Detection ofXanthomonas campestnis pv. Citri bythe Polymerase ChainReaction Method

pFLlisa pUC9derivitive thatcontains a 572-bp EcoRIinsert cloned fromplasmid DNA ofXanthomonas campestris pv.citri XC62.Thenucleotide sequenceofpFL1was determined, andthesequenceinformation was usedtodesign primers forapplication ofthepolymerase chain reaction (PCR)tothedetection ofX.campestris pv.citri, thecausal agentofcitrus bacterial canker disease. Seven18-bp oligonucleotide primers were designed andtested withDNA fromX.campestris pv.citri strains andother strains ofX.campestris associated with Citrus spp.astemplates inthePCR.Fourprimerpairs directed theamplification oftargetDNA fromX. campestris pv.citri strains butnotfromstrains ofX.campestris associated witha different disease, citrus bacterial spot. Primerpair 2-3directed thespecific amplification oftarget DNAfrompathotype A butnotother pathotypes ofX.campestris pv.citri. A pH9.0buffer thatcontained 1% Triton X-100and0.1%gelatin was absolutely required forthesuccessful amplification ofthetarget DNA,whichwas 61% G+C. Limits of detection after amplification andgelelectrophoresis were25pgofpurified target DNA andabout10cells when Southern blots were madeafter gelelectrophoresis andprobed withbiotinylated pFLl.Thislevel ofdetection represents an increase insensitivity ofabout100-fold overthatofdotblotting withthesame hybridization probe. PCRproducts oftheexpected sizes were amplified fromDNA extracted from7-month-old lesions from whichviable bacteria could notbeisolated. Theseproducts wereconfirmed tobespecific forX.campestris pv. citri bySouthern blotting. ThisPCR-based detection protocol willbea useful addition tocurrent methods of detection ofthis pathogen, whichiscurrently thetarget ofinternational quarantine measures.