S. Mhirsi1*, H. Acheche2, S. Fattouch1, G. Boccardo3 and M.Marrakch Marzouki2
1 Laboratoire de Génie Biologie. Institut National des Sciences Appliquées Ã la Technologie, Bd Ali Akid, Centre Urbain Nord, BP N° 676 Tunis, 1080, Tunisie
2 Laboratoire de génétique. Faculté des Sciences de Tunis, Campus Universitaire, 1004, Tunis, Tunisie
3 Istituto di Fitovirologia applicata del C.N.R., Strada delle Cacce 73, I-10135 Torino, Italy
Accepted: 19 Feb 2004
In northern Tunisia, grapevines (Vitis vinifera) were found exhibiting symptoms of grapevine yellows that included plant weakness, incomplete lignification, flexible shoots and drooping. Affected leaves were thicker than normal, brittle, rolled downward and showed veinal yellowing and necrosis (Fig 1A). In addition, grape bunches became dry and shrivelled before fruit could fully develop and ripen (Fig 1B). Given the symptoms that were observed, a phytoplasma infection was suspected.
DNA was extracted using an enrichment procedure (Gundersen et al., 1996) from 27 symptomatic grapevines and was analysed by PCR, using the universal 16S rRNA phytoplasma primer pair R16R2/F2 (Lee et al., 1993). The resulting products were reamplified using the group-specific rRNA primer pair R16(I)R1/F1 (Lee et al., 1994). Seven grapevine samples each yielded a nested rDNA product of 1,100 base pairs. No products were amplified using DNA extracted from healthy grapevines. When the nested-PCR products were digested with either HhaI, MseI or RsaI endonucleases, the restriction profiles obtained were uniform indicating that infected grapevines all contained very similar or co-identical phytoplasmas (Fig. 2).
Likewise, profiles were also indistinguishable from those obtained after HhaI, MseI or RsaI digestion of R16(1)R1/F1 products obtained from control DNA extracts, made from European aster yellows phytoplasma infected samples and included for comparative purposes. Collectively, rDNA restriction profiles delineated phytoplasmas infecting Tunisian grapevine as aster yellows (16SrI-B) subgroup strains (Lee et al, 1998). This is the first report of phytoplasmas in the aster yellows group infecting grapevine in Tunisia.
Gundersen DE, Lee IM, Schaff DA, Harrison NA, Chang CJ, Davis RE, Kingbury DT, 1996. Genomic diversity and differentiation among phytoplasma strains in 16S rRNA group I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). International Journal of Systematic Bacteriology 46, 64-75.
Lee IM, Gundersen DE, Davis RE, Bartoszyk IM, 1998. Revised classification scheme of phytoplasmas based on RFLP analysis of 16S rRNA and ribosomal protein gene sequences. International Journal of Systemic Bacteriology 48, 1153-1169.
Lee IM, Gundersen DE, Hammond RW, Davis RE, 1994. Use of mycoplasmalike organism (MLO) group specific oligonucleotide primers for nested-PCR assays to detect mixed infections in a single host plant. Phytopathology 84, 556-559.
Lee IM, Hammond RW, Davis RE, Gundersen DE, 1993. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma like organisms. Phytopathology 83, 834-842.
©2004 The Authors