Molecular Identification of Beet Curly top Iran Virus Associated with Bean in Zanjan Province

Document Type : Research Article

Authors

• Vahid Hoseini
• Omid Eini

University of Zanjan

10.22067/jpp.v30i4.51762

Abstract

Introduction: Plant diseases caused by geminiviruses are one of the main constraints of legume production in the world. They are responsible for a constraint on production of various crops. Geminiviruses are characterized by their twinned icosahedral particles and their single-stranded DNA genome. The family Geminiviridae is grouped into four genera: Begomovirus, Mastrevirus, Curtovirus and Topocuvirus (Brown and Moriones, 2012). Recent reports for geminiviruses show that viruses such as Spinach curly top Arizona virus (SCTAV) and Beet curly top Iran virus (BCTIV) (Yazdi et al., 2008) can be grouped in a new genus, Becurtovirus (ICTV 2012;http://www.ictvonline.org/virusTaxonomy.asp). Zanjan Province is one of the main regions for growing bean in Iran. Various geminiviruses have been reported from bean included: Bean golden mosaic virus (BGMV), BCTIV, Bean Calico mosaic virus (BCaMV) and Bean dwarf mosaic virus (BDMV). In this study in order to determine the type and distribution of geminivirus/es causing bean diseases, samples were collected from Zanjan province and then tested for the presence of geminiviruses using polymerase chain reaction and rolling circle amplification systems.
Materials and Methods: Some of the geminiviruses cause disease in the bean. To identify geminiviruses in the bean, sixty samples were collected from Zanjan, Khdabadeh, KhoramDareh and Abhar in summer 2014. These samples were collected according to the symptoms such as golden mosaic, curling, malformation, blistering, yellowing of leaves and plant stunting. After DNA extraction, viral infection was tested by polymerase chain reaction (PCR) using degenerate primers, PAL1V 1978/ PAR1C 496 and Primer B/ Primer V181. Based on the disease symptoms and results of PCR, four samples were selected to confirm the presence of geminiviruses and also to amplify the full-length genome using a rolling cycle amplification (RCA) kit. The amplified DNA products were digested with EcoRI, PstI, and EcoRV enzymes. A 2800 bp DNA fragment was isolated from gel and cloned into EcoRI site of a pBlunt vector and then sequenced (Microgen, Korea). The resulted sequence was compared to other reported geminiviruses from databases such as Genbank. To find the phylogenetic relation of the identified virus with other reported geminiviruses, we made a phylogenetic tree using the aligned sequences in MEGA6 software by applying Neighbour-joining method with 1000 replicates.
Results and Discussion: Using degenerate primers more than 15 percent of the collected samples showed amplification of DNA fragments with expected sizes. Gel electrophoresis for PCR products using BC and PCR V181 primers for bean samples showed production of a 550bp fragment. The PCR products size was 900 and 1100 bp using PaR1C 496 and PAL1V 1978 primers. The phenotype for theses samples was included geminivirus-like symptoms such as: abnormal and yellowing (S37), cup shape (S60), yellowing and mosaic (S34), blistering and abnormality (S62). The same abnormal shape of leaves was reported from bean plants infected with geminiviruses such as BCTIV and BGMV (Gharouni K. S., 2012). According to the results of the PCR and type of the symptoms, four samples (S26, S37, S61 and S65) selected to confirm the geminiviral infection using Rolling Circle Amplification (RCA) reactions. The RCA reaction was performed using 200 ng of the DNA and followed the instruction of kit. The RCA product was digested with various restriction enzymes. Based on the digestion patterns of the amplified DNA in the presence of three restriction enzymes EcoRI, EcoRV, Pst1, a 2800 bp fragment from sample S37 was selected for cloning into a vector and sequencing. Analyzing of this sequence showed that the amplified DNA has the highest (98%) similarity to a Beet curly top Iran virus (BCTIV) isolated from sugar beet and a lower (89 %) similarity to an isolate of BCTIV from the bean in Khorasan-Razavi Province.
Conclusion: Geminiviruses are limiting factors for crop production in the bean. The most common geminiviruses are Bean golden mosaic virus and Bean calico mosaic virus. In Iran, BCTIV has been recently identified as a dominant and widespread curly top producing agent in important crops (Heydarnejad et al., 2007; Kardani et al., 2013; Soleimani et al., 2013). This diversity and wide occurrence of BCTIV made a big challenge for breeders to produce resistance or tolerant traits (Strausbaugh et al., 2008). Our results also confirmed the widespread occurrence of BCTIV in Iran and also the genetic variation of virus isolates from the same host plant in various geographical regions.

