Molecular detection of pycnidiospores of Leptosphaeria maculans from tapes from spore sampler

Abstract: Oilseed rape in Poland is exposed to two fungal pathogens, Leptosphaeria maculans and L. biglobosa, which cause stem canker of crucifers and are responsible for considerable yield loss. Considerable reduction of seed yield is attributed primarily to stem infection with L. maculans. The primary source of disease are airborne ascospores and secondary infection is caused by fungal pycnidiospores. Modern decision support systems that allow us to forecast the disease are based on the detection of ascospores using volumetric air samplers, but little is known about the detection limits of the secondary inoculum. Analysis of presence and concentration of airborne fungal spores may be performed either by means of conventional microscopy methods or PCR-based molecular techniques. This experiment was designed to optimise methods used for molecular detection of pycnidiospores of L. maculans from tapes obtained from spore samplers and processed in the same way as for the detection of primary inoculum. The sensitiveness of two methods was compared: traditional end-point PCR and Loop-mediated Isothermal Amplification of DNA (LAMP). The detection was done using pathogen DNA extracted from vaseline-coated Melinex tapes routinely used in 7-day volumetric spore samplers of the Hirst-type. The sensitiveness of the end-point PCR was 100 pycnidiospores and 50 spores using LAMP. Lower numbers of spores were also occasionally detected. The paper for the first time describes the use of the LAMP method for the detection of the secondary inoculum of L. maculans from tapes in volumetric spore samplers.