Barley yellow dwarf virus and Cereal yellow dwarf virus Quantification by Real-Time Polymerase Chain Reaction in Resistant and Susceptible Plants

Abstract

Reliable detection and quantification of barley and cereal yellow dwarf viruses (YDVs) is a critical component in managing yellow dwarf diseases in small grain cereal crops. The method currently used is enzyme-linked immunosorbent assay (ELISA), using antisera against the coat proteins that are specific for each of the various YDVs. Recently, quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR) has been used to detect bacterial and viral pathogens and to study gene expression. We applied this technique to detect and quantify YDVs using primers specific for Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) coat protein genes because of the higher sensitivity of RT-PCR and the advantage of using a real-time PCR instrument. This Q-RT-PCR was used to detect BYDV and CYDV, and to examine disease development in a resistant wheatgrass, a resistant wheat line, a susceptible wheat line, and a susceptible oat line. BYDV-PAV and CYDV-RPV were detected as early as 2 and 6 h, respectively, in susceptible oat compared with detection by ELISA at 4 and 10 days postinoculation. BYDV-PAV RNA accumulated more rapidly and to a higher level than CYDV-RPV RNA in both oat and wheat, which may account for PAV being more prevalent and causing more severe viral disease than CYDV. Q-RT-PCR is reproducible, sensitive, and has the potential to be used for examining yellow dwarf disease and as a rapid diagnostic tool for YDVs.

Timing of Induction of Osmotically Controlled Genes in Salmonella enterica Serovar Typhimurium, Determined with Quantitative Real-Time Reverse Transcription-PCR

“…To our knowledge, this is the first work in which induction of genes has been monitored by Q-RT-PCR in bacteria, although this technique been used to follow the kinetics of accumulation of mRNAs in plants after viral or fungal infection (2,39). The sensitivity of Q-RT-PCR in a bacterial system has been documented by its usefulness in measuring mRNA half-lives at a 0.5-min resolution (41).…”

“…Random hexamer-primed cDNAs were obtained from the RNA preparations by using the Superscript First Strand Synthesis system for RT-PCR (Invitrogen). (2). The RT-PCR and melting-curve analysis of the amplification products were carried out with an ABI PRISM 7700 Sequence Detector (38).…”

Analysis of Accumulation Patterns of Barley yellow dwarf virus-PAV (BYDV-PAV) in Two Resistant Wheat Lines

“…ponticum (Chen et al, 1998). The limited number of available resistant wheat lines and the varied origins of the selected resistance genes have justified investigations of performance of these materials in B/CYDV resistance response, in field and/ or in greenhouses (Chen et al, 1997;Anderson et al, 1998;Ayala et al, 2001;Balaji et al, 2003;Barloy et al, 2003).…”

Salt- and osmotic stress-induced choline monooxygenase expression in Kochia scoparia is ABA-independent

“…Forward (5'-AGTGCATGC ATTTCACAACG-3') and reverse (5'-TGAGTTTCA GCTTGGGTTGC-3') primers were used to amplify a 150-bp amplicon. Normalization of cDNA content in each reaction was accomplished by co-amplifying a 151-bp amplicon from 18S rRNA as described by Balaji et al (2003), using 5'-GTGACGGGTGACGGAGAATT-3' and 5'-GACACTAATGCGCCCGGTAT-3' as reverse and forward primers, respectively. PCR was carried out in a RotorGene 6000 thermal cycler (Corbett Life Science, Mortlake, NSW, Australia).…”