همسانه‌سازی و بررسی تبارزایی جدایه‌های ایرانی narcissus latent virus براساس ترادف نوکلئوتیدی ناحیه ’3 ژنوم

نوع مقاله : مقالات پژوهشی

نویسندگان

1 گروه گیاهپزشکی، دانشکده علوم زراعی، دانشگاه علوم کشاورزی و منابع طبیعی ساری

2 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، خراسان رضوی

چکیده

ویروس نهفته نرگس (narcissus latent virus, NLV) یکی از بیمارگرهای بسیار مهم و خسارت­زای ارقام تجاری گیاه نرگس و زنبق در سراسر جهان می­باشد. به منظور شناسایی این ویروس، گیاهان زنبق با علائم مشکوک ویروسی از شمال شرق کشور جمع‌آوری و ناحیه ´3 ژنوم آنها با استفاده از آغازگرهای اختصاصی با روش رونویسی معکوس و واکنش زنجیره­ای پلی­مراز (RT-PCR) تکثیر و همسانه­سازی شد. ناحیه CP-UTR این جدایه­ها (سه جدایه) به همراه تعدادی از ترادف­های جدایه­های موجود در بانک ژن (24 جدایه) مقایسه و پس از انجام همردیف­سازی چندگانه، مورد تجزیه و تحلیل فیلوژنتیکی قرار گرفتند. جدایه­های مورد مقایسه در درخت فیلوژنتیکی در دو گروه جداگانه قرار گرفتند ( I وII ) و جدایه­های ایرانی در کنار جدایه­هایی از لهستان، نیوزیلند، و بریتانیا در گروه II قرار گرفتند. مقایسه درصد مشابهت ژنتیکی نشان داد که جدایه­های ایرانی با سایر جدایه­های NLV بین 47/77 تا 12/98 درصد در سطح نوکلئوتیدی مشابهت دارند که به ترتیب بیشترین میزان شباهت (بین 65/97 تا 12/98 درصد) با جدایه NLV5_1 (JX270766) از لهستان و کمترین (بین 47/77 تا 95/77 درصد) با جدایه NLV3 (JX270762) از لهستان بود. همچنین تشابه توالی نوکلئوتیدی و آمینواسیدی سه جدایه ایرانی با یکدیگر به ترتیب 84/97-97 و 02/99-38/97 درصد تعیین گردید. میزان تنوع نوکلئوتیدی ناحیه CP-UTR  بین جدایه­های NLV، 130/0 به دست آمد که نشان دهنده تنوع ژنتیکی بالای این ویروس در این ناحیه از ژنوم است. نتایج پژوهش حاضر می­تواند در برنامه­های به­نژادی مولکولی برای تولید ارقام مقاوم به ویروس مفید بوده و خسارت ناشی از بیماری را کاهش دهد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Cloning and Phylogenetic Analysis of Iranian Isolates of Narcissus Latent Virus Based on Sequence of 3´ Region of Genome

نویسندگان [English]

  • Z. Moradi 1
  • M. Mehrvar 2
1 Department of Plant Pathology, Faculty of Crop Sciences, Sari Agricultural Sciences and Natural Resources University
2 Department of Plant Pathology, Faculty of Agriculture, Ferdowsi University of Mashhad
چکیده [English]

