Mikrobiol. Z. 2021; 83(2):64-72.
doi: https://doi.org/10.15407/microbiolj83.02.064

Anti-TMV Activities of Pantoea agglomerans Lipopolysaccharides in vitro

T.V. Bulyhina, A.M. Kyrychenko, M.S. Kharchuk, L.D. Varbanets

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

Today there are no antiviral drugs of chemical nature that can completely cure virus-infected plants. The fact that their effect is limited to minimizing the pathogenic effect of viruses motivates many researchers to look for alternatives. In recent years it has been shown that lipopolysaccharides (LPS) of some bacteria, in particular representatives of the Pseudomonas genus were active against Tobacco mosaic virus (TMV). Therefore, we were interested in the additional study of LPS of phytopathogenic bacteria Pantoea agglomerans as a possible drug acting as antiviral agent. The aim of current study was to evaluate the antiviral activities of LPS obtained from phytopathogenic bacteria P. agglomerans against TMV in vitro. Methods. The antiviral activity of LPS preparations was investigated in vitro and assessed according to the inhibition percentage towards the number of local lesions in Datura stramonium leaves. P. agglomerans LPS was isolated from dry bacterial mass by phenol-water method. LPS mild acid degradation allowed to separate O-specific polysaccharide (OPS) and lipid A, which structures were identified by us earlier. The analysis of TMV and LPS interactions was carried out using a JEM 1400 transmission electron microscope (Jeol, Japan) at an accelerating voltage of 80 kV. Results. The most active were LPS preparations from P. agglomerans P324 and 8488. In vitro inhibitory efficacies of TMV infection by these LPS preparations was 59 and 60% respectively. LPS preparations of P. agglomerans 7969, 7604 and 9637, on the contrary, were inactive. Comparative analysis of the antiviral activity of LPS structural components of two P. agglomerans P324 and 7604 strains showed that the greatest inhibitory effect on the infectivity of TMV was exhibited by P. agglomerans P324 lipid A, the antiviral activity of which practically did not differ from the activity of the LPS molecule (it was lower by 7%). At the same time, the inhibitory effect of P. agglomerans 7604 core oligosaccharide (OG-core) against TMV was slightly higher compared to the effect of the whole LPS molecule. It can be assumed that the OG-core stimulated the defense mechanisms of plants and prevented the development of viral infection. Electron microscopic dates have shown that P. agglomerans P324 LPS at the concentration of 1 mg/ml influenced on freely located virions in the control causing “sticking” thus forming dense clusters, complexes or “bundles” of the virus. The individual structural components of P. agglomerans P324 LPS (lipid A and OG-core) did not have the same effect as a whole molecule. Conclusions. The study of the antiviral activity of LPS in the model system TMV – Datura stramonium L. plants showed that the most active were LPS preparations of only two strains of P. agglomerans (P324 and 8488) while the other seven strains were inactive. Individual structural components: lipid A from P. agglomerans P324 and OG-core from P. agglomerans 7604 decreased the infectivity of TMV by 7 and 15% higher than the initial LPS molecule. According to electron microscopy data the virions sticked together forming the dense clusters in case of the direct LPS-virus contacting in vitro whereas in the control it was observed just a single free virus particles. A more detailed study of the effect of individual structural components will help to understand the regularities of the LPS structure effect on TMV infectivity.

Keywords: Pantoea agglomerans, lipopolysaccharides, lipid A, core oligosaccharide, tobacco mosaic virus, infectivity inhibition, electron microscopy.

