Abstract
The complete genome sequence (4,267 nt) of a Melon necrotic spot virus (MNSV) isolate (ABCA13-01) infecting greenhouse cucumber in Canada was determined through deep sequencing of small RNAs. Its genome sequence was most closely related to MNSV-N (97%) but lacked a 55-nucleotide insertion at the 3′ untranslated region for resistance breaking.
GENOME ANNOUNCEMENT
Melon necrotic spot virus (MNSV), a small isometric virus (30-nm diameter), belongs to the genus Carmovirus in the family Tombusviridae (1). MNSV is naturally transmitted by the soil-inhabiting fungus Olpidium radicale (2, 3) and by seeds (4, 5). It can also be transmissible by mechanical inoculation. MNSV infects mainly cucurbits, including melon, cucumber, and watermelon in the field, causing serious yield loss (6). The virus is widely distributed in Asia, Europe, and North America, including Japan (4), the United States (5), Greece (7), Sweden (8), Italy (9), The Netherlands (10, 11), Tunisia (12), China (13), and Spain (14, 15).
MNSV genomic RNA is about 4.3 kb in size (11) and contains four open reading frames (ORFs). Translation of an MNSV genome yields a polypeptide of 29 kDa (p29) and a read-through polypeptide of 89 kDa (p89) in ORF1. ORFs 2 and 3 code for two small polypeptides, each of about 7 kDa (p7A and p7B), which are involved in virus cell-to-cell movement. ORF4 codes for the 42-kDa coat protein (p42). Currently, a total of 18 complete or near-complete MNSV genome sequences are available in GenBank. From a viral disease outbreak on greenhouse cucumber in 2013 in Alberta, Canada, in addition to the Cucumber green mottle mosaic virus previously identified (16, 17), 8 of the 10 samples analyzed also contained a mixed infection of MNSV. Upon mechanical inoculation, necrotic spots were observed on the inoculated plants in several cucurbit species, including Cucumis sativus, C. metulifer, C. melo, and Citrullus lanatus and confirmed for the presence of MNSV by real-time RT-PCR (R. Li and K.-S. Ling, unpublished data). MNSV was biologically purified through three local lesion passages on C. metulifer. Symptomatic cotyledons were collected for total RNA isolation using the TRIzol reagent (Invitrogen, USA). The small RNA (sRNA) was separated from the total RNA by polyacrylamide gel electrophoresis, and the sRNA library was constructed according to the published protocol (18) and sequenced on an Illumina HiSeq 2000 system. The sRNA sequences were assembled into contigs using a bioinformatics pipeline (19). A single contig (4,267 nt) covering a complete MNSV genome was obtained and deposited in GenBank (no. KR094068). A BLASTn search using the full-genome sequence indicated that this Canadian isolate (ABCA13-01) shares a genome sequence identity of 80% to 97% to the other 18 MNSV isolates. Interestingly, the most closely related isolate is MNSV-N (KF060715), a new virulent isolate in Spain that breaks the host resistance (15).
Although they shared the strongest sequence identity, the 55-nucleotide insertion in the 3′ untranslated region of MNSV-N that is responsible for the resistance breaking (15) was not present in isolate ABCA13-01. The close phylogenetic relationship to the European isolates of MNSV suggests that this Canadian isolate might have been introduced in seeds from Europe. To our knowledge, this is the first complete genome sequence of MNSV in Canada.
Nucleotide sequence accession number.
The full-genome sequence of MNSV Canadian isolate ABCA13-01 was deposited in GenBank under the accession number KR094068.
ACKNOWLEDGMENTS
The sRNA deep sequencing was conducted by the Genomics Resources Core Facility at Weill Cornell Medical College in New York City. We thank Andrea Gilliard and Alan Wilder for their excellent technical assistance. This work was supported in part by USDA, National Institute of Food and Agriculture SCRI 2010-600-25320 to K.-S.L. and SCRI 2012-01507-229756 to K.-S.L. and Z.F.
Footnotes
Citation Li R, Zheng Y, Fei Z, Ling K-S. 2015. Complete genome sequence of an emerging melon necrotic spot virus isolate infecting greenhouse cucumber in North America. Genome Announc 3(4):e00775-15. doi:10.1128/genomeA.00775-15.
