c3518cb17d976b8

شناسایی و بررسی تنوع ژنتیکی ویروس لکه‌حلقوی بافت مرده هسته‌دارها (Prunus necrotic ring spot virus) در درختان میوه هسته‌دار و دانه‌دار استان کردستان

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی سابق کارشناسی ارشد، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه کردستان، سنندج، ایران

2 استادیار، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه کردستان، سنندج، ایران

3 استادیار پژوهش، موسسه تحقیقات ثبت و گواهی بذر و نهال، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران

چکیده

شمار 149 نمونۀ برگی از درختان میوۀ هسته‌دار و دانه‌دار استان کردستان هنگام بهار و تابستان سال‌های 1394 و 1396 به‌منظور شناسایی مولکولی و بررسی گوناگونی ژنتیکی عامل لکه حلقوی بافت مردۀ درختان هسته‌دار جمع‌آوری شدند و با آغازگرهای اختصاصی ژن پروتئین پوششی PNRSV-F3/PNRSV-R3 آزمون RT-PCR شدند. نتیجه‏ها RT-PCR نشان دادند که 8/20 درصد از نمونه‌ها آلوده به PNRSV بودند. سیزده جدایه بر پایۀ نوع میزبان و منطقۀ جغرافیایی گزینش و پس از تکثیر و پیوست بخشی از ژن پروتئین پوششی آن‌ها به پلاسمید pTG-19 و همسانه‌سازی در باکتری E. coli تعیین توالی شدند. توالی‌های به‌دست‌آمده در سطح نوکلئوتیدی به‌طور میانگین 002/0 ± 9/98 درصد با یکدیگر و 006/0 ± 4/94 درصد با دیگر جدایه‌های موجود در بانک ژن همانندی نوکلئوتیدی نشان دادند. در واکاوی تبارزایی بر پایۀ ترادف نوکلئوتیدی جدایه‌های PNRSV در سه گروه تبارزایی PV96، PV32 و PE5 قرار گرفتند که سیزده جدایۀ این پژوهش به همراه بیشتر جدایه‌های ایرانی در گروه تبارزایی PV96 قرار گرفتند. بیشترین همانندی نوکلئوتیدی میان جدایه‌های هلو از کامیاران (KH10)، زردآلو از سنندج (SZ93) و هلو از دهگلان (D7) با جدایۀ شلیل از ایران (KX353935) با 98 درصد و کمترین همانندی نوکلئوتیدی میان جدایۀ زردآلو از سنندج (SZ26) با جدایۀ آلو از لهستان (DQ983499) با 6/83 درصد همانندی دیده شد. نسبت‌های کم جانشینی مترادف به غیر مترادف (dN/dS) در ژن پروتئین پوششی این ویروس روشنگر این نکته است که گزینش منفی نقش بزرگی را در فرگشت این ژن بازی کرده است و بررسی نوترکیبی با نرم‌افزار RDP v.4.63 نیز نشان داد که در جدایه‌های موردبررسی در این پژوهش نوترکیبی در این بخش از ژنوم رخ نداده است.

کلیدواژه‌ها


عنوان مقاله [English]

Phylogenetic analysis of Prunus necrotic ring spot virus in pome and stone fruit trees in Kurdistan province

نویسندگان [English]

  • Mahdi Azaryar 1
  • Mohammad Hajizadeh 2
  • Abdolbaset Azizi 2
  • masoud Naderpour 3
1 Former M.Sc. Student, Department of Plant Pathology, University of Kurdistan, Sanandaj, Iran
2 Assistant Professor, Department of Plant Pathology, University of Kurdistan, Sanandaj, Iran
3 Assistant Professor, Seed and Plant Improvement Institute, Education and Extension Organization, Karaj, Iran
چکیده [English]

