REPOSITORY > RESULTS

Abstract

Plant viruses are very important plant pathogens, causing economic losses by infecting cultivated plants, causing diseases, and consequently reducing crop quality and quantity. In recent years, the development of high throughput sequencing (HTS) technologies has dramatically broadened the possibilities for plant virus research and diagnostics, enabling the discovery of new or obscure viruses and virus strains and the rapid sequencing of their genomes. HTS enables the sequencing of all nucleic acids in the sample; however, due to its generic approach, it may detect the more abundant host nucleic acids while overlooking less abundant yet important viral ones. To overcome this problem, different laboratories use different sequencing platforms and sample preparation protocols. In the first study, we compared the two most widely used sample preparation protocols for viral nucleic acid enrichment (small RNAs vs. ribosomal RNA-depleted total RNA) to perform the generic detection of plant viruses and viroids on the Illumina platform. In the study, we included viruses with different genome organizations and viroids. All selected viruses/viroids were detected with both protocols; however, a putative novel cytorhabdovirus, discovered in this study, was only detected by analyzing the data generated from ribosomal RNA-depleted total RNA and not from the small RNA dataset. The obtained knowledge was then used to investigate tomato sample with unknown etiology symptoms. HTS results identified a mixed infection of three different viruses: potato virus M (Carlavirus, Betaflexiviridae), southern tomato virus (Amalgavirus, Amalgamaviridae), and (for the first time in tomato and in Slovenia) a new strain of henbane mosaic virus (Potyvirus, Potyviridae). In the next step, the complete genomic sequence of henbane mosaic virus (HMV) was assembled from the HTS reads for the first time. By re-inoculation of the infected material on selected test plants, HMV was isolated and a host range analysis was performed, demonstrating that the virus was able to infect and cause symptoms in several plant species, including tomato. Thus, we demonstrated the usability and added value of high throughput sequencing as a diagnostic technique. With this in mind and with the increasing need for fast HTS analysis, the third study was conducted. We compared the most established HTS platform (MiSeq, Illumina) using ribosomal RNA-depleted total RNA as RNA input, with different library preparation protocols using a smaller, more affordable MinION sequencer (Oxford Nanopore Technologies) for the detection of plant viruses and viroids. Protocol comparisons were performed on five selected samples, containing eleven plant viruses with different genome organizations and two viroids, which are the first example of a circular dsRNA pathogen being sequenced using nanopore technology. The results of this study suggested that, upon selection of appropriate library preparation protocols, nanopore MinION sequencing can be used for the detection of plant viruses and viroids with similar performance as Illumina sequencing; this opens the door for implementation in official diagnostics.