Rapid Detection of Airborne Inocula of Grapevine Mildews Using PCR and LAMP Assay

Abstract

Grapes powdery mildew and downy mildew caused by Erysiphe necator and Plasmopara viticola respectively are the most devastating diseases worldwide resulting in significant loss of yield and quality. Epidemics of grapevine mildews are caused by airborne inocula such as conidia and sporangia. Rapid detection of airborne inocula will help to face up timely management strategies under field conditions. The aim of the current study was to design a suction spore trap to trap the airborne mildew inocula and their early detection by molecular methods of PCR and LAMP assay. A total of twelve airborne inocula samples were collected the weekly intervals from 3 to 14 standard weeks of 2021 during the cropping season. The presence of airborne inocula of E. necator was detected on standard weeks 3,6,10 and 13 through PCR assay which yielded an amplicon of 470 bp. Similarly, airborne inocula of P.viticola were detected on standard week 6 only through PCR which yielded an amplicon of 520 bp. A rapid, highly specific , sensitive Loop mediated isothermal amplification (LAMP) assay was performed to detect the E. necator and P. viticola using six sets of LAMP primers constructed by targeting rDNA region of ITS and the 5S rRNA and CesA4 a gene, respectively. LAMP assay efficiently detected the presence of airborne inocula of E.necator in most of the samples collected from standard week 3 – 14 except 7, 8, and 9. However, the presence of airborne inocula of P.viticola from standard week 3 – 14 was confirmed by LAMP assay. The LAMP assay is absolutely the best in identifying airborne inocula of grapevine mildews compared to PCR and phenotypic microscopic observation.

LAMP for in-field quantitative assessments of airborne grapevine downy mildew inoculum

“…Marimuthu et al (2020) also used a LAMP assay, this time based on the CesA4 gene sequence, to detect the presence of P. viticola in leaf samples. Their primer set was recently successfully used to detect airborne P. viticola inoculum in air samples obtained from a suction trap placed in a vineyard plot (Basha et al, 2021).…”