Naked-Eye Molecular Testing for the Detection of Xylella fastidiosa in Mallorca (Balearic Island) Almond Orchards by Colorimetric LAMP

Beyond Xylella, Integrated Management Strategies for Mitigating Xylella fastidiosa Impact in Europe (BeXyl) (Grant Agreement 101060593). Partner/Coordinador principal: Blanca B. Landa del Castillo, Investigadora Científica del Instituto de Agricultura Sostenible (IAS-CSIC).

Xylella fastidiosa (Xf) is a quarantine pathogen heavily affecting economically important crops worldwide. Different sequence types (STs) belonging to Xf subspecies are present in various areas of Spain, including the Balearic Islands, and cause the almond leaf scorch disease (ALSD) in Prunus spp. The increased demand for rapid tests for early detection of the pathogen should enforce strict containment measures. Molecular detection through isothermal amplification reactions enables simplified instrumentation and the use of raw nucleic acid extracts. Colorimetric loop-mediated isothermal amplification (cLAMP) was applied to rapidly detect Xf in naturally infected almonds on Mallorca Island (Spain), using a quick crude sap extraction without DNA purification. Following tissue homogenization, an alkaline treatment for target DNA extraction was conducted before the cLAMP test. The cLAMP assay was able to detect up to 100 CFU/mL of the Xf bacterial suspension diluted in healthy almond sap. The same crude extracts used in the cLAMP test were also tested by qPCR. An overall positive agreement of about 47% was observed between the results of the two techniques, while a decrease in cLAMP sensitivity was evident as the bacterial titer declined in infected plants over Cq > 26–27. This study shows the potential of the cLAMP application as a rapid and low-cost point-of-care diagnostic method for the timely monitoring of Xf directly in the field.

This research was conducted through a joint action of the EU’s Horizon 2020 Projects No. 887281. ‘Biovexo; Biocontrol of Xylella and its vector in olive trees for integrated pest management’ (GA n. 887281), ‘FREE@POC; Towards an instrument-FREE future of molecular diagnostics at the Point-Of-Care’ (GA n. 862840) and also partially supported by ‘BEXYL’ (Beyond Xylella, Integrated Management Strategies for Mitigating Xylella fastidiosa impact in Europe; grant number 101060593).

Peer reviewed