Viromes of Plants Determined by High-Throughput Sequencing of Virus-Derived siRNAs.

Plants growing in open airfields can be infected by several viruses even as a multiple infection. Virus infection in crops can lead to a serious damage to the harvest. In addition, virus presence in grapevine, fruit trees, and tuberous vegetables, propagated vegetatively affects the phytosanitary status of the propagation material (both the rootstock and the variety) having profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Small interfering (si) RNAs produced by the RNA silencing-based host immune system during viral infection can be sequenced by high-throughput techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample and therefore opening new avenues in virus diagnostics. This method is based on Illumina sequencing and bioinformatics analysis of virus-derived siRNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, to ensure success for every user.

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.