Risk assessment of Aphanomyces euteiches root rot disease: quantification of low inoculum densities in field soils using droplet digital PCR

Aphanomyces euteiches Drechs is a pathogenic soil-borne oomycete that causes root rot of legumes, one of the most serious diseases affecting legume production worldwide. There is currently no registered pesticide and no available resistant pea cultivar. Avoidance of infested fields based on disease risk assessment prior to pea sowing remains the main method available to manage the disease, but time-consuming bioassays are required to assess disease severity on susceptible plants grown in field soil samples. Direct quantification of A. euteiches inoculum in the soil by targeting multicopy genomic sequences of the internal transcribed spacer 1 with a qPCR-based method has been proposed as a rapid alternative for disease prediction. However, the method lacks sensitivity to accurately quantify low inoculum levels from naturally infested fields. We developed a suitable methodology based on droplet digital PCR (ddPCR) to quantify low A. euteiches inoculum levels in naturally infested soils. The methodology was validated on 200 soil samples taken from four naturally infested fields in the main pea cropping area in the north of France. The comparative analysis of inoculum density and disease severity of the 50 samples within each of the four fields revealed a non-homogeneous distribution of the A. euteiches population; this explains why the disease is visible in the form of foci. A significant relationship between pea root rot disease severity determined by bioassays and A. euteiches inoculum density was highlighted, and a linear mixed model is proposed to predict disease severity from inoculum density.