Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola

Abstract

A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-μL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estimating the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extraction method, this assay’s total detection time is shortened to approximately one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly associated with disease severity. The LAMP method was also demonstrated to be able to estimate the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew.

Real-time and spatially resolved assessment of pathogens in crops for site-specific pesticide reduction strategies

“…The correlations between temperature/humidity and spore concentration indicate that predicting the number of airborne spores using these proxys is difficult. Similar conclusions were recently reported by Carisse et al [6,7].…”

Loop‐mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

“…The CesA4 gene sequence () of P. viticola was selected as the target site for LAMP primers designing. Kong et al, , have developed an LAMP assay targeting ITS region. We hypothesized that targeting a specific gene may be more appropriate for specific detection.…”

“…The higher sensitivity made LAMP an outstanding tool for the diagnosis of P. viticola even at lower concentrations. Kong et al, , have reported ITS region‐based LAMP analysis for detection of P. viticola . In this assay, LAMP protocol was applied for on‐site detection of airborne sporangia of P. viticola in field condition through spore trap and reported that the LAMP assay is more sensitive and the detection limit of this assay up to 33 fg of genomic DNA when compared to conventional PCR (3.3 pg).…”

“…This indicates the sensitivity of this assay is 100 times more than conventional PCR. The higher sensitivity made LAMP an outstanding tool for the diagnosis of P. viticola even at lower concentrations Kong et al, 2016,. have reported ITS region-based LAMP analysis for detection of P. viticola.…”

Methods for the Diagnosis of Grapevine Viral Infections: A Review

“…The sensitivity of this reaction is either higher than, or similar to that of PCR [107,114]. LAMP was used for the detection of grapevine DNA phytopathogens [107,[115][116][117][118]. Combined with reverse transcription in one tube, LAMP was used for GLRaV-3 detection [113].…”