ردیابی ویروس پسوروز مرکبات با استفاده از dsRNA و هیبریداسیون
محورهای موضوعی :
ویروس شناسی
فایزه فلکی
1
,
فرشاد رخشنده رو
2
,
وحید علوی
3
1 - گروه گیاه پزشکی، دانشکده علوم کشاورزی و صنایع غذایی، واحد علوم و تحقیقات دانشگاه آزاد اسلامی، تهران، ایران
2 - گروه گیاه پزشکی، دانشکده علوم کشاورزی و صنایع غذایی، واحد علوم و تحقیقات دانشگاه آزاد اسلامی، کد پستی: ۱۴۵۱۵-۷۷۵
3 - بخش تحقیقات گیاه پزشکی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی مازندران، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران
تاریخ دریافت : 1401/09/29
تاریخ پذیرش : 1402/02/10
تاریخ انتشار : 1402/03/15
کلید واژه:
ردیابی,
ویروس پسوروز مرکبات (CPsV),
هیبریداسیون,
RNA دورشته‌ای,
الگوی الکتروفورزی,
چکیده مقاله :
سابقه و هدف: ویروس پسوروز مرکبات عامل ایجاد یکی از مهم‌ترین بیماری های ویروسی مرکبات در جهان است که در باغات مرکبات واقع در شرق مازندران شناسایی شده است. به‌دلیل حضور هم زمان نژادهای مختلف ویروس در نمونه های آلوده، روش های شناسایی مولکولی مانند PCR اغلب از توان ردیابی این ویروس با دقت بالا برخوردار نیستند. در این تحقیق، روشی دقیق برای شناسایی ویروس پسوروز مرکبات معرفی شده است.
مواد و روش ها: ردیابی و تشخیص نمونه های آلوده با استفاده از آزمون الایزا به روش ساندویچ غیر مستقیم سه طرفه پـادتن (TAS-ELISA) و آنتی بادی تک همسانه ای از نمونه های پرتقال تامسون و نارنگی انشو با علایم مشکوک به آلودگی به CPsV، در شهرستان های ساری، نکا و قائم شهر از استان مازندارن انجام پذیرفت. آلودگی در نمونه ها با استفاده از RT-PCR تایید و استخراج ds RNA (Double-strand RNA) ویروسCPsV با استفاده از ستون CF-11 انجام شد. برای هیبریداسیون مولکولی از پروب cDNA نشاندار شده با DIG استفاده شد.
یافته ها: بررسی الگوی الکتروفورز حاصل از استخراج dsRNA از نمونه هایی که با الایزا و RT-PCR به CPsV آلوده تشخیص داده شده بودند بیانگر حضور ژنوم CPsV با وزن های مولکولی 2500 و 920 جفت باز در نمونه های آلوده بود. همچنین با استفاده از هیبریداسیون مولکولی و پروب طراحی شده، حضور CPsV در نمونه های آلوده مورد تایید قرار گرفت.
نتیجه گیری: نتایج به دست آمده از آزمون هیبرید سازی مولکولی ارتباط مستقیم بین dsRNA استخراج شده از نمونه های آلوده و حضور ویروس پسوروز مرکبات را در نمونه های علایم دار مرکبات شهرستان ساری اثبات نمود.
چکیده انگلیسی:
Background & Objectives: Citrus psorosis virus is the causal agent of one of the most important viral citrus diseases in the world and has been detected from the citrus orchards in the east of Mazandaran province, IRAN. Because of the simultenious presence of various strains of this virus in infected citrus samples, molecular detection methods like PCR are not able to detect this virus with high accuaracy. In this research, we introduce a method for the precise detection of Citrus psorosis virus.
Materials & Methods: Detection and Identification of infected samples performed by triple antibody sandwich indirect-ELISA (TAS-ELISA) method and monoclonal antibody from symptomatic Thomson orange and Unshiu tangerine samples with suspicious symptoms to CPsV infection in Sari, Neka and Ghaemshahr districts of Mazandaran. Infection in samples tested by ELISA and confirmed by RT-PCR. Extraction of CPsV-dsRNA (Double-strand RNA) from infected samples was done by CF-11 column. Molecular hybridization performed by DIG labeled cDNA probe.
Results: Exploring the electrophoretic pattern of dsRNA extracted from the CPsV infected citrus samples by using ELISA and RT-PCR methods, indicating the presence of CPsV genomes with the molecular weights of 920 bp and 2500 bp in infected samples. The presence of CPsV in the samples was also confirmed by using molecular hybridization and designed probe.
Conclusion: The major advantages of the hybridization method relating to the dsRNA electrophoretic mobility is the simultenious detection of the all citrus psorosis virus strains within the infected citrus samples. This way can separate virus isolates.
