Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported, but little attention has been paid so far to their sensitivity and reliability for diagnostic purposes. We therefore compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large scale performance test ten datasets of 21-24 nt small (s)RNA sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty to detect viral agents when they are novel and/or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases and (iv) the significant level of scientific expertise needed when interpreting pipelines results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

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Tomato chlorosis virus (ToCV) is implicated in tomato yellows disease in many countries worldwide. It has a wide host range, including cultivated species as well as arable weeds, and it is transmitted in a semipersistent manner by at least five whitefly species or biotypes of the genera Trialeurodes and Bemisia. ToCV is not seed transmitted and more than 36 weed species have been recorded as natural reservoirs, acting as unique sources both for the virus and its vectors when susceptible crops are harvested. In this study, experiments were conducted to determine the transmission parameters of ToCV by biotype Q, the most abundant biotype of Bemisia tabaci in Greece. Results showed that biotype Q is an efficient vector of ToCV and it is able to retain the virus for at least 6 days. This vector was then used for the evaluation of four widespread weed species (Solanum nigrum, Sonchus oleraceus, Amaranthus retroflexus, and Chenopodium album) as ToCV sources through transmission experiments. Solanum nigrum was shown to be the most significant viral source among the tested weeds, followed by Sonchus oleraceus, A. retroflexus, and, lastly, C. album. Nevertheless, none of them was as efficient a ToCV source as tomato. This variation could be attributed to differences in virus concentration in each plant species or possible host preference by the whitefly vector.