Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene, On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16Srl-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three subgroups that differ in the RFLP profiles were identified and designated 16Srl-L, 1GSrl-M and 16Srl-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 kp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16Srl-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16Srl-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16Srl-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16Srl-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.