Detached leaf inoculation assay for evaluating resistance to the spinach downy mildew pathogen

Abstract

Downy mildew, caused by the obligate oomycete Peronospora effusa (Pfs), is the most economically important disease of spinach. In recent years, numerous new races of the pathogen have overcome the resistance used in newly released cultivars. Resistance to Pfs in spinach is predominantly governed by dominant major resistance genes (RPFs) that are widely used in commercial spinach hybrid cultivars. Currently, a labor and resource-intensive inoculation test of whole plants, in a large tray format, in a temperature-controlled growth chamber and dew chamber, is employed to evaluate the resistance of spinach germplasm and to characterize races of the downy mildew pathogen. The objective of this work was to evaluate, standardize, and validate a more efficient detached leaf inoculation method to differentiate resistant and susceptible spinach genotypes and characterize Pfs races on a standardized set of spinach differential genotypes. Detached leaves and cotyledons of standard host differentials commonly used for race differentiation were placed on water agar in closed Petri dishes and inoculated by spraying the leaves with a spore suspension of Pfs inoculum. Disease incidence and severity on detached leaves and cotyledons were compared to the response of the corresponding cultivars in the standard whole-plant assay. There was a complete match between the disease reaction on whole plants and the disease reaction on detached leaves for all three races of Pfs examined. Furthermore, the obligate pathogen could infect, sporulate, and maintain pathogenicity by propagation solely on detached leaves. The detached leaf assay could facilitate advances in breeding for Pfs by evaluating resistance, pathogen race identification, and studies on epidemiology and genetics of the pathogen as the tests are less labor, resource-intensive than the whole-plant assay format, and environmental variables can be more accurately controlled.