Molecular characterization and differentiation of cucumber mosaic virus subgroups in Serbia by RT-PCR-RFLP

Authors

  • Katarina Zečević University of Belgrade-Faculty of Agriculture, Institute of Phytomedicine, Department of Phytopathology, Nemanjina 6, 11080 Belgrade, Serbia
  • Ivana Stanković University of Belgrade-Faculty of Agriculture, Institute of Phytomedicine, Department of Phytopathology, Nemanjina 6, 11080 Belgrade, Serbia https://orcid.org/0000-0003-3828-4718
  • Branka Petrović Institute for Science Application in Agriculture, Bulevar despota Stefana 68b, 11108 Belgrade, Serbia https://orcid.org/0000-0003-1557-5408
  • Branka Krstić University of Belgrade-Faculty of Agriculture, Institute of Phytomedicine, Department of Phytopathology, Nemanjina 6, 11080 Belgrade, Serbia

DOI:

https://doi.org/10.2298/ABS230718035Z

Keywords:

cucumber mosaic virus, RFLP analysis, sequencing, population structure

Abstract

Paper description:

  • Restriction enzymes have been used in studies of the differentiation of cucumber mosaic virus (CMV) isolates but often only discriminating between virus subgroups I and II.
  • This study provides the development and validation of RT-PCR-RFLP suitable for differentiating between CMV subgroups IA, IB and II.
  • It describes a reliable and less costly protocol that allows for grouping CMV isolates into appropriate subgroups based on different genes.
  • The protocol allows the analysis of many samples and first insight into the natural genetic population of CMV.

Abstract: Samples of various host plants from different locations in Serbia showing foliar symptoms resembling those caused by the cucumber mosaic virus (CMV) were collected. Samples were considered CMV-infected after detection by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Fifteen CMV isolates were selected for developing a reverse transcription-polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) protocol and a more detailed molecular characterization of all five genes. Based on in silico RFLP, the following restriction enzymes were selected: HindIII, SacII (1a gene), MluI (2a gene), StuI, SalI (2b gene), BaeI (movement protein (MP) gene of CMV), SfcI, and HaeIII (capsid protein (CP) gene), which were capable of distinguishing between subgroups of CMV based on the obtained characteristic restriction patterns. According to in situ RFLP, the predominant haplotype IA; IA, IA; IA, IA was determined in 14, while the distinct haplotype II; II, II; II, II was found in only one isolate. Sequence analyses of two selected Serbian CMV isolates with different restriction patterns and haplotype profiles confirmed the RFLP results, showing that isolates 674-11 and 137-13 belong to subgroups IA and II, respectively. Different restriction patterns after digestion of all five CMV genomic regions proved to be a simple way to investigate the natural population of CMV. This study provides insight into the genetic structure of the CMV population in Serbia.

Downloads

Download data is not yet available.

References

Doolittle SP. A new infectious mosaic disease of cucumber. Phytopathology. 1916;6:145-7.

Jagger IC. Experiments with the cucumber mosaic virus. Phytopathology. 1916;6:149-51.

Palukaitis PM, Roossinck J, Dietzgen RG, Francki RIB. Cucumber mosaic virus. Adv Virus Res. 1992;41:281-348. https://doi.org/10.1016/s0065-3527(08)60039-1

Garcìa-Arenal F, Palukaitis P. Cucumber mosaic virus. In: Mahy BWJ, van Regenmortel MHV, editors. Desk Encyclopedia of Plant and Fungal Virology. Amsterdam: Elsevier; 2008. p. 171-6. https://doi.org/10.1016/B978-012374410-4.00640-3

Roossinck MJ. Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses. J Virol. 2002;76(7):3382-7. https://doi.org/10.1128/jvi.76.7.3382-3387.2002

Brigneti G, Voinnet O, Li WX, Ji LH, Ding SW, Baulcombe DC. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. Embo J. 1998;17(22):6739-46. https://doi.org/10.1093/emboj/17.22.6739

Palukaitis P, Garcìa-Arenal F. Cucumoviruses. Adv Virus Res. 2003;62:241-323. https://doi.org/10.1016/s0065-3527(03)62005-1

