Poster: Diseases of Plants: Disease Detection & Diagnosis
Development, testing and pending deployment of a LAMP diagnostic screening method for the Citrus Black Spot pathogen, Guignardia citricarpa.
K. ZELLER (1), K. Levin (2), G. Wei (2), G. Abad (2), H. Gomez (3), T. Riley (3), Z. Liu (2) (1) USDA-APHIS-PPQ, CPHST, U.S.A.; (2) USDA-APHIS-PPQ, CPHST, U.S.A.; (3) USDA-APHIS-PPQ, CHRP, U.S.A.
Guignardia citricarpa, the causal agent of Citrus Black Spot (CBS) was discovered in Florida during 2010. Today, three FL counties remain under quarantine. G. citricarpa causes unsightly lesions that reduce fruit marketability, and CBS is subject to national and international quarantine restrictions. The affected packing houses must monitor all fruits coming into the facility before they ship to the EU. If CBS symptoms are found on just a single fruit, entire shipments are put on hold where this perishable commodity’s time is extremely valuable and a decision must be made. Current protocol requires that suspect fruit be shipped to PPQ or State Labs for confirmatory diagnosis which include a conventional and qPCR assay. We have developed a diagnostic tool that will enable inspectors to turn a 2-3 day decision into less than 1 hour using a LiNK extraction method followed by Loop-Mediated Isothermal AMPlification (LAMP). The LiNK column allows for rapid DNA extraction for use on the Genie III, a durable, sensitive, and user-friendly isothermal platform. Our LAMP screening assay is able to detect down to 30 fg of DNA with amplification times between 5-20 minutes. The Genie has been able to reproduce qPCR results on all 55 citrus samples tested from Florida as well as 32/33 CBS DNA diagnostic samples submitted to CPHST. Further testing on environmental samples and non-target hosts are underway. We will discuss the advantages and limitations of the method and deployment.