EXTRACHROMOSOMAL DNA ELEMENTS OF PLANT PATHOGENIC MYCOPLASMALIKE ORGANISMS

Extrachromosomal DNA elements of plant pathogenic mycoplasmalike organisms (MLOs) were obtained by two methods: 1) direct lysis of intact MLO cells and 2) differential alkaline denaturation by selective precipitation of high molecular weight plant and mycoplasma chromosomal DNA. The MLOs were obtained from roots of aster plants (Callistephus chinensis) affected with eastern aster yellows (AYE), western aster yellows (AYW), clover phyllody (CP), or clover yellow edge (CYE). Multiple covalently closed circular double-stranded DNA molecules were found in each MLO, ranging in size from 1.5 kb to 7.0 kb as shown by electron microscopy and agarose gel electrophoresis. The extrachromosomal DNA bands can be attributed to nicked, supercoiled, and concatemeric forms of component plasmids with the following size ranges: AYW MLO (1.5,2.5,3.0,4.2,4.7,7.0 kb), AYE MLO (1.7,3.5,4.2 kb), CP MLO (1.8,4.3,4.7 kb), and CYE MLO (3.8 kb). Hybridization of Southern blots of total MLO DNA with either the 2.5 kb plasmid of AYW MLO or the 4.3 kb plasmid of CP MLO detected common sequences among the extrachromosomal elements of AYW, AYE, and CP MLO. The 1.5 kb plasmid of AYW MLO contained sequences common to both strains of aster yellows MLO; however, the 1.8 kb plasmid of CP MLO hybridized only with AYE MLO sequences. The presence of reiterated repeat sequences in AYW MLO extrachromosomal DNA was deduced from the hybridization pattern of either its 1.5 kb or 2.5 kb plasmid. Total CYE MLO-DNA did not show homology with the DNA of the other three MLOs. Thus, the uniqueness of the plasmid banding pattern and hybridization characteristics of selected plasmids can be used to differentiate each of the four MLOs.