Termal olmayan plazma tedavisi ile mikro çoğaltılmış erik bitkilerinde Plum pox virus inaktivasyonu çalışmaları

Öz

Son zamanlarda, soğuk atmosferik plazma (SAP)’ın tıp, gıda teknolojileri, su arıtma teknolojileri ve tarımda patojenlerin inaktivasyonu için uygulanabilme olasılığı araştırılmaktadır. Bu çalışmanın amacı SAP’ın, Plum pox virus’ünün (PPV) M ve D ırkları tarafından doğal olarak enfekte edilen erik ağacından, in vitro koşullar altında elde edilmiş bitkicikler üzerindeki etkisini ve bu bağlamda SAP’ın virüs inaktivasyonunda uygulanması olasılığını araştırmaktır. Çalışmada, biyolojik sistemlerin uygulanmasında “yüzey dalgası-sürekli Argon plazma torçu” ve “su altı diyafram deşarjı” olmak üzere iki tip plazma kaynağı kullanılarak plazma tedavisinin birkaç varyantının gerçekleştirilmesini sağlamıştır. Uygulamadan sonra üçüncü alt kültür üzerindeki bitkiciklerin IC-RT-PCR verilerine dayanarak, en etkili varyantın gaz ortamında yapraksız nodal segmentlere tekrarlanan plazma torç ucu uygulaması olduğu bulunmuştur. SAP ile muamele edilmiş, her iki PPV ırkı ile enfekteli bitkiciklere yapılan ırka spesifik RT-PCR analiz sonucunda, sadece PPV-M tespit edilmiştir. İklimlendirilmiş ex vitro bitkilerinin IC-RT-PCR ve ırka spesifik RT-PCR'dan elde edilen sonuçları, test edilmiş bitkiciklerin moleküler analizlerinden elde edilen verilerle uyumlu olduğu görülmüştür. Elde edilen bu veriler, in vitro koşullarda yapılmış olsa da canlı odunsu bitkilerin dokusundan PPV'nin inaktivasyonu için CAP yeteneği üzerinde yapılan ilk çalışma niteliğindedir. PPV’den ari erik bitkilerinin elde edilmesinde kullanılan bu yaklaşımın son değerlendirmesi, ex vitro bitkilerin daha uzun süre gözlemlenmesi ve test edilmesinden sonra yapılacaktır.

Anahtar Kelimeler

• Plum pox virus,soğuk atmosferik plazma,doku kültürü,virüs inaktivasyonu

Experiments on Plum pox virus inactivation from micropropagated plum plants through non-thermal plasma treatment

Abstract

Recently, cold atmospheric plasma (CAP) is under investigation for possibility to be applied for inactivation of pathogens in medicine, food technologies, water cleaning technologies and agriculture. The aim of the current study is to investigate the effect of CAP on microplants, propagated in vitro from plum tree (Prunus domestica L., cv. ‘Kyustendilska sinya’) naturally co-infected by M and D strains of Plum pox virus (PPV) and in that respect the possibility for CAP application for virus inactivation. In the present work, we have used two types of plasma sources for biological systems treatments: a surface-wave-sustained Argon plasma torch and an underwater diaphragm discharge. These enabled several variants of plasma treatment to be performed. Based on the data of IC-RT-PCR tests of the microplants on the third subculture after treatment, it was found the most effective variant was the reiterated plasma torch tip treatment to nodal segments without leaves in gas medium. The strain specific RT-PCR analysis results of PPV positive CAP-treated microplants showed that only PPV-M strain was identified after treatment, although the starting material was co-infected by both strains. The results obtained from IC-RT-PCR and strain specific RT-PCR of the acclimatized ex vitro plants have been in agreement with the data from molecular analyses of the microplants tested. These are the first experiments on CAP ability for inactivation of PPV from tissue of living woody plants even if in in vitro conditions. The completed estimation of this approach for obtaining of PPV-free plum plants will be made after more prolonged observation and testing of the ex vitro plants.

