VIEW ARTICLE

Molecular Plant Pathology

Molecular Cloning and Detection of Chromosomal and Extrachromosomal DNA of the Mycoplasmalike Organism Associated with Little Leaf Disease in Periwinkle (Catharanthus roseus). R. E. Davis, Microbiology and Plant Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705; I.-M. Lee, S. M. Douglas, and E. L. Dally. Connecticut Agricultural Experiment Station, New Haven, CT 06504. Phytopathology 80:789-793. Accepted for publication 25 January 1990. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1990. DOI: 10.1094/Phyto-80-789.

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 nucleic acid extracted from periwinkle plants infected by periwinkle little leaf, aster yellows, 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 yellows 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 yellows 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.