Cloned DNA probes for detection of grapevine Flavescence dorée mycoplasma‐like organism (MLO)

Abstract

Summary 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 laboratory, FD is transmitted from broadbean to broadbean by the leafhopper Euscelidius variegatus. Total DNA from FD‐diseased broadbean was centrifuged in a bisbenzimide‐CsCl density gradient. Low density DNA was collected from the gradient, digested with Hind III, ligated into plasmid pUC18 and cloned in Escherichia coli. Trans‐formants were differentially screened by colony hybridisation with 32 P‐iabelled healthy and FD‐infected leafhopper DNA as probes. The selected clones were shown to carry inserts which all hybridised with FD‐diseased host DNA and not with DNA from healthy host. These 32 P‐labelled inserts, used as probes in dot blot hybridisation, 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.