Yb. Pan et al., A POLYMERASE-CHAIN-REACTION PROTOCOL FOR THE DETECTION OF CLAVIBACTER-XYLI SUBSP XYLI, THE CAUSAL BACTERIUM OF SUGARCANE-RATOON-STUNTING-DISEASE, Plant disease, 82(3), 1998, pp. 285-290
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.