Detection and differentiation of the mycoplasmalike organism association with sugarcane white leaf disease using cloned extrachromosomal DNA probe

DNA was isolated from sugarcane infected by sugarcane white leaf mycoplasmalike organism [SCWL-MLO]. The MLO DNA was separated from the host plant DNA by repeated CsCl-bisbenzimide buoyant density gradient centrifugation. 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 HindIII 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 8 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 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 SCWL-MLO in 100 pg of purified SCWL-MLO and in 8 ng of total nucleic acids from infected sugarcane.