Cloning and detection of extrachromosomal DNA from mycoplasmalike organism associated with sugarcane white leaf disease
1995
Klinkong, S. | Seemuller, E. (Kasetsart Univ. Kamphaeng Saen Campus, Nakhon Pathom (Thailand). Central Laboratory and Greenhouse Complex)
Purified DNA of mycoplasmalike organisms(MLOs) was obtained by bisbenzimide CsCl buoyant density gradient centrifugation of DNA from sugarcane white leaf disease (WLD). Six bands of extrachromosomal DNA (E-DNA) presumably of plasmid origin were found by agarose gel electrophoresis which seem to be attributed to three different replicons. One of these replicons 2.5 kb in size was linearized with Hind III restriction enzyme and was cloned in Escherichia coli. A probe made from the insert hybridized with two bands of undigested DNA from diseased sugarcane corresponding to the homologous nicked and supercoiled forms of the cloned replicon. The probe hybridized with all eight isolates examined collected in different areas of Thailand and reacted only slightly with E-DNA from bermudagrass and Brachiaria sp. showing white leaf symptoms. It did not hybridize with DNA from correspondingly healthy plant material. Also, no hybridization was observed with DNA from plants infected by MLO associated with 18 different other plant diseases. The probe detected the DNA of the WLD-MLO in 100 pg of purified WLD-MLO-DNA and in 8 ng of total nucleic acids from infected sugarcane.
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