شناسایی مولکولی و بررسی جایگاه تبارزایی ویروس پیسک بادام‌زمینی در مزارع سویای شمال ایران

نوع مقاله : مقالات پژوهشی

نویسندگان

دانشگاه فردوسی مشهد

چکیده

ویروس پیسک بادام‌‌زمینی (Peanut mottle virus, PeMoV) یک پوتی‌‌ویروس است که می‌تواند علاوه بر بادام‌زمینی، با سویا و برخی حبوبات دیگر نیز سازگار شود. این ویروس در بادام‌زمینی و سویا خسارت قابل‌توجهی ایجاد می‌کند و از طریق بذرهای آلوده بادام‌زمینی و شته‌های ناقل به روش ناپایا به گیاهان سالم بادام‌زمینی و سویا منتقل می‌شود. طی فصل زراعی 1399، تعداد 132 نمونه برگی دارای نشانه‌های مشکوک به ویروس شامل موزائیک، پیسک، بدشکلی، کوتولگی، زردی و چروکیدگی برگ، از مزارع سویا در دو استان مازندران و گلستان جمع‌آوری و آران‌ای کل با استفاده از کیت پرومگا استخراج و دی‌ان‌ای مکمل ساخته شد. در ادامه، با استفاده از آغازگرهای دژنره مربوط به نواحی ژنومی cylindrical inclusion (CI) و helper component proteinase (HC-Pro) پوتی‌ویروس‌ها و جفت آغازگر اختصاصی پروتئین پوششی (coat protein, CP) ویروس پیسک بادام‌زمینی، قطعاتی به طول 700 و 1450 جفت باز تکثیر شد. توالی‌یابی سنگر محصول پی‌سی‌آر و بلاست نوکلئوتیدی، وجود این ویروس را در سویا تأیید نمود. نتیجه به‌دست‌آمده نشان داد که ناحیه پروتئین پوششی این جدایه در سطح نوکلئوتیدی، به میزان 56/88 تا 80/95 درصد با توالی‌های دیگری از PeMoV در GenBank، شباهت دارد. این جدایه، با نام PeMoV-soya و رس شمار MW464637 در GenBank ثبت شد. بررسی روابط تبارزایی با روش حداقل تکامل بر اساس توالی نوکلئوتیدی ناحیه پروتئین پوششی نشان داد که جدایه‌های مختلف PeMoV در دو گروه تبارزایی قابل‌تفکیک بوده و جدایه PeMoV-soya در گروه I تبارزایی و در کنار جدایه‌هایی از کنیا، کره جنوبی و ترکیه قرار گرفت. در جدایه soya نیز مانند سایر جدایه‌های PeMoV، موتیف DAG در پروتئین پوششی با موتیف DAA جایگزین شده است. نتایج این مطالعه نشان می‌دهد که PeMoV-soya دارای دامنه میزبانی محدودی در میان گیاهان خانواده Fabaceae است.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Molecular Identification and Phylogenetic Analysis of Peanut mottle virus in Soybean Fields in the North of Iran

نویسندگان [English]

  • J. Ramezani Avval Reiabi
  • M. Mehrvar
Department of Plant Protection, Faulty of Agriculture, Ferdowsi University of Mashhad, Iran
چکیده [English]