REFERENCES
- 1.Rochon D, Lommel S, Martelli GP, Rubino L, Russo M. 2012. Tombusviridae, p 1111–1138. In King AMO, Adams MJ, Carstens EB, Lefkowitz E (ed), Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, London, United Kingdom. [Google Scholar]
- 2.Campbell RN, Sim ST, Lecoq H. 1995. Virus transmission by host-specific strains of Olpidium bornovanus and Olpidium brassicae. Eur J Plant Pathol 101:273–282. doi: 10.1007/BF01874783. [DOI] [Google Scholar]
- 3.Campbell R, Wipfscheibel C, Lecoq H. 1996. Vector-assisted seed transmission of melon necrotic spot virus in melon. Phytopathology 86:1294–1298. doi: 10.1094/Phyto-86-1294. [DOI] [Google Scholar]
- 4.Kishi K. 1966. Necrotic spot of melon, a new virus disease. Jpn J Phytopathol 32:138–144. doi: 10.3186/jjphytopath.32.138. [DOI] [Google Scholar]
- 5.Gonzalez-Garza R, Gumpf D, Kishaba A, Bohn G. 1979. Identification, seed transmission, and host range pathogenicity of a California isolate of melon necrotic spot virus. Phytopathology 69:340–345. doi: 10.1094/Phyto-69-340. [DOI] [Google Scholar]
- 6.Bos L, Van Dorst HJM, Huttinga H, Maat DZ. 1984. Further characterization of melon necrotic spot virus causing severe disease in glasshouse cucumbers in the Netherlands and its control. Neth J Plant Pathol 90:55–69. doi: 10.1007/BF01999953. [DOI] [Google Scholar]
- 7.Avgelis A. 1985. Occurrence of melon necrotic spot virus in Crete (Greece). J Phytopathol 114:365–372. doi: 10.1111/j.1439-0434.1985.tb00631.x. [DOI] [Google Scholar]
- 8.Ryden K, Persson P. 1986. Melon necrotic spot-A new virus disease in Sweden. Vaxtskyddsnotiser 50:130–132. [Google Scholar]
- 9.Tomlinson JA, Thomas BJ. 1986. Studies on melon necrotic spot virus disease of cucumber and on the control of the fungus vector (Olpidium radicale). Ann Appl Biol 108:71–80. doi: 10.1111/j.1744-7348.1986.tb01967.x. [DOI] [Google Scholar]
- 10.Riviere CJ, Pot J, Tremaine JH, Rochon DM. 1989. Coat protein of melon necrotic spot carmovirus is more similar to those of tombusviruses than those of carmoviruses. J Gen Virol 70:3033–3042. doi: 10.1099/0022-1317-70-11-3033. [DOI] [PubMed] [Google Scholar]
- 11.Riviere CJ, Rochon DM. 1990. Nucleotide sequence and genomic organization of melon necrotic spot virus. J Gen Virol 71:1887–1896. doi: 10.1099/0022-1317-71-9-1887. [DOI] [PubMed] [Google Scholar]
- 12.Yakoubi S, Desbiez C, Fakhfakh H, Wipf-Scheibel C, Marrakchi M, Lecoq H. 2008. First report of Melon necrotic spot virus on melon in Tunisia. Plant Pathol 57:386. doi: 10.1111/j.1365-3059.2007.01719.x. [DOI] [Google Scholar]
- 13.Gu Q-S, Bao WH, Tian YP, Prins M, Yang HX, Lu J, Liu LF, Peng B. 2008. Melon necrotic spot virus newly reported in China. Plant Pathol 57:765. doi: 10.1111/j.1365-3059.2008.01847.x. [DOI] [Google Scholar]
- 14.Díaz JA, Bernal JJ, Moriones E, Aranda MA. 2003. Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus. Arch Virol 148:599–607. doi: 10.1007/s00705-002-0927-y. [DOI] [PubMed] [Google Scholar]
- 15.Miras M, Sempere RN, Kraft JJ, Miller WA, Aranda MA, Truniger V. 2014. Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking. New Phytol 202:233–246. doi: 10.1111/nph.12650. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Ling K-, Li R, Zhang W. 2014. First report of Cucumber green mottle mosaic virus infecting greenhouse cucumber in Canada. Plant Dis 98:701. doi: 10.1094/PDIS-09-13-0996-PDN. [DOI] [PubMed] [Google Scholar]
- 17.Li R, Zheng Y, Fei Z, Ling KS. 2015. First complete genome sequence of an emerging cucumber green mottle mosaic virus isolate in North America. Genome Announc 3(3):e00452-15. doi: 10.1128/genomeA.00452-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Chen Y-R, Zheng Y, Liu B, Zhong S, Giovannoni J, Fei Z. 2012. A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters. Plant Methods 8:41. doi: 10.1186/1746-4811-8-41. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Li R, Gao S, Hernandez AG, Wechter WP, Fei Z, Ling K-S. 2012. Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation. PLoS One 7:e37127. doi: 10.1371/journal.pone.0037127. [DOI] [PMC free article] [PubMed] [Google Scholar]