Rizos H, Gunn LV, Pares RD, Gillings MR. Differentiat ion of Cucumber mosaic virus isolates using the polymerase chain reaction. J Gen Virol. 1992;73(8):2099-103. https://doi.org/10.1099/0022-1317-73-8-2099

Wahyuni WS, Dietzgen RG, Hanada K, Francki RIB. Serological and biological variation between and within subgroup-1 and subgroup-II strains of Cucumber mosaic virus. Plant Physiol. 1992;41(3):282-97. https://doi.org/10.1111/j.1365-3059.1992.tb02350.x

Singh Z, Jones RAC, Jones MGK. Identification of Cucumber mosaic virus subgroup I isolates from banana plants affected by infectious chlorosis disease using RT-PCR. Plant Dis. 1995;46(7):633-43.

Roossinck MJ, Zhang L, Hellwald KH. Rearrangements in the 5’ nontranslated region and phylogenetic analyses of Cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. J Virol. 1999;73(8):6752-8. https://doi.org/10.1128/JVI.73.8.6752-6758.1999

Gallitelli D. The ecology of Cucumber mosaic virus and sustainable agriculture. Virus Res. 2000;71(1-2):9-21. https://doi.org/10.1016/S0168-1702(00)00184-2

Lin HX, Rubio L, Smythe AB, Jiminez M, Falk BW. Genetic diversity and biological variation among California isolates of Cucumber mosaic virus. J Gen Virol. 2003;84(1):249-58. https://doi.org/10.1099/vir.0.18673-0

Eiras M, Boari AJ, Colariccio A, Chaves ALR, Briones MRS, Figueira AR, Harakava R. Characterization of isolates of the cucumovirus Cucumber mosaic virus present in Brazil. J Plant Pathol. 2004;86(1):61-9.

Bonnet J, Fraile A, Sacristan S, Malpica JM, Garcìa-Arenal F. Role of recombination in the evolution of natural populations of Cucumber mosaic virus, a tripartite RNA plant virus. Virology. 2005;332(1):359-68. https://doi.org/10.1016/j.virol.2004.11.017

Sclavounos AP, Voloudakis AE, Arabatzis CH, Kyriakopoulou PE. A severe Hellenic CMV tomato isolate: Symptom variability in tobacco, characterization and discrimination of variants. Eur J Plant Pathol. 2006;115(2):163-72. https://doi.org/10.1007/s10658-006-0010-8

Kayode KT, Odu BO, Ako-Nai KA, Alabi OJ. Occurrence of Cucumber mosaic virus Subgroups IA and IB Isolates in Tomatoes in Nigeria. Plant Dis. 2014;98(12):1750. https://doi.org/10.1094/PDIS-08-14-0844-PDN

Li N, Yu C, Yin Y, Gao S, Wang F, Jiao C, Yao M. Pepper Crop Improvement Against Cucumber Mosaic Virus (CMV): A Review. Front Plant Sci. 2020;11:598798. https://doi.org/10.3389/fpls.2020.598798

Nikolić D, Vučurović A, Stanković I, Radović N, Zečević K, Bulajić A, Krstić B. Viruses affecting tomato crops in Serbia. Eur J Plant Pathol. 2018;152(1):225-35. https://doi.org/10.1007/s10658-018-1467-y

Stanković I, Vučurović A, Zečević K, Petrović B, Nikolić D, Delibašić G. Characterization of cucumber mosaic virus and its satellite RNAs associated with tomato lethal necrosis in Serbia. Eur J Plant Pathol. 2021;160(2):301-13. https://doi.org/10.1007/s10658-021-02241-8

Petrović D, Bulajić A, Stanković I, Ignjatov M, Vujaković M, Krstić B. Presence and distribution of pepper viruses in Serbia. Field Veg Crop Res. 2010;47(2):567-76.

