Amplification of the 23S rRNA gene and its application in differentiation and detection of phytoplasmas

The 23S rRNA gene from phytoplasmas was amplified by using the oligonucleot ide primer pair P23S5F3 (5'-GTGGATGCCTTGGCACTAAGAGCC-3') and A23S3R3 (5'-AC TTACACACCTGGCCTATCAACC-3'), designed from the conserved 23S rRNA gene seque nces of various mollicutes identified in GenBank. The amplified product fro m a representative phytoplasma strain, eastern X-disease (CX) phytoplasmas, was sequenced and found to be 2798 base pairs in size, with a G + C conten t of 44.5%, representing about 97% of the entire length of the 23S rRNA gen e. The product was confirmed to include a 23S rRNA gene by sequence homolog y between the amplified fragment and the 23S rRNA genes of other mollicutes and walled bacteria. The 23S rRNA gene from CX phytoplasmas has higher seq uence homologies with genes of 23S rRNA from other bacteria (72.6-74.7%) th an with those from mollicutes (68.9-71.1%). The primer pair was used to suc cessfully amplify the 23S rRNA gene sequences from phytoplasma strains belo nging to 10 different groups or subgroups, but no polymerase chain reaction products were amplified from DNA preparations from healthy plants, suggest ing that the primer pair is useful for phytoplasma detection. The 23S rRNA genes of these phytoplasmas were compared by restriction fragment length po lymorphism (RFLP) analysis using four DNA restriction enzymes, Alu I, Hpa I I, Mse I, and Rsa I. Among the phytoplasmas examined, each phytoplasma grou p showed a unique RFLP pattern with each of four enzymes. CX phytoplasmas, western X-disease (WX) phytoplasmas, goldenrod yellows (GR1) phytoplasmas, and chokecherry X-disease (ChX) phytoplasmas had identical RFLP patterns us ing all four enzymes, supporting previous reports that they belong to a sin gle group.