Bean yellow mosaic virus: from Koch's postulates to next generation sequencing and their use to unravel the cause of black pod syndrome of narrow-leafed lupin

[Truncated] Black pod syndrome (BPS) causes devastating losses in Lupinus angustifolius (narrow-leafed lupin) crops in Australia, and late infection with Bean yellow mosaic virus (BYMV) (genus Potyvirus) was suggested as a possible cause. In 2011, an end of growing season survey of L. angustiolius plants with BPS from six locations in south western Australia was conducted. Tissue samples from different positions on each of these symptomatic plants were tested for BYMV and generic potyvirus by ELISA and RT-PCR. Detection was most reliable when RT-PCR with generic potyvirus primers was used on tissue taken from the main stem of the plant just below the black pods. An initial glasshouse experiment revealed that mechanical inoculation of L. angustifolius plants with BYMV after pods had formed caused pods to turn black. A subsequent experiment in which plants were inoculated at eight different growth stages confirmed that BPS was only induced when L. angustifolius plants were inoculated after first flowering when pods were forming. Thus, BYMV was isolated from a symptomatic L. angustifolius survey sample, inoculated to and maintained in culture hosts, inoculated to healthy L. angustifolius test plants inducing BPS and then successfully re-isolated from them. As such, Koch’s postulates were fulfilled for the hypothesis that late infection with BYMV causes BPS in L. angustifolius plants.

One of the questions remaining was whether any other related plant viruses may also cause BPS and be a threat to Lupinus species. Hardenbergia mosaic virus (HarMV), genus Potyvirus, belongs to the Bean common mosaic virus (BCMV) potyvirus lineage found only in Australia. The original natural host of HarMV is Hardenbergia comptoniana, family Fabaceae, which is indigenous to the South-West Australian Floristic Region (SWAFR), where Lupinus spp. are grown as introduced grain legume crops or exist as naturalized weeds. Two plants of H. comptoniana and one of L. cosentinii, each with mosaic and leaf deformation symptoms, were sampled from a small patch of disturbed vegetation at an ancient ecosystem-recent agroecosystem interface. Potyvirus infection was detected in all three samples by ELISA and RT-PCR. After sequencing on an Illumina HiSeq 2000, three complete and two nearly complete HarMV genomes from H. comptoniana and one complete HarMV genome from L. cosentinii were obtained. Phylogenetic analysis which compared either whole genomes or coat protein genes revealed that three of the complete and one of the nearly complete new genomes