Loop-mediated isothermal amplification (LAMP) assays for rapid detection of Pyrenopeziza brassicae (light leaf spot of brassicas)

Pyrenopeziza brassicae (anamorph Cylindrosporium concentricum) is an ascomycete fungus that causes light leaf spot (LLS) disease of brassicas. It has recently become the most important pathogen of winter oilseed rape (Brassica napus) crops in the UK. The pathogen is spread by both asexual splash-dispersed conidia and sexual wind-dispersed ascospores. Such inoculum can be detected with existing qualitative and quantitative PCR diagnostics, but these require time-consuming laboratory-based processing. This study describes two loop-mediated isothermal amplification (LAMP) assays, targeting internal transcribed spacer (ITS) or -tubulin DNA sequences, for fast and specific detection of P. brassicae isolates from a broad geographical range (throughout Europe and Oceania) and multiple brassica host species (B. napus, B. oleracea and B. rapa). Neither assay detected closely related Oculimacula or Rhynchosporium isolates, or other commonly occurring oilseed rape fungal pathogens. Both LAMP assays could consistently detect DNA amounts equivalent to 100 P. brassicae conidia per sample within 30minutes, although the -tubulin assay was more rapid. Reproducible standard curves were obtained using a P. brassicae DNA dilution series (100ng-10pg), enabling quantitative estimation of amounts of pathogen DNA in environmental samples. In planta application of the -tubulin sequence-based LAMP assay to individual oilseed rape leaves collected from the field found no statistically significant difference in the amount of pathogen DNA present in parts of leaves either with or without visible LLS symptoms. The P. brassicae LAMP assays described here could have multiple applications, including detection of symptomless host infection and automated real-time monitoring of pathogen inoculum.