منابع و مأخذ:
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El Sayed TA, Saker KE, El Shorbagy AE, Soliman AM. Elimination of Citrus psorosis virus using shoot tip grafting and electrotherapy techniques. Egypt. J. Agric. Res. 2022; 100 (4): 641-652.
Alas T, Akın A, Kahramanoğlu i. Symptomological identification of Citrus Psorosis Virus (CPsV) in citrus orchards of Northern Cyprus. Tr Prikl Bot Genet Sel. 2022;183(2):149-158. DOI: 10.30901/2227-8834-2022-2-149-158.
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14. Sofy AR, Moussa AA, Fahmy H, Ghazal SA, El-Dougdoug KA. Anatomical and ultrastructural changes in Citrus leaves infected with Citrus psorosis virus Egyptian isolates. J Appl Sci Res. 2007; 3(6):485–94.
15. Roistacher CN. Psorosis-Areview. Proc. 12th Conf. IOCV. 1993; 139-154.
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18. Valverde RA, La Torre-Almaraz R. Extraction and purification of large dsRNAs from virusinfected plants and fungi: Applications in virus detection and identification. Rev Mex Fitopatol. 2017; 35:80-105. doi: 10.18781/R.MEX.FIT.1606-9.
19. Ma Y, Marais A, Lefebvre M, Theil S, Svanella-Dumas L, Faure C, Candresse T. Phytovirome analysis of wild plant populations: comparison of double-stranded RNA and virion-associated nucleic acid metagenomic approaches. J Virol. 2020; 94: e01462-19. https://doi .org/10.1128/JVI.01462-19.
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23. Falaki F, Alavi SV, Rakhshanderoo F, Mousavi A. Partial Biological and Molecular Characterization of Citrus psorosis virus Infecting Satsuma mandarin (Citrus unshiu Marc) in the east of Mazandaran province. Iranian JAEP. 2019; 87:2 (195-207). (In Persian with English summary).
24. Rocha-Pena MA, Lee RF, Niblett CL. Development of a dot-immunobinding assay for detection of citrus tristeza virus. J Virol Methods. 1991; 34: 297-309
25. Huang C, Yu Y. Synthesis and Labeling of RNA in Vitro. Curr Protoc Mol Biol. 2013; Pp. 19.
26. Martin S, Alioto D, Milne RG, Garnsey SM, Garcia ML, Grau O, Guerri J, Moreno P. Detection of Citrus psorosis virus by ELISA, molecular hybridization, RT-PCR and immunosorbent electron microscopy and its association with citrus psorosis disease. Eur J Plant Pathol. 2004; 110: 747–757.
27. Morris TJ, Dodds JA. Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology. 1979; 69: 854-858.
28. Tzanetakis IE, Martin RR. A new method for extraction of double-stranded RNA from plants. J Virol Methods. 2008; 149:167–170. doi:10.1016/j.jviromet.2008.01.014
29. Tabein S, Jansen M, Noris E, Vaira AM, Marian D, Behjatnia SAA, Accotto GP, Miozzi L. The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region. Front. Plant Sci. 2020; 11:533338. doi: 10.3389/fpls.2020.533338
30. Loconsole G, Fatone MT, Savino V. Specific digoxigenin-labelled riboprobes for detection of citrus psorosis virus and citrus variegation virus by molecular hybridization. Plant Pathol. 2009; 91 (2): 311-319.
_||_
1. Anonymous. Production and yield per hectare ofmandarin crop by province. Agricultural Statistics ofthe Crop Year 2016-2017. Department of Statisticsand Information of Ministry of Agriculture Jihad. 2018; 241
2. FAO STAT. National Agricultural Statistics Service. 2016; http://www.faostat.fao.org.
3. Anonymous. Production and yield per hectare ofmandarin crop by province. Agricultural Statistics ofthe Crop Year 2006-2007. Department of Statisticsand Information of Ministry of Agriculture Jihad. 2008; 154
4. Khoii S. The Principales of citrus nutrition. Author Press. 1993; 266pp.
5. Behdad, E. Pests and diseases of fruit trees. Yadbood Press. 2009;350pp.
6. Sanchez De La Torre ME, Garcia ML, Riva O, Dal Bo E, Jones L, Zandomeni R, Grau O. Genome Organization of the Top Component of Citrus Psorosis Virus and Identification of the Coat Protein Gene. Fourteenth IOCV Conference. 2000; 345-346.
7. Garnsey SM, Timmer LW. Local Lesion Isolate of Citrus Ringspot Virus Induces Psorosis Bark Scaling. 10th Conf. IOCV. 1988; 334-339.
