CLASSIFICATION OF NEW PHYTOPLASMAS ASSOCIATED WITH DISEASES OF STRAWBERRY IN FLORIDA BASED ON ANALYSIS OF 16S RRNA AND RIBOSOMAL PROTEIN GENE OPERON SEQUENCES

Authors: Jomantiene, Rasa; Davis, Robert; Maas, John; Dally, Ellen

Submitted to: International Journal of Systematic and Evolutionary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Phytoplasmas (tiny microbes formerly called mycoplasmalike organisms, MLOs) cause many plant diseases, reducing product quality, hindering exports, and reducing farmer profits in fruit crops including strawberry. Some are responsible for quarantine restrictions on movement of critically important germplasm and planting stock. This research was initiated to learn whether phytoplasmas might be involved in a disease problem that is present in winter production fields of strawberries in Florida. Diseased strawberry plants included green petal and multiplier type symptoms, resulting in poor fruit quality and low plant yield. The results of our experiments indicated that at least two previously undescribed phytoplasmas were involved in the Florida disease problem. Although DNA from one of the phytoplasma was amplified in polymerase chain reactions (PCR) that were primed by a primer pair previously described (by others) as specific for European stolbur disease phytoplasma, that phytoplasma clearly was not stolbur phytoplasma based on our study of its DNA. Our results also indicated that the identity of phytoplasma present in a diseased plant could not be reliably predicted on the basis of the type of disease symptom in the plant. This work contributes valuable progress toward unraveling the cause of the strawberry disease problem. The results provide pathogen targets for disease management in the field, for indexing to produce disease-free planting stock, and for managing quarantined plant germplasm. The results form a basis for investigating whether the phytoplasmas studied could present a threat to crops in addition to strawberry. The findings will be of interest to strawberry growers, diagnostic companies, and APHIS.

Technical Abstract: Strawberry plants exhibiting symptoms of stunting and abnormally small leaves were observed in production fields in central Florida. Since the symptoms were suggestive of possible phytoplasma infection, plants were assayed for presence of phytoplasma by polymerase chain reaction (PCR) amplification of 16S rDNA and ribosomal (r) protein (p) gene sequences. Amplification of phytoplasma-specific DNA sequences in PCR indicated infection of the diseased strawberry plants by phytoplasma. 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 16SrI-C). Both phytoplasmas were members of 16S rRNA gene group I (16SrI). Based on RFLP analysis of amplified 16S rDNA and rp gene sequences, one was classified in group 16SrI subgroup I and new rp subgroup 16SrI-I(rp); its 16S rRNA-ribosomal protein subgroup was designated 16SrI-K(rr-rp). The second phytoplasma represented a previousl undescribed subgroup, designated K, in 16S rRNA group I but belonged to rp subgroup 16SrI- J(rp); this phytoplasma's 16S rRNA-ribosomal protein subgroup was designated 16SrI-J(rr-rp). Results of the 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 16SrI-K(rr-rp) strawberry phytoplasma, Mexican periwinkle virescence phytoplasma, and stolbur phytoplasma shared sequence homologies that enabled amplification of DNA from all three in PCR using primers previously designed as stolbur-specific.