Development of a quantitative loop-mediated isothermal amplification assay for the field detection ofErysiphe necator

Abstract

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (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 (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six 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, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one 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.

Use of LAMP for Assessing Botrytis cinerea Colonization of Bunch Trash and Latent Infection of Berries in Grapevines

“…When coupled with spore trap systems, LAMP assays have been used for the detection and quantification of airborne pathogen inoculum, including the conidia of Alternaria solani and sporangia of P. infestans [70], and the conidia of Magnaporthe oryzae [56]. LAMP assays have been also developed for the quantification of airborne Erysiphe necator inoculum and have been evaluated for potential implementation by vine growers for the initiation of fungicide programmes [55,71]. LAMP has recently been applied for the detection of B. cinerea resistance to benzimidazole fungicides [60] and for the on-site detection of B. cinerea resistance to quinone outside inhibitors [61].…”

Comparative Evaluation of the LAMP Assay and PCR-Based Assays for the Rapid Detection of Alternaria solani

“…Additionally, the LAMP reaction can be assessed visually ( Liu et al, 2017 ). LAMP technologies may be useful in airborne inoculum detection and quantification assays performed with a spore trap system, as described recently for grapevine powdery mildew ( Thiessen et al, 2016 ) and rice blast pathogen ( Villari et al, 2017 ). In our study, the assays other than conventional PCR (10 ng) were very sensitive and able to detect 1.36 pg, 13.6 pg and 1 ng genomic DNA of the target fungi in the case of real time-qPCR, nested PCR and LAMP assay, respectively.…”

Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology

“…Recently, the development of LAMP portable devices has allowed a 'real-time' detection pathogen directly on-field [39,164], facilitating their diagnosis during the routine surveys or in sanitary selection or eradication programs.…”

“…In addition, it improves the accuracy of pathogens detection, and allows their quantification at the same time. Several portable devices based on this technology are commercially available, for example, "Bioranger" (Diagenetix, Inc., Honolulu, HI, USA) and "Genie II and III" (Optigene Ltd., West Sussex, UK), which are used by growers or crop consultants, especially in nursey or phytosanitary program activities [164].…”