AS  Vol.6 No.11 , November 2015
Antiviral Effect of Ribonuclease from Bacillus pumilus against Phytopathogenic Rna-Viruses
Abstract: Background: Viruses can cause different diseases in plants. To prevent viral infections, plants are treated with chemical compounds and antiviral agents. Chemical antiviral agents usually have narrow specificity, which limits their wide application. Alternative antiviral strategy is associated with the use of microbial enzymes, which are less toxic and are readily decomposed without accumulation of harmful substances. The aim of this work is to study the effect of Bacillus pumilus ribonuclease on various phytopathogenic viruses with specific focus on the ability of enzyme to eliminate them from plant explants in vitro. Materials and methods: Extracellular ribonuclease of B. pumilus is tested as an antiviral agent. To study the antiviral effect of RNase, depending on concentration and the time of application several plant-virus model systems are used. Virus detection is conducted by serological testing and RT-PCR. Results: Bacillus pumilus ribonuclease possesses antiviral activity against plant Rna-viruses RCMV (red clover mottle virus), PVX (Potato Virus X) and AMV (Alfalfa Mosaic Virus). The maximum inhibitory effect against actively replicating viruses is observed when plants are treated with the enzyme in the concentration of 100 ug/ml prior to infection. In case of local necrosis ribonuclease in the concentration of 1 ug/ml completely inhibits the development of RCMV virus on bean plants. The enzyme is able to penetrate plants and inhibit the development of viral infection, inhibiting effect for untreated surfaces decreased on average for 20%. It is also found that B. pumilus ribonuclease protects apical explants of sprouts of potato tubers from PVM and PVS viruses. Conclusion: B. pumilus ribonuclease possesses antiviral activity against plant Rna-viruses and produces viruses-free plants in the apical meristem culture.
Cite this paper: Sharipova, M. , Rockstroh, A. , Balaban, N. , Mardanova, A. , Toymentseva, A. , Tikhonova, A. , Vologin, S. and Stashevsky, Z. (2015) Antiviral Effect of Ribonuclease from Bacillus pumilus against Phytopathogenic Rna-Viruses. Agricultural Sciences, 6, 1357-1366. doi: 10.4236/as.2015.611130.

[1]   Halterman, D., Charkowski, A. and Verchot, J. (2012) Potato, Viruses, and Seed Certification in the USA to Provide Healthy Propagated Tubers. Pest Technology, 6, 1-14.

[2]   Conrad, P.L. (1991) Potato Virus S-Free Plants Obtained Using Antiviral Compounds and Nodal Segment Culture of Potato. American Journal of Potato Research, 68, 507-511.

[3]   Brown, F. (2001) Inactivation of Viruses by Aziridines. Vaccine, 20, 322-327.

[4]   Nascimento, L.C., Pio-Ribeiro, G., Willadino, L. and Andrade, G.P. (2003) Stock Indexing and Potato Virus Y Elimination from Potato Plants Cultivated in Vitro. Scientia Agricola, 60, 525-530.

[5]   Trifonova, E.A., Komarova, M.L., Leonova, N.S., Shcherban, A.B., Kochetov, A.V., Malinovskii, V.I. and Shumnyi, V.K. (2004) Transgenic Potato (Solanum tuberosum L.) Plants Expressing the Gene of Secretory Nuclease from Serratia marcescens. Doklady Biochemistry and Biophysics, 394, 39-41.

[6]   Zhou, W.W. and Niu, T.G. (2009) Purification and Some Properties of an Extracellular Ribonuclease with Antiviral Activity against Tobacco Mosaic Virus from Bacillus cereus. Biotechnology Letters, 31, 101-105.

[7]   Zhou, W.W., He, Y.L., Niu, T.G. and Zhong. J.J. (2010) Optimization of Fermentation Conditions for Production of Anti-TMV Extracellular Ribonuclease by Bacillus cereus Using Response Surface Methodology. Bioprocess and Biosystems Engineering, 33, 657-663.

[8]   Fedorova, A.A., Azzami, K., Ryabchikova, E.I., Spitsyna, Y.E., Silnikov, V.N., Ritter, W., Gross, H.J., Tautz, J., Vlassov, V.V., Beier, H. and Zenkova, M.A. (2011) Inactivation of a Non-Enveloped RNA Virus by Artificial Ribonucleases: Honey Bees and Acute Bee Paralysis Virus as a New Experimental Model for in Vivo Antiviral Activity Assessment. Antiviral Research, 91, 267-277.

[9]   Leshchinskaia, I.B., Kleiner, G.I., Volkova, T.I., Balaban, N.P. and Sharipova, М.R. (1981) Isolation and Purification of Alkaline Ribonuclease from Bacillus Intermedius. PriklBiokhimMikrobiol, 17, 241-246.

[10]   Kluge, S. and Marcinka, K. (1979) The Effects of Polyacrylic Acid and Virazole on the Replication and Component Formation of Red Clover Mottle Virus. Acta Virologica, 23, 148-152.

[11]   Murashige, T.A. (1962) Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Plant Physiology, 15, 473-497.

[12]   Crosslin, J.M. and Hamlin, L.L. (2011) Standardized RT-PCR Conditions for Detection and Identification of Eleven Viruses of Potato and Potato Spindle Tuber Viroid. American Journal of Potato Research, 88, 333-338.

[13]   Sharipova, M.R. (2002) Late Stages of Protein Secretion in Bacilli. Biochemistry, 67, 1207-1216.

[14]   Grishina, I.B., Makarov, A.A., Kuznetsov, D.V., Vasil’ev, D.G., Palovskii, A.G., Aizenmenger, F. and Esipova, N.G. (1993) Ionic Pairs in Bacillus Intermedius 7P Ribonuclease. Biofizika, 38, 22-30.

[15]   Demchuk, I.V., Zaritsky, N.M. and Volkova, I.V. (2011) Efficiency of Biotechnology Disease Eradication System for the Potato Varieties. POTATO-GROWING: Proceedings, Vol. 19, RUE: Research and Practical Center of NAS of Belarus for Potato, Fruit and Vegetable Growing, Minsk, 221-230.

[16]   Malinovsky, V.I. (2010) Resistance Mechanisms of Plants to Viruses. Dalnauka, Vladivostok, 191-192.

[17]   Mercer, J.L., Schelhaas, M. and Helenius, A. (2010) Virus Entry by Endocytosis. Annual Review of Biochemistry, 79, 803-833.

[18]   Fartais, L. (1998) The Regeneration Capacity of Potato Explains Stored on Media with Growth Inhibitors. Vol. 44, Analele Stiintificeaie Universitatii Al. I. Cuza din lasi (Romania) Sec.2a, 69-73.