Abstract
Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/μl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025–0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.
Highlights
Tomato brown rugose fruit virus (ToBRFV), a member of the genus Tobamovirus, family Virgaviridae, is an emerging and highly virulent virus, mainly affecting tomato crops worldwide
We have developed a rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-Loop-mediated isothermal amplification (LAMP)) and a visual RT-LAMP assay for the specific detection of ToBRFV RNA for the first time in tomato and pepper seeds using a single-tube one-step RT-LAMP and visual RT-LAMP, as well as comparing the sensitivity and specificity of the developed methods with those of reverse transcription polymerase chain reaction (RT-PCR) and realtime RT-PCR
A one-step LAMP assay for the rapid detection of ToBRFV was developed using a set of six primers designed from a highly conserved region of the RNA-dependent RNA polymerase gene (RdRp) gene (Table 1)
Summary
Tomato brown rugose fruit virus (ToBRFV), a member of the genus Tobamovirus, family Virgaviridae, is an emerging and highly virulent virus, mainly affecting tomato crops worldwide. Tomato brown rugose fruit virus constitutes an emerging threat of global concern to tomato crops, as it is able to overcome the resistance gene Tm-22 routinely used by breeders for the constitution of tomato hybrids, with special reference to those destined for protected cultivation, as it is effective in controlling other tomato tobamoviruses, such as tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV; Zhang et al, 2013). Pepper varieties harboring the L1, L3, or the L4 alleles of the L resistance gene to tobamoviruses have displayed hypersensitivity response (HR) when inoculated with ToBRFV, allowing for some control of the virus. A severe outbreak of ToBRFV in a red sweet pepper (Capsicum annum) variety not harboring the resistance gene, has been recorded in Sicily (south Italy), with an incidence of the viral disease of about 85% (Panno et al, 2020b)
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