A novel synthetic quantification standard including virus and internal report targets: application for the detection and quantification of emerging begomoviruses on tomato

Abstract

Background: Begomovirus is a genus of phytopathogenic single-stranded DNA viruses, transmitted by the whitefly Bemisia tabaci. This genus includes emerging and economically significant viruses such as those associated with Tomato Yellow Leaf Curl Disease, for which diagnostic tools are needed to prevent dispersion and new introductions. Five real-time PCRs with an internal tomato reporter gene were developed for accurate detection and quantification of monopartite begomoviruses, including two strains of the Tomato yellow leaf curl virus (TYLCV; Mld and IL strains), the Tomato leaf curl Comoros virus-like viruses (ToLCKMV-like viruses) and the two molecules of the bipartite Potato yellow mosaic virus. These diagnostic tools have a unique standard quantification, comprising the targeted viral and internal report amplicons. These duplex real-time PCRs were applied to artificially inoculated plants to monitor and compare their viral development.

Results: Real-time PCRs were optimized for accurate detection and quantification over a range of 2 × 10(9) to 2 × 10(3) copies of genomic viral DNA/μL for TYLCV-Mld, TYLCV-IL and PYMV-B and 2 × 10(8) to 2 × 10(3) copies of genomic viral DNA/μL for PYMV-A and ToLCKMV-like viruses. These real-time PCRs were applied to artificially inoculated plants and viral loads were compared at 10, 20 and 30 days post-inoculation. Different patterns of viral accumulation were observed between the bipartite and the monopartite begomoviruses. Interestingly, PYMV accumulated more viral DNA at each date for both genomic components compared to all the monopartite viruses. Also, PYMV reached its highest viral load at 10 dpi contrary to the other viruses (20 dpi). The accumulation kinetics of the two strains of emergent TYLCV differed from the ToLCKMV-like viruses in the higher quantities of viral DNA produced in the early phase of the infection and in the shorter time to reach this peak viral load.

Conclusions: To detect and quantify a wide range of begomoviruses, five duplex real-time PCRs were developed in association with a novel strategy for the quantification standard. These assays should be of a great interest for breeding programs and epidemiological surveys to monitor viral populations.

Figure 1

Presentation of the 435 bp sequence synthetic quantification standard. Alignment of qPCR targets of TYLCV-Mld, TYLCV-IL, ToLCKMV-like, PYMV (DNA-A and DNA-B), tomato internal control Solanum lycopersicum 25S ribosomal RNA gene (Sl25S; GenBank: X13557) with Taqman-MGB probes and forward and reverse primers.

Figure 2

Amplification plots and standard curves of the five duplex real-time PCRs. Typical amplification plots for each viral target and the corresponding internal report in theirs linear dynamic ranges are represented. The corresponding standard curves are obtained by linear regression analysis of the threshold cycle (Ct) value of each of the two standard-dilution replicates over the log₁₀ of the copies of DNA targets. The slope and the correlation coefficients are mentioned for each standard curve.

Figure 3

Average virus accumulation of TYLCV-IL, TYLCV-Mld, ToLCKMV and PYMV (DNA-A and DNA-B) in tomato plants. Data were obtained from 8 plants inoculated per virus. Vertical bars around each point represent the 95% confidence interval and the bars topped by the same letter (a to h) are not significantly different at p = 0.01 (Tuckey's HSD test). None of the inoculated plants with the ToLCKMV-[YT:Dem:03] were detected as infected at 10 dpi.