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Title: DETECTING CLAVIBACTER XYLI SUBSP. XYLI USING TISSUE BLOT HYBRIDIZATION WITHA PCR-DERIVED DNA PROBE.

Author
item Pan, Yong-Bao
item Grisham, Michael
item Burner, David
item WEI, Q - OICD

Submitted to: International Society of Sugar Cane Technologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 5/11/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: The objective of this study was to develop a DNA-based diagnostic tool for the detection of Clavibacter xyli subsp. xyli (Cxx), the causal agent of ratoon stunting disease. Clavibacter xyli subsp. xyli bacteria were isolated from infected sugarcane tissue and pure culture verified by Gram- and immunofluorescence staining. Polymerase chain reaction with universal primers L1/G1 on Cxx genomic DNA amplified a 566 bp major DNA product from the 16S and 23S ribosomal intergenic transcribed spacer region. The 566 bp PCR product was gel purified, labeled with 32P, and a probed in Southern blot hybridization experiments. We found that the radioactive probe hybridized only to Cxx-derived PCR products and not to other L1/G1-primed PCR products from Xanthomonas albilineans, other Xanthomonas species, and other unknown sugarcane bacterial saprophytes. Due to this sequence specificity, the 566 bp PCR-derived DNA probe has been used to detect Cxx in tissue- and dot- blotted sugarcane samples. A duplicated set of tissue blots was tested by immunoassay using polyclonal anti-Cxx antibodies. Preliminary results indicated that the DNA-based tissue blot hybridization procedure detected the RSD bacterium as effectively as the protein-based method. The two methods may compliment each other thereby enhancing the sensitivity and accuracy of Cxx detection