SENSITIVE AND SPECIFIC DETECTION OF CLAVIBACTER-XYLI SUBSP XYLI, CAUSAL AGENT OF RATOON STUNTING DISEASE OF SUGARCANE, WITH A POLYMERASE CHAIN REACTION-BASED ASSAY

A sensitive, specific polymerase chain reaction-based assay was develo ped for the detection of the causal agent of ratoon stunting disease o f sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleo tide primers derived from the internal transcribed spacer region betwe en the 16S and 23S rRNA genes oi the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplificat ion product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was su ccessfully applied to the detection of C. xyli subsp. xyli in fibrovas cular fluid extracted from sugarcane and was sensitive to approximatel y 22 cells per PCR assay. A multiplex PCR test was also developed whic h identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.