Development of loop-mediated isothermal amplification assays for rapid detection of blackleg pathogens in Swedish winter oil seed rape

Blackleg is a devastating disease of winter oil seed rape (WOSR) caused by the two species Leptosphaeria maculans and L. biglobosa “brassicae”. In this study, Loop-Mediated Isothermal Amplification (LAMP) assays were developed using two primer sets, SirP and PKS5, to identify and monitor L. maculans “brassicae”, and one primer set PKS21 to monitor L. biglobosa “brassicae”. The three primer sets were tested in real-time LAMP with DNA templates from 38 Leptosphaeria spp. reference isolates. The SirP and PKS5 primer sets exclusively amplified DNA from L. maculans isolates whereas, the PKS21 primers amplified DNA solely from L. biglobosa. Further specificity tests with DNA templates from other ascomycetes showed that both PKS primers were highly specific to Leptosphaeria. The SirP primer set produced in a few cases non-specific amplifications, but they were easily differentiated from the positive reactions by the differences in melting curve temperatures. The detection limits of LAMP assays with PKS21, PKS5 and SirP primers were at 100 fg, 30 pg and 180 fg DNA, respectively. DNA from leaf segments with typical L. maculans lesions was successfully amplified using primer set SirP, as was visually indicated by the colour change of phenol red from pink to yellow. This feature makes the method suitable for simple screening of L. maculans in WOSR leaves and in spores collected by spore traps.