Turunçgil Psorosis Virüsü (CPsV)’nün Çukurova bölgesinde dağılımı ve kılıf protein genine göre moleküler olarak tanılanması

Öz

Bu çalışmada, Çukurova Bölgesi’nde Turunçgil kavlama hastalığı etmeni Citrus psorosis virus (CPsV)’nin portakal, mandarin ve altıntop bahçelerinde yaygınlığı belirlenmiş, alınan bitki örneklerinin biyolojik ve moleküler karakterizasyonu yapılmıştır. CPsV izolatlarının arasındaki farklılıklar nükleotid dizilimleri analiz edilerek belirlenmiştir. CPsV’nin tanılanması ve karakterizasyonu için 50 farklı turunçgil ağacından şiddetli kabuk kavlama lezyonu ve genç yapraklarda damar bantlaşması, meşe yaprağı deseni simptomu, yaşlı yaprak simptomları gösteren farklı izolatlar toplanmıştır. Toplanan örnekler RT-PCR çalışmalarıyla analiz edilmiştir. Kullanılan farklı primer çiftleri ile CPsV’nin kılıf protein bölgesi belirlenmiştir. Yapılan biyolojik indeksleme çalışmalarında ise tohumdan yetiştirilen Pineapple portakal (Citrus sinensis) fidanlarına CPsV’nin inokulasyonu yapılmış ve enfekteli indikatör bitkilerin ilk gelişen sürgünlerinde CPsV’nin damar bantlaşması ve şok simptomları gözlemlenmiştir. Blast analizi ve NCBI gen bankasındaki CPsV’nin referans genomlarının karşılık gelen bölgeleri ile nükleotid dizilimlerinin %98 oranında benzerliğe sahip olduğu belirlenmiştir.

Anahtar Kelimeler

• turunçgil,CPsV,RT-PCR,biyolojik indeksleme,Türkiye

Distribution and molecular detection of Citrus psorosis virus (CPsV) based on coat protein gene in Cukurova region

Abstract

This study was conducted in the Cukurova region to determine the prevalence of Citrus psorosis virus (CPsV), a citrus disease agent in orange, mandarin, and grapefruit orchards. Biological and molecular characterization of collected plant samples were also carried out and the differences between the CPsV isolates were separated by analyzing nucleotide sequences. For the identification and characterization of CPsV, isolates were collected from 50 different citrus trees showing severe barking lesions, vein banding, and oak leaf pattern symptoms on young leaves, old leaves symptoms. The leaf samples were tested by RT-PCR analysis using different primer pairs to identify the coat protein region of CPSV. For the biological indexing studies, CPsV was inoculated to Pineapple orange (Citrus sinensis) plants grown from the seed. Vein banding symptoms and shock reactions of CPsV were observed in the first growth flushes of infected indicator plants during biological indexing assays. As a result of Blast analysis, CPsV in the NCBI GenBank had 98% similarity to nucleotide sequences with corresponding regions of the reference genomes.

Keywords

• citrus,CPsV,RT-PCR,biological indexing,Turkey

References

1. Achachi, A., 2014. Recent advances in Citrus psorosis virus. VirusDisease, Volume 25, Issue 3, pp 261–276
2. Barthe, G. A., Ceccardi, T. L., Manjunath, K. L. and Derrick, K. S., 1998. Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RT–PCR. Journal of General Virology, 79, 1531–1537.
3. Derrick, K. S., Brlansky, R. H., da Graça, J. V., Lee, R. F., Timmer, L. W. and Nguyen, T. K., 1988. Partial characterization of a virus associated with citrus ringspot. Phytopathology, 78, pp. 1298–1301.
4. Derrick, K. S., Lee, R. F., Hewitt, B. J. and Barthe, G. A., 1993. Spiroviruses: A new group of serologically diverse plant viruses associated with citrus psorosis and ringspot. In: Proc 12th Conf. IOCV, 428-429. IOCV, Riverside, CA.
5. Fawcett, H.S., 1933. New symptoms of psorosis indicating a virus disease of citrus. Phytopath., 23: 930. (Abstract)
6. Fawcett, H. S., and Bitancourt, A. A., 1943. Comparative symptomatology of psorosis varieties on citrus in California. Phytopath., 33; 837-864.14 figs.
7. Fawcett, H. S., and Klotz, L. J., 1938. Types and symptoms of psorosis and psorosis-like diseases of citrus. Phytopath 28:670.
8. Fawcett, H. S., and Klotz, L. J., 1941. Prevention of brown rot gummosis in planting and growing young citrus trees. Univ. Calif. Citrus Experiment Station. ( Mimeo.) 3 pp.

