A Colorimetric LAMP Detection of <i>Xylella fastidiosa</i> in Crude Alkaline Sap of Olive Trees in Apulia as a Field-Based Tool for Disease Containment

Abstract

Xylella fastidiosa subsp. pauca (Xfp) infects olive trees and other hosts in Southern Apulia (Italy), devastating agriculture and landscape. A containment strategy of the disease requires quick and sensitive detection tools. Therefore, a colorimetric LAMP protocol was developed using as a template a crude alkaline sap obtained from incubation of 50–60 mg of thin slices of olive twigs in a NaOH-containing buffer. This rapid molecular assay can be performed directly in the field, as it needs only a portable isothermal block. Tissues of the same olive trees analysed by this technique were also compared to qPCR (using purified total plant DNA as template) as well as digital droplet PCR (on the same crude alkaline extracts used in cLAMP). A titration of the cLAMP reaction with healthy olive sap, spiked with dilutions of in vitro cultivated Xfp cells and plasmid DNA containing the target sequence, gave positive detection results as low as 102 CFU/mL and up to 169.2 target copies/µL, equivalent to about 0.9 pg of the genomic DNA. A portable, sensitive and target-specific Xfp field test was developed, which has a 40 min sample-to-answer time and does not require any DNA isolation procedure or laboratory equipment. The application of this detection assay could help the monitoring and containment of the disease spread.

Keywords

• <i>Xylella fastidiosa</i>
• quarantine pathogen
• alkaline crude extract
• isothermal amplification
• colorimetric LAMP
• point-of-care