Virus-like diseases of rose plants of uncertain aetiology have been widely distributed throughout the world (4). Symptoms include dieback, rose rosette, witches' broom, and bud proliferation. Research conducted in England (4) and the United States (1) could not reveal the etiology of the diseases. Disease symptoms including stunted growth, leaf and flower malformation, and shoot and flower proliferation were observed in rose plants in Poland (2). In this previous work, we reported cases of phytoplasma closely related to group 16SrI in rose plants with shoot proliferation as determined by nested polymerase chain reaction (PCR) with ribosomal primers R16F1/R0 followed by rA/fA or R16(I)F1/R1. In this study, we examined 48 symptomatic rose plants of 12 cultivars using nested PCR primed by P1/P7 and followed by universal primer pairs R16F2n/R2, fA/rA, or group 16SrI-specific R16(I)F1/R1. To detect potential mixed infection in roses, the group 16SrX-specific primer pairs fAT/rAS, fAT/rPRUS, and fPD/rAT were used for nested PCRs. Samples of rose plants with disease symptoms and nonsymptomatic, samples of Catharanthus roseus, healthy and inoculated by grafting with the reference strain of aster yellows phytoplasma (AY1, group 16SrI-B, kindly supplied by I.-M. Lee, Beltsville, MD) and the reference strain of apple proliferation phytoplasma (AP, group 16SrX-A, kindly supplied by A. Bertaccini, Bologna, Italy), were tested for the presence of phytoplasma rDNA by PCR. Phytoplasma identification was accompanied by digestion with restriction endonucleases, AluI, HhaI, HpaII, MseI, and RsaI, and restriction fragment length polymorphism (RFLP) analysis of a 1.2-kb fragment of rDNA (3). A DNA amplification product was observed in all nested PCRs containing template DNA of samples collected from diseased roses and the reference strains but not from control plants. On the basis of RFLP analysis of PCR products and comparison of the RFLP patterns with those of the reference strains, we demonstrated the presence of aster yellows phytoplasma belonging to phytoplasma group 16SrI-B in roses of 11 cultivars. RFLP profile of samples collected from rose cv. Red Champ was identical to those obtained for reference AP strain (group 16SrX-A). Mixed RFLP profiles were observed in samples collected from rose cv. Memory, which were doubly infected by phytoplasmas belonging to groups 16SrI-B or 16SrX-A. These results were confirmed by PCR with group 16SrX-specific primer pairs. The target DNA was amplified when amplifications were conducted with subgroup 16SrX-A-specific primer pair fAt/rAS, whereas no observable PCR products were obtained with subgroup 16SrX-B- (fAT/rPRUS) or 16SrX-C- (fPD/rAT) specific primer pairs. This report confirms infection of roses by aster yellows phytoplasma belonging to group 16SrI-B, and to our knowledge, records for the first time, infection by phytoplasma of group 16SrX-A.
References: (1) A. H. Epstein and J. H. Hill. J. Phytopathol. 143:353, 1995. (2) M. Kami ska et al. J. Phytopathol. 149:3, 2001. (3) I.-B. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1968. (4) B. J. Thomas. Ann. Rep. Glasshouse Crops Res. Inst. 1979:178, 1981.