Stanković I, Bulajić A, Vučurović A, Ristić D, Milojević K, Berenji J, Krstić B. Status of tobacco viruses in Serbia and molecular characterization of Tomato spotted wilt virus isolates. Acta Virol. 2011;55(4):337-47. https://doi.org/10.4149/av_2011_04_337

Vučurović A, Bulajić A, Stanković I, Ristić D, Berenji J, Krstić B. Characterization of Cucumber Mosaic Virus Originating from Cucurbits in Serbia. Pestic Phytomed. 2011;26(4):325-36. https://doi.org/10.2298/PIF1104325V

Vučurović A, Bulajić A, Stanković I, Ristić D, Berenji J, Jović J, Krstić B. Non-persistently aphid-borne viruses infecting pumpkin and squash in Serbia and partial characterization of Zucchini yellow mosaic virus isolates. Eur J Plant Pathol. 2012;133(4):935-47. https://doi.org/10.1007/s10658-012-9964-x

Milojević K, Stanković I, Vučurović A, Ristić D, Nikolić D, Bulajić A, Krstić B. Biological and molecular characterization of cucumber mosaic virus infecting watermelon in Serbia. Plant Prot. 2013;64(1):14-25.

Milošević D, Ignjatov M, Nikolić Z, Gvozdanović V, Tamindžić G, Stanković I, Krstić B. The Presence of Cucumber Mosaic Virus in Pot Marigold (Calendula officinalis L.) in Serbia. Field Veg Crop Res. 2015;52(2):67-73. https://doi.org/10.5937/ratpov52-7814

Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673-80. https://doi.org/10.1093/nar/22.22.4673

Kumar S, Stecher G, Tamura T. MEGA 7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870-4. https://doi.org/10.1093/molbev/msw054

Lin HX, Rubio L, Smythe AB, Falk BW. Molecular Population Genetics of Cucumber mosaic virus in California: Evidence for Founder Effects and Reassortment. J Virol. 2004;78(12):6666-75. https://doi.org/10.1128/JVI.78.12.6666-6675.2004

Liu YY, Yu SL, Lan YF, Zhang CL, Hou SS, Li XD, Chen XZ, Zhu XP. Molecular variability of five Cucumber mosaic virus isolates from China. Acta Virol. 2009;53(2): 89-97. https://doi.org/ 10.4149/av_2009_02_89

Tamarzizt HB, Montarry J, Girardot G, Fakhfakh H, Tepfer M, Jacquemond M. Cucumber mosaic virus populations in Tunisian pepper crops are mainly composed of virus reassortants with resistance-breaking properties. Plant Pathol. 2013;62(6):1415-28. https://doi.org/10.1111/ppa.12032

Nouri S, Arevalo R, Falk BW, Groves RL. Genetic Structure and Molecular Variability of Cucumber mosaic virus Isolates in the United States. PLoS One. 2014;9(5):e96582. https://doi.org/10.1371/journal.pone.0096582

Chen Y, Chen J, Zhang H, Tang X, Du Z. Molecular evidence and sequence analysis of a natural reassortant between Cucumber mosaic virus subgroup IA and II strains. Virus Genes. 2007;35(2): 405-413. https://doi.org/10.1007/s11262-007-0094-z

Koundal V, Haq Q MR, Praveen S. Characterization, genetic diversity, and evolutionary link of Cucumber mosaic virus strain New Delhi from India. Biochem Genet. 2011;49(1-2):25-38. https://doi.org/10.1007/s10528-010-9382-8

Fakhtakh H, Gorsane F, Marrakchi M, Makni M. Differentiation of Tunisian isolates of Cucumber mosaic virus using biological and molecular properties. Phytopathol. Mediterr. 1999;38(2):61-7.

Ryu KH, Park HW, Choi JK. Characterization and sequence analysis of a lily isolate of Cucumber mosaic virus from Lilium tsingtauense. Plant Pathol. J. 2002;18(2):85-92. https://doi.org/10.5423/PPJ.2002.18.2.085

Choi GS, Kim JH, Kim JS, Choi JK. Characterization of Cucumber mosaic virus Isolated from Water Chickweed (Stellaria aquatica). J Plant Pathol. 2004;20(2):131-4. https://doi.org/10.5423/PPJ.2004.20.2.131

Srivastava A, Raj SK. High molecular similarity among Indian isolates of Cucumber mosaic virus suggests a common origin. Curr. Sci. 2004;87(8):1126-31.

