Detection and Diagnosis of Xylella fastidiosa by Specific Monoclonal Antibodies

María Teresa Gorris,Ester Marco-Noales,Javier Peñalver,Mario Colomer,Antonio Sanz,María M López

doi:10.3390/agronomy11010048

María Teresa Gorris, Ester Marco-Noales + Show 4 more

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https://doi.org/10.3390/agronomy11010048

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Abstract

Monoclonal antibodies (MAb) specific to Xylella fastidiosa were obtained through hybridoma technology using heat-treated somatic O antigens from LMG 17159strain. Ten stable hybrydoma clones secreting MAb were selected and their isotype was determined. The MAbs 2G1/PPD, IgG1 showed specificity for X. fastidiosa, detecting all the analyzed strains representing different subspecies, STs and hosts. Polyclonal antibodies (PAb) against X. fastidiosa were also produced and antiserum 17159-O/IVIA was selected for the highest titre and its excellent detection capability. MAb 2G1/PPD was tested against strain IVIA 5235 in PBS and in spiked raw extract samples from almond, olive, citrus, and other hosts and its sensitivity by DAS-ELISA was 104 CFU mL−1. The MAb also reacted with high affinity and avidity against X. fastidiosa by DASI-ELISA and Tissue print-ELISA. The diagnostic parameters of DAS-ELISA based on MAb were calculated and compared with the gold standard real-time PCR. The diagnostic specificity of MAb2G1/PPD was 100%, the diagnostic sensitivity was 88.5% compared to Harper’s real-time PCR and 89.9% compared to Francis’ real-time PCR. The agreement between the techniques was almost perfect according to the estimated Cohen’s kappa-index, even in symptomless almond trees. The developed immunological techniques represent sustainable and low-cost analysis tools, based on specific, homogeneous, and well-characterized MAbs, which can be obtained in unlimited quantities in a reproducible way and constitute a guarantee for the standardization of commercial kits. They are a valuable option within a polyphasic strategy for the detection of X. fastidiosa.

Highlights

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.license.Xylella fastidiosa is one of the fifteen quarantine bacteria of highest phytopathological interest in the European Union (EU) [1]
That is 2G1/PPD, 1C6/PPD (IgG1 ), and 9F7/PPD (IgG2 ) were initially selected for their high affinity to react against the bacterial antigens, high avidity as well as wide reaction spectrum against different X. fastidiosa strains
Immunoglobulins of the three Monoclonal antibodies (MAb) were purified by affinity chromatography in columns, conjugated with alkaline phosphatase, and combinations of those three MAb tested by DAS-ELISA MAb

Summary

Introduction

Xylella fastidiosa is one of the fifteen quarantine bacteria of highest phytopathological interest in the European Union (EU) [1]. It causes damaging diseases in strategic crops of socio-economic importance, and in ornamental and wild plants of a wide variety of botanical species [2]; the overall number of host plants in the last update by EFSA reached. X. fastidiosa is currently present in several European countries, mainly in Italy, France, and Spain [7], where a number of areas are under eradication or containment strategies in order to avoid dissemination of this quarantine organism [8]. The situation is of great concern to international organizations such as the European and Mediterranean

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