A qPCR approach to quantify the growth of basil downy mildew pathogen Peronospora belbahrii during infection

Abstract

Sweet basil (Ocimum basilicum) is an economically important herb crop. Its production has been severely threatened by the downy mildew disease caused by the obligate biotrophic oomycete Peronospora belbahrii. Deployment of disease resistant cultivars would be the most cost effective, environmentally friendly control strategy. However, transferring resistance found only in wild Ocimum species to sweet basil via traditional breeding has been extremely challenging. To apply cutting-edge biotechnological approaches to breeding for basil downy mildew resistance, it is essential to better understand the molecular basis of basil-P. belbahrii interactions, for which a set of experimental tools, including a sensitive disease assessment method, are required. Here we described a quantitative PCR (qPCR) approach to quantify P. belbahrri growth during infection. The approach utilizes two pairs of primers that specifically and effectively amplify the internal transcribed spacer 2 (ITS2) region of P. belbahrii and basil β-tubulin gene for pathogen detection, and normalization of pathogen DNA relative to plant biomass, respectively. This approach was shown to be able to detect increased pathogen growth during infection time course and differentiate disease levels of different sweet basil cultivars at an early infection stage. This qPCR approach is expected to facilitate the dissection of the molecular basis of basil-P. belbahrii interactions and it can also be used for pathogen detection in propagating materials to ensure the growth of disease-free materials and prevent disease spread. Keywords: Basil downy mildew, Peronospora belbahrii, Ocimum basilicum, Pathogen quantification, qPCR