Search PMN  

 

PDF version
for printing

Peer Reviewed
Impact
Statement




© 2013 Plant Management Network.
Accepted for publication 21 June 2013. Published 1 September 2013.


First Report of Arabis mosaic virus Infecting Vinca minor in Ohio


John R. Fisher, 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. 2013. First report of Arabis mosaic virus infecting Vinca minor in Ohio. Online. Plant Health Progress doi:10.1094/PHP-2013-0901-02-BR.


Arabis mosaic virus (ArMV) is a Subgroup A member of the Nepovirus genus of the family Secoviridae. The virus has a bipartite, single-stranded positive-sense RNA genome that is translated into two polyproteins. The RNA 1 polyprotein has six domains and includes the RNA dependent RNA polymerase (RdRp) at its 3' end. The RNA 2 polyprotein has three domains and includes the capsid protein (CP) at its 3' end (3). ArMV has a broad host range, is widely distributed, and is transmitted by the dagger nematode Xiphenema diversicaudatum (4). The virus is also readily sap transmissible, transmissible by dodder, and is seed-borne in at least fifteen species in twelve plant families (4).

In the spring of 2012, a single container-grown periwinkle (Vinca minor) sample collected from a small nursery block of approximately 75 plants was submitted to the Ohio Plant Diagnostic Network as part of a Farm Bill funded survey of viruses infecting ornamental hosts. The plant exhibited mottle and marginal/interveinal chlorosis symptoms (Fig. 1) resembling a nutritional problem and was representative of approximately ten percent of the block. The sample tested positive for ArMV and negative for the Potyvirus group, Alfalfa mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Lily symptomless virus, Tobacco mosaic virus, Tobacco ringspot virus, Tobacco streak virus, Tomato mosaic virus, Tomato ringspot virus, and Tomato spotted wilt virus by ELISA using commercially produced antibodies. The sample also tested negative for Tobacco rattle virus by RT-PCR.



A
 
B

Fig. 1. Leaf mottle and marginal/interveinal chlorosis symptoms observed on Vinca minor infected with ArMV.


For immunocapture reverse transcription (IC-RT), magnetic beads conjugated with sheep anti-rabbit IgG were incubated with polyclonal rabbit anti-ArMV IgG (Agdia Inc., Elkhart, IN) as previously described (2). Leaf tissue samples were extracted, incubated with ArMV-IgG conjugated beads, washed, and cDNAs were synthesized from bound virions (2). Three full length ArMV RNA 1 sequences (accession numbers AY303786.1, GQ369528.1, NC_006057.1) and two full length ArMV RNA 2 sequences (accession numbers HQ834962.1, GQ369529.1) were used to design two sets of primers to amplify the RdRp region of RNA 1 (ArMVRdRpfwd6070, 5'- GGAGTATTCAGAAAGYGTCCGARATTTC-3'; ArMVRdRprev7185, 5'- GGTTATTTAWYGATGGTTATCCCAG-3') and the CP region of RNA 2 (ArMVCPfwd2511, 5'-GCCCCAAACTTCATTTCACATG-3'; ArMVCPrev3327, 5'-CCATGRTGGAGTCCATGACAAG-3'; Integrated DNA Technologies Inc., Coralville, IA). Five µl of cDNA or sterile water were used as a template for PCR reactions. Amplification was performed in 25 µl reactions [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); 30 cycles of 94°C (45 sec); 54°C (30 sec); 51°C (30 sec); 47°C (30 sec); 72°C (60 sec); and a final extension of 72°C (10 min). Both primer pairs amplified distinct products of the expected size (Fig. 2). The amplicons were excised, purified from the agarose, and ligated into pGEM-T vector as previously described (2). Colonies were screened for inserts which were subsequently sequenced. Sequences were assembled, subjected to pairwise and multiple sequence alignments (2), and the open reading frames (ORFs) were translated (Genedoc v. 2.6.001, 2000).


 

Fig. 2. PCR detection of ArMV from cDNAs synthesized from immunocaptured virions with CP-specific (Lane 1) and RdRp-specific (Lane 2) primers. Water controls with CP (Lane 3), RdRp (Lane 4), and M13 sequencing (Lane 5) primers. Tobacco streak virus (TSV) Hosta isolate movement protein (MP) clone (Lane 6) and TSV-Hosta CP clone (Lane 7) with M13 primers used as positive PCR controls. 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. CP and RdRp amplicons are 817 bp and 1062 bp, respectively.

 

Four RdRp and five CP clones were sequenced (The Ohio State University Plant Microbe Genomics Facility) and the ORF sequences deposited in GenBank (accession numbers KC816725-KC816733). The RdRp amplicon clones were 1062 nt and encompassed the 3’ 1018 nt of the RNA 1 polyprotein ORF representing aa 1946-2283 (338 aa) of the predicted RNA 1 polyprotein. The CP amplicon clones were 817 nt and encompassed nt 2510-3326 of the CP region of the RNA 2 polyprotein ORF representing aa 741-1011 (271 aa) of the predicted RNA 2 polyprotein. The RdRp ORF clones shared 99.5-100% nt identity with a 99.7% mean, and when translated shared 99.4-100% predicted aa identity with a 99.7% mean. The CP clones shared 98.5-99.8% nt identity with a 99.2% mean, and when translated shared 97.8-99.6% predicted aa identity with a 98.7% mean. BLASTn searches of the NCBI database using default settings (100% query coverage) with the ArMV-Vinca RdRp ORF sequences resulted in an 85% match with an ArMV isolate from winter barley (accession number GQ369528.1), and BLASTn searches using the ArMV-Vinca CP ORF sequences resulted in a 92% match with an ArMV isolate from Ligustrum vulgare (accession number EU617327.1). These results represent the first confirmed report of ArMV infecting Vinca minor in Ohio. They also demonstrate that the ArMV-Vinca isolate is only 85% and 92% identical to other ArMV sequences in GenBank with respect to its 3’ RNA 1 and RNA 2 terminal regions. Growers should be aware of ArMV as a threat to the industry due to its wide host range and ease of transmission, and of Vinca minor as a potential perennial reservoir for the virus since periwinkle is propagated by seed, division, and by cuttings (1).


Literature Cited

1. Dirr, M. A. 2009. Manual of woody landscape plants. Their Identification, Ornamental Characteristics, Culture, Propagation, and Uses. p. 1230-1231.

2. 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.

3. King, A. M. Q., Adams, M. J., Carstens, E. B., and Lefkowitz, E. J. 2012. Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Secoviridae, pp. 881-899. Elsevier Academic Press, Waltham, MA.

4. Murant, A. F. 1970. Arabis mosiac virus. CMI/AAB Descriptions of Plant Viruses No. 16. Kew, Surrey, UK.