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.

Paper-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the identification of latent Colletotrichum in harvested fruit.

Paper-based analytical devices (PADs) have gained enormous attention because of their low-cost, simple fabrication, and portability. Here, we propose a paper-based device for performing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with real-time simultaneous detection of C. gloeosporioides latent infections in tomatoes. RT-LAMP-based PAD platform comprises a paper substrate on which the DNA amplification reaction occurs. Among different types of tested papers, cellulose membrane (grade 4) enabled effective visualization of the amplification result. The assay was found highly selective for the latent stage of C. gloeosporioides with lower limit of detection (LOD) of 0.5 pg of total extracted RNA. The developed assay generated the results within 40 min and hence can be efficiently employed for identifying C. gloeosporioides in resource-limited settings.

Rapid and simple colorimetric detection of quiescent Colletotrichum in harvested fruit using reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) technology.

Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is one of the major causes of postharvest decay of fruits and vegetables. Detection of the pathogen at an early stage of infection is crucial to developing a disease management strategy. In this work, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of C. gloeosporioides targeting the transcript enoyl-CoA hydratase (ECH) that significantly upregulates only during C. gloeosporioides quiescent stage. The assay enabled a naked-eye detection of C. gloeosporioides RNA within 23 min based on a color change of LAMP products from pink to yellow. The detection limit of the LAMP assay was 1 pg of total RNA extracted from fruit peel in a 25 µL reaction. Positive results were obtained only in samples carrying the ECH gene, whereas no cross-reaction was observed for a different quiescent marker (histone deacetylase (HDAC)) or an appressorium marker (scytalone dehydratase, (SD)), indicating the high specificity of the method. Hence, the results indicate that the developed LAMP assay is a rapid, highly sensitive, and specific tool for the early detection of quiescent C. gloeosporioides and could be employed to manage postharvest diseases.