A POLYMERASE-CHAIN-REACTION PROTOCOL FOR THE DETECTION OF CLAVIBACTER-XYLI SUBSP XYLI, THE CAUSAL BACTERIUM OF SUGARCANE-RATOON-STUNTING-DISEASE

A polymerase chain reaction (PCR) protocol was developed that specific ally detected Clavibacter xyli subsp. xyli, the causal agent of sugarc ane ratoon stunting disease. Generic PCR products from the intergenic transcribed spacer (ITS) region of 16S-23S ribosomal DNA of C. xyli su bsp. xyli and C. xyli subsp. cynodontis were cloned and sequenced. Bas ed on a multiple sequence alignment among these two sequences and othe r nonredundant highly homologous sequences from the database, two C. x yli subsp, xyli-specific PCR primers were designed, Cxx1 (5' CCGAAGTGA GCAGATTGACC) and Cxx2 (5' ACCCTGTGTTGTTTTCAACG). These two 20-mer olig onucleotides primed the specific amplification of a 438-bp DNA product from genomic DNA samples of 21 C. xyli subsp. xyli strains. Amplifica tion was not observed with genomic DNA of one C. xyli subsp. cynodonti s strain, five strains of four other Clavibacter species, and two stra ins of two Rathayibacter species. The 438-bp PCR product also was ampl ified directly from cultured C. xyli subsp. xyli cells and from C. xyl i subsp. xyli-infected sugar cane vascular sap with a unique reaction buffer containing polyvinylpyrrolidone and ficoll. Extraction of genom ic DNA was not necessary prior to PCR assay.