Grapevine flavescence dorée phytoplasma

Abstract

EPPO BulletinVolume 37, Issue 3 p. 536-542 Free Access Grapevine flavescence dorée phytoplasma First published: 07 December 2007 https://doi.org/10.1111/j.1365-2338.2007.01161.xCitations: 8 European and Mediterranean Plant Protection Organization Organisation Européenne et Méditerranéenne pour la Protection des Plantes AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat PM 7/79 Specific scope This standard describes a diagnostic protocol for Grapevine flavescence dorée phytoplasma. Specific approval and amendment Approved in 2005-09. Introduction Grapevine flavescence dorée phytoplasma (FD) belongs to the elm yellows group (16 SrV). It is one of a complex of disorders of Vitis vinifera, known as grapevine yellows, associated with the presence of phytoplasmas (Caudwell et al., 1971). Though characterized by similar symptomatology, these disorders (most of which are localized in distribution) are caused by different phytoplasmas belonging to six out of the twenty subclades so far distinguished on the basis of molecular evidence (Table 1) (Boudon-Padieu, 2003, 2005). The other yellows diseases of grapevine occurring in Europe (Table 1) are widespread in other hosts, and are not considered to be quarantine pests. Spread of flavescence dorée occurs through infected grapevine planting material and through its vector, the cicadellid Scaphoideus titanus. Table 1. Current status of molecular characterization, biology and vectors of phytoplasmas causing grapevine yellows diseases (Boudon-Padieu, 2003, 2005) Grapevine yellows disease Phytoplasma name Ribosomal group (subgroup) Known insect vector to grapevine Preferred host plants of vector Occurrence Flavescence dorée FD 16SrV (-C, -D) or EY Scaphoideus titanus Ball Vitis sp. France, Italy, Spain, Serbia, Slovenia, Switzerland Palatinate grapevine yellows PGY 16SrV or EY Oncopsis alni Schrank Alnus glutinosa L. Germany Bois noir, Legno nero, Vergilbungskrankheit Schwarzholzkrankheit stolbur 16SrXII-A or stolbur Hyalesthes obsoletus Sign Convolvulus arvensis L. Urtica dioica L., Ranunculus, Solanum, Lavandula Europe, Israel, Lebanon Australian grapevine yellows Candidatus Phytoplasma australiense 16SrXII-B ND ND Australia Australian grapevine yellows Candidatus Phytoplasma australasia 16SrII FBP ND ND Australia Buckland valley grapevine yellows (Aus) BVGY 16SrI-related or AY- ND ND Australia grapevine yellows Aster yellows 16SrI (-B, -C) or AY ND ND Italy, Chile North American grapevine yellows (NAGY) Virginia grapevine yellows I (VGY I) 16SrI-A or AY ND ND Virginia (UA) Western X Virginia grapevine yellows III (VGYII)I 16SrIII-I or WX ND ND New York (US) Virginia (US) Identity Name: Grapevine Flavescence dorée phytoplasma Taxonomic position: Bacteria, Firmicutes, Mollicutes, Acholeplasmatales, Acholeplasmataceae Provisional taxon: Phytoplasma Elm Yellows (EY) Group or16SrV Phytosanitary categorization: EPPO A2 list N° 94; EU Annex designation II/A2. Detection Disease symptoms Flavescence dorée can be recognized in the field by the following symptoms, which develop mainly in summer (July onwards). Leaves turn yellow or red depending on the cultivar. They roll downward and become brittle (1-3). The interveinal areas of leaves may become necrotic. Shoots show incomplete lignification and rows of black pustules develop on the green bark along the diseased branches; they are thin, rubbery and hang pendulously. During winter they blacken and die. The inflorescences dry out and fall off. Fruit setting is reduced. In later infections, bunches are irregular and berries become shrivelled. They have a