ARS | Publication request: DETECTING CLAVIBACTER XYLI SUBSP. XYLI BY TISSUE BLOT HYBRIDIZATION WITH A POLYMERASE CHAIN REACTION-DERIVED DNA PROBE

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 5, 1998
Publication Date: N/A

Interpretive Summary: Ratoon stunting disease (RSD) is believed to cause more yield loss in sugarcane than any other disease. The problem is that plants infected with the RSD bacterium show no obvious external symptoms. Infected plants may have reduced vigor that is not noticed or may be attributed to weather, fertilizer, or other factors. What is needed is a reliable and economical method to detect the RSD bacterium in infected plants. A laboratory assay procedure was developed that can detect a portion of DNA that is unique to the RSD bacterium even when very few bacteria are present. The impact of this newly developed biotechnology tool on the sugarcane industry is that testing of the disease can be more accurate, and probably more sensitive than other testing methods. It is important to be able to detect infections in stalks to know if control practices are effective and to prevent spread of the bacterium when stalks are used to plant new fields. The assay can be immediately applicable to many sugarcane pathology diagnostic laboratories.

Technical Abstract: A tissue blot DNA hybridization assay was developed to detect Clavibacter xyli subsp. xyli (Cxx), the causal agent of sugarcane ratoon stunting disease, from infected sugarcane stalks. The assay used a 560 bp PCR product as the probe which was amplified from the intergenic transcribed spacer region of the 16S/23S ribosomal DNA of Cxx with two universal primers. The sequence of this 560 bp PCR product proved useful for the detection of Cxx in Southern blot experiments. It hybridized equally well to itself and to a PCR product of about 540 bp from Clavibacter xyli subsp. cynodontis isolated from Bermudagrass, and it hybridized weakly with the PCR products of about 520 bp from C. michiganensis isolates. The probe did not hybridize to the PCR products from Xanthomonas albilineans, the leaf scald bacterial pathogen, other Xanthomonads, and saprophytic bacteria commonly found in sugarcane. Assays on 346 duplicate sets of sugarcane tissue blots indicated that the tissue blot DNA hybridization assay detected Cxx as effectively as a tissue blot immunobinding assay. The tissue blot DNA hybridization assay can be immediately applicable in many sugarcane diagnostic laboratories to compliment current diagnostic methods, thereby enhancing the sensitivity and accuracy of Cxx detection.