Molecular Cloning and Detection of Chromosomal and Extrachromosomal DNA of the Mycoplasmalike Organism Associated with Little Leaf Disease in Periwinkle (Catharanthus roseus)

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Abstract
Preparations enriched for periwinkle little leaf mycoplasmalike organism (MLO) were obtained by a method involving isolation of sieve cells from diseased plants of periwinkle (Catharanthus roseus). DNA was extracted from the preparations, ligated into plasmids pSP64 and pSP65, and cloned in Escherichia coli JM83. Selected recombinant plasmids and cloned DNA inserts were labeled with biotin and employed as probes in dot hybridizations. The probes hybridized with neucleic acid extracted from periwinkle plants infected by periwinkle little leaf, aster yelows, or tomato big bud MLOs, but not with extracts from healthy plants. Some hybridized also with nucleic acid from plants infected by other MLOs. Southern hybridization analyses revealed the occurrence of extrachromosomal DNA in periwinkle little leaf, tomato big bud, and aster yellow MLOs. These and other results indicate genetic relatedness at the level of chromosomal DNA among these MLOs, and nucleotide sequence homology among their extrachromosomal DNAs. Biotinylated cloned DNA probes were successfully applied in dot hybridizations for detection of an Oklahoma strain of aster yellow MLO in the insect vector Macrosteles fascifrons and for detection of this and other MLos in plant species which serve as their hosts in nature.