Introduction
Iris spp. is reported to be affected by several viruses in the family Potyviridae including iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV), iris fulva mosaic virus,  bean yellow mosaic virus (BYMV), turnip mosaic virus (TuMV), ornithogalum mosaic virus (OrMV), narcissus latent virus (NLV), butterfly flower mosaic virus (BFMV), and gladiolus mosaic virus (tentative name). Narcissus latent virus (NLV) is a member of the genus Macluravirus in the family Potyviridae. It has non-enveloped flexuous filamentous virions of 657 nm long and 13 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 8,000 nt long. NLV is distributed widely throughout the major planting areas of Japan, New Zealand, and European countries. It is one of the most common viruses infecting narcissus, iris, gladiolus, and nerine, causing significant yield losses and quality deterioration in their bulbs and flowers. Due to the presence of asymptomatic infection of NLV in iris and narcissus, the relevance of its infection in host plants may be severely underrated. As Khorasan Razavi province is one of the major producing areas of ornamental plants in Iran, identification of this virus is a concern. In this study, we attempted to identify NLV infecting iris plants and compare Iranian NLV isolates with other sequences from different geographical regions to provide the first detailed information of phylogenetic characterization of this virus in Iran.
Materials and Methods
Iris leaf samples showing virus-like symptoms of leaf chlorosis and mosaic were collected from field-grown plants in Khorasan Razavi province. Total RNA was extracted from the field samples using Promega SV Total RNA Isolation Kit (USA). Reverse-transcription polymerase chain reaction (RT-PCR) was performed using specific primer pair CPU-F (5΄-CATTACACCCGACCTGGAACT-3΄) and CPU-R (5΄-CCATTTCAGGGCATTGGAGGA-3΄), which were designed to amplify a 1066 bp fragment of the 3΄-region of NLV genome (encompassing partial NIb (25 nt), complete CP (894 nt), and partial 3'UTR (147 nt)). PCR products and DNA ladder were separated by agarose gel electrophoresis, visualized using DNA Green viewer staining, and photographed with ultraviolet-illumination. Amplified fragments of the expected size were purified, cloned into pTG19-T vector and bi-directionally sequenced. Obtained sequences were phylogenetically compared with the corresponding isolates available in the GenBank after multiple alignments. The phylogenetic tree was constructed based on the nucleotide sequences of the CP-UTR using the neighbor-joining method by MEGA11.
 
Results and Discussion
Amplification product (1066 bp) was obtained from five infected samples, but not from healthy samples. The most typical symptoms in positive samples were mosaic, and interveinal chlorosis. Three selected PCR positive samples were cloned into the pTG19-T vector and sequenced. BLASTn analysis of the sequenced data revealed that the PCR-amplified fragments belonged to NLV. Three selected isolates which are referred to as IR, IR2, and IR3 were deposited in GenBank. The previously identified and conserved amino acid sequence motifs described in CP of macluraviruses were present in Iranian CP sequences. The phylogenetic tree placed the NLV sequences into two distinct phylogroups I and II; the Iranian isolates clustered together with isolates from Poland, New Zealand, and United Kingdom into group II. Phylogenetic analysis showed that Iranian isolates shared 77.47 to 98.12% nucleotide sequence identity and 77.70-99.34% amino acid sequence identity with other isolates of NLV. Also, identity of these three isolates in the nucleotide and amino acid levels ranged between 97 to 97.84% and 97.38 to 99.02%, with each other, respectively. Iranian isolates showed the highest nucleotide sequence identity with NLV5_1 isolate (JX270766) from Poland (between 97.65 to 98.12 %) and the lowest with NLV3 isolate (JX270762) from Poland (between 77.47 to 77.95 %).
 
Conclusion
NLV is a major constraint to iris and narcissus production worldwide. The phylogenetic analysis showed a low correlation between genetic and geographic distances which further emphasizing the importance of the exchange and use of virus-free propagating organs in preventing the dissemination of this virus. It seems that contaminated vegetative organs from some European countries (e.g. Netherlands), which are the major producer and the largest exporters of flowers and ornamentals in the world, can play a significant role in the worldwide distribution of the virus. Identification and the use of more isolates are recommended for a better understanding of the genetic structure and variation of NLV populations on a large geographical scale. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran.

کلیدواژه‌ها [English]

  • Cloning
  • CP-UTR
  • Iran
  • Narcissus latent virus
  • Phylogenetic analysis
  1.  

