High-Throughput Sequencing of Small RNAs for the Sanitary Certification of Viruses in Grapevine

Abstract

Biological indexing is the method generally recognized for the certification of propagative grapevines in many countries, and it is mandatory in the European Union. It consists of the evaluation of the plant material after grafting on indicators that are inspected for symptom development. This is a lengthy process that requires well-trained workers, testing field, etc. Alternative diagnostic methods such as serology and RT-qPCR have been discarded for certification because of their intrinsic drawbacks. In turn, high-throughput sequencing (HTS) of plant RNA has been proposed as a plausible alternative to bioassay, but before it is accepted, different aspects of this process must be evaluated. We have compared the HTS of small RNAs with bioassays and other diagnostic methods from a set of 40 grapevine plants submitted for certification. The results allowed the authors the identification of numerous grapevine viruses in the samples, as well as different variants. Besides, relationships between symptom expression and viromes were investigated, in particular leafroll-associated viruses. We compared HTS results using analytical and bioinformatics approaches in order to define minimum acceptable quality standards for certification schemes, resulting in a pipeline proposal. Finally, the comparison between HTS and bioassay resulted favorable for the former in terms of reliability, cost, and timing.

Application of High-Throughput Sequencing for Comprehensive Virome Profiling in Grapevines Shows Yellows in Iran

“…The automated VirusDetect package has already been used to identify viruses in grapevine, soybean, sweet potato, cherry tree, and lettuce [ 49 , 50 , 51 , 52 ]. Also, the highest number of viruses was detected in contigs assembled with k-mer = 15 in Velvet, which is consistent with previous studies [ 53 , 54 ]. Therefore, when more than one assembler or k-mer was used, the chance of identifying viruses with low concentrations or those that do not have many reads in the library increased [ 55 ].…”

Characterization of Grapevine Fanleaf Virus Isolates in ‘Chardonnay’ Vines Exhibiting Severe and Mild Symptoms in Two Vineyards

“…Another criterion for cross-protection that should be carefully examined is the eventual presence of other viruses in the infected host plant, potentially responsible for specific intra host virus-virus interactions that might affect the ability of the mild strain at protecting the crop [ 66 , 67 ]. This is particularly important for grapevine for which multi-infection is common generally displaying a complex virome in the field [ 20 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 ]. With our RNA-Seq data, we showed that several viroids and viruses were present in the studied vines samples.…”

Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants

“…The sRNA method explicitly targets the products of the host antiviral response mediated by plant Dicer-like enzymes (DCLs) that cleave viral RNA and replication intermediates into 21–24 nt-long fragments (reviewed in [ 14 ]). Moreover, the reduced costs of sRNA-seq and its ability to flag actively replicating viral pathogens compared to RNA-seq have made it a widely adopted approach for viral pathogen diagnostics [ 10 , 15 , 16 , 17 ].…”