In order to study molecular identification and genetic diversity of Prunus necrotic ringspot virus (PNRSV), 149 leaf samples from 37 orchards of pome and stone fruit trees of Kurdistan province were collected and tested by RT-PCR using a PNRSV-F3/PNRS-R3 specific primer pair. Results showed that 20.8% of the samples were infected by PNRSV.In the next stage of the study, 13 isolateswere selected based on the host and geographic region and their amplified fragments were ligated into the pTG19-T plasmid, transformed to E. coli DH5α, and sequenced. Phylogenetic analysis showed that PNRSV isolates formed three clades PV96, PV32, and PE5, and all new-sequences from Kurdistan (in this research) were categorized in PV96 phylogenetic clade, which is close to other Iranian isolates. The new isolates shared 99 ± 0.002 identities together and 94/9 ± 0.005 with other previously PNRSV reported isolates at the nucleotide level. Pairwise comparisons of sequences showed that isolates peach from Kamyaran (KH10), pear from Sanandaj (SZ93), and peach from Dehgolan (D7) had the highest nucleotide similarity (98%) with Iranian isolate nectarine (KX353935) whereas isolate pear from Sanandaj (SZ26) had the lowest nucleotide identity with isolate plum from Poland (DQ983499). The low dN/dS ratio in all populations of the virus showed that negative selection plays important role in PNRSV-CP evolution and recombination. There is no recombination event in this domain of PNRSV genome of these isolates.

کلیدواژه‌ها [English]