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  1. Hansen AF. Antiviral chemicals for plant disease control. Critical Reviews in Plant Sciences. 1989; 8:45–88. https://doi.org/10.1080/07352688909382270
  2. Wang ZW, Wang L, Ma S, Liu YX, Wang LZ, Wang QM. Design synthesis, antiviral activity, and SARs of 14-aminophenanthroindolizidines. J Agric Food Chem. 2012; 2(60):5825–5831. https://doi.org/10.1021/jf3013376
  3. Wang ZW, Wei P, Liu YX, Wang QM. D and E rings may not be indispensable for antofine: discovery of phenanthrene and alkylaminechain containing antofine derivatives as novel antiviral agents against tobacco mosaic virus (TMV) based on interaction of antofine and TMV RNA. J Agric Food Chem. 2014; 62:10393–10404. https://doi.org/10.1021/jf5028894
  4. Yucheng Yao, Xueshun Yu, Zhide Yu. Plant antiviral agent, preparation method and application thereof . WO 2007/1090014 AO 1N65/08.
  5. Shuster G, Kluge Z, Kovalenko AG. [Sredstvo borby s virusami rasteniy]. Patent RF N 2036583 AO 1N 63/00. Russian.
  6. Yuki D, Kiso A. Method and agent for controlling plant disease using bacteria of genus Bacillus. United States Patent N US 2009/0175837 C 12N 1/20, AO 1N 63/00.
  7. Miller SH, Mark GL, Franks AO, Gara F. Pseudomonas – Plant Interactions: Pseudomonas. Model Organism, Pathogen, Cell Factory. B.H. Rehm, editor. Weinheim: Wiley-VCH Verlag; 2008. P. 353–370.
  8. Naoto S, Hanae K, Yoshitake D. Resistance-inducing agent for disease of monocotyledonae. JP 2007-03-29 N 2007077065 (A), AO1N63/00, AO1N63/02.
  9. Kiprianova EA, Varbanets LD, Shepelevich VV. Voichuk SI. Antiviral activity of lipopolysaccharides of Pseudomonas chlororaphis subsp. aureofaciens. Biotechnologia acta. 2013; 6(2):68–73. https://doi.org/10.15407/biotech6.02.068
  10. Avdeeva LV, Balko OI, Kiprianova EA, Kovalenko OG. Pseudomonas aureofaciens and biopreparation Haupsin. New aspects of biological activity. Abstracts of the XII Congress of Vynograds’kyj Society of Microbiologists of Ukraine. 2009:282.
  11. Balko OI, Kiprianova EA, Kovalenko AG, Shepelevych VV, Avdeeva LV. Antiphytoviral activity of Gaupsin biopreparat. Microbiology and Biotechnology. 2010; 2(10):12–17. https://doi.org/10.18524/2307-4663.2010.2(10).98806
  12. Galanos C, Tanaka A. Biological activities of chemically modifiend endotoxins. Eur J Biochem. 1971; 22:218–224. https://doi.org/10.1111/j.1432-1033.1971.tb01535.x
  13. Dubois M, Gilles KA, Hamilton JK. Colorimetric method for determination of sugars and related substances. Anal Chem. 1956; 28(3):350–356. https://doi.org/10.1021/ac60111a017
  14. Hebert TT. Precipitation of plant viruses by polyethylene glycol. Phytopathology. 1963; 53:362. 
  15. Chen J, Yan XH, Dong JH, Sang P, Fang X, Di YT, Zhang ZK, Hao XJ, Tobacco Mosaic Virus (TMV) Inhibitors from Picrasma quassioides Benn. J Agric Food Chem. 2009; 57:6590–6595. https://doi.org/10.1021/jf901632j
  16. Yan XH, Chen J, Di YT, Fang X, Dong JH, Sang P, Wang YH, He HP, Zhang ZK, Hao XJ. Anti-tobacco mosaic virus (TMV) quassinoids from Brucea javanica (L.) Merr. J Agric Food Chem. 2010; 58:1572−157. https://doi.org/10.1021/jf903434h
  17. Zdorovenko EL, Kadykova AA, Shashkov AS, Varbanets LD, Bulyhina TV, Knirel YuA. Lipopolysaccharides of Pantoea agglomerans 7604 and 8674 with structurally related O-polysaccharide chains: Chemical identification and biological properties. Carbohydr Polymers. 2018; 181:386–393. https://doi.org/10.1016/j.carbpol.2017.10.087
  18. Zdorovenko EL, Kadykova AA, Shashkova AS, Varbanets LD, Bulyhina TV, Knirel YA. Lipopolysaccharide of Pantoea agglomerans 7460: O-specific polysaccharide and lipid A structures and biological activity. Carbohydrate Research. 2020; 496:108132. https://doi.org/10.1016/j.carres.2020.108132
  19. Zdorovenko EL, Kadykova AA, Shashkov AS, Varbanets LD, Bulyhina TV. Pantoea agglomerans P1a lipopolysaccharide: Structure of the O-specific polysaccharide and lipid A and biological activity. Carbohydrate Research. 2019; 484:1–5. https://doi.org/10.1016/j.carres.2019.107767
  20. Zdorovenko EL, Kadykova AA, Shashkov AS, Varbanets LD, Bulyhina TV, Toukach PV. Structure and Biological Properties of the O-specific Polysaccharide and Lipid A from Pantoea agglomerans P 324. Microbiology. 2021; 90(1):96–105. https://doi.org/10.1134/S0026261721010124
  21. Bulyhina TV, Zdorovenko EL, Varbanets LD, Shashkov AS, Kadykova AA, Knirel YuA, Lushchak OV. Structure of O-Polysaccharide and Lipid A of Pantoea agglomerans 8488. Biomolecules. 2020; 10(5):804. https://doi.org/10.3390/biom10050804
  22. Zdorovenko EL, Kadykova AA, Shashkova AS, Varbanets LD, Bulyhina TV, Knirel YA. Lipopolysaccharide of Pantoea agglomerans 7969: chemical identification, function and biological activity. Carbohydrate Polymers. 2017; 165:351–358. https://doi.org/10.1016/j.carbpol.2017.02.053
  23. Bulyhina TV, Varbanets LD, Pasichnyk LA. Lipopolysaccharide of Pantoea agglomerans 9649: chemical identification and biological activity. Mikrobiol Z. 2018; 80(2):56–66. https://doi.org/10.15407/microbiolj80.02.056
  24. Dijkstra J, de Jager CP. Infectivity Assay on Local-Lesion Hosts. In: Practical Plant Virology. Springer Lab Manual. Springer, Berlin, Heidelberg. 1998. https://doi.org/10.1007/978-3-642-72030-7_10
  25. Price W.C. Thermal inactivation rates of four plant viruses. Archiv Virusforschung. 1940; 1:373–386. https://doi.org/10.1007/BF01245548
  26. Seydel U, Schromm A, Blunck, Branderburg K. Chemical structure, molecular conformation, and bioactivity of endotoxins. Chem Immunol. 2000; 74:5–24. https://doi.org/10.1159/000058754