Keywords

• Bean
• Beet curly top Iran virus
• Geminiviruses
• Zanjan

20.1001.1.20084749.1395.30.4.7.3

---

References

1- Adams M.J., King A.M., and Carstens E.B. 2013. Ratification vote on taxonomic proposals to international committee on taxonomy of viruses. Archives of Virology, 158: 2023-2030.

2- Anonymous. 2013. The statistics of crop production in Zanjan Province. Ariculture Jihad organization of Zanjan Province. Avalble at http://agrizanjan.ir/amaar/1392 (visited 5 November 2014).

3- Baliji S., Sunter J., and Sunter G. 2007. Transcriptional analysis of complementary sense genes in spinach curly top virus and functional role of c2 in pathogenesis. Molecular Plant Microbe Interaction, 20: 194-206.5.

4- Bolok Yazdi H., Heydarnejad J., and Massumi H. 2008. Genome characterization and genetic iversity of beet curly top iran virus: A geminivirus with a novel nonanucleotide. Virus Genes, 36: 539-545

5- Briddon R.W., Heydarnejad J., Khosrowfar F., Massumi H., Martin D.P., and Varsani A. 2010. Turnip curly top virus, a highly divergent geminivirus infecting turnip in iran. Virus Research, 152: 169-175.

6- Brown J. K., Fauquet C.M., Briddon R. W., Zerbini M., Moriones E., and Navas-Castillo J. 2012. Virus taxonomy: Ninth report of the International Committee on Taxonomy of Viruses. Academic press, London.

7- Chasan R. 1995. Geminiviruses: A twin approach to replication. Plant Cell, 7: 659-661.

8- Eini O., Sahraei G.E., and Behjatnia S.A.A. 2016. Molecular characterization and construction of an infectious clone of a pepper isolate of Beet curly top Iran virus. Molecular Biology Research Communications, 5: 101-113.

9- Farzadfar S., Pourrahim R., Golnaraghi A.R., Hahraeen N., and Makkouk M. 2002. First report of sugar beet and bean as natural hosts of Chickpea chlorotic dwarf virus in Iran. Plant Pathology, 51: 795.782.

10- Gharouni Kardani S.G., Heydarnejad J., Zakiaghl M., Mehrvar M., Kraberger S., and Varsani A. 2013. Diversity of beet curly top Iran virus isolated from different hosts in Iran. Virus Genes, 46: 571-575.

11- Gharouni Kardani S.G., Heydarnejad J., Zakiaghl M., and Mehrvar M. 2012. Identification of Beet curly top Iran virus in Razavi province. . P862. 20th Congree of Plant Protection, Iran

12. Gharouni Kardani S. G., Jafarpour B., Mehrvar M., and Tarighi S. 2013. Identification and sequencing for coat protein of Tomato yellow leaf curl virus in Razavi Province. Plant Protection, 27: 427-433.

13- Gilbertson R. L., Faria J.C., Hanson S.F., Morales F.J., Ahlquist P.G., Maxwell D.P., and Russell D.R. 1991. Cloning of the complete DNA genomes of four bean-infecting geminiviruses and determining their infectivity by electric discharge particle acceleration. Phytopathology, 81: 980-985.

14- Ghorbani S.G.M., Shahraeena N., and Elahinia S.A. 2010. Distribution and impact of virus associated diseases of common bean (Phaseolus vulgaris L.) in Northern Iran. Archives of Phytopathology and Plant Protection, 43: 1183-1189.

15- Ha C., Coombs S., Revill P., Harding R., Vu M., and Dale J. 2008. Molecular characterization of begomoviruses and DNA satellites from vietnam: Additional evidence that the new world geminiviruses were present in the old world prior to continental separation. Jornal of General Virology, 89: 312-326.

16- Hernandez C., and Brown J.K. 2010. First report of a new curtovirus species, spinach severe curly top virus, in commercial spinach plants (spinacia oleracea) from south-central arizona. Plant Disease, 94: 94-97.

17- Heydarnejad J., Keyvani N., Razavinejad S., Massumi H., and Varsani A. 2013. Fulfilling koch’s postulates for beet curly top iran virus and proposal for consideration of new genus in the family geminiviridae. Archives of Virology, 158: 435-443.

18- Lam N., Creamer R., Rascon J., and Belfon R. 2009. Characterization of a new curtovirus, pepper yellow dwarf virus, from chile pepper and distribution in weed hosts in new mexico. Archives of Virology, 154: 429-436.

19- Liu L., van Tonder T., Pietersen G., Davies J.W., and Stanley J. 1997. Molecular characterization of a subgroup I geminivirus from a legume in south africa. Journal of General Virology, 78: 2113-2117.10.

20- MacDowell S.W., Macdonald H., Hamilton W.D. O., Coutts R.H. A., and Buck K. W. 1985. The nucleotide sequence of cloned wheat dwarf virus DNA. The EMBO Journal, 4: 2173-2180.

21- Malmrot A. 2010. Begomovirus prevalence and diversity in the TYLCV resistant tomato cultivar Shanty in Nicaragua. Epsilon Archive, 144: 1-16.

22- Martin P., Willment J., Billharz R., Velders R., and Odhiambo B. 2001. Sequence diversity and virulence in zea mays of maize streak virus isolates. Virology, 288: 247.

23- Morales F. J., and Anderson P. K. 2001. The emergence and dissemination of whitefly-transmitted geminiviruses in Latin America. Archives of Virology, 146: 415-441

24- Pakniat A., Behjatnia S.A.A., Kharazmi S., Shahbazi M., and Izadpanah K. 2010. Molecular characterization and construction of an infectious clone of a new strain of Tomato yellow leaf curl virus in southern Iran. Iranian journal of plant pathology, 46: 101-115.

25- Rodriguez-Pardina P.E., Zerbini F.M., and Ducasse D.A. 2006. Genetic diversity of begomoviruses infecting soybean, bean and associated weeds in Northwestern Argentina. Fitopatologia Brasileira, 31: 342-348.

26- Rojas M.R. 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. Plant Disease, 77: 340-348

27- Rojas M.R., Gilbertson R.L., Russell D.R., and Maxwell D.P. 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. Plant Disease, 77: 340-347.

28- Rouhibakhsh A., Priya J., Periasamy M., Haq Q.M.I., and Malathi V.G. 2008. An improved DNA isolation method and PCR protocol for efficient detection of multicomponents of begomovirus in legumes. Journal of Virological Methods, 147: 37-42.

29- Stanley J., and Gay M. 1983. Nucleotide sequence of cassava latent virus DNA. Nature, 301: 260.

30- Stanley J., Markham P. G., Callis R. J., and Pinner M. S. 1986. The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus. EMBO Journal, 5: 1761 - 1767.

31- Soleimani R., Matic S., Taheri H., Behjatnia S.A.A., Vecchiati M., Izadpanah K., and Accotto G.P. 2013. The unconventional geminivirus Beet curly top Iran virus: satisfying Koch's postulates and determining vector and host range. Annals of Applied Biology, 162: 174-181.

32- Varsani A., Shepherd D.N., Dent K., Monjane A.L., Rybicki E.P., and Martin D.P. 2009. A highly divergent south african geminivirus species illuminates the ancient evolutionary history of this family. Virology Journal, 6: 1-12.

33- Varsani A., Navas-Castillo N., Moriones E., Hernandez-Zepeda C., Idris A., Brown J.K., Murilo Zerbini F., and Martin F.D. 2014. Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus. Archives of Virology, 159: 2193-2203

34- Wu C.Y., Lai Y.C., Lin N.S., Hsu Y.H., Tsai H.T., Liao J.Y., and Hu C.C. 2008. A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification. Journal of Virological Methods, 147: 355-359.

35- Zhou X., Liu Y., Calvert L., Munoz C., Otim-Nape G., Robinson D., and Harrison B. 1997. Evidence that DNA-A of a geminivirus associated with severe cassava mosaic disease in Uganda has arisen by interspecific recombination. Journal of General Virology, 78: 2101-2111.