High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing

Abstract

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.

Viruses of Apple Are Seedborne but Likely Not Vertically Transmitted

“…Next, total RNA was isolated using the GenCatch Plant Total RNA Miniprep Kit (Epoch Life Science, Missouri City, TX, USA) following the manufacturer’s guidelines, quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA), and stored at −80 °C for up to three weeks prior to virus screening. Each sample was tested for the presence of 19 viruses and viroids of pome fruit trees ( Table 1 ) via multiplex PCR-based amplicon sequencing, as previously described by Costa et al [ 23 ].…”

PhytoPipe: a phytosanitary pipeline for plant pathogen detection and diagnosis using RNA-seq data

“…HTS-based plant pathogen detection involves two major strategies: amplicon sequencing, which uses the power of PCR to amplify specific standardized genetic marker(s), such as 16S rRNA gene for bacteria [ 21 ] or unique genomic regions of virus and viroid genomes [ 22 ]; or shotgun sequencing, which captures the complete nucleic acids present in a sample [ 20 , 23 ]. Amplicon sequencing is popularly used for identification and comparison of entire microbial communities while shotgun sequencing has wider applications for uncovering novel and emerging pathogens [ 23 ].…”