SUMMARY Amplification of ribosomal phytoplasma DNA by di- rect or nested PCR and stolbur-specific primers, fol- lowed by restriction length polymorphism analysis were used to detect and identify stolbur phytoplasmas in symptomatic alfalfa, celery, field bindweed, grapevine, olive and tomato plant samples from different regions of Italy. Alfalfa appears to be a new host for this phytoplas- ma. All isolates showed RFLP patterns indistinguishable from one another and from the one of the Serbian refer- ence isolate from pepper. In Southern hybridization ex- periments, EcoRI and HindIII restricted DNAs from stolbur-infected herbaceous hosts hybridized with two riboprobes from random-cloned chromosomal DNA fragments of a Sardinian stolbur isolate from tomato, yielding more or less complex patterns. None of the probes hybridized with DNAs from stolbur-unrelated phytoplasma infected or healthy plants or olives known to harbour stolbur phytoplasma. With one of the probes, two types of molecular hybridization profiles were detected among stolbur-infected samples. Based on the nucleotide sequence of this probe, a new non-ri- bosomal primer pair was designed. These primers am- plified a stolbur-specific fragment of 720 bp from stol- bur-infected samples, but not from symptomatic olives, healthy plants or plants infected with unrelated phyto- plasmas. To verify whether absence of amplified frag- ments in gels was due to lack of priming or to unde- tectably low amplification of stolbur DNA, negative PCR products were tested in hybridization assays with the primers parental probe. Positive hybridization sig- nals were obtained with such samples but not with products from healthy DNAs or negative PCR controls. The same probe was also used to detect stolbur DNA on membranes printed with tissues from field-infected tomato plants or from glasshouse-maintaned stolbur-in- fected periwinkles or young asymptomatic tomato seedlings grafted with infected field material. Corresponding author: G. Boccardo Fax: +39.011.343809 E-mail: g.boccardo@ifa.to.cnr.it Key words: stolbur-specific PCR primers, molecular hybridization, Southern-hybridization, dot-blot, tissue printing. INTRODUCTION Based on restriction fragment length polymorphism (RFLP) analysis of 16S rRNA and ribosomal protein genes the monophyletic clade of the phytoplasmas was divided into 14 major 16S rDNA groups (I to XIV) and 32 subgroups within the class Mollicutes (Lee et al., 1998). In Italy, numerous plant diseases affecting trees, weeds, shrubs, vegetable, ornamentals and forage crops, characterized by symptoms of yellowing, witch- es’-broom, epinasty, slow growth and/or decline have been associated with phytoplasmas of groups I, II, III, V, X and XII (Lee et al., 1993; Davis and Sinclair, 1998; Lee et al., 1998; Seemüller et al., 1998). Although phy- toplasmas of group I appear to be the commonest in various parts of Italy (Bertaccini et al., 1992; Vibio et al., 1996; Marcone et al., 1997b; Marzachì et al., 1999), members of group XII have also been found in several naturally infected plant species. Actinidia deliciosa (chi- nensis) Planch. (kiwi), Allium ampeloprasum L. var. por- rum (leek), Apium graveolens L. (celery), Catharanthus roseus (L.) G. Don (periwinkle), Convolvolus arvensis L. (field bindweed), Hydrangea macrophylla (Thunb.) Ser. (French hydrangea), Jasminum officinale L. (jas- mine), Lavandula officinalis Chaix (lavender), Lycopersi- con esculentum Mill. (tomato), Olea europea L. (olive tree), Persica laevis DC (=Amygdalus persica nectarina Maxim, A. nuci-persica Reich) (nectarine), Pyrus com- munis L. (pear), Viola odorata L. (violet) and Vitis vinifera L. (grapevine) have been reported to harbour stolbur phytoplasmas (Bertaccini et al., 1995; Lee et al., 1995; Albanese et al. , 1996; ; Marcone et al., 1996; Vibio et al., 1996; Boudon-Padieu et al., 1997; Marcone et al., 1997a, b; Minucci and Boccardo, 1997; Schneider et al. , 1997; Albanese et al. , 1998; Bertaccini et al. , 1999; Marzachì et al., 1999; Poggi Pollini et al., 1999a, b). Group XII is divided into two subgroups (A and B), Journal of Plant Pathology (2000), 82 (3), 201-212 Edizioni ETS Pisa, 2000 201 MOLECULAR HYBRIDIZATION AND PCR AMPLIFICATION OF NON-RIBOSOMAL DNA TO DETECT AND DIFFERENTIATE STOLBUR PHYTOPLASMA ISOLATES FROM ITALY C. Marzachì, F. Veratti, M. d’Aquilio, A. Vischi, M. Conti and G. Boccardo Istituto di Fitovirologia Applicata del C.N.R., Strada delle Cacce 73, I-10135 Torino, Italy