significantly lower sugar content and higher acidity compared to healthy grapes. Flavescence dorée occurs randomly in vineyards (this is perhaps associated with vine-to-vine transmission by the vector). It may occur in rootstocks, but without conspicuous symptoms. Most grapevine cultivars are affected by flavescence dorée. Cv ‘Chardonnay’ is particularly susceptible and sensitive to all grapevine yellows, especially flavescence dorée. Figure 1Open in figure viewerPowerPoint Leaves of grapevine cv. Chardonnay affected by flavescence dorée showing typical yellowing of the laminar blade and rolling of margins. Leaves present a triangular shape [photograph courtesy of Dr. Federico Bondaz, Plant Protection Unit of Val d’Aosta region (IT)]. Figure 2Open in figure viewerPowerPoint Leaves of grapevine cv. Bellone affected by stolbur phytoplasmas. Symptoms are indistinguishable from the ones observed in grapevine affected by flavescence dorée. To perform molecular diagnosis is the only way to discriminate phytoplasmas belonging to different groups (Photograph courtesy of Dr. Federico Bondaz, Plant Protection Unit of Val d’Aosta region (IT)). Figure 3Open in figure viewerPowerPoint Leaves of red grapevine cultivars affected by flavescence dorée showing rolling and reddening of the laminae. Symptoms are often present on a single shoot of the plant (Photograph courtesy of Dr. Federico Bondaz, Plant Protection Unit of Val d’Aosta region (IT)). Identification of flavescence dorée sensu stricto Biological assay The biological assay proposed by OEPP/EPPO (1994) is not suitable for reliable or specific detection or identification of flavescence dorée, and is no longer recommended. Serological assay ELISA with polyclonal and monoclonal antibodies have been used for detection of flavescence dorée in vector (Boudon-Padieu et al., 1989) and grapevine (Caudwell & Kuszala, 1992; Kuszala et al., 1993; Kuszala, 1996), but the method relies on availability of antibodies which are not sold commercially. It has been replaced in practice by PCR, which is versatile, specific and sensitive. Molecular methods Two methods can be used, the first one with two variants. In the first method, direct PCR with generic primers for phytoplasma 16S rDNA amplification is followed by nested PCR using group-specific primers (a), or by nested PCR using a second generic primer pair (b). In this latter case, the amplification product can be submitted to RFLP analysis for identification of the causal phytoplasma. The second method is a multiplex nested-PCR assay that allows direct identification of phytoplasmas in the elm yellows (16SrV) (Flavescence dorée) and stolbur (16SrXII) (Bois noir) groups, the two main grapevine yellows present in Europe (c). These methods are described in Appendix 1. Reference material Institut national de la recherche agronomique, Equipe phytoplasmes, UMR PME, INRA, BP 86510, 21065 DIJON Cedex, France. Institut national de la recherche agronomique, UMR GDPP Bordeaux, BP 81, 33883 VILLENAVE D’ORNON Cedex, France. Reporting and documentation Guidelines on reporting and documentation are given in EPPO Standard PM 7/77 Documentation and reporting on a diagnosis. Further information Further information on this organism can be obtained from: Dr E. Boudon-Padieu, Institut national de la recherche agronomique, Equipe phytoplasmes, UMR PME, INRA, BP 86510, 21065 DIJON Cedex, France. Dr. G. Pasquini, CRA-Istituto Sperimentale per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Rome, Italy. Acknowledgements This protocol was originally drafted for EPPO by: Dr Paola Del Serrone, Istituto Sperimentale per la Patologia Vegetale, Rome (IT). References Ahrens U & Seemüller E (1992) Detection of plant pathogenic mycoplasmalike organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82, 828– 832. Angelini E, Clair D, Borgo M, Bertaccini A & Boudon-Padieu E (2001) Flavescence dorée in France and Italy – Occurrence of closely related phytoplasma isolates and their near relationships to Palatinate grapevine yellows and an alder yellows phytoplasma. Vitis 40, 79– 86. Berges R, Rott M & Seemüller E (2000) Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology 90, 1145– 1152. Boudon-Padieu E, Larrue J & Caudwell A (1989) ELISA and dot-blot detection of flavescence doree-MLO in individual leaf hopper vectors during latency and inoculative state. Current Microbiology 19 (6), 357– 364. Boudon-Padieu E (2003) The situation of Grapevine yellows and current research directions: distribution, diversity, vectors, diffusion and control. 14th Meeting of the ICVG. Locorotondo (BARI), Italy. 12–17 Sept. 2003. Extended abstracts session 3, 47– 53. http://www.agr.uniba.it/ICVG2003/ (accessed on 2007-09-07). Boudon-Padieu E (2005) Phytoplasmes de la vigne et vecteurs potentiels/ Grapevine phytoplasmas and potential vectors. Bulletin O.I.V. 78 (891–892), 299– 320. Boudon-Padieu E, Béjat A, Clair D, Larrue J, Borgo M, Bertotto L et al . (2003) Grapevine yellows: comparison of different procedures for DNA extraction and amplification for routine diagnosis of phytoplasmas in grapevine. Vitis 42 (3), 141– 149. Caudwell A, Giannotti J, Kuszala C & Larrue J ((1971) Etude du rôle des particules de type ‘Mycoplasme’ dans l’étiologie de la Flavescence dorée de la vigne. Examen cytologique des plantes malades et des cicadelles infectieuses. Annales de Phytopathologie 3, 107– 123. Caudwell A & Kuszala C (1992). Mise au point d’un test ELISA sur les tissus de vignes atteintes de Flavescence dorée. Research in Microbiology 143, 791– 806. Clair D, Larrue J, Aubert G, Gillet J, Cloquemin G & Boudon-Padieu E (2003) A multiplex nested-PCR assay for sensitive and simultaneous detection and direct identification of phytoplasma in the Elm yellows group and Stolbur group and its use in survey of grapevine yellows in France. Vitis 42, 151– 157. Constable FE, Gibb KS & Symons RH (2003) Seasonal distribution of phytoplasmas in Australian grapevines. Plant Pathology 52, 267– 276. Daire X, Clair D, Reinert W & Boudon-Padieu E (1997) Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and the stolbur subgroup by PCR amplification of non-ribosomal DNA. European Journal of Plant Pathology 103, 507– 514. Davis RE, Dally EL, Gundersen DE, Lee IM & Habili N (1997) ‘Candidatus phytoplasma australiense’, a new phytoplasma taxon associated with Australian grapevine yellows. International Journal of Systematic Bacteriology 47, 262– 269. Deng S & Hiruki C (1991) Amplification of 16S rRNA genes from culturable and non-culturable mollicutes. Journal of Microbiological Methods 14, 53– 61. Doyle JJ & Doyle JI (1990) Isolation of plant DNA from fresh tissue. Focus 12, 13– 15. Gibb K, Padovan AC & Mogen BD (1995) Studies on sweet potato little-leaf phytoplasma detected in sweet potato and other plant species growing in northern Australia. Phytopathology 85, 169– 174. Kollar A, Seemüller E, Bonnet F, Saillard C & Bové JM (1990) Isolation of the DNA of various plant pathogenic Mycoplasmalike organisms from infected plants. Phytopathology 80, 233– 237. Kuszala C (1996) Influence du milieu d’extraction sur la détection du Bois noir et de la Flavescence dorée de la vigne, par des anticorps poly et monoclonaux dirigés contre les phytoplasmes du stolbur et de la Flavescence dorée. Agronomie 