Location: Horticultural Crops ResearchTitle: Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator Author
Submitted to: PeerJ
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/18/2018
Publication Date: 4/20/2018
Citation: Thiessen, L.D., Neill, T.M., Mahaffee, W.F. 2018. Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator. PeerJ. 6:e4639. https://doi.org/10.7717/peerj.4639.
DOI: https://doi.org/10.7717/peerj.4639 Interpretive Summary: The development and testing of a molecular assay to quantify, in the field, the number of grape powdery mildew spore from spore samplers is presented. While the assay was sensitive and accurate to a single spore in the lab setting it was less sensitive and accurate in the field for quantification but performed similar to other methods for detection. The new assay was easier for growers to accurately assess presence of grape powdery mildew spores in the air but not suitable for their quantification.
Technical Abstract: Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a LAMP assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP assay using a FRET-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and 6 commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix Inc., Honolulu, HI), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a qPCR assay was used as a gold standard to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the E. necator inoculum quantification was unreliable using the beta-SMART-dart devices. The qLAMP assay developed was sensitive to 1 spore in early testing of the assay, but decreased to > 20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other more reliable molecular tools.