Detection and Differentiation of the Mycoplasmalike Organism Associated with Apple Proliferation Disease Using Cloned DNA Probes. F. Bonnet. Laboratoire de Biologie Cellulaire et Moléculaire, Institut National de la Recherche Agronomique and Université de Bordeaux II, Domaine de la Grande Ferrade, 33883 Villenave d’Ornon Cedex, France. C. Saillard(1), A. Kollar(2), E. Seemüller(2), and J. M. Bové(1). (1)Laboratoire de Biologie Cellulaire et Moléculaire, Institut National de la Recherche Agronomique and Université de Bordeaux II, Domaine de la Grande Ferrade, 33883 Villenave d’Ornon Cedex, France. (2)Biologische Bundesanstalt, Institut für Pflanzenschutz im Obstbau, D-6915 Dossenheim, Federal Republic of Germany.. MPMI 3:438-443. Accepted 3 August 1990. Copyright 1990 The American Phytopathological Society.

An enriched preparation of chromosomal DNA of the mycoplasmalike organism (MLO) associated with apple proliferation (AP) disease was obtained by CsCl buoyant density gradient centrifugation of the total DNA extracted from periwinkle plants infected with AP. The MLO-enriched DNA was digested with HindIII or EcoRI restriction enzymes. The fragments were ligated into plasmids pBR322 or pBR329 and amplified in Escherichia coli. Nineteen recombinant plasmids, each containing a different AP-MLO DNA fragment, were obtained. Four of these fragments were radioactively labeled and evaluated as probes for the detection of the AP-MLO in plant material. All four probes hybridized with DNA extracted from apple trees or periwinkle plants infected with the AP-MLO but not with DNA from correspondingly healthy plant material. Also, no hybridization was observed with DNA from plants infected by MLOs associated with 18 different plant diseases, with DNAs of two other mollicutes, including Spiroplasma citri, and with the extract from apple trees infected by rubbery wood disease. The probes detected the DNA of the AP-MLO in 7-15 ng of DNA from periwinkle infected with AP and in 15-30 ng of DNA from tissue of apple infected with AP. All five symptomatic apple trees examined and one of three asymptomatic apple trees gave a positive hybridization reaction with the probes. Southern hybridization of one of the probes with HindIII-restricted DNA from two different geographic and plant host isolates of the AP-MLO (grown in either periwinkle plants or apple trees) revealed the presence of a restriction fragment length polymorphism that indicates the occurrence of genetically different strains.