Monitoring of Peronospora destructor Primary and Secondary Inoculum by Real-Time qPCR

Abstract

Onion downy mildew (ODM), caused by Peronospora destructor, is a serious threat for onion growers worldwide. In southwestern Québec, Canada, a steady increase in occurrence of ODM has been observed since the mid-2000s. On onion, P. destructor can develop local and systemic infections producing numerous sporangia which act as initial inoculum locally and also for neighboring areas. It also produces oospores capable of surviving in soils and tissues for a prolonged period of time. A recent study showed that ODM epidemics are strongly associated with weather conditions related to production and survival of overwintering inoculum, stressing the need to understand the role of primary (initial) and secondary inoculum. However, P. destructor is an obligate biotrophic pathogen, which complicates the study of inoculum sources. This study aimed at developing a molecular assay specific to P. destructor, allowing its quantification in environmental samples. In this study, a reliable and sensitive hydrolysis probe-based assay multiplexed with an internal control was developed on the internal transcribed spacer (ITS) region to quantify soil- and airborne inoculum of P. destructor. The assay specificity was tested against 17 isolates of P. destructor obtained from different locations worldwide, other members of the order Peronosporales, and various onion pathogens. Validation with artificially inoculated soil and air samples suggested a sensitivity of less than 10 sporangia g−1 of dry soil and 1 sporangium m−3 of air. Validation with environmental air samples shows a linear relationship between microscopic and real-time quantitative PCR counts. In naturally infested soils, inoculum ranged from 0 to 162 sporangia equivalent g−1 of dry soil, which supported the hypothesis of overwintering under northern climates. This assay will be useful for primary and secondary inoculum monitoring to help characterize ODM epidemiology and could be used for daily tactical and short-term strategic decision-making.