological tests DASI-ELISA (Cambra et al., 1994) 1 Prepare an appropriate dilution of rabbit-PPV polyclonal immunoglobulins (usually 1–2 µg mL−1) in carbonate buffer pH 9.6 (Appendix 1). Add 200 µL to each well. Incubate at 37 °C for 4 h or at 4 °C for 16 h. Wash the wells three times with PBS-Tween (washing buffer) (Appendix 1). 2 Add 200 µL per well of the plant extract (see sample preparation). Use two wells of the plate for each sample or positive controls and at least two wells for negative controls. Incubate at 4 °C for 16 h. Wash as before. 3 Add specific monoclonal antibodies for universal detection of any PPV isolate or for selective detection of PPV-D or PPV-M (Appendix 1: prepare an appropriate dilution of the monoclonal antibodies 5B-IVIA (0.1 µg mL−1 in PBS with 0.5% bovine serum albumin-BSA) for universal PPV detection (Appendix 1. For specific PPV-D detection with 4D monoclonal antibody or PPV-M detection with AL monoclonal antibody (Appendix 1), proceed as above. Add 200 µL to each well. Incubate at 37 °C for 2 h. Wash as before. 4 Add antimouse immunoglobulins conjugated with alkaline phosphatase: prepare an appropriate dilution of antimouse immunoglobulins conjugated with alkaline phosphatase (Appendix 1) in PBS plus 0.5% BSA. Add 200 µL to each well. Incubate at 37 °C for 2 h. Wash as before. 5 Prepare 1 mg mL−1 alkaline phosphatase solution (p-nitrophenylphosphate) in substrate buffer (Appendix 1). Add 200 µL to each well. Incubate at room temperature and read at 405 nm after 30, 60 and 90 min. The ELISA test is negative if the absorbance of the sample is less than two times the absorbance of the healthy control. The ELISA test is positive if the absorbance of the sample is equal or greater than two times the absorbance of the healthy control. DAS-ELISA (Clark & Adams, 1977) conventional or biotin/streptavidin system 1 Prepare an appropriate dilution of polyclonal antibodies or monoclonal antibody 5B-IVIA (Appendix 1) (usually 1–2 µg mL−1) in carbonate buffer pH 9.6 (Appendix 1). Add 200 µL to each well. Incubate at 37 °C for 4 h or at 4 °C for 16 h. Wash the wells three times with PBS-Tween (washing buffer) (Appendix 1). 2 Add 200 µL per well of the plant extract (see sample preparation). Use two wells of the plate for each sample or positive controls and at least two wells for negative controls. Incubate at 4 °C for 16 h. Wash as before. 3 Add specific monoclonal antibodies 5B-IVIA or polyclonal antibodies linked with alkaline phosphatase or biotin (Appendix 1) for universal detection of any PPV isolate: prepare an appropriate dilution of the conjugated antibodies (about 0.1 µg mL−1 in PBS with 0.5% bovine serum albumin-BSA). Add 200 µL to each well. Incubate at 37 °C for 3 h. Wash as before. 4 When antibodies are linked with biotin, use an appropriate dilution of streptavidin-alkaline phosphatase conjugated (Appendix 1). Add 200 µL to each well. Incubate at 37 °C for 30 min and wash as before. For both methods (conventional or biotin/streptavidin), prepare 1 mg mL−1 alkaline phosphatase solution (p-nitrophenylphosphate) in substrate buffer. Add 200 µL to each well. Incubate at room temperature and read at 405 nm after 30, 60 and 90 min. The ELISA test is negative if the absorbance of the sample is less than two times the absorbance of the healthy control. The ELISA test is positive if the absorbance of the sample is equal or greater than two times the absorbance of the healthy control. Appendix 3. Detailed protocols for molecular tests IC-RT-PCR (Wetzel et al., 1991b, 1992; Olmos et al., 1997) Immunocapture phase (IC) according to Wetzel et al. (1
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