Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction

A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed. This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two-step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X fastidiosa. A total of 347 leafhoppers representing 16 species were captured and sampled from American elm (Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X. fastidiosa occurs. Two of these leafhopper species, Graphocephala coccinea and G. versuta, regularly tested positive for X. fastidiosa using this technique. These insects are therefore potential vectors of X. fastidiosa. Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.