Introduction
 Soybean (Glycine max) is one of the major oilseeds of the Fabaceae family. Peanut mottle virus (PeMoV) is a Potyvirus that can infect not only peanuts, but also soybeans, and some other legumes. Aphids and contaminated peanut seeds transmit the virus to healthy peanut and soybean plants, causing significant damage. Soybeans infected with the virus have been found to exhibit symptoms such as mosaic, mottling, yellowing, and stunting.
Materials and Methods
 From May to October 2020, 132 leaf samples with presumptive symptoms of the virus, such as mosaic, mottling, deformation, stunting, yellowing, and wrinkling, were collected from soybean fields in Mazandaran and Golestan provinces of Iran. Total RNA was isolated using Promega SV Total RNA Isolation Kit (USA). Complementary DNA (cDNA) was synthesized using random hexamer primers and reverse transcriptase enzyme, M-MuLV thermo-resistant H (ParsTous, Iran). Subsequently, the cDNA library was amplified in the presence of Taq DNA polymerase with degenerate primers related to the genomic regions of the cylindrical inclusion (CI) and Helper component proteinase (HC-Pro) of potyviruses and the coat protein (CP)-specific primer pairs of PeMoV for Sanger sequencing. Phylogenetic analysis was performed using MEGAX (Kumar et al., 2018). Multiple alignments were performed using the MAFFT software version 7. A phylogenetic tree was constructed using the Minimum Evolution (ME) and Tamura-Nei methods with 1000 bootstraps.
Results and Discussion
 RT-PCR tests with degenerate primers for potyviruses (HC-Pro and CI) and specific primer pairs for PeMoV virus (CP), followed by Sanger sequencing, confirmed the presence of this virus in soybean samples. The RT-PCR amplified fragments of 700 and 1450 bp with degenerate primers for potyviruses and CP-specific primer pairs for PeMoV, respectively, whereas no fragment was amplified from the healthy plants. The nucleotide sequence of the coat protein region of this isolate shared 88.56-95.80% identity with the corresponding sequence of other GenBank isolates. Phylogenetic trees based on coat protein alignments of the PeMoV-soya isolate with 16 other PeMoV isolates in the GeneBank grouped these isolates into two phylogroups I and II. Group I included isolates from China, Israel, Brazil, Iran, Kenya, South Korea, Turkey, Colombia, Australia, and the USA isolated from soybean, Arachis pintoi, and peanut. Group II were consisted of two isolates from Zambia (Phaseolus vulgaris). The Iranian isolate (PeMoV-soya) was placed in Group I and formed a clade with isolates from Kenya, South Korea, and Turkey. Based on phylogenetic analysis, the results showed that isolates from different hosts with different geographical origins belonged to the same phylogenetic group and probably shared a common ancestor. It indicates that geographical distribution and host do not play a role in grouping of Iranian isolate. Accordingly, it can be assumed that the multiple transmissions from one host to another (peanuts, beans, and soybeans) or host jumping, has led to the accumulation of mutations in its coating protein gene. In the study of motifs in this region (CP), the functional DAA motif at the amino terminus of the PeMoV coat protein replaced the DAG motif which is involved in aphid transmission. All PeMoV isolates contain the DAA motif, allowing their identification. A repetitive conserved sequence (EK)3 was observed upstream of this motif. PeMoV isolates (distributed across Africa, Asia, and the United States) may be geographically widespread due to this conserved sequence. In bioassay, there was a limited host range for this isolate, which was limited to soybean (Williams 82 and Essex) and peanut.
Conclusion
 For effective disease management, viruses must be correctly identified. Virus detection and disease management are difficult as viruses can evolve and produce molecular and biological variations. A well-established approach that is usually used when a new virus-like disease appears is to test for known viruses. PCR is the most widely used technique for detection of plant viruses. Based on the results of this study, the presence of the peanut mottle virus was confirmed in soybean fields in the north of Iran using the RT-PCR test. Based on the CP gene, the molecular characteristics of the virus were determined. According to this, unlike mutations, recombination plays no role in the evolution of the new PeMoV. Since the Peanut mottle virus can infect peanut plants, soybeans, beans, and most legumes, avoiding cultivating soybeans, peanuts, and beans close to each other will assist in management of the disease. Using integrated cultivation and avoiding crofting will effectively reduce the creation of recombinant isolates and appearance of the resistance-breaking isolates.

کلیدواژه‌ها [English]

