Molecular Identification of Potyviruses Infecting Bulbous Ornamentals by the Analysis of Coat Protein (CP) Sequences

Potyviruses (genus Potyvirus, family Potyviridae) are transmitted by aphids in a non-persistent manner and cause significant losses in many crops including bulbous ornamentals. Host range, symptoms, physical and biochemical properties of many potyviruses in bulbous ornamentals are reported, but, especially for viruses infecting ornamentals of minor economical importance, sequence data are still lacking. We used molecular techniques for the identification, characterization and detection of these viruses. Leaf material of several ornamental crops showing viruslike symptoms were tested in indirect ELISA, using monoclonal antibodies specific for potyviruses. Generic potyvirus primers were used in an RT-PCR to amplify the 3’ terminal region of these viruses. The fragments encode the viral coat protein (CP) gene and comprise the 3’-untranslated region (3’-UTR). Nucleotide sequences of the obtained fragments were determined and compared with potyvirus sequences present in the NCBI database using the BLAST algorithm. We have characterized some potyviruses previously accepted by the International Committee on Taxonomy of Viruses (ICTV), including Freesia mosaic virus (FreMV), Gloriosa stripe mosaic virus (GSMV), Hippeastrum mosaic virus (HiMV), Hyacinth mosaic virus (HyaMV), Iris mild mosaic virus (IMMV) and Nerine yellow stripe virus (NeYSV). Additionally, the identities of other potyviruses infecting ornamentals such as Anemone, Galtonia, Muscari, Ornithogalum, Allium, Stenomesson and Veltheimia have been determined. These viruses, however, have not yet been reported by the ICTV. The virus-specific sequence information generated in this research project can subsequently be used to develop PCR-based detection methods. INTRODUCTION The genus Potyvirus is the largest plant virus genus, and includes more than 200 definitive and tentative species. Potyviruses are transmitted by aphids in a non-persistent manner and cause significant losses in many crops. Potyviruses have particles of 700 to 900 nm in length harboring a ssRNA genome of approximately 10 kb in length. The single open reading frame (ORF) encodes a large polyprotein which is processed into 10 smaller proteins, including the coat protein (CP). The coat protein encoding region is located at the 3’end. More than 200 distinct potyviruses were reported and accepted in the NCBI database; the complete genome sequence is only available for about 55 species. The remaining potyviruses are currently only characterized based on the coat protein encoding sequences. Host range, symptoms, physical and biochemical properties of many potyviruses in bulbous ornamentals were reported, but sequence data are still lacking, especially of viruses infecting ornamentals of minor economical importance. In this study potyviruses infecting bulbous ornamentals were identified based on their reaction to a monoclonal antibody specific to the potyvirus group and their coat protein sequence. We have used serological and molecular techniques for the identification, characterization and detection of these viruses. CP nucleotide sequences were determined and compared with potyvirus sequences present in the NCBI database using the BLAST algorithm. For species demarcation we used the criteria described by Adams et al. (2005) and listed in the current ICTV Report which states: “potyvirus species are characterized Proc. XII IS on Virus Diseases of Ornamental Plants Eds.: A.F.L.M. Derks et al. Acta Hort. 901, ISHS 2011 168 by a CP amino acid sequence identity less than about 80% and/or a nucleotide sequence identity less than about 85% over the whole genome” (Fauquet et al., 2005). MATERIALS AND METHODS Virus Sources Leaf samples of different bulbous ornamentals showing characteristic symptoms of virus infection were used for this study. Most samples are from our own collection, especially samples of viruses previously reported by the ICTV (Fauquet et al., 2005). Samples were tested in indirect ELISA using antibodies detecting a large group of members of the genus Potyvirus (Agdia Inc., IN, USA). RNA was extracted for further molecular analysis from the samples with positive reactions. RNA Extraction and RT-PCR with Degenerate Primers RNA was extracted from leaf samples using the plant tissue protocol (10 mg) of the Purescript RNA isolation Kit (Gentra Systems Inc., Minneapolis, MN, USA). The potyvirus viral RNA was detected by amplifying the 3’ terminal region by RT-PCR with a potyvirus-specific degenerate forward primers: S-primer (Chen et al., 2001) or primer U880 (Langeveld et al., 1991), in combination with an oligo-dT reverse primer. First strand cDNA synthesis was performed on 1 to 2 μg RNA in 20 μl volume at 37°C for 1 hour, with 200 ng oligo-dT primer and 200 units M-MLV reverse transcriptase (Invitrogen, California, USA). For PCR, 2 μl first strand cDNA solution was added to 23 μl amplification mixture containing 12.5 μl PCR Mastermix (Promega, Madison, USA), and 100 ng of each primer (Oligo-dT and either S-primer or U880). PCR reactions were done by first incubating for 4 min at 94°C, followed by 40 cycles each of 0.5 min at 94°C, 1 min at 48°C and 2 min at 72°C, with a final extra 10 min at 72°C. PCR products were examined by electrophoresis in 1% agarose gels. Cloning, Sequencing, and Pairwise Sequence Analysis The PCR products were ligated to pCR2.1 TOPO vector from the TA-TOPO Cloning kit (Invitrogen, California, USA). Clones containing an insert op appropriate size were identified by using EcoRI restriction digestion and selected for sequencing the insert. Nucleotide and protein sequences were analyzed using software program BLASTn or BLASTp of the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/Blast.cgi). RESULTS AND DISCUSSION The results are separated in two sections: (1) characterization of potyviruses that were previously accepted by the ICTV and (2) characterization of potyviruses that are not yet accepted by the ICTV. Characterization of Potyviruses Accepted by the ICTV 1. Freesia mosaic virus (FreMV). This virus (ICTV code: 00.057.0.01.078) was first identified in Freesia refracta in Lisse, The Netherlands by Van Koot et al. (1954). Nucleotide sequence and pairwise sequence analyses showed that the FreMV isolate (1020nt, EF203688) from cultivar Golden Yellow shared more than 98% identity with Spiranthes mosaic virus 2 (SpiMV-2) from Spiranthes (AY685219) (Guaragna et al., 2006) and from Freesia (AM748701) (Kumar et al., 2009). However, SpiMV-2 is not accepted yet by ICTV. Based on the high sequence homology between FreMV and SpiMV-2 it can be concluded that SpiMV-2 is not a distinct virus species but a synonym of FreMV. 2. Gloriosa stripe mosaic virus (GSMV). GSMV (ICTV code: 00.057.0.01.026) was first reported in Gloriosa in Germany by Koenig and Lesemann (1974). Nucleotide sequence of the GSMV isolate CP in our collection (1600 nt; EU042761) showed more than 81% identity with Glory lily mosaic virus from Chinese Taipei (EU250360). Based on the