Keywords

• Plum pox virus,cold atmospheric plasma,tissue culture,virus inactivation

References

1. Aboubakr H. A., Gangal U., Youssef M. M., Goyal S. M. and Bruggeman P. J. 2016. Inactivation of virus in solution by cold atmospheric pressure plasma: Identification of chemical inactivation pathways. J. Phys. D: Appl. Phys., 49 204001 (17pp) doi:10.1088/0022-3727/49/20/204001
2. Atanasoff D., 1933. Sharka po slivata. Godishnik na Sofiiskiya Universitet, XI, 49-70.
3. Chirkov S., Sheveleva A., Ivanov P., Zakubanskiy A., 2018. Analysis of genetic diversity of Russian sour cherry Plum pox virus isolates provides evidence of a new strain. Plant Dis 102, 569–575.
4. Dobnik D., Junkar I., Primc G. and Ravnikar Maja. 2016. Plasma treatment of potato plants and plant extracts infected with PVYNTN. Workshop on Application of Advanced Plasma Technologies in Central Europe Agriculture 17th-21st April, Ljubljana , Slovenia. Primc, G. (Ed.). Published by: Slovenian Society for Vacuum Technique (DVTS), Ljubljana, 48 p. http://www.plasmadis.com/wp/waapt-in-cea.
5. Filipic A., Primc G., Zaplotnic R., Mehle N., Gutierrez‑Aguirre I., Ravnicar M., Mozetic M., Zel J., Dobnik D. 2019. Cold atmospheric plasma as a novel method for inactivation of Potato virus Y in water samples. Food and Environmental Virology, 11, 220–228.
6. Gabova R., 1989. In vitro thermotherapy and virus elimination in fruit crops. Proceedings of First national conference “Application of in vitro technics in agricultural plants”, 12 October, 1989, Plovdiv, Bulgaria, pp. 74-78.
7. Guo L, Xu R, Gou L, Liu Z, Zhao Y, Liu D, Zhang L, Chen H, Kong M.G., 2018. Mechanism of virus inactivation by cold atmospheric-pressure plasma and plasma activated water. Appl Environ Microbiol, 84, e00726-18. https://doi.org/10.1128/AEM.00726-18.
8. Hauptmanova A, Polak J., 2011. The elimination of Plum pox virus in plum cv. Bluefree and apricot cv. Hanita by chemotherapy of in vitro cultures. 2011. Hort. Sci., 38 (2), 49–53.

9. James D., Varga A. and Sanderson D., 2013. Genetic diversity of Plum pox virus: strains disease and related challenges for control. Can. J. Plant Pathol., 35, 431-441.
10. James D., 2017. Perspective on strategies for controlling the spread of Plum pox virus, the causal agent of Sharka/plum pox disease. Acta Hort, 1163, 129-136.
11. Kamenova I., Borisova A., Dragoyski K., Milusheva S., Stefanova B., Dallot S., and Glasa M., 2015. Plum pox virus strains in Bulgaria. Acta Hort, 1063, 47-54.
12. Koubouris G., Maliogka V., Efthimiou K., Katis N., Vasilakakis M., 2007. Elimination of Plum pox virus through in vitro thermotherapy and shoot tip culture compared to conventional heat treatment in apricot cultivar Bebecou. J Gen Plant Pathol, 73, 370-373.
13. Kozakova Z., Krčma F., Vašíček M., Hlavatá L., Hlochová L., 2015. Generation of dc pin-hole discharges in liquids: comparison of discharge breakdown in diaphragm and capillary configuration. Eur. Phys. J. D, 69: 100.
14. Krčma F., Tsonev I., Smejkalová K, Truchlá D., Kozáková Z., Zhekova M., Marinova P., Bogdanov T. and Benova E., 2018. Microwave micro torch generated in argon based mixtures for biomedical applications. J. Phys. D: Appl. Phys. 51, 414001
15. Murashige, T., Skoog, F. (1962). A revised medium for rapid growth and bio assays for tobacco tissue cultures. Physiol. Plant., 15, 473–497.
16. Nacheva L., Milusheva S. Ivanova K., 2002. Elimination of Plum pox potyvirus (PPV) in plum (Prunus domestica L.) cvs Kyustendilska sinja and Veljevka through in vitro techniques. Acta Hort., 577: 289-291.
17. Nacheva L. and Milusheva S., 2008. Preliminary results of the effect of ribavirin on in vitro cultivated apple plants with the aim of eliminating some viruses. Journal of Mountain Agriculture on the Balkans, 11(1), 129-137.
18. Olmos A., Cambra M., Dasi M.A., Candresse T., Esteban O., Gorris M.T, and Asensio M., 1997. Simultaneous detection and typing of Plum pox potyvirus (PPV) isolates by hemi-nested PCR and PCR-ELISA. Journal of virological methods, 68, 127-137.
19. Scholthof K.B., Adkins S., Czosnek H., Palukaitis P., Jacquot E., Hohn T., Hohn B., Saunders K., Candresse T., Ahlquist P., Hemenway C., Foster G.D.m 2011. Top 10 plant viruses in molecular plant pathology. Molecular Plant Pathology, 12, 938-954.
20. Subr Z., Pittnerova S., Glasa M., 2004. A simplified RT-PCR–based detection of recombinant Plum pox virus isolates. Acta Virologica , 48, 173-176.
21. Wetzel, T., Candresse, T., Ravelonandro, M. & Dunez., J., 1991. A polymerase chain reaction adapted to Plum pox potyvirus detection. J. Virol. Methods, 33, 355 - 366.
22. Wetzel, T., Candresse, T., Masquiare, M., Ravelonandro, M. & Dunez., J., 1992. A hightly sensitive immunocapture polymerase chain reaction for Plum pox virus detection. J. Virol. Methods, 39, 27 - 37.
23. Wu Y., Liang Y.D., Wei K., Li W., Yao M.S, Zhang J., Grinshpun S.A., 2015. MS2 virus inactivation by atmospheric-pressure cold plasma using different gas carriers and power levels. Appl Environ Microb, 81, 996-1002.
24. Vyhnankova E.J., Hammer M.U., Reuter S. and Krcma F., 2015. DC diaphragm discharge in water solutions of selected organic acids. Eur. Phys. J. Appl. Phys., 71(2), 20809.