ABSTRACT
Pepper mild mottle virus (PMMoV) was first reported as a latent strain in the United States and then later reported in other countries around the world. Here, we report the first complete genome sequence of a PMMoV isolate (BL14) that was collected from chili pepper during the 2014 growing season in Oklahoma.
GENOME ANNOUNCEMENT
Chili and bell peppers are economically important cash crops grown in the United States (1). Viral infection causes destructive effects in Capsicum species, resulting in significant economic loss (2). Pepper mild mottle virus (PMMoV) is one of the important pepper viruses that infect pepper plants worldwide. PMMoV (genus Tobamovirus, family Virgaviridae) (3, 4) is a single-stranded positive-sense RNA virus with a 6.3-kb genome and is one of the viruses highly transmitted by seeds (5).
PMMoV was first identified in the early 1950s as a latent strain in the United States (5) and then in 1984 as a distinct strain in Italy (6). Since then, it has been reported in many countries around the world. In the United States, PMMoV has been isolated from peppers in several states, including Colorado, Florida, Georgia, Oregon (7, 8), South Carolina (6, 7, 9, 10), and Texas (11). Recently, we reported for the first time a PMMoV infection during the 2014 growing season in Oklahoma (12).
Although PMMoV is one of the important pepper viruses, to our knowledge no complete genome sequence has been reported from the United States. In this study, we report the first complete genome sequence of a PMMoV isolate collected from a commercial pepper field in Oklahoma.
In our previous work, a sample showing typical PMMoV symptoms was collected in Blaine County, Oklahoma (designated PMMoV isolate BL14), and brought to the laboratory. Virus-infected leaves were tested by DIBA against the antisera of PMMoV and mechanically inoculated on the leaves of healthy pepper seedlings using extracted sap from infected tissue (12). The virus culture was maintained in peppers in the growth chambers.
Total RNA was extracted according to the Tri-Reagent procedure (13) from young leaves of pepper plants systemically infected with PMMoV isolate BL14. Six pairs of overlapping primers were designed from the complete genome sequences of PMMoV isolates available from GenBank (https://www.ncbi.nlm.nih.gov). Seven genome fragments were amplified by reverse transcription-PCR, as described previously (13). PCR products of each fragment were confirmed on 1% agarose gel and then purified from the gel and cloned into a pGEM-T Easy vector. At least three to five recombinant clones for each PCR fragment were sequenced in both forward and reverse directions using an Applied Biosystems model 3130 analyzer at the Department of Biological Science, The University of Tulsa, Oklahoma.
Sequence alignment to generate consensus nucleotide sequences of each fragment was done using both Clustal W (14) and Muscle alignment (15). The complete genome of PMMoV was generated by joining overlapping sequences among adjacent fragments using both Clustal W and Muscle alignment. Sequence alignments were generated using MEGA version 7.0 software (16).
The complete genome sequence of PMMoV isolate BL14 is 6,353 nucleotides long. Nucleotide BLAST searches showed that isolate BL14 shared 94% to 99% nucleotide sequence identity with available PMMoV complete genomes in the GenBank database. Maximum nucleotide (99%) and amino acid sequence similarities were observed with a Spanish-S isolate (GenBank accession no. M81413).
Accession number(s).
The complete genome sequence of PMMoV isolate BL14 was deposited in GenBank under the accession no. MH063882.
ACKNOWLEDGMENTS
This work was supported by the Beta Beta Beta National Biological Honor Society and the Office of Research and Sponsored Programs at The University of Tulsa.
Footnotes
Citation Secrist K, Ali A. 2018. First complete genome sequence of Pepper mild mottle virus from chili pepper in the United States. Genome Announc 6:e00331-18. https://doi.org/10.1128/genomeA.00331-18.
REFERENCES
- 1.USDA-NASS. 2018. Vegetables: 2017 summary. U.S. Department of Agriculture, National Agricultural Statistics Service (USDA-NASS), Washington, DC: http://usda.mannlib.cornell.edu/usda/current/VegeSumm/VegeSumm-02-13-2018.pdf. [Google Scholar]
- 2.Alonso E, Garcia-Luque I, de la Cruz A, Wicke B, Avila-Rincon MJ, Serra MT, Castresana C, Diaz-Ruiz JR. 1991. Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper. J General Virology 72:2875–2884. doi: 10.1099/0022-1317-72-12-2875. [DOI] [PubMed] [Google Scholar]
- 3.Tsuda S, Kubota K, Kanda A, Ohki T, Meshi T. 2007. Pathogenicity of Pepper mild mottle virus is controlled by the RNA silencing suppression activity of its replication protein but not the viral accumulation. Phytopathology 97:412–420. doi: 10.1094/PHYTO-97-4-0412. [DOI] [PubMed] [Google Scholar]
- 4.King A, Adams M, Carstens E, Lefkowitz E. 2012. Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Academic Press, London, UK. [Google Scholar]
- 5.McKinney HH. 1952. Two strains of tobacco mosaic virus, one of which is seed-borne in an etch-immune pungent pepper. Plant Dis Report 36:184–187. [Google Scholar]
- 6.Wetter C, Conti M, Altschuh D, Tabillion R, Van Regenmortel MHV. 1984. Pepper mild mottle virus, a tobamovirus infecting pepper cultivars in Sicily. Phytopathology 74:405–410. doi: 10.1094/Phyto-74-405. [DOI] [Google Scholar]
- 7.CABI. 2018. Invasive species compendium. CABI, Wallingford, UK. [Google Scholar]
- 8.Hamm PB, Jaeger JR, MacDonald L. 1995. Virus diseases of pepper in northeast Oregon. Plant Dis 79:968. doi: 10.1094/PD-79-0968A. [DOI] [Google Scholar]
- 9.Greenleaf WH, Cook AA, Heyn ANJ. 1964. Resistance to tobacco mosaic virus in capsicum, with reference to the Samsun latent strain. Phytopathology 54:1367–1371. [Google Scholar]
- 10.McKinney HH. 1968. Further studies of the latent strain of the tobacco mosaic virus. Plant Dis Report 52:919–922. [Google Scholar]
- 11.European and Mediterranean Plant Protection Organization. 2017. PQR database. European and Mediterranean Plant Protection Organization, Paris, France: https://www.eppo.int/DATABASES/pqr/pqr.htm. [Google Scholar]
- 12.Ali I, Ali A. 2015. First report of Pepper mild mottle virus in peppers in Oklahoma. Plant Dis 99:736. doi: 10.1094/PDIS-11-14-1144-PDN. [DOI] [Google Scholar]
- 13.Ali A, Mohammad O, Khattab A. 2012. Distribution of viruses infecting cucurbit crops and isolation of potential new virus-like sequences from weeds in Oklahoma. Plant Dis 96:243–248. doi: 10.1094/PDIS-05-11-0419. [DOI] [PubMed] [Google Scholar]
- 14.Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. doi: 10.1038/msb.2011.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. doi: 10.1093/nar/gkh340. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. doi: 10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed] [Google Scholar]