ࡱ> VXU`Ibjbj 4J#""""R R R R T 3)  ((((((($)hQ,(9("" ("l  ((R&,:( *gR 'b(,)03)'fC-(C-<:(C-:((]((3)R R """""" Diversity of 16SrXII phytoplasmas detected in grapevine growing areas worldwide Assunta Bertaccini1*, Samanta Paltrinieri1, Simona Botti1, Bojan Duduk2, Nicola Fiore3, Maria Kolber4, Dijana Skoric5, Ester Torres6, Maurizio Conti7 1DiSTA, Patologia vegetale, Alma Mater Studiorum, University of Bologna, viale Fanin, 42, 40127 Bologna, Italy Telephone/Fax ++390512096723 E-mail Bertaccini_a@biblio.cib.unibo.it 2 Agricultural Research Institute Serbia, Pesticide and Environmental Research Centre, Banatska 31b, 11080 Zemun, Belgrade, Serbia 3 Departamentos de Produccin Agrcola y Sanidad Vegetal, Facultad de Ciencias Agronmicas, Universidad de Chile. Casilla 1004, Santiago. Chile 4 Central Service for Plant Protection and Soil Conservation, Budapest, Hungary 5 Department of Biology, Faculty of Science, University of Zagreb, Maruliev trg 9a, Zagreb, Croatia 6 Laboratori de Sanitat Vegetal, DARP, Generalitat de Catalunya, Barcelona, Spain 7 Istituto di Fitovirologia vegetale, CNR, Strada delle Cacce, 73, Torino , Italy Introduction During the last five years, severe spreading of Bois Nois (BN) phytoplasmas Candidatus Phytoplasma solani, has been described (Bertaccini et al., 2003) in several grapevine-growing areas, where samples of grapevines, other plant species as well as insects vectors, or potential BN vectors were collected. Molecular identification of BN-related phytoplasmas performed on 16S ribosomal gene on grapevine samples, allowed to find some variability that do not appear to be related to the epidemiology of this disease (Bertaccini et al., 2004; Botti et al., 2005). Preliminary results performed on grapevine and on insects Hyalesthes obsoletus Signoret and Reptalus panzeri Lw collected in heavily BN-infected Italian vineyards, indicated that the use of Tuf gene could help in understanding spreading of BN phytoplasmas (Botti et al., 2005). Further research was performed in several grapevine growing regions worldwide, in order to evaluate the usefulness of Tuf gene polymorphism in BN epidemiological studies. Material and Methods Grapevine samples collected since 2002 onwards in several regions of Northern Italy [Emilia Romagna (MO, RE, BO, RA), Veneto (PD, VE, VR), Lombardy (BS), Piedmont (AT) and of Central and Southern Italy [Umbria (PG), Tuscany (SI), and Apulia,( TA)] were analyzed together with samples from Spain (Catalonia), Hungary (Eger, Tolna and Mecsekalja regions), Croatia, Serbia and Chile. Seventy-eight samples of different plant species growing in, or in close proximity of BN infected vineyards in Modena (MO), Reggio Emilia (RE), and Asti (AT) Italian provinces, as well as from Rasina County in Serbia were tested. Two hundred and seventy samples of H. obsoletus, and 190 of R. panzeri (frequently captured in vineyards in which BN phytoplasmas were detected: Palermo et al., 2004), collected in MO and RE provinces were also tested. Nucleic acids of plants and insects were extracted following different protocols, from 1 g of phloem tissue, or insect batches, respectively. Direct PCR with universal primer pair P1/P7, followed by nested PCRs using primer pairs F1/B6, R16F2/R2, and/or R16(I)F1/R1 was carried out. PCR products were digested with TruI, RsaI, HhaI, and Tsp509I restriction enzymes. In some cases cloning and sequencing of amplified products were also performed following described procedures (Botti and Bertaccini, 2004). Phytoplasmas showing affinity to the ribosomal subgroup 16SrXII were subjected to molecular characterization using Tuf gene primers: Tuf1f/r in direct PCR, and primers TufAYf/r (Schneider et al., 1997), and TufINT1f/TufINT4r (Andersen et al., 2004) in nested PCR reactions. RFLP analyses of all obtained amplicons were performed with HpaII restriction enzyme. Results and Discussion 16S rRNA. In the majority of the tested samples (200 grapevine, 18 other plant samples, and 140 insects) no polymorphism was observed after RFLP analyses of 16S ribosomal gene confirming that Ca. P. solani (stolbur) is associated with grapevine yellows in all the geographical areas surveyed. In only two cases the presence of polymorphisms was ascertained: grapevine samples collected in Chile contained, together with phytoplasmas clearly referable to subgroup 16SrXII-A, one peculiar strain showing, after TruI