Abstract
Loose smut of wheat caused by the basidiomycete fungus Ustilago tritici, a seed-borne disease, is difficult to control because of the expanse of wheat planting area and difficulty in pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays are used to rapidly amplify the DNA of U. tritici. Five pairs of primers for qPCR and two series primers for LAMP were designed. Primarily, the specificity of the primer was assessed by using genomic DNA of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Further, the amplification systems were optimized. Finally, the sensitivity of qPCR and LAMP assays were evaluated. The results showed that the primer Y-430 F/R, Y-307 F/R, Y-755 F/R, and Y-139 F/R for qPCR and primers L-139 and L-988 for LAMP could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay was identified as 10 pg μL−1 of genomic DNA, the detection limit for LAMP assay was 100 fg μL−1. We successfully performed qPCR and LAMP assays on wheat loose smut wheat samples. This paper establishes two methods for U. tritici detection, which can be used for diagnosis of wheat loose smut in the laboratory and in the field.
Highlights
Wheat is one of the most important food crops in the world, and it is the staple food for most of the world's population (Garg et al, 2014)
Based on the preliminary screening results, primers are further screened by quantitative PCR (qPCR) using the control strains as temples, and determined whether the primers were specific by the Ct value reflected with the amplification curves (Fig. 2)
In qPCR assays, in order to determine whether the designed primers are specific, we performed PCR
Summary
Wheat is one of the most important food crops in the world, and it is the staple food for most of the world's population (Garg et al, 2014). Its global planting area is as high as 220 107 600 hectares (Zhao et al., 2018). With the popularization of wheat planting area and the immatureness seed detection technology, the damage of wheat loose smut has become more serious, and it has gradually become one of the most important diseases in wheat field (Knox & Menzies, 2012). The most effective control method of wheat loose smut disease is seed dressing sterilization before seeding (Singh et al, 2014; Duan et al, 2016). The detection of pathogen Ustilago tritici is very necessary. The original detection method of U. tritici is serological identification, but it is time-consuming and can only be concluded by professional. Its accuracy and sensitivity are not satisfactory (Walcott, 2003; Munkvold, 2009)
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