Development of Grower Performed LAMP PCR for Detection-Based Management Programs for Grapevine Powdery Mildew in Vineyards
Inoculum detection for timing fungicide applications against grape powdery mildew has been shown to work using qualitative and quantitative PCR approaches. However, these approaches require expensive equipment, specialized skills, and labor costs that impede implementation by viticulturist. Loop mediated isothermic PCR (LAMP) is a robust method for the detection of DNA that can be performed with minimal equipment and skill. A set of LAMP primers were designed against the ITS2 segment of the ribosomal DNA region of Erysiphe necator that are specific and can detect less than one spore or less than 5 copies of target DNA in a purified plasmid. Spores were trapped from vineyard air using by continuously running an impaction trap with 40 ? 1.5mm stainless steel rods coated with vacuum grease and replacing sample rods every 3 to4 days. DNA extraction was accomplished by placing rods in 100 ?l of TE buffer, centrifuging for 1 min, boiling for 5 min, vortexing for 10 sec and then placing 5 ?l DNA extract in PCR tube with mastermix. The PCR tube was then placed at 65?C for 45 min followed by 80?C for 5 min. Positive detection was determined by the formation of white precipitate. Grower implementation was tested by placing 3 traps at each vineyard with one processed by the grower using LAMP and the others processed in the lab for LAMP and quantitative PCR (qPCR). The results of the grower implementation was that participating growers were more than 50%accurate in detecting 1 or 10 spores and 100%accurate in detecting 100 spores in spiked samples. They had 74%agreement in detecting E. necator compared to our LAMP-PCR results, and our LAMP-PCR results were 96%in agreement with our qPCR results.