Sequencing viral siRNAs to identify previously undescribed viruses and viroids in a panel of ornamental plant samples structured as a matrix of pools

• Published in: Virus research 2017-09, Vol.241, p.19-28
• Main Authors: Verdin, Eric, Wipf-Scheibel, Catherine, Gognalons, Patrick, Aller, François, Jacquemond, Mireille, Tepfer, Mark
• Format: Article
• Language: English
• Subjects: Agricultural sciences; Life Sciences; Microbiology and Parasitology; Next-generation sequencing; Ornamental plant; siRNA; Viroid; Virology; Virus
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2017.05.019

•Deep sequencing of siRNAs allowed de novo assembly of contigs to identify viruses.•Samples could be pooled before deep sequencing without loss of virus detection.•Detection of 27 previously unknown viruses, including all types of viral genomes.•Ornamental sector could be the source of novel plant diseases in all types of crops. Ornamental plants constitute a largely unknown and potentially important source of pathogens affecting not only ornamental plants, but also major crop species. We have carried out studies using high-throughput sequencing of 21–24 nt RNAs from potentially virus-infected ornamental plants, followed by assembly of sequence scaffolds, to identify the virus and viroid genomes present in a panel of 67 plant samples representing 46 species belonging to the main sectors of the ornamental plant industry (cut flowers, pot plants, bulbs). A pilot study demonstrated that samples could be pooled (5 samples per pool), and the overall process simplified without loss of detection of important known pathogens. In a full-scale study, pools of 5 samples were organized in a 5×5 matrix to facilitate attribution of a sequence to a precise sample directly from analysis of the matrix. In the total of 67 samples analyzed in the two studies, partial sequences suggesting the presence of 25 previously unknown viruses and viroids were detected, including all types of virus and viroid genomes, and also showed four cases of known viruses infecting previously undescribed hosts. Furthermore, two types of potential mis-assembly were analyzed, and were shown to not affect the conclusions regarding the presence of the pathogens identified, but show that mis-assembly can affect the results when the objective is determining complete bona fide viral genome sequences. These results clearly confirm that ornamental plants constitute a potential source of unknown viruses and viroids that could have a major impact on agriculture, and that sequencing siRNAs of potentially virus- or viroid-infected ornamental plants is an effective means for screening for the presence of potentially important pathogens.