restriction, a double phytoplasma infection profile, referable to 16SrXII and to 16SrI phytoplasmas respectively. After RFLP with RsaI on R16(I)F1/R1 amplicons, a 16SrXII-A profile was distinguishable from the typical stolbur RFLP profile. After cloning the 16Sr amplicon of this sample and screening by RFLP, ten recombinant colonies were obtained which showed 3 different profiles. Their sequencing revealed that one phytoplasma was referable to the subgroup 16SrI-A (tomato big bud) while the other two (Chile a and b) were homologous with members of 16SrXII phytoplasma group. In particular, Chile a (AY739654) and Chile b ( HYPERLINK "http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=52789115" AY739653) showed 99% and 98% sequence identity to Ca. P. solani, respectively, and the latter showed also 97% identity to Ca. P. australiense. Virtual digestion of these sequences confirmed both the RFLP profiles obtained from cloned amplicons: Chile b profile was identical to the one detected in the original PCR product from grapevine. A 16S ribosomal gene molecular variant was identified also by RFLP with TruI in some of the R. panzeri samples collected in RE and MO provinces. The sequenece of this molecular variant showed 98% identity with Ca. P. solani, however its economic importance remains uncertain since it has not detected so far in either grapevine or other plant species. Tuf gene. Only about 70% of all 16SrXII phytoplasmas detected in the samples tested were amplified with the tuf gene system, however the R. panzeri molecular variant was amplified, and its HpaII restriction profile resulted different from those previously reported (Langer and Maixner, 2004). Therefore, it was designed as the type D. RFLP analyses performed on amplicons from grapevine collected in all the areas investigated from Medicago sativa, Galega spp., Urtica dioica R. panzeri, H. obsoletus from MO and RE provinces, and on Convolvolus arvensis from MO, RE, and Rasina county in Serbia, allowed phytoplasma identification of the type B. Type A phytoplasmas were detected in a very high percentage of samples from grapevine tested in MO, RE, Verona (VR) and Venice (VE) provinces. The same phytoplasma type was sporadically detected in Padua (PD), Bologna (BO), Ravenna (RA), Brescia (BS), Perugia (PG), Siena (SI) provinces in Italy, and in Brodski-Stupnik, Jazbina and Jaska in Croatia. It was also detected in Italy in Parthenocissus quinquefolia from Asti province in mixed infection with type B, and in H obsoletus and R. panzeri from MO and RE provinces. The type A phytoplasma was never detected in herbaceous samples even though it appeared to be the most frequent BN molecular variant in grapevine and H. obsoletus samples collected in BN epidemic areas in Italy. Acknowledgements Work performed with sponsorships of MiPAF- Italy under the project GIAVI, and of CRPV Emilia-Romagna under the project Studio sui giallumi da fitoplasma della vite. References Andersen, M.T., Beever, R.E. & Newcomb, R.D., 2004. Polymorphism in the tuf gene of Candidatus Phytoplsma australiense. In: 15th Congress of IOM, July 2004, Athens Georgia, USA, p. 129. Bertaccini, A., Mori, N., Botti, S., Castiglioni, A., Cavallini, G., & Malossi, A. 2003. Survey on Bois Noir phytoplasmas spreading in vineyards of Modena province (Italy). In: Extended abstracts of the 14th Meeting of the ICVG, September 2003 Locorotondo (Bari), Italy, pp. 104-105. Bertaccini, A., Botti, S., Fiore, N., Gajardo, A. & Montealegre, J., 2004. Identification of a new phytoplasma(s) infecting grapevine with yellows in Chile. In: 15th Congress of IOM July 2004, Athens Georgia, USA, pp. 63-64. Botti, S. & Bertaccini, A., 2004. Flavescence dore associated phytoplasmas: identification of molecular variants and epidemiological implications. In: 15th Congr. of IOM, July 2004, Athens Georgia, USA, p. 122. Botti, S., Paltrinieri, S., Mori, N., Milanesi, L., Bondavalli, R. & Bertaccini, A., 2005. Variabilita molecolare di fitoplasmi 16SrXII in vigneti delle province di Modena e Reggio Emilia. In: III Incontro Nazionale sulle Malattie da Fitoplasmi Milano, Italy, giugno 2005, pp. 73-75. Langer, M. & Maixner, M., 2004. Molecular characterisation of grapevine yellows associated phytoplasmas of the stolbur-group based on RFLP-analysis of non ribosomal DNA. Vitis 43(4), 191-199. 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