9. García ML, Grau O, Sarachu AN, 1991. Citrus psorosis is probably caused by a bipartite ssRNA virus. Research in Virology, 142, 303–11.
10. Güllü, M., 1989. Doğu Akdeniz Bölgesi Navel Grubu Portakalı ve Satsuma Mandarin Ağaçlarında Yaygın Virus ve Virus Benzeri Hastalıkların Surveyi ve İndekslenmesi Üzerinde Çalışmalar. Doktora Tezi, 70. Araştırma Yayınları Serisi, Ankara.
11. Kayım, M. 2010. Biological and Molecular Detection of Citrus Psorosis Virus in Citrus in the Eastern Mediterrenean Region of Turkey. J. Plant Biochemistry & Biotechnology Vol. 19(2)
12. Knorr, L.C. and Child, J.F.L., 1968. Incidence and Spread of Citrus Viruses: Control Methods and Programs. Production of Virus-free Budwood in CitrusPast, Present, and Future. In W.C. Price (ed.), Proc. 4 th. Conf. Intern. Organization. Citrus Virol. Univ. Florida Press, Gainesville.
13. Kütük, B., Çevik, B., 2011. Antalya ili turunçgil alanlarında psorosis virüsü'nün belirlenmesi, biyolojik ve moleküler karakterizasyonu. Süleyman Demirel Üniversitesi, Fen Bilimleri Enstitüsü, Bitki Koruma Anabilim Dalı, Yüksek lisans tezi, 65 pp
14. Li, R., Mock, R. , Huang, Q. , Abad, J. , Hartung, J. , G. and Kinarda, G.(2008).A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. J.Virol.Methods.154,48-55.
15. Martin S, Alioto D, Milne RG et al., 2004. Detection of Citrus psorosis virus by ELISA, molecular hybridization, RT-PCR and immunosorbent electron microscopy and its association with citrus psorosis disease. European Journal of Plant Pathology 110, 747–57.
16. Martín, S., López, C., García, M. L., Naum-Onganía, G., Grau, O., Flores, R.,Moreno, P. and Guerri, J., 2005. The complete nucleotide sequence of a Spanish isolate of Citrus psorosis virus: comparative analysis with other ophioviruses. Arch Virol, 150, pp. 167–176.
17. Murray, M.G. and W.F. Thompson, 1980. Rapid isolation of high molecular weight plant DNA. Nuc. Acids Res. 8:4321-4325.
18. Naum-Onganía, G., Gago-Zachert, S., Peña, E., Grau, O. and García, M. L., 2003. Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase. Virus Res, 96, pp. 49–61.
19. Önelge N, 1997. Direct nucleotide sequencing of Citrus exocortis viroid (CEV). Turkish Journal of Agriculture and Forestry 21, 419-422.
20. Roistacher CN, 1993. Psorosis – a review. In: Moreno P, da Grac¸a JV, Timmer LW, eds. Proceedings of the 12th Conference of the International Organization of Citrus Virologists, Riverside, CA, USA: IOCV, 139–54
21. Sánchez de la Torre E, Riva O, Zandomeni R, Grau O, Garcı´a ML, 1998. The top component of citrus ringspot virus contains two ssRNAs, the smaller encodes the CP. Molecular Plant Pathology Online [http://194.247.68.33/mppol/1998/1019 sanchez/].
22. Sánchez de la Torre, M. E., M. L. García, O. Riva, E. Dal Bó, Jones, L., Zandomeni,R. and Grau, O., 2000. Genome organization of the top component of Citrus psorosis virus and identification of the coat protein gene. In: Proc. 14th Conf. IOCV, Riverside, CA, pp. 345-346.
23. Sánchez de la Torre E, Lo´pez C, Grau O, Garcı´a ML, 2002. RNA2 of Citrus psorosis virus is of negative polarity and has a single open reading frame in its complementary strand. Journal of General Virology 83, 1777–81.
24. Timmer, L. W. and Beiiatena, H. N., 1977. Comparison of psorosis and other viruses causing leaf flecking in citrus. Proc. Int. Soc. Citriculture 3: 930-935.
25. Timmer LW, Garnsey SM, 1980. Natural spread of citrus ringspot virus in Texas and its association with psorosis-like diseases in Florida and Texas. In: Calavan EC, Garnsey SM, Timmer LW, eds. Proceedings of the 8th Conference of the International Organization of Citrus Virologists, Riverside, CA, USA: IOCV, 167–93.
26. TUİK, 2017. www.tuik.gov.tr ( erişim tarihi 07.06.2019)
27. Velázquez K, Pina JA, Navarro L, Moreno P, Guerr J. Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA2. Plant Pathol. 2012;61:448–456.
28. Wallace, J. M., 1968. Citrange stunt and ringspot, two previously undescribed virus diseases of citrus, p. 177-183. In Proc. 4th Conf. IOCV. Univ. Florida Press, Gainesville.