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© 2013 Plant Management Network.
Accepted for publication 16 October 2013. Published 17 October 2013.


Identification of Lily mottle virus and Lily symptomless virus Co-infecting Asiatic Lily in Ohio


John R. Fisher, Savannah C. Rutherford, and Nicole K. Fleming, Ohio Department of Agriculture, Plant Health Diagnostic Laboratory, Plant Health Division, Reynoldsburg, OH 43068


Corresponding author: John R. Fisher.  jfisher@agri.ohio.gov


Fisher, J. R., Rutherford, S. C., and Fleming, N. K. 2013. Identification of Lily mottle virus and Lily symptomless virus co-infecting Asiatic lily in Ohio. Online. Plant Health Progress doi:10.1094/PHP-2013-1017-01-BR.


Lily mottle virus (LMoV) and Lily symptomless virus (LSV) are two viruses that plague bulb production operations in the Netherlands (2). LMoV is a member of the Potyvirus genus of the Potyviridae family. Potyvirus genomes are a single molecule of single-stranded RNA approximately 9.7 Kb in length encoding a single, large open reading frame (ORF) that is translated into a polyprotein which undergoes proteolytic cleavage into ten individual products. The viral replicase and capsid protein regions of the polyprotein are located near the 3’ terminus of the genome (6). LSV is a member of the Carlavirus genus of the Betaflexiviridae family. The LSV genome is a single molecule of single-stranded RNA approximately 8.4 Kb in length encoding six ORFs that include the viral replicase ORF at the 5’ terminus and the capsid protein ORF at the 3’ terminus (5). Both viruses are transmitted non-persistently by aphids (1,5,6), cause varying symptoms when occurring singly or together (1,2), and may reduce bulb yields by as much as 20% or more (2).

In the spring of 2013, an Asiatic lily cv. Sunny After Eight plant showing severe mosaic/mottle symptoms (Fig. 1) was submitted to the Ohio Plant Diagnostic Network for virus testing. The plant was collected from a garden center from a small block of ten container grown lilies, all showing symptoms, originally imported from Europe. The sample tested positive for LSV and the Potyvirus group, and negative for Alfalfa mosaic virus, Apple mosaic virus, Arabis mosaic virus, Broad bean wilt virus, Carnation latent virus, Carnation mottle virus, Carnation necrotic fleck virus, Carnation ringspot virus, Cowpea mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Peanut stunt virus, Pelargonium flower break virus, Prunus necrotic ringspot virus, Tobacco etch virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato aspermy virus, Tomato bushy stunt virus, Tomato mosaic virus, Tomato ringspot virus, Tomato spotted wilt virus, and Watermelon mosaic virus by ELISA using commercially available antibodies (Agdia Inc., Elkhart, IN).



A
 
B

Fig. 1. Mosaic/mottle symptoms observed on Asiatic lily ‘Sunny After Eight’ coinfected with LMoV and LSV.


For immunocapture reverse transcription (IC-RT), magnetic beads conjugated with sheep anti-mouse IgG were incubated with monoclonal mouse anti-Potyvirus IgG (Agdia Inc., Elkhart, IN) as previously described (4). Leaf tissue samples were extracted, incubated with Potyvirus-IgG-conjugated beads, washed, and cDNAs synthesized from captured virions (4). Two LMoV sequences (accession numbers: AB570195.1 and AB054886.1), and four Tulip breaking virus (TBV) sequences (accession numbers: S44147.1, AB674535.2, S60808.1, and S60804.1) were used to design two sets of primers to amplify overlapping regions of the 3’ terminus of the LMoV polyprotein gene. TBV/LMoVfwd540: 5’-GGTGATGACCTATTGCTGGCAG-3’ and TBV/LMoVrev1370: 5’-CGTACTCATCTTTCACKGCGTTG-3’ amplify the region corresponding to nucleotides (nt) 8116-8945 (nt 7963-8792 in polyprotein ORF) and TBV/LMoVfwd900: 5’-GGTAAAGCTCCATATTATCGGAG-3’ and TBV/LMoVrev2004: 5’-GGAGCTCGGTTCCCAMCTAC-3’ (Integrated DNA Technologies Inc., Coralville, IA) amplify the region corresponding to nt 8476-9578 (nt 8323 of polyprotein ORF to 9425 of 3’ UTR) of the full-length LMoV genome. Five µl of cDNA or sterile water was used as a template for PCR reactions. Amplification was performed in 25-µl reactions containing 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 µM primer pair, 0.625 units GoTaq Flexi polymerase (Promega Inc., Madison, WI) with the cycling parameters: 94°C (2 min), 40 cycles of 94°C (45 sec), 55°C (30 sec), 51°C (30 sec), 72°C (60 sec), and a final extension of 72°C (10 min). Both primer pairs amplified distinct products of expected size; approximately 830 bp and 1100 bp, respectively (Fig. 2). The amplicons were cloned into pGEM-T vector as previously described (4), clones were screened and sequenced bidirectionally with M13 vector primers, individual clone sequences were assembled into a consensus contig (Vector NTI Advance 11.5; Invitrogen Inc.), and the partial ORF was translated (Genedoc v. 2.6.001, 2000). IC/RT-PCR was also performed for LSV using RNA-dependent RNA polymerase (RdRp) and capsid protein (CP) specific primers as previously described (3). Both primer pairs amplified distinct products of approximately 1050 bp and 925 bp respectively (data not shown), which were cloned, sequenced, and assembled as above.


