Loop-mediated isothermal amplification as a good tool to study changing Leptosphaeria populations in oilseed rape plants and air samples

Abstract

A b s t r a c t LAMP is an innovative, simple, rapid, specific and cost--effective nucleic acid amplification method. Due to the use of a special enzyme -GspSSD polymerase, the reaction takes a short time and can be performed at isothermal conditions. The sensitivity and specificity of LAMP technique is significantly higher, than standard PCR techniques, as two or three specific primer pairs are used. The technique is regarded as a useful tool for the detection and identification of plant pathogens. In this work, LAMP was used to study the composition of the population of fungi of the genus Leptosphaeria, causing a damaging disease of oilseed rape, called blackleg or stem canker. The detection concerned DNA present in fungal spores contained in air samples obtained using Hirst-type volumetric trap, in Pomerania (north Poland) in 2010. The results achieved using the LAMP technique were similar to these obtained with previously used, highly specific method of Real-time PCR. Conducting LAMP reaction was much easier and less time-and cost-consuming, due to a simplified method of DNA isolation of pathogens from plant tissues. Then, the LAMP technique was used to assess the composition of the population of Leptosphaeria spp. in plants of oilseed rape collected from the field in the Opole region (south-western part of Poland) in 2013. In contrast to studies conducted in [2002][2003], the analysis of leaf symptoms showed a higher proportion of L. maculans compared to L. biglobosa, what reflects changes in the composition of pathogen population of fungi causing blackleg on oilseed rape in this part of Poland.

Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation ofNosema apisandN.ceranaein honeybees

“…Poon et al ., ; Njiru et al ., ) and fungi (e.g. Inácio et al ., ; Jędryczka et al ., ; Suebsing et al ., ).…”

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

“…These advantages include reportedly improved sensitivity and specificity, fast reaction times (<30 min), use at a single constant temperature, thus reducing power requirements, portable equipment and the possible use of lyophilized (freeze‐dried) reaction reagents (Beissner et al ., ; Dickinson, ; http://www.optigene.co.uk). LAMP assays are now being reported for a range of fungal plant pathogens including those of oilseed rape such as Leptosphaeria maculans / L. biglobosa (phoma stem canker; Jędryczka et al ., ), Plasmodiophora brassicae (clubroot; Kaczmarek et al ., ) and Sclerotinia sclerotiorum (sclerotinia stem rot; Duan et al ., ). However, no LAMP assay has been developed for P. brassicae despite its economic importance, especially in the UK but also elsewhere (e.g.…”

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

“…Zhou (Zhou et al 2011) developed a real-time PCR method which detected L. maculans from samples collected in Australia, Ukraine and Canada with a detection limit of 4 pg. Until now, the most promising approach was a highly sensitive detection method for L. maculans and L. biglobosa using loop-mediated isothermal amplification (LAMP) (Jedryczka et al 2013). The subsequent DNA extraction was performed using a Tiangen biotech box (Tiangen Biotech, Beijing Co Ltd, Beijing, China) and followed by a real-time PCR assay.…”