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The distribution of  Ilarvirus  species populations amongst 61 Australian  Prunus  trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the  Ilarvirus  RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of  Ilarvirus  sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS.  Prunus necrotic ringspot virus  (PNRSV) was the most frequently detected  Ilarvirus , occurring in 48 of the 61  Ilarvirus -positive trees and  Prune dwarf virus  (PDV) and  Apple mosaic virus  (ApMV) were detected in three trees and one tree, respectively.  American plum line pattern virus  (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of  Ilarvirus -like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel  Ilarvirus -like sequences. Mixed infections of ilarviruses were also detected in seven  Prunus  trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of  Ilarvirus -like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full  Ilarvirus  genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the need for a standardized approach to accurately determine what constitutes an active, viable virus infection after detection by molecular based methods.

Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus