An Advanced One-Step RT-LAMP for Rapid Detection of Little cherry virus 2 Combined with High-Throughput Sequence-Based Phylogenomics Reveal Divergent Flowering Cherry Isolates.
[en] Little cherry virus 2 (LChV-2, genus Ampelovirus) is considered to be the main causal agent of the economically damaging little cherry disease, which can only be controlled by removal of infected trees. The widespread viral disease of sweet cherry (Prunus avium L.) is affecting the survival of long-standing orchards in North America and Europe, hence the dire need for an early and accurate diagnosis to establish a sound disease control strategy. The endemic presence of LChV-2 is mainly confirmed using laborious time-consuming reverse-transcription (RT-PCR). A rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting a conserved region of the coat protein was developed and compared with conventional RT-PCR for the specific detection of LChV-2. This affordable assay, combined with a simple RNA extraction, deploys desirable characteristics such as higher ability for faster (<15 min), more analytically sensitive (100-fold), and robust broad-range diagnosis of LChV-2 isolates from sweet cherry, ornamental flowering cherry displaying heterogenous viral etiology and, for the first time, newly identified potential insect vectors. Moreover, use of Sanger and total RNA high-throughput sequencing as complementary metaviromics approaches confirmed the LChV-2 RT-LAMP detection of divergent LChV-2 isolates in new hosts and the relationship of their whole-genome was exhaustively inferred using maximum-likelihood phylogenomics. This entails unprecedented critical understanding of a novel evolutionary clade further expanding LChV-2 viral diversity. In conclusion, this highly effective diagnostic platform facilitates strategical support for early in-field testing to reliably prevent dissemination of new LChV-2 outbreaks from propagative plant stocks or newly postulated insect vectors. Validated results and major advantages are herein thoroughly discussed, in light of the knowledge required to increase the potential accuracy of future diagnostics and the essential epidemiological considerations to proactively safeguard cherries and Prunus horticultural crop systems from little cherry disease.
Disciplines :
Biotechnology
Author, co-author :
Tahzima, Rachid ; Université de Liège - ULiège > Département GxABT > Gestion durable des bio-agresseurs ; Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), 9820 Merelbeke, Belgium
Foucart, Yoika; Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), 9820 Merelbeke, Belgium
Peusens, Gertie; Department of Zoology, Proefcentrum Fruitteelt vzw, 3800 Sint-Truiden, Belgium
Massart, Sébastien ; Université de Liège - ULiège > TERRA Research Centre > Gestion durable des bio-agresseurs
Beliën, Tim; Department of Zoology, Proefcentrum Fruitteelt vzw, 3800 Sint-Truiden, Belgium
De Jonghe, Kris ; Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), 9820 Merelbeke, Belgium
Language :
English
Title :
An Advanced One-Step RT-LAMP for Rapid Detection of Little cherry virus 2 Combined with High-Throughput Sequence-Based Phylogenomics Reveal Divergent Flowering Cherry Isolates.
Funding: This work was supported by the Federal Flemish Agency for Innovation and Entrepreneurship (Agentschap Innoveren en Ondernemen) under grant no. IWT LA-140967.
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