    1. Adams, M.J., Antoniw, J.F., & Fauquet C.M. (2005). Molecular criteria for genus and species discrimination within the family Potyviridae. Archives of Virology 150: 459–479. https://doi.org/10.1007/s00705-004-0440-6.
    2. Asjes, C.J. (1979). Viruses and virus diseases in Dutch bulbous irises (Iris hollandica) in the Netherlands. Netherlands Journal of Plant Pathology 85: 269–279.
    3. Attari, S., Shoor, M., Ghorbanzadeh Neghab, M., Tehranifar, A., & Malekzadeh Shafaroudi, S. (2016). Evaluation of genetic diversity of iris genotypes (Iris) Using ISSR. Journal of Horticultural Science 30(3): 376-382. https://doi.org/10.22067/jhorts4.v30i3.28044.
    4. Atreya, C.D., Raccah, B., & Pirone, T.P. (1990). A point mutation in the coat protein abolishes aphid transmission of a potyvirus. Virology 178: 161–165. https://doi.org/1016/0042-6822(90)90389-9.
    5. Atreya, P.L., Lopez-Moya, J.J., Chu, M., Atreya, C.D., & Pirone, T.P. (1995). Mutational analysis of the coat protein N-terminal amino acids involved in potyvirus transmission by aphids. Journal of General Virology 76: 265–270. https://doi.org/1099/0022-1317-76-2-265.
    6. Ateka, E., Alicai, T., Ndunguru, J., Tairo, F., Sseruwagi, P., Kiarie, S., Makori, T., Kehoe, M.A., & Boykin, L.M. (2017). Unusual occurrence of a DAG motif in the Ipomovirus Cassava brown streak virus and implications for its vector transmission. PLoS One 12: e0187883. https://doi.org/1371/journal.pone.0187883.
    7. Badge, J., Robinson, D.J., Brunt, A.A., & Foster, G.D. (1997). 3′-Terminal sequences of the RNA genomes of narcissus latent and maclura mosaic viruses suggest that they represent a new genus of the Potyviridae. Journal of General Virology 78: 253-257. https://doi.org/1099/0022-1317-78-1-253.
    8. Barnett, O.W. (1991). Iris fulva mosaic virus, AAB Descriptions of Plant Viruses No. 310. Association of Applied Biologists, Wellesbourne.
    9. Barnett, O.W., & Alper, M. (1977). Characterization of Iris fulva mosaic virus. Phytopathology 67: 448–454. https://doi.org/1094/Phyto-67-448.
    10. Berger, P.H., Wyatt, S.D., Shiel, P.J., Silbernagel, M.J., & Druffel, K. (1997). Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses. Archives of Virology 142: 1979±1999. https://doi.org/1007/s007050050216.
    11. Berniak, H., Komorowska, B., & Sochacki, D. (2013). Detection of Narcissus latent virus isolates using one-step RT-PCR assay. Journal of Horticultural Research 21: 11–14. https://doi.org/2478/johr-2013-0002.
    12. Brunt, A.A., & Atkey, P.T. (1967). Rapid detection of narcissus yellow stripe and two other filamentous viruses in crude negatively- stained narcissus sap. Report of the Glasshouse Crops Research Institute for 1966, pp. 155-159.
    13. Brunt, A.A. (1977). Some hosts and properties of narcissus latent virus, a carlavirus commonly infecting narcissus and bulbous iris. Annals of Applied Biology 87: 355-364. https://doi.org/1111/j.1744-7348.1977.tb01900.x.
    14. Chen, J., Shi,H., Li, M.Y., Adams, M.J., & Chen, J.P. (2008). A new potyvirus from butterfly flower (Iris japonica Thunb.) in Zhejiang, China. Archives of Virology 153: 567–569. https://doi.org/10.1007/s00705-007-0014-5.
    15. Clark, V.R., & Guy, P.L. (2000). Five viruses in Narcissus plants from New Zealand. Australasian Plant Pathology 29: 227-229. https://doi.org/10.1071/AP00044.