16, 355– 365. Kuszala K, Cazelles J, Boulud J, Credi R, Granata G, Kriel G et al . (1993) Contribution à l’étude des jaunisses de la vigne dans le monde. Prospection par test ELISA spécifique du mycoplasma-like organism (MLO) de la Flavescence dorée. Agronomie 13, 929– 933. Lee IM, Bertaccini A, Vibio M & Gundersen DE (1995) Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology 85, 728– 735. Maixner M, Reinert W & Darimont H (2000) Transmission of grapevine yellows by Oncopsis alni (Schrant) (Auchenorrhyncha: Macropsinae). Vitis 39, 83– 84. Marzachì C, Alma A, D’Aquilio M, Minuto G & Boccardo G (1999) Detection and identification of phytoplasmas infecting cultivated and wild plants in Liguria (Italian Riviera). Journal of Plant Pathology 81 (2), 127– 136. OEPP/EPPO (1994) EPPO Standards PM 3/57. Mycoplasma-like organisms in fruit trees and grapevine. Inspection and test methods. Bulletin OEPP/EPPO Bulletin 24, 339– 342. Padovan AC, Gibb KS, Bertaccini A, Vibio M, Bonfiglioli RE, Magarey PA et al . (1995) Molecular detection of the Australian grapevine yellows phytoplasmas and comparison with grapevine yellows phytoplasmas from Italy. Australian Journal of Grape and Wine Research 1, 25– 31. Palmano S (2001) A comparison of different phytoplasma DNA extraction methods using competitive PCR. Phytopathologia mediterranea 40, 99– 107. Schneider B, Cousin MT, Klinkong S & Seemüller E (1995) Taxonomic relatedness and phylogenetic positions of phytoplasmas associated with disease of faba bean, sunhemp, sesame, soybean and eggplant. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 102, 225– 232 (in German). Appendix 1 Molecular methods Sampling The titer of phytoplasmas is usually low in woody hosts (Berges et al., 2000) and it varies in grapevine according to organ and season (Constable et al., 2003). Samples should be collected in July-September, selecting leaves showing symptoms but in good condition (no necrotic areas) and not affected by other pests. At least 20 leaves per plant should be randomly collected, and midribs and veins (to a total of about 1.5 g) separated. Material for diagnostic assays should be used fresh, or else ground in liquid nitrogen and stored at a maximum of –20°C (or lower depending on the storage time e.g. –80°C for more than two years). DNA extraction Several methods have been developed and compared (Palmano, 2001; Boudon-Padieu et al., 2003). Two methods are described below. The first one uses tissue disruption and phytoplasma enrichment (Ahrens & Seemüller, 1992) then DNA extraction is performed following a method from Doyle & Doyle (1990) and slightly modified by Marzachìet al., 1999. The second method which is more rapid consists of grinding grapevine tissues in CTAB buffer. Originally used at lower concentrations of Tris and CTAB for extraction of nucleic acids from periwinkle (Kollar et al., 1990), a CTAB concentration of 3% in 1M Tris (Boudon-Padieu et al., 2003) has been found to be optimum for grapevine and other woody plants that contain high quantities of phenolics, tannins and acids. Phytoplasma enrichment procedure and DNA extraction The following procedure has been validated during a ring test organised in Italy to establish a common protocol for DNA extraction from grapevine. Grind 1.5 g of the fresh midveins in a sterile cold mortar and pestle with 7–8 mL of phytoplasma grinding buffer (PGB) freshly prepared (see Appendix 2) and 50 mg of sterile quartz sand (Sigma, cod. S9887). Incubate 10–15 min in ice. Add another 5 mL of PGB and homogenize thoroughly. Transfer into 15 mL tubes (Corex) and centrifuge in a pre-cooled rotor (Beckman JA 20) at 