Detecting Clavibacter xyli subsp. xyli by tissue blot DNA hybridization.
A tissue blot DNA hybridization assay was developed to detect C. 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 a probe which was amplified from the intergenic transcribed spacer region of the 16S/23S ribosomal DNA of Cxx with 2 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 C. xyli subsp. cynodontis isolated from bermudagrass (Cynodon spp.). 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, other Xanthomonads, or saprophytic bacteria commonly found in sugarcane. Comparative assays on duplicate sets of sugarcane tissue blots suggested that the tissue blot DNA hybridization assay (TB-DHA) detected Cxx in mid-July, which was 2 weeks earlier than the tissue blot immunobinding assay (TB-IBA). The DNA hybridization assay also detected Cxx as effectively as TB-IBA in later dates. It is suggested that TB-DHA can be immediately applied in many sugarcane diagnostic laboratories to compliment current diagnostic methods.