A novel method for cloning DNA of plant-pathogenic mycoplasmalike organisms

Abstract

A novel method was developed for cloning the DNA from a representative of plant-pathogenic mycoplasmalike organisms (MLOs). This procedure utilized random amplified polymorphic DNA (RAPD) and basic recombinant DNA techniques. It consisted of amplification of total DNA from diseased plants using one oligonucleotide primer with arbitrary sequence and separation of RAPD products in agarose gels. Unique RAPD band(s) of MLO origin was (were) then recovered from the gel and cloned into the specifically designed vector pCRTM II. With this method, a DNA fragment of the SA2 isolate of grapevine yellows MLO was cloned. Southern blot hybridizations revealed that most of the DNA in the unique RAPD band was derived from MLO. Results from dot-blot hybridizations used for screening showed that approximately 60% of transformants harbored MLO-specific recombinant plasmids. Our approach is relatively simple, quite efficient, and not limited by the amount of diseased material available. It does not depend on DNA sequence information for primer design and does not rely on restriction endonucleases for cloning. In addition, it can be used directly for disease diagnosis and for differentiation of closely related MLOs. Our system may serve as a model for cloning DNAs of other fastidious plant pathogens.Key words: mycoplasmalike organism, DNA, cloning, RAPD, detection.