5000 rpm for 5 min (4°C). Transfer the supernatant in a cold clean Corex tube. Centrifuge at 19 000 rpm for 20 min (Beckman JA 20). Dry the pellet and re-suspend with 2 mL of 3% CTAB buffer (see Appendix 2), pre-warmed at 60°C. Incubate 10–20 min at 60°C with gentle agitation. Transfer 1 mL into a 2 mL clean Eppendorf tube and extract DNA by adding 1 mL of chloroform:isoamyl alcohol (24:1). Vortex and centrifuge at 6000 rpm for 10 min. Collect the aqueous phase and place it into a clean Eppendorf tube. Add 1 mL of cold isopropanol and incubate 5 min in ice. Centrifuge at 12 000 rpm for 20 min, discard the supernatant and wash the pellet with 1 mL of 70% ethanol. Centrifuge at 12 000 rpm for 10 min and dry the pellet. Re-suspend the pellet DNA in 400 µL TE (see Appendix 2) and precipitate in 900 µL of 95% ethanol and 40 µL of 3 M sodium acetate pH 5.2 at –80°C for 40 min or at –20°C overnight. Centrifuge at 12 000 rpm for 20 min, discard the supernatant and wash the pellet with 1 mL of 70% ethanol. Centrifuge at 12 000 rpm for 10 min and then re-suspend the pellet in 100 µL TE or dH2O. As an alternative commercial kits (e.g. DNeasy, Qiagen) can be used for DNA extraction. CTAB procedure for Nucleic acids extraction (Boudon-Padieu et al., 2003) This method was found to be almost as efficient as the above method in comparisons (Boudon-Padieu et al., 2003), however it is much more rapid and does not require multiple precipitation steps. Nucleic acids can be extracted from fresh or frozen (–20°C or –80°C) tissues (preferably veins or petioles) of grapevine. Grind 1 g of tissue using a ball-bearing apparatus in 7 mL of extraction buffer at room temperature. Transfer 1 mL of the suspension to an Eppendorf tube and incubate for 20 min at 65°C. Then add an equal volume of chloroform. Recover the aqueous phase and precipate the nucleic acids with an equal volume of cold isopropanol. Centrifuge to recover the precipitate, wash the pellet with 70% ethanol, dry and dissolve in 150 µL of TE buffer. Amplification a) Direct generic PCR followed by nested group-specific PCR • Direct generic PCR The P1/P7 primers are recommended (Deng & Hiruki, 1991; Schneider et al., 1995), with the following sequences: P1: 5′-AAG AGT TTG ATC CTG GCT CAG GAT T-3′ P7: 5′-CGT CCT TCA TCG GCT CTT-3′ These primers amplify the whole length of 16S and intergenic 16S–23S and a small part of 23S rRNA gene. Reaction mixture is as follows: 10X PCR buffer 5 µL; 25 mM MgCl2 3 µL, 10 mM dNTPs 4 µL; primer P1 20 µM 1 µL; primer P7 20 µM 1 µL; Taq polymerase 5 U µL−1 0.3 µL; DNA extract 2 µL diluted 1:10; water to 50 µL. PCR is then conducted as follows: 1 cycle at 95°C for 3′; 35 cycles as follows: 94°C for 1 min, 50°C for 2 min; 72°C for 3 min; final extension 72°C for 5 min. This step confirms the presence of a phytoplasma. • Nested PCR with group 16SrV specific primers This step uses the specific primers R16(V)F1 and R16(V)R1 (Lee et al., 1995), with the following sequences: R16(V)F1: 5′-TTA AAA GAC CTT CTT CGG-3′ R16(V)R1: 5′-TTC AAT CCG TAC TGA GAC TAC C-3′ Reaction mixture and PCR cycles are as for the direct PCR, substituting the new primers in the same amounts. The DNA is provided as 2.0 µL of the product of the P1/P7 PCR, diluted 1:40. The product is visualized on a 1.0% agarose gel stained with ethidium bromide. If necessary, RFLP analysis, using Bfa I enzyme, can be performed to distinguish grapevine flavescence dorée sensu stricto from other phytoplasmas belonging to the same group (elm yellow group), in particular Palatinate grapevine yellows. b) Direct generic PCR followed by nested generic PCR and RFLP analysis After the first