First Molecular Evidence of the Occurrence of a Pea Mosaic Strain of Bean Yellow Mosaic Virus in Verbena x Hybrida

Verbena x hybrida is an ornamental annual used in rock gardens, as an edging plant, and in hanging baskets. It comes in a variety of colors and grows approximately 6 to 10 inches tall. Verbena 'Lavender Shades' plants from California showing leaf mosaic symptoms tested positive for potyvirus in an antigen-coated plate ELISA using our potyvirus broad spectrum reacting PTY-1 monoclonal as the detecting antibody. The virus was transmitted mechanically to Nicotiana benthamiana from infected verbena plants by sap inoculation. Further serological analysis with our panel of Bean yellow mosaic virus (BYMV)-specific, BYMVsubgroup, and potyvirus cross-reactive monoclonal antibodies suggested that the verbena potyvirus might be a member of the BYMV subgroup. Total RNA extractions from infected verbena and tobacco leaves were used in RT-PCR assays with generic potyvirus-specific primers, which amplify highly conserved 700bp or 1600bp fragments from the 3' terminus of most potyviruses. This region includes the potyviral coat protein (CP) and the 3' non-coding region (3'NCR). The PCR amplified fragments were cloned using standard 'TA cloning' procedures and sequenced using dye-terminator chemistry. The cloned nucleotide and putative coat protein amino acid sequences from the infected verbena and tobacco plants were compared to the corresponding regions of other potyviruses. Amino acid comparison of the CP region of the verbena potyvirus showed 95-96% identity to four pea mosaic strains (PMV) of BYMV, 85-89% identity to 20 other strains of BYMV, 7476% identity with six strains of Clover yellow vein virus (CYVV), and only 50-64% identity with 28 other potyviruses. Additionally, similar pairwise analysis of the 3'NCR of the verbena potyvirus showed 98-99% identity to PMV strains, 81-94% to other BYMVs, 68-75% to CYVVs, and 52-64% with other potyviruses. These and other phylogenetic analysis of the CP and 3'NCR sequences of PMV, BYMV, CYVV and other potyviruses confirmed the designation of the verbena potyvirus isolate as a pea mosaic strain of BYMV. INTRODUCTION Verbena, the common name for some members of the Verbenaceae, is a family of herbs, shrubs, trees, and vines (often climbing forms) of warmer regions of the world. Well-known wild and cultivated members of the family include species of the shrubby Lantana and of Verbena. Many cultivated verbenas have fragrant blossoms and leaves that are sometimes used as condiments, for distillation of oils or for tea. Verbena x hybrida is an ornamental annual that offers both upright and trailing habits and is useful as a groundcover or specimen plant used in rock gardens, as an edging plant, and in hanging baskets. It comes in a variety of colors and grows approximately 6 to 10 inches tall. Greenhouse and nursery crops in the U.S. constitute a third of the total farm cash receipts from horticulture crops and were estimated at $14.3 billion in 2003 (USDA, 2004). Viruses infecting ornamentals are a serious threat to the industry as they reduce vigor of infected plants and decrease foliage and flower quality. Previously documented viruses of verbena include Alfalfa mosaic virus, Apple mosaic virus, Arabis mosaic virus, Broad bean wilt virus, Carnation ringspot virus, Chrysanthemum virus B, Clover yellow mosaic virus, Cucumber mosaic virus, Impatiens Proc. XI tn IS on Virus Diseases in Ornamentals Ed. C.A. Chang Acta Hort. 722, ISHS 2006 necrotic spot virus, Melon necrotic spot virus, Melilotus mosaic virus, Prunus necrotic ringspot virus, Ribgrass mosaic virus, Tobacco etch virus, Tobacco mosaic virus, Tobacco ringspot virus, Tobacco streak virus, Tomato