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Title: COMPARATIVE CLASSIFICATION OF ASTER YELLOWS-GROUP PHYTOPLASMAS BASED ON RFLP ANALYSIS OF RIBOSOMAL RNA AND TUF GENE SEQUENCES

Author
item MARCONE, C - INSTIT PFLANZENSCHUTZ, GE
item Lee, Ing Ming
item Davis, Robert
item RAGOZZINO, A - UNIV. DI NAPOLI, ITALY
item SEEMULLER, E - INSTIT PFLANZENSCHUTZ, GE

Submitted to: International Journal of Systematic and Evolutionary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Phytoplasmas, formerly called mycoplasmalike organisms, are associated with diseases in several hundred plant species. Thus far, none has been cultured in vitro. The development of molecular-based tools, especially, polymerase chain reaction (PCR) assays using universal primers designed based on conserved gene sequences, has made it possible for us to detect and identify a wide array of phytoplasmas associated with infected plants or insects. A comprehensive classification scheme based on fingerprinting of 16S rRNA and ribosomal protein (rp) gene sequences was recently proposed from our laboratory. Among the 14 phytoplasma groups proposed, the aster yellows (AY) group phytoplasmas are most diverse. The AY phytoplasmas are associated with numerous diseases in many important crops worldwide. For an accurate diagnosis there is a need to further differentiate this large group into subgroups. Several subgroups have been identified based on analysis of 16S rRNA or rp gene in our laboratory. In this study another conserved gene encoding the elongation factor TU (tuf gene) was analyzed and used to compare 16S rRNA gene for classification of phytoplasmas in the AY group. The results indicated mutual consistency of the two genetic markers for classification of subgroups within the AY groups. Several new subgroups were recognized among European strains which are not present in the United States. Information about genomic diversity of phytoplasmas and their geographical distribution is indispensable for preventing the spread of disease through international exchange of germplasm and for formulating an effective control measure to combat phytoplasmal diseases. The information will be a benefit to diagnosticians and to APHIS for implementation of new quarantine regulations.

Technical Abstract: More than fifty phytoplasma isolates of the aster yellows (AY) group, most of them previously not, or not sufficiently characterized, were examined by RFLP analysis of PCR-amplified ribosomal DNA (rDNA) and tuf gene sequences. Based on rDNA restriction profiles, five of six established 16S rDNA (16SrI) subgroups were recognized in the material examined. In addition, three new subgroups were identified which were designated 16rI-L, 16rI-M, and 16SrI-N. Of the two 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-bp fragment of the 16S rRNA gene. One of the new subgroups could only be identified when the 1800-bp fragment was examined. Many differences in the rDNA profiles between the subgroups can most like be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. Several subgroups seem to differ only in this respect. The subgroups identified b RFLP analysis of a 940-bp tuf gene fragment corresponded to the 16SrI subgroups. However, 16SrI subgroups D, L, and M showed the same tuf gene restriction profiles as 16SrI subgroup B. These subgroups seem to differ less from 16SrI subgroup B than the other subgroups.