generic PCR with primers P1 and P7 as described above, the obtained amplicon product is diluted 1:100 and used as the target in a nested PCR procedure with primers fU5 and rU3 (Lorenz et al., 1995) or with primers 16r758f (Gibb et al., 1995) and M23Sr (Padovan et al., 1995) with the following sequences: fU5: 5′-CGG CAA TGG AGG AAA CT-3′ rU3: 5′-TTC AGC TAC TCT TTG TAA CA-3′ 16f758f: 5′-GTC TTT ACT GAC GCT GAG GC-3′ M23Sr: 5′-TAG TGC CAA GGC ATC CAC TGT G-3′ PCR conditions are as follows: Reaction mixture: 0.5 µM each primer, 0.25 mM each dNTP, 1.5 U Appligen Taq polymerase and buffer supplied with the enzyme, 1 µL of the diluted first PCR product, in a total reaction volume of 25 µL. Amplification is carried out for 35 cycles under the conditions described in Table 2. PCR products are then submitted to digestion with restriction enzymes for phytoplasma group or subgroup identification. Table 2. Amplification conditions for direct generic PCR followed by nested generic PCR and RFLP analysis Primer pair Predenaturation Denaturation Annealing Elongation Final elongation fU5/rU3 92°C 120 s 92°C 30 s 57°C 30 s 72°C 50 s ” 16r758f/M23Sr 92°C 120 s 92°C 60 s 50°C 120 s 72°C 180 s ” RFLP analysis for characterization of phytoplasma group on the fU5/rU3 amplicon Five to 20 µL of the fU5/rU3 amplification product is submitted to hydrolysis with Tru9I restriction enzyme at 65°C following the manufacturers instruction. Restriction products are analysed with 10% acrylamide gel electrophoresis and size of products is evaluated using MW ladder pBR 322/HaeIII (Appligene) (Fig. 4). Figure 4Open in figure viewerPowerPoint Polyacrylamide gel (10%) RFLP analysis of phytoplasma rDNA fU5/rU3 fragment after digestion with Tru9I restriction enzyme. Lanes: pBR322/HaeIII, Appligene ladder; ULW, elm yellows (Morvan isolate from France); FD70 and FD92, FD reference isolates from France; FD-D and FD-C, FD isolates from Italy (FD92 and FD-D are similar in all respects up to now); EAY, Eastern aster yellows; STOL, stolbur; BN, Bois noir –affected grapevine (stolbur phytoplasma). RFLP analysis for characterization of 16SrV subgroup on the 16r758f/M23Sr amplicon The 16r758f/M23Sr PCR product is submitted to a RFLP analysis with TaqI enzyme and the product is visualized with 10% acrylamide gel electrophoresis or 3% agarose gel, stained with ethidium bromide (Angelini et al., 2001) (Fig. 5). The size of the products is evaluated using MW ladder pBR 322/HaeIII (Appligene). Figure 5Open in figure viewerPowerPoint Polyacrylamide gel (10%) RFLP analysis of rDNA fragment 16r758f/M23Sr after digestion with TaqI restriction enzyme. Lanes: pBR322/HaeIII, Appligene ladder; ULW, elm yellows (Morvan isolate from France); ALY, alder yellows (Marcone isolate from Italy); FD70 and FD92, FD reference isolates from France; FD-D and FD-C, FD isolates from Italy (FD92 and FD-D are similar in all respects up to now); PGY, Palatinate grapevine yellows (Maixner PGY-A isolate), naturally transmitted from alder to grapevine by Oncopsis alni (Maxiner et al. 2000). c) Multiplex nested-PCR for simultaneous FD and BN detection This bi-specific multiplex nested-PCR procedure was developed (Clair et al., 2003) to amplify simultaneously two non-ribosomal DNA fragments, of 1150 bp and 720 bp in length, which are specific for elm yellows-group (16SrV) and stolbur-group (16SrXII) phytoplasmas, respectively. Phytoplasma in groups 16SrV and 16SrXII are then identified using agarose gel electrophoresis of amplification products on the basis of the size of the band obtained. The method is the official method in France (JORF N°112, May 14th, 2004, p. 08635). RFLP