Classification of new phytoplasmas associated with diseases of strawberry in Florida, based on analysis of 16S rRNA and ribosomal protein gene operon sequences

Abstract

Strawberry plants exhibiting symptoms of stunting and abnormally small leaves were observed in production fields in central Florida, USA. Since the symptoms were suggestive of phytoplasma infection, plants were assayed for presence of phytoplasma by PCR amplification of 16S rDNA and ribosomal protein (rp) gene sequences. Amplification of phytoplasma-specific DNA sequences by PCR indicated infection of the diseased strawberry plants by phytoplasmas. RFLP analyses of amplified 16S rDNA revealed that the plants were infected by two mutually distinct phytoplasmas that differed from strawberry green petal phytoplasma (group 16Srl-C). Both phytoplasmas were members of 16S rRNA gene group I (16Srl). Based on RFLP analysis of amplified 16S rDNA and rp gene sequences, one was classified in group 16Srl subgroup I and new rp subgroup 16Srl-l(rp); its 16S rRNA-rp subgroup was designated 16Srl-K(rr-rp). The second phytoplasma represented a previously undescribed subgroup, designated K, in 16S rRNA group I but belonged to rp subgroup 16Srl-J(rp); this phytoplasma's 16S rRNA-rp subgroup was designated 16Srl-J(rr-rp). Results of RFLP analyses agreed with putative restriction site maps based on nucleotide sequences determined for the amplified 16S rDNAs and rp gene operon DNAs. Further evidence indicated that the 16Srl-K(rr-rp) strawberry phytoplasma, Mexican periwinkle virescence phytoplasma and stolbur phytoplasma shared sequence homologies that enabled amplification of DNA from all three by PCR using primers previously designed as stolbur-specific.