First Report of Apple of Peru (Nicandra physalodes) Infected with Pepper Mottle Virus in Hawaii

Abstract

Apple of Peru (Nicandra physalodes [L.]) is a member of the Solanaceae family and one of the serious weed threats in crop fields. It is tolerant to commonly used herbicides and competes with cultivated crops (Watanabe et al. 2002). Additionally, apple of Peru and other solanaceous weeds are known as an alternative host of several viruses (Barbosa et al. 2009; Hobbs et al. 2000). Pepper mottle virus (PepMoV) in the genus Potyvirus (family Potyviridae) is a monopartite, positive-sensed, single-stranded RNA virus. PepMoV causes economic losses to peppers and tomatoes in Honduras, South Korea, and Hawaii (Kim et al. 2009; Melzer et al. 2012). In February 2018, we observed apple of Peru plants with severe virus-like symptoms: mosaic, mottling, and crinkling of leaves in a fallow area at the University of Hawaii in Manoa, HI. Six samples with severe symptoms and three asymptomatic samples were collected and tested for potyvirus infection using a potyvirus-specific ELISA (Agdia, Elkhart, IN) and universal potyvirus-specific reverse transcription polymerase chain reaction (RT-PCR) (Zheng et al. 2010). All of the symptomatic apple of Peru plants tested positive for potyviruses in both tests, whereas three asymptomatic plants tested negative. To identify the specific potyvirus involved, amplicons (328 bp) of the nuclear inclusion body (NIb) region of potyviruses that were generated by RT-PCR using universal potyvirus-specific primers were directly sequenced (GenBank accession MH476197). BLASTn analysis of the sequences showed that this virus isolate shared 95% nucleotide sequence identity with a PepMoV isolate from Florida (AF501591). Two additional primer pairs were used to further characterize the isolate: PepMoV coat protein (CP) gene primers 900/S (Melzer et al. 2012) and PepMoV NIb gene primers PMoVFP (5′-GTGATTTCAACAACCAGTTT-3′)/PMoVRP (5′-CCACTGTTATTGCCTCTGAA-3′) designed from the sequence generated by the universal potyvirus-specific primers (GenBank accession MH476197). The cDNA (1 µl) was subjected to PCR in a 20-µl reaction mixture containing 10 µl of GoTaq Master Mix (Promega) and 10 pmol of PMoVFP and PMoVRP primers. The thermal cycler program was as follows: 94°C for 5 min; followed by 35 cycles of amplification at 94°C for 30 s, 48°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. BLASTn analysis of the 670-bp CP amplicon (GenBank accession MH499458) shared 98% nucleotide sequence identity to a Hawaii isolate of PepMoV (JQ429788) infecting tomato. BLASTn analysis of the 310-bp NIb amplicon (GenBank accession MH476198) shared 97% nucleotide sequence identity to a PepMoV isolate from Florida (AF501591). These samples also tested positive for PepMoV by triple-antibody sandwich (TAS) ELISA using PepMoV-specific antibodies (Agdia). In June 2018, nine new apple of Peru samples with severe symptoms and three asymptomatic samples were collected from the same area and tested again. All of the symptomatic apple of Peru plants tested positive for PepMoV in TAS-ELISA using PepMoV-specific antibodies, whereas three asymptomatic plants tested negative. This study is the first report of apple of Peru as a host of PepMoV in Hawaii. Apple of Peru is an invasive weed in tomato and pepper fields in Hawaii, and our results indicate eliminating solanaceous weeds is an important management step to mitigate potential losses by PepMoV in these commercial crops.