Poster: Diseases of Plants: Disease Detection & Diagnosis
Development of recombinase-polymerase amplification assay for the detection of Western X phytoplasma (‘Candidatus Phytoplasma pruni’) in sweet cherry
D. VILLAMOR (1), H. Ferguson (1), K. Eastwell (1) (1) Washington State University, U.S.A.
Resurgence of Western X disease (WX) of sweet cherry (P. avium), caused by WX phytoplasma (‘Candidatus Phytoplasma pruni’), has been observed in Washington State (WA) in recent years. The disease presents a major economic impact in WA sweet cherry production as affected trees not only decline but produce unmarketable small, insipid fruits. In this study, an isothermal recombinase-polymerase amplification assay (RPA) for the detection of WX phytoplasma was developed. Using sequence data generated by high-throughput sequencing of three isolates of WX phytoplasma from WA along with sequence available in the GenBank database, primers and probe were selected within the immunodominant protein (idpA) coding region. The RPA assay system has three attributes that make it a very useful tool for detection of WX phytoplasma by cherry growers: (1) it is fast and simple – results are obtained in 20 minutes and because it uses only crude sap preparation, no sophisticated equipment for nucleic acid extraction is necessary; (2) it is sensitive – the RPA assay showed similar sensitivity with PCR both in temporal detection throughout the growing season and the amount of bacterial DNA detected; and (3) it is specific only to WX phytoplasma. Additionally, RPA detected WX phytoplasma from total DNA extracts of three known leafhopper vectors of the pathogen; this makes it a valuable tool in pathogen vector research.