Guignardia citricarpa is a fungal pathogen which causes Citrus Black Spot disease, affecting a number of economically important citrus crops including oranges, mandarins, lemons and grapefruits. Imported consignments are inspected at ports in the EU and USA and any fruits found to be infected are rejected. Diagnosis can be made based on visual assessment only if characteristic structures known as pycnidia are present in the black spot lesions.
However, pycnidia are not always present, and symptoms can be variable, ambiguous and sometimes confused with physical damage. Laboratory testing to confirm suspected black spot can be time-consuming. The Food and Environment Research Agency (Fera) has developed a G. citricarpa-specific LAMP detection method using the field-portable Genie® II instrument which can provide results in less than 30 minutes. This tool has the potential to be deployed by Plant Health Inspectors at ports.
G. citricarpa lesions were excised from the surface of infected fruit using a sterile scalpel and placed into a small heavy-gauged plastic bag containing extraction buffer. The material was lightly crushed using a hammer, and the resulting crude extract was added directly to the LAMP reaction. The assay was run on the Genie® II at 65°C for 30 minutes.
Positive detection of G. citricarpa in crude extracts was observed in less than 20 minutes; a characteristic annealing temperature peak in the range 86-87oC of the amplification product serves as a confirmation for specific amplification of the correct pathogen. This rapid and easy to use G.citricarpa sampling and detection method in conjunction with the Genie® II makes it an invaluable diagnostic tool especially for importers and inspectorate services.
This work was funded by a Plant Health Division of Defra.