Effect of inoculum density and quantitative PCR-based detection of Rhizoctonia solani AG-2-1 and Fusarium avenaceum on canola

Abstract

Canola seedling blight, caused by Rhizoctonia solani, and Fusarium spp., can result in large yield losses to canola (Brassica napus) at high inoculum pressure. The effect of inoculum density was studied by mixing different amounts of R. solani AG-2-1 and Fusarium avenaceum into a sterilized natural soil and soil-less mix (2:1, v:v) separately, and recording seedling emergence, damping-off and seedling height within ten days after seeding; root rot severity at 12 days after seeding and seed yield at harvest on canola cultivars ‘45H29’ and ‘73-77RR’. Root rot severity increased and emergence, plant height and seed yield decreased with increased inoculum density of both R. solani and F. avenaceum. For quantification of R. solani AG-2-1, a primer and TaqMan probe set (Rs21F/Rs21R/Rs21P) was designed based on the nuclear ribosomal internal transcribed spacer (ITS) region of R. solani AG-2-1. From a conventional PCR amplification, an 88-bp product was amplified from all isolates classified as AG-2-1 with the primers Rs21F and Rs21R. No product was amplified with DNA from isolates belonging to other anastomosis groups of R. solani, other pathogens or the host plant. By using quantitative PCR, DNA amounts as low as 100 fg of R. solani AG-2-1 were detected. The quantity of DNA from soil samples with different inoculum densities estimated using qPCR was highly correlated to the number of colony-forming units (cfu) obtained from the same soil samples for both R. solani AG-2-1 and F. avenaceum.