Detection of Tilletia caries, Tilletia laevis and Tilletia controversa wheat grain contamination using loop-mediated isothermal DNA amplification (LAMP)

Abstract

The study describes a novel diagnostic protocol based on a loop-mediated isothermal DNA amplification (LAMP) for identification of wheat grains infection by Tilletia laevis, Tilletia caries (common bunt) and Tilletia controversa (draft bunt). The presented data showed that the LAMP analysis is a simple, specific and rapid method that could be used for detection of Tilletia spp. in contaminated grain samples. The lowest DNA concentration required for the successful detection of Tilletia spp. strains were estimated to be 0.001 ng/μl. Simultaneously the detection limit for wheat grain contamination by T. caries and T. laevis teliospores was estimated at 20 μg per 100 g of grain. For T. controversa detection limit was lower and was approximately 20 mg of teliospores per 100 g of grain. The negative results of the LAMP reactions were achieved for the most common fungal species colonizing wheat grain like Fusarium spp., Alternaria sp., Cladosporium sp., Helminthosporium sp., and Penicillium sp.

Keywords: Common bunt; Loop-mediated isothermal DNA amplification; Seed testing.; Tilletia spp..