Detection of Xylella fastidiosa in Host Plants and Insect Vectors by Droplet Digital PCR

Serafina Serena Amoia, Angelantonio Minafra, Angela Ligorio, Vincenzo Cavalieri, Donato Boscia, Maria Saponari and Giuliana Loconsole ()
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Serafina Serena Amoia: Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy
Angelantonio Minafra: Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy
Angela Ligorio: Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy
Vincenzo Cavalieri: Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy
Donato Boscia: Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy
Maria Saponari: Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy
Giuliana Loconsole: Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy

Agriculture, 2023, vol. 13, issue 3, 1-15

Abstract: Xylella fastidiosa ( Xf ) is a Gram-negative plant bacterium that causes severe diseases affecting several economically important crops in many countries. To achieve early detection of the pathogen, a droplet digital PCR (ddPCR)-based approach was used to detect the bacterium at low concentrations in different plant species and insect vectors. In this study, we implemented the reaction conditions of a previously developed ddPCR assay, and we validated its use to detect Xf in insect vectors as well as in a broader list of host species. More specifically, the sensitivity and accuracy of the protocol were assessed by testing five plant matrices ( Olea europaea , Nerium oleander , Vitis vinifera , Citrus sinensis , and Prunus dulcis ), and for the first time, the insect vector ( Philaenus spumarius ), was either naturally infected or artificially spiked with bacterial suspension at known concentrations. The lowest concentrations detected by ddPCR were 5 ag/µL of bacterial DNA and 1.00 × 10 2 CFU/mL of bacterial cells. Both techniques showed a high degree of linearity, with R 2 values ranging from 0.9905 to 0.9995 and from 0.9726 to 0.9977, respectively, for qPCR and ddPCR. Under our conditions, ddPCR showed greater analytical sensitivity than qPCR for O. europea , C. sinensis , and N. oleander . Overall, the results demonstrated that the validated ddPCR assay enables the absolute quantification of Xf target sequences with high accuracy compared with the qPCR assay, and can support experimental research programs and the official controls, particularly when doubtful or inconclusive results are recorded by qPCR.

Keywords: droplet digital PCR; ddPCR; qPCR; Xylella fastidiosa; quarantine pest; molecular diagnosis (search for similar items in EconPapers)
JEL-codes: Q1 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18 (search for similar items in EconPapers)
Date: 2023
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