High-Throughput Bioassay for Detection of Latent Fungi in Postharvest Produce.

Enormous fresh agricultural produce is wasted annually due to rots caused by pathogenic microorganisms. Most pathogenic fungi attack the harvested produce by penetrating the fruit at the field and remaining quiescent or latent until the fruit ripens or senescence. In this work, a recently developed simple, cost-effective, and high-throughput 96-well plate-based assay was applied to determine the presence of pathogenic fungi in their latent stage. The surface strands immobilized on the 96-well plate, only with the presence of the complementary RNA marker (enoyl-CoA hydratase (ECH)) of the latent fungal-pathogen Colletotrichum gloeosporioides will create a complex with the target and reporter (labeled with the horseradish peroxidase (HRP) enzyme) strands for positive signal generation. The developed assay demonstrated 3.1-fold higher specificity for the latent marker (ECH) of C. gloeosporioides compared to latent markers of other pathogenic fungi. A 2 nM detection limit of target strands was demonstrated, showing a high plate sensitivity, and was further validated with biological samples extracted from latent infection in tomato fruit. The developed assay provides a new economical tool for detecting the presence of latent RNA markers of pathogenic fungi in agricultural produce, ultimately improving postharvest decision-making and reducing postharvest losses.