    16. Derks, A.F.L.M., Hollinger, T.H.C., & Vink-van den Abeele, J.L. (1985). Identification and symptom expression of four elongated viruses infecting bulbous irises. Acta Horticulturae 164: 309-318.
    17. Farahbakhsh, F., Masumi, M., Afsharifar, A.R., Izadpanah, K., & Rahpeyma Sarvestani, N. (2013). Genetic variation of Bermuda grass southern mosaic virus isolates based on sequence of 3’ region of genome. Iranian Journal of Plant Pathology 49(1): 61-75. (In Persian with English abstract)
    18. Flasinski, S., & Cassidy, B.G. (1998). Potyvirus aphid transmission requires helper component and homologous coat protein for maximal efficiency. Archives of Virology 143: 2159±72. https://doi.org/1007/s007050050449.
    19. Gao, F., Shen, J., Liao, F., Cai, W., Lin, S., Yang, H., & Chen, S. (2018). The first complete genome sequence of narcissus latent virus from Narcissus. Archives of Virology 163(5): 1383-1386. https://doi.org/1007/s00705-018-3741-x.
    20. Hammond, J., Derks, A.F.L.M., Barnett, O.W., Lawson, R.H., Brunt, A.A., Inouye, N., & Allen, T.C. (1985). Viruses infecting bulbous iris: a clarification of nomenclature. ISHS Acta Horticulturae 164: 395–397.
    21. Inouye, N., & Mitsuhata, K. (1978). Turnip mosaic virus isolated from Iris. Nogaku Kenkyu 57: 1–16. (In Japanese with English abstract)
    22. Jin, J., Shen, J.G., Cai, W., Xie, G.H., Liao, F.R., Gao, F.L., Ma, J.F., Chen, X.H., & Wu, Z.J. (2017). Narcissus yellow stripe virus and narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay. Journal of Applied Microbiology 122: 1299–1309. https://doi.org/1111/jam.13422.
    23. Johansen, I.E., Keller, K.E., Dougherty, W.G., & Hampton, R.O. (1996). Biological and molecular properties of a pathotype P-1 and a pathotype P-4 isolate of pea seed-borne mosaic virus. Journal of General Virology 77: 1329–1333. https://doi.org/1099/0022-1317-77-6-1329.
    24. King, A.M., Lefkowitz, E., Adams, M.J., & Carstens, E.B. (2012). Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, Amsterdam. pp 1272. ISBN: 978-0-12-384684-6.
    25. Kulshrestha, S., Hallan, V., Raikhy, G., Ram, R., Zaidi, A.A., & Garg, I.D. (2006a). Incidence of Bean yellow mosaic virus on Iris. Acta Horticulturae 722: 235–240. https://doi.org/10.17660/ActaHortic.2006.722.29.
    26. Kulshrestha, S., Hallan, V., Raikhy, G., Ram, R., Garg, I.D., Haq, Q.M.R., & Zaidi, A.A. (2006b). Occurrence of iris mild mosaic potyvirus in cultivated iris in India. Indian Journal of Biotechnology 5: 94–98.
    27. Lopez-Moya, J.J., Wang, R.Y., & Pirone, T.P. (1999). Context of the coat protein DAG motif affects potyvirus transmissibility by aphids. Journal of General Virology 80 (Pt 12): 3281±8. https://doi.org/10.1099/0022-1317-80-12-3281.
    28. Moradi, Z., Mehrvar, M., Nazifi, E., & Zakiaghl, M. (2017a). Identification and molecular analysis of sugarcane mosaic virus (SCMV) in Mazandaran province. Journal of Iranian Plant Protection Research 30(4): 639-645. (In Persian with English abstract). https://doi.org/22067/jpp.v30i4.50484.
    29. Moradi, Z., Mehrvar, M., Nazifi, E., & Zakiaghl, M. (2017b). Iranian johnsongrass mosaic virus: the complete genome sequence, molecular and biological characterization, and comparison of coat protein gene sequences. Virus Genes 53: 77-88. https://doi.org/1007/s11262-016-1389-8.