 

Fig. 2. PCR detection of LMoV from cDNAs synthesized from immunocaptured virions with TBV/LMoVfwd540+rev1370 (Lane 1) and TBV/LMoVfwd900+rev2005 (Lane 2) primers. Water controls with TBV/LMoVfwd540+rev1370 (Lane 3), TBV/LMoVfwd900+rev2005 (Lane 4), LSV-RdRp (Lane 5), and LSV-CP (Lane 6) primers. LSV clones with LSV-RdRp (Lane 7) and CP (Lane 8) primers used as positive PCR controls because of compatible reaction and cycling conditions. M = 1 Kb DNA ladder (250, 500, 750, 1000, and 1500 bp markers indicated). Electrophoresis was performed in 0.8% agarose at 100 volts for 60 min in 1X TAE buffer. Gel was stained with ethidium bromide. LMoV amplicons are 829 bp and 1102 bp, respectively.

 

Seven TBV/LMoVfwd540+rev1370 and six TBV/LMoVfwd900+rev2004 clones were sequenced (The Ohio State University Plant Microbe Genomics Facility) and all were 829 and 1102 nt long, respectively. The assembled consensus contig was 1463 nt and corresponded to nt 8116-9578 of the LMoV genome encompassing the 3’ 1325 nt of the polyprotein ORF (nt 7963-9288). When aligned with a full length LMoV sequence (accession number AB570195.1) the consensus sequence was 99% identical and when translated, the predicted 440 amino acid (aa) sequence was 99.5% identical. A BLASTn search of the NCBI database using default settings (100% query coverage) resulted in four sequences with 99% nt identity (accession numbers: AB570195.1, AF531458.1, FJ618539.1, AJ564636.1). A representative of the LMoV consensus sequence was deposited in GenBank under accession number KF553658.

Six LSV-CP and four LSV-RdRp clones were used to assemble consensus sequences for those regions of the LSV genome. The CP ORF is 876 nt and encodes a predicted 291 aa protein. When aligned with the CP ORF of an LSV isolate we recently reported from Lilium martagon (3), the Asiatic lily isolate CP nt sequence was 98.4% identical and the predicted aa sequence was 100% identical. A BLASTn search of the NCBI database (default settings; 100% query coverage) resulted in several sequence matches with 99% nt identity (accession numbers JX962776.1, U43905.1, X15343.1, GQ150682.1, AJ564638.1, AJ516059.1, AJ564641.1, AJ564640.1, and AF103784.1). A representative of the LSV-CP consensus sequence was deposited in GenBank under accession number KF553659. The 5’ 1025 nt of the LSV RdRp ORF was 98.6% identical to the L. martagon isolate and the predicted 341 aa sequence was 99.4% identical. A BLASTn search using the Asiatic lily isolate partial RdRp nt sequence also resulted in several matches with 99% nt sequence identity (accession numbers AM236208.1, HM222522.1, AM422452.1, and AJ564638.1). A representative of the partial LSV-RdRp consensus sequence was deposited in GenBank under accession number KF553660.

LMoV symptoms differ according to the susceptibility of the cultivar and generally range from fairly mild to severe, whereas those caused by LSV range from symptomless to mild. LMoV symptoms can vary from leaf mottle and mosaic (such as we observed), to vein clearing, yellow streaking, leaf curling and narrowing, necrotic spots and flower breaking, to milder leaf symptoms or lack thereof. Symptoms on plants coinfected with LMoV and LSV are generally more severe than on those infected with LMoV alone (2). The results presented here demonstrate that the Potyvirus detected in Asiatic lily is in fact LMoV, and represent the first confirmed occurrence of LMoV in Ohio as well as the first confirmed detection of an LMoV-LSV coinfection. An observant garden center manager scouting plant material recognized the symptoms as potentially viral in nature and brought it to the attention of a nursery inspector so all of the remaining plants were destroyed before they could be sold. The test plant was traced back to a supplier in Europe, specifically the Netherlands. Perennial and bulb growers should benefit from these findings by gaining awareness of LMoV as yet another potential threat to their operations. It is important to be aware of both of these viruses and scout for symptomatic plants since both are mechanically and aphid transmitted and could potentially be spread to other members of the Liliaceae family.


Literature Cited

1. Allen, T. C. 1972. Lily symptomless virus. CMI/AAB Descriptions of Plant Viruses No. 96, Kew, Surrey, UK.

2. Asjes, C. J. 2000. Control of aphid-borne Lily symptomless virus and Lily mottle virus in Lilium in the Netherlands. Virus Res 71:23-32.

3. Fisher, J. R. 2013. Identification of Lily symptomless virus infecting Lilium martagon ‘PinkTaurade’ in Ohio. Online. Plant Health Progress doi:10.1094/PHP-2013-0814-01-BR.

4. Fisher, J. R. 2012. Identification of three distinct classes of satellite RNAs associated with two Cucumber mosaic virus serotypes from the ornamental groundcover Vinca minor. Online. Plant Health Progress doi:10.1094/PHP-2012-0412-01-RS

5. King, A. M. Q., Adams, M. J., Carstens, E. B., and Lefkowitz, E. J. 2012. Betaflexiviridae. Pages 920-941 in: Virus Taxonomy, 9th Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, Waltham, MA.

6. King, A. M. Q., Adams, M. J., Carstens, E. B., and Lefkowitz, E. J. 2012. Potyviridae. Pages 1069-1089 in: Virus Taxonomy, 9th Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, Waltham, MA.