DETECTION OF DNA OF PLANT PATHOGENIC MYCOPLASMALIKE ORGANISMS BY A POLYMERASE CHAIN-REACTION THAT AMPLIFIES A SEQUENCE OF THE 16S RNA GENE

A polymerase chain reaction (PCR) method has been developed with a 558-bp fragment of the 16S rRNA gene as template DNA and two oligonucleotide primers from conserved regions of this gene. The suitability of the system has been tested with 17 isolates of mycoplasmalike organisms (MLOs) maintained in periwinkle (Catharanthus roseus) and with nine MLO samples from field-grown woody plants. With DNA preparations enriched in MLO DNA, an amplification product was obtained after 24 cycles from all MLOs maintained in periwinkle. No amplified DNA was detected under these conditions in the samples from healthy plants. However, after 40 cycles a DNA fragment similar in size to those from MLO-infected plants was detected. This amplification could clearly be distinguished from the MLO fragments by restriction fragment length polymorphism (RFLP) analysis with AluI restriction endonuclease. Amplified DNA was also obtained with DNA preparations from MLO-diseased and healthy woody plants after 40 cycles. In this case, too, MLO fragments could be distinguished from the amplification of DNA from healthy plants by RFLP analysis. According to the restriction profiles, four distinct groups could be differentiated among the MLOs examined. With the PCR system developed, a MLO fragment was detected after amplification of approximately 18 pg of DNA from diseased periwinkle and 170 pg of DNA from infected woody plants.