Abstract:In order to develop quick and sensitive detection technique for the causal agent of ratoon stunting disease of sugarcane, Leifsonia xylixyli, this study developed a nested-PCR for rapid and accurate detection of L.xylixyli using bacterial universal primer pair ITS1/ITS2, based on the intergenic transcribed spacer (ITS) region of 16S-23S ribosomal DNA of bacterium, as the first-round primers followed by a L.xylixyli specific primer pair Lxx1/Lxx2. The nested-PCR could amplify a unique 438 bp specific fragment whose nucleotide sequence sharing 100% and 99.8% nucleotide identity with Brazil, Australia and USA isolates of L.xylixyli, and its detection sensitivity was 10 fg of L.xylixyli genomic DNA in 20 μL reaction solution, which increased the detection sensitivity by 100-fold compared to the simple PCR method. Detection results from samples also showed that detection sensitivity of the nested-PCR was significantly better than that of the simple PCR. The nested-PCR could be used to detect L.xylixyli from some sugarcane tissue with very few L.xylixyli, such as+1 young leaf and the youngest leaf above growing point.