A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.
A new RNA virus was discovered from a horse nettle plant using high throughput sequencing (HTS) and its full genome was characterized consisting of two molecules: RNA1 and RNA2 which are 7522 and 4710 nucleotides in length, respectively. Each molecule encodes a single open reading frame flanked by 5’ and 3’ untranslated regions (UTRs) followed by a poly(A) tail at the 3’ end. Genome organization and the phylogenetic analysis revealed its close relationship with subgroup B of nepoviruses, sharing minimal similarity with any known nepoviruses, and the recombination analysis also revealed its evolutionary history within the same subgroup. These results suggest the new virus, provisionally named as horse nettle virus A, represents a new species within the genus Nepovirus.
A new RNA virus was discovered from a horse nettle plant using high throughput sequencing (HTS) and its full genome was characterized consisting of two molecules: RNA1 and RNA2 which are 7522 and 4710 nucleotides in length, respectively. Each molecule encodes a single open reading frame anked by 5' and 3' untranslated regions (UTRs) followed by a poly(A) tail at the 3' end. Genome organization and the phylogenetic analysis revealed its close relationship with subgroup B of nepoviruses, sharing minimal similarity with any known nepoviruses, and the recombination analysis also revealed its evolutionary history within the same subgroup. These results suggest the new virus, provisionally named as horse nettle virus A, represents a new species within the genus Nepovirus.
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