Cloned DNA probes for detection of grapevine flavescence dorée mycoplasma-like organism (MLO).
Flavescence dorée (FD), a yellows disease of grapevine is caused by a non-cultivable mycoplasma-like organism (MLO) transmitted in vineyards by the leafhopper vector Scaphoideus titanus. In the lab., FD is transmitted from faba bean to faba bean by the leafhopper Euscelidius variegatus. Total DNA from FD-diseased faba bean was centrifuged in a bisbenzimide-CsCl density gradient. Low density DNA was collected from the gradient, digested with HindIII, ligated into plasmid pUC18 and cloned in Escherichia coli. Transformants were differentially screened by colony hybridization with 32P-labelled healthy and FD-infected leafhopper DNA as probes. The selected clones were shown to carry insects which all hybridized with FD-diseased host DNA and not with DNA from healthy host. These 32P-labelled inserts, used as probes in dot blot hybridization, enabled detection of FD-MLO in field-collected samples of grapevine. However, because of the low MLO titre in this plant, an MLO enrichment procedure using tissue from main leaf veins was necessary to ensure efficient DNA extraction.