Detection of X-Disease Mycoplasma-Like Organisms in Plant and Insect Hosts Using Cloned, Disease Specific DNA

Abstract

DNA was extracted from MLO enriched extracts derived from Colladanus montanus leafhoppers infected with the peach yellow leafroll (PYLR) strain of X-disease. Equilibrium centrifugation in CsCl/ethidium bromide gradients produced two distinct bands. The upper DNA band was removed and sequentially digested with HindIII and EcoR1. The resulting fragments were size fractionated on agarose gels and recovered from the gel using DE 81 cellulose paper. Digested DNA was inserted into the plasmid UC8 and cloned in E. coli (strain JM83). Recombinant clones were screened by colony, dot and Southern blot hybridization using ³²P-nick translated DNA extracted from healthy and PYLR infected C. montanus and celery. Cloned, disease specific DNA hybridized with DNA extracted from Vinca major or celery infected with either the PYLR or Green Valley (GV) strain of X-disease but not with healthy plant DNA. Leafhoppers infected with either the PYLR or GV strains of X-disease were readily detected using ³²P-nick translated cloned probe, while no hybridization occurred with healthy leafhoppers. PYLR MLOs were readily detected in individual leafhoppers 2 wk after feeding on diseased celery. When tested in groups of ten, the X-disease MLO was detected in leafhoppers after a 1 wk acquisition access period. No hybridization has been detected with aster yellows infected aster or Vinca.