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.
An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape.
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