aspermy virus, Tomato ringspot virus, Tomato spotted wilt virus, and Verbena latent virus (Agdia, 2003; Baker et aI., 2004; Breman et aI., 1995; Brunt, et aI., 1996, 1997; Cohen, et aI., 2003). Chrysanthemum stunt viroid and Iresine viroid have also been detected singlyor doubly-infected in Verbena (Bostan et aI., 2004). Here we report the results of our investigations to determine the identity of a potyvirus detected by our potyviral broad-spectrum monoclonal antibody in various Verbena x hybrida 'Lavender Shades' plants showing leaf mosaic symptoms. We have used electron microscopy, serology and reverse transcription polymerase chain reaction (RT-PCR) methods, and cloning and sequence analysis, to identify this potyvirus as a pea mosaic strain of Bean yellow mosaic virus. Preliminary results have been reported (Guaragna et aI., 2004). MATERIALS AND METHODS Plant Sources, dsRNA Analysis, Electron Microscopy, Inoculations and ELISA Symptomatic plants of Verbena x hybrida 'Lavender Shades' were provided to us by a commercial grower from California. Plant samples were tested for the presence of viruses by electron microscopy and dsRNA analysis as previously described (Jordan and Dodds, 1985; Jordan et aI., 2002). Samples were tested in a modified indirect ACPELISA using potyvirus-specific and broad-spectrum monoclonal antibodies (Jordan and Hammond, 1991; Jordan, 1992), or purchased polyclonal antisera (Agdia, Inc., Elkhart, IN; DSMZ, Braunschweig, Germany) as previously described (Jordan and Hammond, 1991). Representative samples from symptomatic and asymptomatic Verbena were also sent to Agdia, Inc for DAS-ELISA testing. Samples from symptomatic, PTY 1 positive Verbena leaves containing elongated particles were homogenized in 1% K2HP04 and the sap extracts were mechanically inoculated to seedlings of Chenopodium quinoa, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, Helianthus annuus, Hordeum vulgare, Lactuca sativa, Lolium perenne, Lycopersicon esculentum, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. tabacum, Phaseolus lunatus, Phaseolus vulgaris, Raphanus sativus, Tagetes erecta. Sample Preparation and RT-PCR One hundred mg of fresh leaf tissue was ground in liquid nitrogen to a fine powder and total RNAs were isolated using the RNeasy Plant Mini Kit (Qiagen, Inc., Valencia, CA) following the manufacturer's recommendations with minor modifications. The reverse transcription (RT) synthesis and PCR reactions were done using the RETROscript® First Strand Synthesis Kit for RT-PCR (Ambion, Inc., Austin, TX) according to manufacturer's instructions. cDNA was synthesized (42 for 1 hr) from ca. 1 ug total RNA using random hexamers or an 0Iigo(dT)17(A/C/G)-3' primer (PV1) (Pappu et aI., 1993). For amplification of a 335 bp fragment representing a 3' potyvirus conserved central core (ca. one-third) of the coat protein (CP) gene, 5 ul RT mix was added to 45 ul of a PCR mix [final = IX PCR Buffer (Ambion), 125 ~M each dNTP, 2.5 ~M forward primer, 2.5 ~M reverse primer, and 2.5 U of SuperTaqTM Plus (Ambion)] containing the primers U335 and 0335 (Langeveld et aI., 1991). The following PCR amplification regime was used: 94°C for 2 min; followed by 35 cycles of 94 °c for 1 min, 50°C for 3 min, 72°C for 4 min; and finally 72 °c for 10 min and overnight at 4°C. For amplification of a ca. 700 bp fragment representing the 3' half of the potyvira1 CP gene and the 3' noncoding region (3' NCR), a PCR mix using a PV1-primed RT reaction contained the PV1 and U335 primers and was amplified following the regime above. For amplification of a ca. 1600 bp fragment representing the 3' terminal portion of potyviral NIb gene, all of the CP gene and 3' NCR, the PV1-primed RT reaction PCR mix contained the PVl primer