Development of a novel real-time quantitative PCR method for detection of Ilyonectria robusta, the predominant species causing ginseng rusty root rot

Abstract

Rusty root rot is the most destructive soil-borne disease of ginseng caused by pathogenic Ilyonectria spp., and predominantly I. robusta, in China. However, there remains no effective strategy to control the disease. Current control of the disease requires that soil and ginseng seeds and seedlings infected with I. robusta are avoided during planting. Therefore, rapid and accurate detection of I. robusta would be indispensable in disease control programs. A one-step polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) assay was developed to detect I. robusta in ginseng seeds, roots, and soil. The species-specific primers HIS H3-F and HIS H3-R, designed based on a partial histone gene sequence of I. robusta, yielded a 268 bp product using the optimized PCR and qPCR protocol. DNA of I. robusta was detected by qPCR in all diseased soil and ginseng roots and seeds resulting from artificial inoculation and sampled from natural fields. Ilyonectria robusta was detected at an abundance of 1.42 fg/μL at 12 h post-inoculation and 191.31 fg/μL at 7 days post-inoculation in ginseng roots that showed disease symptoms. In naturally infected soil sampled from ginseng fields, pathogen abundances ranging from 13.23 to 503.39 fg/g were detected, which were 2.04–11.01 times higher than that in ginseng roots. The pathogen was first detected and was more abundant on the surface of the ginseng seed coat compared with that in the seed kernel. This study provides a high-efficiency detection technique for early diagnosis of I. robusta and real-time disease prevention and control strategies.