A Rapid Electrochemical Biosensor Diagnostic for Botrytis ssp. Causing Botrytis Gray Mold of Temperate Legumes

Abstract

Botrytis gray mold (BGM) caused by Botrytis cinerea or B. fabae is a destructive foliar fungal disease of temperate legumes such as chickpea, lentil, and fava bean. With little to no robust host resistance, fast, accurate, and quantifiable diagnosis would help to prevent disease establishment and costly overspraying. For this, gold nanoparticle-based PCR-free assays, comprising inexpensive, portable screen-printed carbon electrodes (SPCEs) and species-specific biotinylated capture probes, were developed to detect, discriminate, and quantify the causal organisms. Initially, probe specificities and sensitivities were determined (100 fg/μL ∼2 genome copies/μL) in pure fungal backgrounds using multiplexed quantitative PCR, detecting as few as 100 spores on artificially infected legume leaves. Subsequently, electrocatalytic (EC) assays were developed using functionalized magnetic nanoparticles and assessed on three lentil cultivars under quasi-field conditions. Using biotinylated capture probes, the charge densities were correlated with pathogen quantity. The limits of detection (LOD) were 10 fg for both species, 10 times more sensitive than qPCR and able to detect a single spore in a plant background. The new diagnostic tools were subsequently validated on naturally infected field material and offer substantial advances for application in advanced informed BGM management.