Optimisation of molecular-genetic methods for diagnosing fungi of Genus Sclerotinia

Phytopathogenic fungi are the causative agent for disruptive crop plant diseases, besides causing significant losses in crop yield annually. Sclerotinia is one of the most common and dangerous pathogens. Species of the Sclerotinia genus cause widespread disease across a broad range 40 of economically important agricultural crops. Regular screening of Sclerotinia in plant material is necessary due to wide host-plants range and absence of resistance to this phytopathogen. The purpose of this study was to test the developed systems of primers and probes to identify the most dangerous species of Sclerotinia genus, such as S. sclerotiorum, S. nivalis, S. borealis and S. minor. This system has been tested on positive samples of fungal cultures. Screening study and determination of the infection level with S. borealis were carried out on winter wheat. The material for the study was 24 samples of fungi belonging to the genus Sclerotinia obtained from Syngenta LLC and 37 winter wheat samples from different places of growth. Species identification of fungal cultures was carried out by Sanger sequencing using the developed pairs of primers for the β-tubulin (tub) gene region and a fragment of the rRNA gene cluster. Additionally, oligonucleotides were designed for the identification of S. borealis by real-time polymerase chain reaction (qPCR). In determining the analytical characteristics of the kit, the absence of false positive and false negative results was demonstrated. Screening studies of 37 winter wheat samples showed the presence of nucleic acids of the studied pathogen in 24.3% among the analyzed samples. There were no severely infected samples among the tested, which indicates that the pathogen is in the initial stage of its development.

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