analysis of the FD9 product permits characterization of subgroup within group 16Sr V. Sequences of primers for amplification of FD9 and STOL11 non-ribosomal DNA fragments are indicated in Table 3. Table 3. Sequences of primers for amplification of FD9 and STOL11 non-ribosomal DNA fragments Primer name Sequence bp Reference Fragment size FD9f1 5′-GAATTAGAACTGTTTGAAGACG-3′ 22 Daire et al. (1997) 1300 bp FD9r1 5′-TTTGCTTTCATATCTTGTATCG-3′ 22 Daire et al. (1997) STOL11f2 5′-TATTTTCCTAAAATTGATTGGC-3′ 22 Daire et al. (1997) 830 bp STOL11r1 5′-TGTTTTTGCACCGTTAAAGC-3′ 20 Daire et al. (1997) FD9f3b 5′-TAATAAGGTAGTTTTATATGACAAG-3′ 25 Clair et al. (2003) 1150 bp FD9r2 5′-GACTAGTCCCGCCAAAAG-3′ 18 Angelini et al. (2001) STOL11f3 5′-ACGAGTTTTGATTATGTTCAC-3′ 21 Clair et al. (2003) 720 bp STOL11r2 5′-GATGAATGATAACTTCAACTG-3′ 21 Clair et al. (2003) Reaction mixture for direct PCR are as follows: extracted DNA 1 µL, each dNTP 150 µM, primer FD9f1/r1 0.375 µM, primer STOL11f2/r1 0.0625 µM, Tris-HCl Buffer pH 9.0 10 mM, MgCl2 2.5 mM, KCl 50 mM, Triton X100 0.1%, BSA 0.2 mg mL−1, Taq Polymerase (Q. Biogene) 0.2 U in a total volume of 20 µL. Reaction mixture for Nested-PCR are as follows: extracted DNA,1 µL (1:1000 first amplification product), each dNTP 150 µM, Primer FD9f3b/r2 0.375 µM, primer STOL11f3/r2 0.375 µM, Tris-HCl Buffer pH 9.0 10 mM, MgCl2 2.5 mM, KCl 50 mM, Triton X100 0.1%, BSA 0.2 mg mL−1, Taq Polymerase (Q. Biogene) 0.2 U in a total volume of 20 µL. PCR conditions: Predenaturation 92°C for 90 s, then 30 cycles for the first PCR or 35 cycles for the nested PCR, with denaturation 92°C for 40 s, hybridization 55°C for 40 s, elongation 72°C for 70 s. Separate amplicons with 1.2% Agarose gel electrophoresis, visualize with UV light after staining with ethidium bromide (Fig. 6). Figure 6Open in figure viewerPowerPoint Agarose gel electrophoresis of amplicons obtained with Multiplex-nested PCR for flavescence dorée and bois noir (BN) detection in grapevines. Lane M: 1 kb ladder. Lane 1–13: suspected GY-infected grapevines from different cultivars (FD: lane 1, 2, 4, 5, 10–12; BN: lane 3, 6, 7, 8, 13; double negative: lane 9). Lane 14: healthy grapevine extract. Lane 15: water control. Lane 16: double FD+stolbur (BN)-infected periwinkle (by D. Clair, INRA Dijon (FR), according to Clair et al., 2003). RFLP analysis for characterization of the 16SrV subgroup on the FD9 amplicon The FD9f3b/r2 PCR product is submitted to a RFLP analysis with Tru9I enzyme and the product is visualized after 10% acrylamide gel electrophoresis or 3% agarose gel, stained with ethidium bromide (Angelini et al., 2001) (Fig. 7). Figure 7Open in figure viewerPowerPoint Polyacrylamide gel (10%) RFLP analysis of 16SrV-group phytoplasma DNA fragment FD9 f3b/r2 after digestion with Tru9I restriction enzyme. Lanes : pBR322/HaeIII, Appligene ladder; ULW, elm yellows (Morvan isolate from France); FD70 and FD92, FD reference isolates from France; FD-D and FD-C, FD isolates from Italy (FD92 and FD-D are similar in all respects up to now); PGY, Palatinate grapevine yellows (Maixner PGY-A isolate), naturally transmitted from alder to grapevine by Oncopsis alni. Appendix 2 Buffers Phytoplasma grinding buffer (PGB) for 100 mL K2HPO4 anhydre, 1.67 g KH2PO4, 0.41 g Saccharose, 10 g BSA (frac V), 0.15 g PVP P.M. 10.000, 2 g Ascorbic acid, 0.53 g pH 7.6 with KOH drops. Keep in ice until use. CTAB buffer 3% 3% CTAB (cethyl-trimethyl-ammonium bromide) in 1 M Tris-HCl pH8 10 mM EDTA 1.4 M NaCl, 0.1% 2-mercaptoethanol. TE buffer 10 mM Tris, 1 mM EDTA, pH 7.6. Citing Literature Volume37, Issue3December 2007Pages 536-542 FiguresReferencesRelatedInformation