Development of loop-mediated isothermal amplification assay for rapid detection of genetically different wheat dwarf vir
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Development of loop‑mediated isothermal amplification assay for rapid detection of genetically different wheat dwarf virus isolates Katarzyna Trzmiel1 · Beata Hasiów‑Jaroszewska1 Received: 14 July 2020 / Accepted: 15 September 2020 © The Author(s) 2020
Abstract Wheat dwarf virus (WDV) is considered as one of the most common viruses on cereal crops. Recently, severe outbreaks of WDV have been observed especially on winter wheat in southwestern part of Poland. Moreover, the presence of genetically different WDV-barley-specific and WDV-wheat-specific forms (WDV-B and WDV-W, respectively) was confirmed. In this study, a loop-mediated isothermal amplification assay (LAMP) was developed for the first time for efficient and rapid detection of WDV-B and WDV-W in infected plants. The reaction was performed using a set of three primer pairs: WDVF3/ WDVB3, WDVFIB/WDVBIP and WDVLoopF/WDVLoopB specific for coat protein coding sequence. The amplified products were analyzed by direct staining of DNA, gel electrophoresis and real-time monitoring of the amplification curves. The sensitivity of optimized reaction was tenfold higher in comparison with conventional PCR. LAMP assay developed here is a useful and practical method for the rapid detection of different WDV isolates and can be implemented by phytosanitary services. Keywords WDV-W · WDV-B · Wheat · Barley · Identification · LAMP technique
Introduction Wheat dwarf virus (WDV) is a member of the genus Mastrevirus in the family Geminiviridae. The virus is transmitted by leafhoppers Psammotettix alienus (Dahlb.) [1] and P. provincialis [2] and it infects numerous species within the family Poaceae including important cereals (mainly wheat and barley) [3]. WDV induces yellowing of leaves, severe stunting and, in case of early infection, death of infected plants which may lead to yield losses up to 80% [4]. The virus was first reported in Czechoslovakia [5] and subsequently in many countries in Europe, Asia and North Africa [6]. Severe outbreaks of WDV have been reported in Sweden [7], Czech Republic [8], Austria [6] and recently also in Poland [9]. The virus has monopartite single-stranded circular (ss) DNA genome of ~2.7 kb which encodes four proteins: the movement protein (MP), the coat protein (CP) and two others * Katarzyna Trzmiel [email protected] 1
Department of Virology and Bacteriology, Institute of Plant Protection–National Research Institute (IPP-NRI), ul. Wł. Węgorka 20, 60‑318 Poznań, Poland
associated with replication (Rep and RepA). Large and small intergenic regions (LIR and SIR, respectively) contain regulatory elements for viral replication and transcription [3]. The recently published results of the phylogenetic studies confirmed division of the global WDV population into two clearly separated WDV-wheat and WDV-barley-specific groups, according to their host. The studies have shown that the crucial regions for this division were MP and LIR [10]. Moreover, six WDV strains (A-E) have been distinguished based on sequence sim
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