Submitted to: Systematic and Applied Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/27/2000
Publication Date: 10/1/2000
Citation: Chen, J., Jarret, R.L., Qin, X., Hartung, J.S., Chang, C.J., Hopkins, D. 2000. 16s rdna analysis of xylella fastidiosa. Systematic and Applied Microbiology. Interpretive Summary: Strains of the bacterium Xylella fastidiosa cause important diseases of grapevine, citrus, and stone fruits. Many other tree species also are susceptible to diseases caused by this bacterium, which is transmitted by common insects. Because of the many hosts for this bacterium and the importance of the diseases that it causes, we wanted to determine whether the strains of the bacterium that cause disease on different hosts could be considered to be different species. We focused on a DNA segment that has been used for taxonomic studies in bacteria for many years. We cloned and sequenced this "ribosomal DNA gene" from 16 strains of Xylella fastidiosa isolated from nine hosts. Our data show that the nearest bacterial relative to Xylella fastidiosa is the well known plant pathogen Xanthomonas. Overall, the level of diversity was lower than observed for closely related but different species of bacteria, suggesting that this DNA sequence analysis is valuable in providing a framework for future classification of X. fastidiosa at the sub-species level. Four distinct groups could be identified by this sequence: the coffee and citrus strains from Brazil, grapevine strains from Florida and California, stone fruit strains from the U.S., and shade tree strains from the U.S. Our results will be of interest to scientists interested in bacterial systematics as well as to regulatory officials who must make biologically sound judgments when enforcing quarantine regulations. Although most of the strains tested are endemic in the United States, the citrus and coffee strains occur only in South America and are subject to international quarantine measures.
Technical Abstract: The 16S rDNA genes encoding the small subunit ribosomal RNA were amplified by PCR, cloned, and sequenced from 16 strains of Xylella fastidiosa originating from nine different hosts. Sequence heterogeneities among the nearly complete 16S rDNA gene sequences were analyzed among strains of X. fastidiosa and in comparison with those from Xanthomonas and Stenotrophomonas. Strains of X. fastidiosa exhibited low levels of sequence heterogeneity. In pair-wise comparisons, X. fastidiosa strains showed a maximum variation of 1.0% or 14 nucleotide positions. When all 16 sequences were considered as a set, 54 variable positions were found. Cladistic analysis (neighbor-joining method) of the sequence data indicated that the X. fastdiosa strains formed four phylogenetic clades: 1) Pierce's disease-mulberry leaf scorch; 2) citrus variegated chlorosis-coffee leaf scorch; 3) periwinkle wilt-plum leaf scald-phony peach; and 4) oak leaf scorch-elm leaf scorch. Four multi-strain variable positions appeared to be critical for formation of the four clades. All X. fastidiosa strains exhibited significantly higher levels of sequence heterogeneity with distance values ranging between 4.3% and 5.2% (63-76 nucleotides) when compared to Xanthomonas (four species, five strains), and between 5.2% and 5.7% (76-83 nucleotides) when compared to Stenotrophomonas maltophilia LMG 958T. Cladistic analysis indicated that Xylella is phylogenetically distinct from both Xanthomonas and Stenotrophomonas, but closer to Xanthomonas. Our data demonstrate that 16S rDNA sequence analysis is valuable in providing a phylogenetic framework for future classification of X. fastidiosa at the sub-species level.