  • PeMoV
  • Phylogeny
  • Potyvirus
  • Soybean

مراجع

  1. Adams, D., & Kuhn, C. (1977). Seed transmission of Peanut mottle virus in peanuts. Phytopathology 67(9): 1126-1129.
  2. Ahmadi, K., Ebadzadeh, H.R., Hatami, F., Mohammadnia Afroozi, S., Esfandiaripour, E., & Abbas-Taghani, R. (2021). Iran Agricultural Statistics of 2019-2020, Vol 1: Field Crops (In Persian). Ministry of Agriculture, Statistics and Information Technology Office, Tehran, Iran.
  3. Baradar, A., Hosseini, A., Abdani Babaki, S., & Hosseini Farhangi, S.A. (2020). Structure and phylogenetic analysis of the coat protein of Bean yellow mosaic virus isolates from Iranian faba bean farms. Iranian Journal of Plant Protection Science 51(1): 147-159. https://dx.doi.org/10.22059/ijpps.2020.299695.1006933.
  4. Beikzadeh, N., Hassani-Mehraban, A., & Peters, D. (2015). Molecular identification of an isolate of Peanut mottle virus (PeMoV) in Iran. Journal of Agricultural Science and Technology 17(3): 765-776.
  5. Bock, K. (1973). Peanut mottle virus in East Africa. Annals of Applied Biology 74(2): 171-179. https://doi.org/10.1111/j.1744-7348.1973.tb07736.x.
  6. Demski, J., Smith, D., & Kuhn, C. (1975). Incidence and distribution of Peanut mottle virus in peanut in the United States. Peanut Science 2(2): 91-93. https://doi.org/10.3146/i0095-3679-2-2-14.
  7. Demski, J.W., & Harris, H. (1974). Seed transmission of viruses in soybean. Crop Science 14(6): 888-890. https://doi.org/10.2135/cropsci1974.0011183X001400060034x.
  8. Demski, J.W., & Kuhn, C.W. (1977). A soybean disease caused by the Peanut mottle virus. Research Bulletin-Georgia Experiment Station (USA).
  9. Dunleavy, J.M., Ross, J.P., & Chamberlain, D.W. (1966). Soybean diseases. Agricultural Research Service, US Department of Agriculture.
  10. Golnaraghi, A., Shahraeen, N., Pourrahim, R., Farzadfar, S., & Ghasemi, A. (2002). First report of the natural occurrence of eight viruses affecting soybeans in Iran. Plant Pathology 51(6): 794-794.
  11. Golnaraghi, A., Shahraeen, N., Pourrahim, R., Farzadfar, S., & Ghasemi, A. (2004). Occurrence and the relative incidence of viruses infecting soybeans in Iran. Plant Disease 88(10): 1069-1074. https://doi.org/10.1094/PDIS.2004.88.10.1069.
  12. Ha, C., Coombs, S., Revill, P.A., Harding, R.M., Vu, M., & Dale, J.L. (2008). Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Archives of Virology 153(1): 25-36. (In eng). https://doi.org/10.1007/s00705-007-1053-7.
  13. Hull, R. (2013). Plant virology. Academic Press.
  14. Katoh, K., Rozewicki, J., & Yamada, K.D. (2019). MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20: 1160-1166. https://doi.org/10.1093/bib/bbx108.
  15. Khalil, S., Erskine, W., & No, G.L. (2001). Combating disease problems of grain legumes in Egypt. Grain Legumes 32: 24-26.
  16. Kuhn, CW. (1965). Symptomatology, host range, and effect on yield of a seed-transmitted Peanut virus. Phytopathology 55(8): 880-884.
  17. Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549. (In eng). https://doi.org/10.1093%2Fmolbev%2Fmsy096.
  18. Lim, S., Lee, Y.-H., Igori, D., Zhao, F., Yoo, R., Lee, S.-H., Baek, I., & Moon, J.S. (2014). First report of Peanut mottle virus infecting soybean in South Korea. Plant Disease 98(9): 1285-1285. https://doi.org/10.1094/PDIS-04-14-0356-PDN.
  19. Shahraeen, N., & Bananej, K. (1995). Occurrence of Peanut mottle virus in Gorgan province. In proceeding of the 12th Iranian Plant Protection Congress 2-7 September 1995 Karaj (Iran Islamic Republic)
  20. Sinclair, J.B., & Backman, P. (1989). Compendium of soybean diseases. American Phytopathological Society.
  21. Spiegel, S., Sobolev, I., Dombrovsky, A., Gera, A., Raccah, B., Tam, Y., Beckelman, Y., Feigelson, L., Holdengreber, V., & Antignus, Y. (2008). Note: Characterization of a Peanut mottle virus isolate infecting peanut in Israel. Phytoparasitica 36(2): 168-174. https://doi.org/10.1007/BF02981329.
  22. Srinivasulu, M., & Gopal, D.S. (2011). Coat protein sequence comparison of south Indian isolates of Papaya ringspot virus with other Indian subcontinent isolates. Phytopathologia Mediterranea 50(3): 359-367.
  23. Strange, R.N., & Scott, P.R. (2005). Plant disease: a threat to global food security. Annual Review of Phytopathology 43: 83-116. https://doi.org/10.1146/annurev.phyto.43.113004.133839.
  24. Wylie, S.J., Adams, M., Chalam, C., Kreuze, J., López-Moya, J.J., Ohshima, K., Praveen, S., Rabenstein, F., Stenger, D., & Wang, A. (2017). ICTV virus taxonomy profile: Potyviridae. The Journal of general virology 98(3): 352. https://doi.org/10.1099%2Fjgv.0.000740.
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