  • Coat protein
  • negative selection
  • recombination
  • Phylogenetic analysis
Abtahi, F. S., Shams-Bakhsh, M., Mahdiyeh, M. & Safaie, N. (2015). Detection and distribution of the important viruses infecting Damask rose in floricultures of Isfahan, Markazi and Kerman provinces. Modern Genetics Journal, 1, 11-20.
Aparicio, F. & Pallás, V. (2002). The molecular variability analysis of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus sheds light on the minimal requirements for the synthesis of its subgenomic RNA. Virus Genes25(1), 75-84.
Aparicio, F., Myrta, A., Di Terlizzi, B. & Pallas, V. (1999). Molecular variability among isolates of Prunus necrotic ringspot virus from different Prunus spp. Phytopathology, 89, 991-999.
Aparicio, F., Pallás, V. & Sánchez-Navarro, J. (2010). Implication of the C terminus of the Prunus necrotic ringspot virus movement protein in cell-to-cell transport and in its interaction with the coat protein. Journal of General Virology, 91(7), 1865-1870.
Boulila, M. (2009). Molecular Evidence for Recombination in Prunus Necrotic Ringspot Virus. Plant Molecular Biology Report, 27, 189-198.
Boulila, M., Ben Tiba, S. & Jilani, S. (2013). Molecular adaptation within the coat protein-encoding gene of Tunisian almond isolates of Prunus necrotic ringspot virus. Journal of Genetics, 92(1), 11-24.
Bujarski, J., Figlerowicz, M., Gallitelli, D., Roossinck, M. J. & Scott, S. W. (2012). Family Bromoviridae. In: Virus Taxonomy. Ninth report of the international committee on taxonomy of viruses. Elsevier Academic Press, San Diego, CA, pp, 965-976.
Cochran, L. C. & Hutchins, L. M. (1941). A severe ring-spot virus on peach. Phytopathology, 31, 860.
Cui, H. G., Liu, H. Z., Chen, J., Zhou J. F., Qu, L. N., Su, J. M., Wang, G. P. & Hong, N. (2012). Genetic diversity of Prunus necrotic ringspot virus infecting stone fruit trees grown at seven regions in China and differentiation of three phylogroups by multiplex RT-PCR. Crop protection, 74, 30-36.
Delport, W., Poon, A. F., Frost, S. D. W. & Pond, K. S. L. (2010). Datamonkey: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics, 26(19), 2455-2457.
Fajardo, T. V. M., Nascimento, M. B., Eiras, M., Nickel, O. & Pio-Ribeiro, G. (2015). Molecular characterization of Prunus necrotic ringspot virus isolated from rose in Brazil. Ciência Rural, 45(12), 2197-220
Fallah, T. & Nasrollanejad, S. (2009). Detection and distribution of PNRSV on stone fruits in Golestan provinces. Journal of Plant Production, 16, 88-98. (in Farsi)
Fiore N., Fajardo T. V., Prodan, S., Herranz, M. C., Aparicio, F., Montealegre, J. & Sánchez-Navarro, J. (2008). Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus. Archives of Virology, 153(5), 909-919.
‏Foissac, X., Savalle-Dumas, L., Gentit, P., Dulucq, M. J. & Candresse, T. (2000). Polyvalent detection of fruit tree Tricho, Capillo and Faveaviruses by nested RT-PCR using degenerated and inosine containing primers (PDO RT-PCR). Acta Horticulture, 357, 52-59.
Gao, F., Lin, W., Shen, J. & Liao, F. (2016). Genetic diversity and molecular evolution of arabis mosaic virus based on the CP gene sequence. Archives of Virology, 161, 1047-1051.
Gao, R., Xu, Y., Candresse, T., He, Z., Li, S., Ma, Y. & Lu, M. (2017). Further insight into genetic variation and haplotype diversity of Cherry virus A from China. PLOS ONE, 12(10), e0186273.
Garcı´a-Arenal, F., Fraile, A. & Malpica, J. M. (2003). Variation and evolution of plant virus populations. International Microbiology, 6, 225-232.
García-Arenal, F., Fraile, A. & Malpica, J. M. (2001). Variability and genetic structure of plant virus populations. Annual Review of Phytopathology, 39, 157-186.
Hammond, R. W. & Crosslin, J. M. (1998). Virulence and molecular polymorphism of Prunus necrotic ringspot virus isolates. Journal of General Virology, 79, 1815-1823.
King, A. M., Lefkowitz, E., Adams, M. J. & Carstens, E. B. (2011). “Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses”. Academic Press, London, UK.1327 pp.89.
Librado, P. & Rozas, J. (2009). Dnasp V5: A software for Comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451-1452.
Martin, D. P., Murrell, B., Golden, M., Khoosal, A., & Muhire, B. (2015). RDP4: Detection and analysis of recombination patterns in virus genomes, Virus Evolution, 1(1), vev003, doi: 10.1093/ve/vev003.
Mink, G. I. (1992). Prunus necrotic ringspot virus, Pp 335-356. In: J. Kumer, H. S. Chaube, U. S. Singh, and A. N. Mukhadpadhyay (Eds). Plant Diseases of International Importance. Vol. III. Prentice Hall, New York.
Moini, A. & Izadpanah, K. (2000). Serological identification of Prunus necrotic ring spot virus and PPV in Dasht-e-Moghan. In: Proceedings of the 14th Plant Protection Congress of Iran, 5-8 September, Isfahan, Iran, p. 338.
Moury, B., Cardin, L., Onesto, J. P., Candresse, T. & Poupet, A. (2001). Survey of Prunus necrotic ringspot virus in rose and its variability in rose and Prunus spp. Phytopathology, 91, 84-91.
Pallas, V., Aparicio, F., Herranz, c. M., Sanchez-Navarro, A. J. & Scott, W. S. (2013). The molecular biology of Ilarviruses. Advances in Virus Research, 87, 139-181.
Rahmanian, P., Rakhshandehroo, F. & Zamanizadeh, H. (2013). Detection and distribution of Prunus necrotic ringspot virus (PNRSV) on stone fruits in some gardens located in Fars and Golestan provinces. Iranian Journal of Plant Protection Science, 44, 129-139. (in Farsi)
Rakhshandehroo, F., Zamani Zadeh, H. R., Modarresi, A. & Hajmansoor, S. (2006). Occurrence of Prunus necrotic ringspot virus and Arabis mosaic virus on rose in Iran. Plant Disease, 90, 975.
Sokhandan-Bashir, N., Kashiha, Z., Koolivand, D. & Eini, O. (2017). Detection and phylogenetic analysis of Prunus necrotic ringspot virus isolates from stone fruits in Iran. Journal of Plant Pathology, 99(3), 717- 723.
Tajima, F. & Nei, M. (1984). Estimation of evolutionary distance between nucleotide sequences. Molecular Biology and Evolution, 1, 269-285.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30(12), 2725-2729.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673-80.
Vaskova, D., Pertzik, K., Spak, J. & Karesova, R. (2000). Variability and molecular typing of the woody tree infecting Prunus necrotic ringspot virus. Archive of Virology, 145(4), 699-709.
Vives, M. C., Rubio, L., Galipienso, L., Navarro, L., Moreno, P. & Guerri, J. (2002). Low genetic variation between isolates of Citrus leaf blotch virus from different host species and of different geographical origins. Journal of General Virology, 83(10), 2587-2591.
Uyemoto, J. K. & Scott, S. (1992). Important diseases of prunus caused by viruses and other graft transmissible pathogens in California and South Carolina. Plant Disease, 76, 5-11.
Zindović, J., Autonell, C. R. & Ratti, C. (2015). Molecular characterization of the coat protein gene of Prunus necrotic ringspot virus infecting peach in Montenegro. European Journal of Plant Pathology, 143(4), 881-891.