Rapid detection of the pathogenic fungi causing blackleg of Brassica napus using a portable real-time fluorescence detector

Abstract

The fungus Leptosphaeria maculans leading to Phoma stem canker (blackleg) of Brassica napus (oilseed rape, canola) produces the phytotoxin sirodesmin PL, which is responsible for major yield losses of oilseed rape worldwide. Polymerase chain reaction (PCR) remains the gold standard diagnostic tool for L. maculans, but the required expensive equipment and long time make it inappropriate for fast field test. Herein, a portable system for rapid assaying L. maculans and L. biglobosa is designed around recombinase polymerase amplification (RPA) with fluorescent probe as the signal indicator, which allowed the real-time assay of amplification performed on a portable device between 37 and 42 °C. The time needed to observe the positive reaction results is controlled within 30 min. The proposed assay system is a good choice for on-site disease screening of oilseed rape plant where rapid detection is valuable, including port quarantine, agriculture quality testing, and pathogen spreading control.

Keywords: Fluorescence; Isothermal; L. maculans; Oilseed rape; Rapid detection; Recombinase polymerase amplification.