Submitted to: Plant Disease
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/12/1998
Publication Date: N/A
Citation: Interpretive Summary: Polymerase chain reaction (PCR) is a DNA-based tool in plant diseases diagnosis. Guided by two 18 to 22 base pair long DNA primers, PCR multiplies a piece of DNA millions of times from a targeted organism and the amplified DNA can easily be detected. We developed a PCR protocol for the diagnosis of sugarcane leaf scald disease by the amplification of a unique DNA product from the leaf scald bacterium. However, the protocol also amplified pieces of DNA from other bacterial species. Although these non- specific DNA pieces from other bacteria were different in size, they could cause confusion and mistake in interpreting test data. As a result, additional tests were often required. To ensure that the PCR protocol amplifies a DNA product only from the leaf scald bacterium, new primers were needed. In doing this, we determined the DNA sequence of all the PCR products from the leaf scald bacterium and other bacteria. We also made use of the DNA sequence information from the Genbank database. Finally, we used computer programs to compare all these DNA sequences and to design two new PCR primers with their DNA sequences only found in the leaf scald bacterium. Using the two new primers, the PCR protocol amplified a unique DNA product only from the leaf scald bacterium. It did not amplify any pieces of DNA from many other bacterial species. The new PCR protocol is being used routinely to detect the leaf scald bacterium in infected sugarcane tissues. It can be used with confidence by technicians at quarantine and diagnostic facilities.
Technical Abstract: New primers were developed that greatly improved the specificity of the polymerase chain reaction (PCR) protocol for Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Length-polymorphic PCR products, amplified under the current PCR protocol from the 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) of X. albilineans and three unidentified sugarcane saprophytic bacterial species, were cloned and sequenced. Fourteen other non-redundant ITS sequences were retrieved from the GenBank database that were highly homologous to the sequence of X. albilineans. Two X. albilineans-specific PCR primers, namely, PGBL1 (5' CTT TGG GTC TGT AGC TCA GG) and PGBL2 (5' GCC TCA AGG TCA TAT TCA GC), were designed based on a multiple sequence alignment among these 18 sequences. These two primers permitted the specific PCR amplification of a 288 bp DNA product from all 71 diverse X. albilineans strains tested. No amplification product was observed from any other bacterial species tested, including the three unidentified sugarcane saprophytes. The new PCR protocol has been routinely used to detect the leaf scald pathogen from infected sugarcane tissues.