Molecular Cloning and Detection of Chromosomal and Extrachromosomal DNA of Mycoplasmalike Organisms Associated with Witches’-Broom Disease of Pigeon Pea in Florida. Nigel A. Harrison. Fort Lauderdale Research and Education Center, University of Florida, Institute of Food and Agricultural Sciences, 3205 College Avenue, Fort Lauderdale 33314 U. S. A.. James H. Tsai, Carol M. Bourne, and Patricia A. Richardson. Fort Lauderdale Research and Education Center, University of Florida, Institute of Food and Agricultural Sciences, 3205 College Avenue, Fort Lauderdale 33314 U. S. A.. MPMI 4:300-307. Accepted 27 February 1991. Copyright 1991 The American Phytopathological Society.

A Florida isolate of the pigeon pea witches’-broom (WB) mycoplasmalike organism (MLO) was transmitted from naturally infected pigeon pea (Cajanus cajan) to periwinkle (Catharanthus roseus) and induced symptoms typical of a “yellows” group disease. Preparations enriched with MLOs were obtained from periwinkle by differential centrifugation after tissues were ground in an osmotically augmented buffer. Total DNA was extracted from these preparations and MLO DNA was separated from host DNA by cesium chloride-bisbenzimide buoyant density gradient centrifugation. EcoRI-HindIII restriction fragments of MLO-associated DNA were cloned in pUC19 and transformed Escherichia coli DH5α. Selected recombinant plasmids or their excised inserts were labeled with [³²P]dATP and used as probes in dot and Southern hybridizations with total DNAs from plants affected by various MLO-associated diseases. Nine of 14 probes hybridized with DNA from WB-affected periwinkle and pigeon pea but not with DNA from plants infected with Eastern aster yellows, Western dwarf aster yellows, periwinkle witches’-broom, maize bushy stunt, beet leafhopper-transmitted virescence, Western-X, and lethal yellowing MLOs, nor with DNA from healthy plants. Southern hybridization analyses also demonstrated the presence of extrachromosomal DNA associated with the WB MLO. Probes containing fragments of chromosomal DNA hybridized more broadly to DNA of other MLOs than did extrachromosomal DNA probes.