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

Abstract

The 23S rRNA gene from phytoplasmas was amplified by using the oligonucleotide primer pair P23S5F3 (5'-GTGGATGCCTTGGCACTAAGAGCC-3') and A23S3R3 (5'-ACTTACACACCTGGCCTATCAACC-3'), designed from the conserved 23S rRNA gene sequences of various mollicutes identified in GenBank. The amplified product from a representative phytoplasma strain, eastern X-disease (CX) phytoplasmas, was sequenced and found to be 2798 base pairs in size, with a G + C content of 44.5%, representing about 97% of the entire length of the 23S rRNA gene. The product was confirmed to include a 23S rRNA gene by sequence homology between the amplified fragment and the 23S rRNA genes of other mollicutes and walled bacteria. The 23S rRNA gene from CX phytoplasmas has higher sequence homologies with genes of 23S rRNA from other bacteria (72.6-74.7%) than with those from mollicutes (68.9-71.1%). The primer pair was used to successfully amplify the 23S rRNA gene sequences from phytoplasma strains belonging to 10 different groups or subgroups, but no polymerase chain reaction products were amplified from DNA preparations from healthy plants, suggesting that the primer pair is useful for phytoplasma detection. The 23S rRNA genes of these phytoplasmas were compared by restriction fragment length polymorphism (RFLP) analysis using four DNA restriction enzymes, Alu I, Hpa II, Mse I, and Rsa I. Among the phytoplasmas examined, each phytoplasma group 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 using all four enzymes, supporting previous reports that they belong to a single group.