Ilarviruses can have significant economic impacts on stone fruit and almond production, yet their distribution and population diversity amongst commercial Australian Prunus species is unknown. PCR amplicon next generation sequencing (NGS) is a powerful approach to sequence a high number of virus amplicon samples in a single run and also detect populations of both high and low-level occurring plant viruses for diversity estimation.
In this study, NGS of genus-specific amplicons from a 371 bp conserved region of the Ilarvirus RNA2 polymerase gene was used to determine the diversity of Ilarvirus species infecting 61 Prunus trees in Australia. Apple mosaic virus (ApMV), Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) were each detected in at least 52 trees. American plum line pattern virus (APLPV) was also detected in one Prunus tree and this is a first report of this virus 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 generic amplicon NGS also allowed the detection of mixed infections of ilarviruses in seven Prunus trees and distinct RNA2 sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences.
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 correlated 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 plant samples.