Rapid and specific detection of Peronosclerospora sorghi in maize seeds by conventional and real-time PCR

Abstract

Downy mildew, caused by Peronosclerospora sorghi (Weston & Uppal) Shaw is a major constraint in maize production (Zea mays L.) throughout the world. The primary source of inoculum is infected seeds and airborne spores play an important role in dispersal of the pathogen. Detection of seed-borne infection of this pathogen is necessary to limit the spread of this pathogen. Hence, a conventional and SYBR Green real-time PCR assays based on sequence characterized amplified region (SCAR) marker were developed for detection of this oomycete pathogen from maize seeds. In the conventional PCR, the primer pair SCAR-F/SCAR-R was able to detect P. sorghi and shown to be highly specific, with a detection sensitivity of 5 ng of DNA from P. sorghi. The PCR assays could detect the pathogen in naturally-infected maize seeds. A real-time PCR detection protocol was standardized for rapid and sensitive detection of P. sorghi from infected maize seeds. The detection limit of the real-time PCR assay for P. sorghi was 32 fg of DNA per reaction. The standard curve obtained showed a linear correlation between input DNA and cycle threshold (Cq) with R2 of 0.99. The results demonstrated that the developed PCR technique is a highly specific and sensitive method that can be used to detect P. sorghi in seeds.