Identification and first report of a potato tuber necrosis-inducing isolate of Alfalfa mosaic virus in Canada

In the 2012 harvest season, several potato tubers exhibiting extensive internal necrosis were collected in a field of cv. Innovator in New Brunswick, Canada. ELISA tests for eight viruses including Potato virus Y (PVY), Potato virus S (PVS), Potato virus X, Potato virus M, Potato virus A, Potato leaf-roll virus, Potato mop-top virus, and Potato latent virus detected PVS and PVY. RT-PCR assay for 10 viruses, including the eight above plus Tobacco rattle virus and Potato spindle tuber viroid, detected the potato tuber necrosis strain of PVY (PVYNTN) and PVS. Progeny plants of the symptomatic tubers developed pale mosaic symptoms and tested positive for PVS and PVYNTNby ELISA and RT-PCR. Tubers from these plants developed severe internal necrosis. The presence of PVYNTNin the plants was confirmed by tobacco (cv. Samsun)- and potato (cv. Yukon Gold)-based bioassay (Hu et al. 2011) as typical veinal/petiole necrosis and characteristic necrotic ringspots were induced in tobacco plants and Yukon Gold potatoes, respectively. To investigate whether the internal tuber necrosis was caused by single or mixed infections with PVS and PVY, particularly PVYNTN, virus-free (VF) plants of Innovator were mechanically inoculated with PVS and various strains of PVY including PVYO, PVYN, PVYN:O, and PVYNTNsingly or mixed. Surprisingly, except tubers of plants inoculated with the yet-to-identify viral inoculum, no internal or external necrosis was induced by any of the PVS and/or PVY infections, suggesting that a novel virus/virus strain was likely the causal agent of the symptoms. Next generation sequencing for detection of virus-derived small RNAs was thereafter performed on samples as described previously (De Koeyer et al. 2013). Small RNA cDNA library was constructed following the instructions for TruSeq Small RNA Sample Preparation Kit (Illumina, San Diego, CA), sequenced on a MiSeq instrument (Illumina) and analyzed. Results revealed that, in addition to PVY and PVS, Alfalfa mosaic virus (AMV) was present in the progeny plants of the original tubers. RT-PCR using primers AMV-F (5′-CCATCATGAGTTCTTCACAAAAG-3′) and AMV-R (5′-TCGTCACGTCATCAGTGAGAC-3′) (Xu and Nie 2006) resulted in a single fragment of ∼351 bp, consistent with the predicted amplicon size of AMV. The presence of AMV was further confirmed by ELISA with an AMV-specific antibody. To prove that AMV was indeed the causal agent of internal tuber necrosis in the cultivar, the inoculum was first passed through tobacco (cv. Samsun) to eliminate PVS and then through a PVY-resistant breeding clone F87084 (Nie et al. 2015) to get rid of PVY. Once the sole presence of AMV was confirmed by RT-PCR and ELISA, the VF plantlets were mechanically inoculated with the purified AMV inoculum. Foliage symptoms including necrotic spots, mosaic, and mild calico symptoms were induced by the virus. Moreover, tubers of the AMV inoculated plants developed internal necrosis, thus demonstrating that AMV was the causal agent of internal necrosis in the field tuber samples of the cultivar. The first potato tuber necrosis-inducing AMV isolate was reported by Oswald in 1950 (Oswald 1950). To our knowledge, this is the first report of an AMV potato tuber necrosis isolate in Canada.