8. Achachi A, Barka EA, Ibriz M. Recent advances in Citrus psorosis virus. Virusdisease. 2014; 25(3): 261-276.
9. Elahinia SA. Diseases of fruit trees and some garden plants and methods of combating them. 3th Ed, Guilan University Press. 2012; 565pp
10. Fotohi-Ghazvini R. Citrus Growing in Iran. 1th Ed, Guilan University Press. 1999; 150pp.
El Sayed TA, Saker KE, El Shorbagy AE, Soliman AM. Elimination of Citrus psorosis virus using shoot tip grafting and electrotherapy techniques. Egypt. J. Agric. Res. 2022; 100 (4): 641-652.
Alas T, Akın A, Kahramanoğlu i. Symptomological identification of Citrus Psorosis Virus (CPsV) in citrus orchards of Northern Cyprus. Tr Prikl Bot Genet Sel. 2022;183(2):149-158. DOI: 10.30901/2227-8834-2022-2-149-158.
Simeone M, Gómez C, Bertalmío A, Ruiz E, Hauteville C, Suarez LG, Tito B, García ML. Detection of citrus psorosis virus by RT-qPCR validated by diagnostic parameters. Plant Pathol. 2021;70:980–986. https:// doi.org/10.1111/ppa.13341.
14. Sofy AR, Moussa AA, Fahmy H, Ghazal SA, El-Dougdoug KA. Anatomical and ultrastructural changes in Citrus leaves infected with Citrus psorosis virus Egyptian isolates. J Appl Sci Res. 2007; 3(6):485–94.
15. Roistacher CN. Psorosis-Areview. Proc. 12th Conf. IOCV. 1993; 139-154.
16. Falaki F, Alavi SV, Rakhshanderoo F. Citrus psorosis virus, causal agent of ring pattern disorder in Thomson Navel trees in east of Iranian JAEP. 2013; 80 (2): 161-172. (In Persian with English summary).
17. Gafar YZA, Ziebell H. Comparative study on three viral enrichment approaches based on RNA extraction for plant virus/viroid detection using high-throughput sequencing. PLoS ONE. 2020; 15 (8): e0237951. doi: 1371/journal. pone.0237951.
18. Valverde RA, La Torre-Almaraz R. Extraction and purification of large dsRNAs from virusinfected plants and fungi: Applications in virus detection and identification. Rev Mex Fitopatol. 2017; 35:80-105. doi: 10.18781/R.MEX.FIT.1606-9.
19. Ma Y, Marais A, Lefebvre M, Theil S, Svanella-Dumas L, Faure C, Candresse T. Phytovirome analysis of wild plant populations: comparison of double-stranded RNA and virion-associated nucleic acid metagenomic approaches. J Virol. 2020; 94: e01462-19. https://doi .org/10.1128/JVI.01462-19.
20. Holeva MC, Sklavounos A, Rajeswaran R, Pooggin MM, Voloudakis AE. Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection. Plants. 2021; 10: 963.https://doi.org/10.3390/plants10050963
21. Alavi SV, Teymuri P. Detection of Citrus Viroids by CF-11 Nucleic Acid Extraction Method. Iranian JAEP. 2017; 85:1 (77-86). (In Persian with English summary).
22. Farrell RE. RNA Methodologies. Academic press. 2010; 261-282.
23. Falaki F, Alavi SV, Rakhshanderoo F, Mousavi A. Partial Biological and Molecular Characterization of Citrus psorosis virus Infecting Satsuma mandarin (Citrus unshiu Marc) in the east of Mazandaran province. Iranian JAEP. 2019; 87:2 (195-207). (In Persian with English summary).
24. Rocha-Pena MA, Lee RF, Niblett CL. Development of a dot-immunobinding assay for detection of citrus tristeza virus. J Virol Methods. 1991; 34: 297-309
25. Huang C, Yu Y. Synthesis and Labeling of RNA in Vitro. Curr Protoc Mol Biol. 2013; Pp. 19.
26. Martin S, Alioto D, Milne RG, Garnsey SM, Garcia ML, Grau O, Guerri J, Moreno P. Detection of Citrus psorosis virus by ELISA, molecular hybridization, RT-PCR and immunosorbent electron microscopy and its association with citrus psorosis disease. Eur J Plant Pathol. 2004; 110: 747–757.
27. Morris TJ, Dodds JA. Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology. 1979; 69: 854-858.
28. Tzanetakis IE, Martin RR. A new method for extraction of double-stranded RNA from plants. J Virol Methods. 2008; 149:167–170. doi:10.1016/j.jviromet.2008.01.014
29. Tabein S, Jansen M, Noris E, Vaira AM, Marian D, Behjatnia SAA, Accotto GP, Miozzi L. The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region. Front. Plant Sci. 2020; 11:533338. doi: 10.3389/fpls.2020.533338
30. Loconsole G, Fatone MT, Savino V. Specific digoxigenin-labelled riboprobes for detection of citrus psorosis virus and citrus variegation virus by molecular hybridization. Plant Pathol. 2009; 91 (2): 311-319.
