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Detection and Differentiation of Plant-Pathogenic Mycoplasmalike Organisms Using Polymerase Chain Reaction. Shigetou Namba, Associate professor, Faculty of Agriculture, University of Tokyo, Tanashi, Tokyo 188, Japan; Shosuke Kato(2), Setsuo Iwanami(3), Hiroshi Oyaizu(4), Hiroyasu Shiozawa(5), and Tsuneo Tsuchizaki(6). (2)(3)Chief and senior researcher, respectively, National Agriculture Research Center, Tsukuba, Ibaraki 305, Japan; (4)(6)associate professor and full professor, respectively, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan; (5)director, Japan Plant Protection Association, Toshima-ku, Tokyo 170, Japan. Phytopathology 83:786-791. Accepted for publication 11 March 1993. Copyright 1993 The American Phytopathological Society. DOI: 10.1094/Phyto-83-786.

Specific oligonucleotide primers were synthesized using sequence data of the 16S rRNA gene from three plant-pathogenic mycoplasmalike organism (MLO) groups (I-III). When used as primers in the polymerase chain reaction (PCR), four sets of primer pairs were identified: 1) mollicute-specific, 2) MLO-specific, 3) MLO-group I-specific, and 4) MLO-group III-specific. MLO 16S rRNA genes were selectively amplified by PCR from infected plants and vector insects. Using these primers and thermal cycling conditions, this technique was effective for detecting MLO 16S rRNA genes from diseased plants and infected insect vectors and for differentiating MLO 16S rRNA genes from similar rRNA genes of eukaryotic host organelles and other microorganisms associated with healthy vector insects. Using group-specific primers, the 16S rRNA gene of each MLO group was also differentially amplified. A modified PCR method (recycled PCR [RPCR]) was developed to perform several different PCR reactions simultaneously in one tube by adding the second primer to the tube containing the first PCR products. RPCR enabled detection of mollicute-specific DNA fragments and MLO-specific or group-specific DNA fragments as multiple bands. Each MLO group could be identified reliably by RPCR. These results show the utility of PCR for diagnosing MLO diseases and the development of a phylogenetically based MLO taxonomy.

Additional keywords: prokaryotes; Mollicutes; MLO-groups I, II, and III; diagnosis.