    30. Moradi, Z., Mehrvar, M., & Nazifi, E. (2018). Genetic diversity and biological characterization of sugarcane streak mosaic virus isolates from Iran. VirusDisease 29: 316–323. https://doi.org/10.1007/s13337-018-0461-5.
    31. Moradi, Z., & Mehrvar, M. (2022). First report of Narcissus latent virus infecting iris in Iran. New Disease Reports 46(1): e12114. https://doi.org/10.1002/ndr2.12114.
    32. Naseri, A., Moradi, Z., Mehrvar, M., & Zakiaghl, M. (2022) Genomic characterization and phylogenetic analysis of two potyviruses infecting iris in Iran. Journal of Agricultural Science and Technology 24(5): 1233-1249.
    33. Nigam, D., LaTourrette, K., Souza, P.F.N., & Garcia-Ruiz, H. (2019). Genome wide variation in potyviruses. Frontiers in Plant Science 10: 1439. https://doi.org/3389/fpls.2019.01439.
    34. Ohshima, K., Nomiyama, R., Mitoma, S., Honda Y., Yasaka, R., & Tomimura, K. (2016). Evolutionary rates and genetic diversities of mixed potyviruses in Narcissus. Infection. Genetics and Evolution 45: 213–223. https://doi.org/1016/j.meegid.2016.08.036.
    35. Rahmana, A., Nasima, S., Baig, I., Jalil, S., Orhan, I., Sener, B., & Choudhary, M.I. (2003). Anti-inflammatory isoflavonoids from the rhizomes of Iris germanica. Journal of Ethnopharmacology 86: 177-180. https://doi.org/1016/s0378-8741(03)00055-2.
    36. Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 38: 3022–3027. https://doi.org/1093/molbev/msab120.
    37. Van der Vlugt, C.I.M., Derks, A.F.L.M., Boonekamp, P.M., & Goldbach, R.W. (1993). Improved detection of Iris severe mosaic virus in secondarily infected iris bulbs. Annals of Applied Biology 122: 279–288. https://doi.org/10.1111/j.1744-7348.1993.tb04033.x.
    38. Van der Vlugt, C.I.M. (1994). Distribution and multiplication of iris severe mosaic potyvirus in bulbous Iris in relation to metabolic activity: implications for ISMV detection, Ph.D. thesis. Bulb Research Centre Lisse, Dutch Flower Bulb Industry, The Netherlands.
    39. Wei, T., Pearson, M.N., & Cohen, D. (2006). First report of ornithogalum mosaic virus and ornithogalum virus 2 in New Zealand. Plant Pathology 55: 820. https://doi.org/10.1111/j.1365-3059.2006.01375.x.
    40. Wei, T., Pearson, M.N., & Cohen, D. (2007). First report of narcissus latent virus in New Zealand. Plant Pathology 56: 720. https://doi.org/10.1111/j.1365-3059.2007.01579.x.
    41. Wylie, S.J., Kueh, J., Welsh, B., Smith, L.J., Jones, M.G.K., & Jones, R.A.C. (2002). A non-aphid-transmissible isolate of bean yellow mosaic potyvirus has an altered NAG motif in its coat protein. Archives of Virology 147: 1813–1820. https://org/10.1007/s00705-002-0846-y.
    42. Wylie, S.J., & Jones, M.G.K. (2012). Complete genome sequences of seven carlavirus and potyvirus isolates from Narcissus and Hippeastrum plants in Australia, and proposals to clarify their naming. Archives of Virology 157: 1471–1480. https://doi.org/10.1007/s00705-012-1319-6.
    43. Wylie, S.J., Tran, T.T., Nguyen, D.Q., Koh, S.H., Chakraborty, A., Xu, W., Jones, M.G.K., & Li, H. (2019). A virome from ornamental flowers in an Australian rural town. Archives of Virology 164: 2255–2263. https://doi.org/10.1007/s00705-019-04317-7.
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