Bashir NS, Kalhor MR, Zarghani SN. Detection, differentiation and phylogenetic analysis of cucumber mosaic virus isolates from cucurbits in the northwest region of Iran. Virus Genes. 2006;32(3):277-88. https://doi.org/10.1007/s11262-005-6912-2

Akhtar KP, Ryu KH, Saleem MY, Asghar M, Jamil FF, Haq MA. Occurrence of Cucumber mosaic virus subgroup IA in tomato in Pakistan. J Plant Dis Prot. 2008;115(1):2-3. https://doi.org/10.1007/BF03356231

Berniak H, Malinowski T, Kamińska M. Comparison of ELISA and RT-PCR assays for detection and identification of cucumber mosaic virus (CMV) isolates infecting horticultural crops in Poland. J. Fruit Ornam. Plant Res. 2009;17(2):5-20

Parrella G, Sorrentino D. Identification of a Cucumber mosaic virus Isolate from Passiflora edulis in Southern Italy and Validation of Subgroup Identification by In Silico Restriction Fragment Length Polymorphism. J Phytopathol. 2009;157(11-12):762-7. https://doi.org/10.1111/j.1439-0434.2009.01604.x

Raj SK, Snehi SK, Gautam KK, Khan MS. First report of association of Cucumber mosaic virus with blister and leaf distortion of castor bean (Ricinus communis) in India. Phytoparasitica. 2010;38:283-9. https://doi.org/10.1007/s12600-010-0096-6

Koolivand D, Bashir NS, Mozafari J. Serological and molecular detection of newly isolated Cucumber mosaic virus variants from Iran. Int J Agric Res. 2012;2:933-41.

Moury B. Differential selection of genes of Cucumber mosaic virus subgroups. Mol Biol. Evol. 2004;21(8):1602-11. https://doi.org/10.1093/molbev/msh164

Jacquemond M. Cucumber mosaic virus. Adv Virus Res. 2012;84:439-504. https://doi.org/10.1016/B978-0-12-394314-9.00013-0

Mochizuki T, Ohki ST. Cucumber mosaic virus: viral genes as virulence determinants. Mol Plant Pathol. 2012;13(3):217-25. https://doi.org/10.1111/j.1364-3703.2011.00749.x

Finetti Sialer MM, Cillo F, Barbarossa L, Gallitelli D. Differentiation of Cucumber mosaic virus subgroups by RT-PCR-RFLP. J Plant Pathol. 1999;81(2):145-8.

Kim MK, Seo JK, Kwak HR, Kim JS, Kim KH, Cha BJ, Choi HS. Molecular genetic analysis of Cucumber mosaic virus populations infecting pepper suggests unique patterns of evolution in Korea. Phytopathology. 2014;104(9):993-1000. https://doi.org/10.1094/PHYTO-10-13-0275-R

Milojević K, Stanković I, Vučurović A, Nikolić D, Ristić D, Bulajić A, Krstić B. First report of Cucumber mosaic virus in Tulipa sp. in Serbia. Plant Dis. 2014;98(10):1449.

Milojević K, Radović N, Stanković I, Vučurović A, Nikolić D, Bulajić A, Krstić B. First report of Cucumber mosaic virus infecting Wisteria sinensis in Serbia. Plant Dis. 2016;100(8):1799. https://doi.org/10.1094/PDIS-05-14-0527-PDN

Balaji S, Bhat AI, Eapen SJ. A phylogenetic reexamination of Cucumber mosaic virus isolates based on 1a, 2a, 3a and 3b proteins. Indian J Virol. 2008;19(1):17-25.

Downloads

Published

2023-12-13

How to Cite

1.
Zečević K, Stanković I, Petrović B, Krstić B. Molecular characterization and differentiation of cucumber mosaic virus subgroups in Serbia by RT-PCR-RFLP. Arch Biol Sci [Internet]. 2023Dec.13 [cited 2024Apr.22];75(4):431-42. Available from: https://serbiosoc.org.rs/arch/index.php/abs/article/view/8894

Issue

Section

Articles