DEVELOPMENT AND APPLICATION OF CLONED DNA PROBES FOR A MYCOPLASMALIKE ORGANISM ASSOCIATED WITH SWEET-POTATO WITCHES-BROOM

EcoRI restriction fragments of genomic DNA from the mycoplasmalike organism (MLO) associated with sweetpotato witches'-broom (SPWB) were cloned in plasmid pGEM3Zf(+). Cloned inserts from 14 SPWB-MLO-specific recombinant plasmids were EcoRI-excised, labeled with digoxigenin, and used as probes. Probes hybridized with DNA derived from SPWB-MLO-affected periwinkle and sweetpotato but not with DNA from healthy plants or plants infected with MLOs associated with loofah, paulownia, and Ipamoea obscura witches'-broom, elm and aster yellows, rice yellow dwarf, and bamboo little leaf disease. Twelve of 14 probes also hybridized with the serologically related peanut witches'-broom (PNWB) MLO. In Southern hybridizations, nine of 12 probes crossreacted to PNWB MLO but could be used to differentiate the SPWB MLO from PNWB MLO readily according to the different band patterns. The probes could detect SPWB MLO DNA with as little as 0.10 ng and 0.39 ng of DNA from periwinkle and sweetpotato, respectively. SPWB MLO was also detected by hybridizing diseased-tissue blots on nitrocellulose membrane with a digoxigenin-labeled probe. A minimum of 10 pg of total DNA from diseased periwinkle and sweetpotato was needed as template to amplify SPWB MLO DNA by the polymerase chain reaction using either of two primer pairs synthesized according to the sequence of a cloned 1.4-kb insert.