Cloned DNA Probes Specific for Detection of a Mycoplasmalike Organism Associated with Ash Yellows. Robert E. Davis. Microbiology and Plant Pathology Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705. Wayne A. Sinclair(2), Ing-Ming Lee(1), and Ellen L. Dally(1). (1)Microbiology and Plant Pathology Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705; and (2)Department of Plant Pathology, Cornell University, Ithaca, NY 14853.. MPMI 5:163-169. Accepted 6 December 1991. 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, 1992.

DNA was isolated from periwinkle (Catharanthus roseus) infected with a mycoplasmalike organism (MLO) that originated in white ash (Fraxinus americana) affected by ash yellows. The DNA was digested with EcoRI and HindIII, ligated to plasmid vector pIBI30, and cloned in Escherichia coli DH5α. Cloned DNA inserts were excised from four ash yellows-specific recombinant plasmids, labeled with biotin, and employed as probes in dot and Southern hybridizations. None of the probes hybridized with nucleic acid from healthy plants; all hybridized with nucleic acid from periwinkle infected by the ash yellows MLO. Southern hybridization analyses showed the probes to contain MLO chromosomal DNA. In dot hybridizations performed at 42° C, probe AA13I hybridized only with nucleic acid from plants infected by the ash yellows MLO, whereas the other three probes, designated AA82I, AA157I, and AA176I, hybridized with nucleic acid from plants infected by any of several MLOs, including the ash yellows MLO. Under conditions of high stringency (52° C), all four probes hybridized only with nucleic acid from plants infected by the ash yellows MLO. In diagnostic tests on naturally diseased plants, probes AA13I and AA176I hybridized with nucleic acid extracted from leaves, twigs, trunk phloem, and roots of white ash with symptoms of ash yellows but not with nucleic acid from healthy ash grown from seed. The findings provide means for specific detection and identification of ash yellows MLO and support the concept that this MLO represents a distinct strain cluster.