Pseudomonas chlororaphis subsp. Aureofaciens native AND MODIFIED BY COMPLEXES OF Ge(IV) AND Sn(IV) LIPOPOLYSACCHARIDE ANTIVIRAL ACTIVITY

form of a ligand are more active than with enol O(C-O)–N(CH=N) form of isonicotinoyl hydrazones of the same aldehydes (I,% 32-63)]. Introduction of two substituents (OH- and Br-) into the hydrazide fragment of hydrazone molecule significantly increases the activity of Sn(IV) complexes with enol form of the ligand (I,% 79). proteolytical

Viral plant diseases are widespread in agro-and biocenoses, therefore they play an important role in crops yield reducing, which leads to significant economic losses in crop production.There are various methods of plants viral disease control; disease prevention and resistant to viruses species breeding are the most effective among them.All stages of viruses reproduction are closely related to the metabolic, energy and enzymatic reactions of the cell, so chemicals usage to virus infections control is often ineffective because drugs that inhibit the reproduction of viruses tend to inhibit the normal metabolic processes in plants.Such compounds are characterized by a significant phytotoxicity, so the problem of plants therapy and viral infections prophylaxis by biological agents usage is urgent nowadays.At the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine on the basis of two strains of Pseudomonas chlororaphis subsp.aureofaciens, earlier [1] a complex biopreparation gaupsin, inhibiting the growth of pathogenic bacteria and fungi, possessing as antimicrobial activity as strong entomopathogenic activity, has been created and patented.In subsequent years it has been found that gaupsin is characterized by pronounced antiviral properties as it inhibits by 80-97% the progression of induced by tobacco mosaic virus (TMV) necroses [2].The authors have shown that gaupsin antiviral activity is associated with exometabolites synthesis; in particular, it has been hypothesized that high molecular weight polysaccharides, primarily lipopolysaccharide (LPS) and exopolysaccharides, probably participate in these antiviral mechanisms.We have found [3] that lipopolysaccharide of Р. chlororaphis subsp.aureofaciens B-111 and B-306 are highly active antiviral agents.It is interesting that lipopolysaccharides derived from microorganisms belonging to other genera and species (Rahnella aquatilis and Ralstonia solanacearum), were not active against TMV, and sometimes even stimulated necrosis formation.
One of the methods for biopolymers activity alteration is their modification, in particular by binding to activators (inhibitors) such as biometals and bioligands complexes.It is known [4] that the complex compounds of germanium with nitrogen-containing compounds of the purine row provide a high level of biological activity against herpes viruses of the 1 st and the 2 nd types and also are effective when used in the comprehensive treatment of HIV-infected and cancer patients; for a series of germanium (IV) complexes the effect on the activity of glycosidases [5] and proteases [6,7] is revealed.The effect of tin complex compounds is less studied; as it is known [7], tin is a part of the gastric enzyme gastrin, affects the activity of flavin enzymes, and is also an effective inhibitor of hemeoxidase [8].

Materials and Methods
The object of the study was the strain of Pseudomonas chlororaphis subsp.аureofaciens UCM B-306, isolated from the rhizosphere of cabbage and was deposited in Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, number IMV B-7096.Cultivation of the strain was performed in semi-synthetic King medium of the following composition (g/l): peptone -20, К 2 SO 4 -20, glycerol -20, MgCl 2 -7, in Erlenmeyer flasks of 750 ml filled with 150 ml of culture medium, for 24 hours, on a shaker (220 rev / min) at 28 C.
Lipopolysaccharides were obtained by extraction from the cells, dried with acetone and ether, with 45% aqueous phenol solution at 65-68 C.The resulting aqueous fractions were dialyzed against tap water, and then against distilled water to remove phenol, hereafter clarified from nucleic acids by precipitation with trichloroacetic acid and also by ultracentrifugation (104 000 g, 4 h).Purified LPS was lyophilized [21].
To modify the LPS of P. chlororaphis subsp.аureofaciens UCM-306 a wide range of coordination compounds was selected: -germanium (IV) with 2-R-benzoyl (R- The choice of these compounds makes it possible to trace the several factors (complexing agent (Ge, Sn), coordination site composition and variety of substituents presence in the ligand molecule) impact on the investigated LPS antiviral activity.
The antiviral activity of Р. chlororaphis subsp.aureofaciens was studied in a model of tobacco mosaic virus (TMV U1 strain), derived from systemically infected Nicotiana tabacum L. varieties Immune 580 (revertant) plants according to [22,23].Investigations were carried out on leaves of hypersensitive to TMV datura plant Datura stramonium L., reacting to virus inoculation by the formation of local necrosis.Experimental halves of leaves previously carborundum dusting were inoculated with a mixture of virus and each of the test preparations (1-35) in a ratio of 1:1, and control halves of leaves -with virus only.LPS preparations at concentrations of 1.5, 1.0 or 0.75 mg/ml were added to TMV (1 or 0.75 g/ml) for 30 min before plants inoculation (in control distilled water was added to TMV).Lipopolysaccharides antiviral action in vitro against TMV was evaluated by the number of local necrosis induced by virus.The inhibitory effect (I) of preparations was determined using the formula: where E and C -an average number of necrosis formed on the experimental and control halves of leaves respectively.
To assess the reliability of experimental data the parametric criteria of normal distribution were used; calculating the arithmetic mean (X a ), mean square inaccuracy measure (S x a ) at a significance level of 0.05 or 0.01, the Student's t test (t) was determined and according to the table for small samples the levels of significance (P) of the mean values difference or ratio were found.Results of statistical processing were represented in the form of confidence intervals or X a ± S x a .The relative mean values error was obtained by the processing of 15-20 repetitions [24].

Results and Discussion
Previously [3] we have shown that lipopolysaccharides (ЛПС) of Р. chlororaphis subsp.aureofaciens B-111 and B-306 (gaupsin components) are highly active antiviral agents.In studies in 2010-2012 they invariably showed efficacy against TMV on the model of three types of indicator plants of the family Solanaceae.Inhibition of virus infectivity was 98-100% by LPS at a concentration of 1-10 mg/ml, 57-69% -at a concentration of 0.1 mg/ml, 43-44% -at a concentration of 0.01 mg/ml.LPS at a concentration of 1 μg / ml caused reduction of virus infectivity at various indicator datura (Datura stramonium L.) and tobacco (Nicotiana sanderae H., Nicotiana tabacum L.) plants ranged from 10.2 to 46.0%.LPS activity of both Р. chlororaphis subsp.aureofaciens strains was approximately the same.Therefore, in this study the effect of LPS modified by different complexes was carried out using LPS of one strain only -Р.chlororaphis subsp.aureofaciens B-306.
It is known [25] that the complexes of biopreparations with metals may induce non-specific resistance of plants to viruses or enhance the inhibitory effect of the drug.Therefore, it was interesting to study the antiviral activity of the modified LPS (LPS-m).Along with the modified LPS we investigated the original unmodified LPS (LPS-o) antiviral activity as positive control.
It should be noted that all LPS preparations in a concentration of 1.5 mg/ml possessed phytotoxicity that appeared on the treated leaves as burns.The first signs of burns in the form of whitish or brown spots on the leaves appeared 2-3 days after treatment (Figure ).Preparations 6-10, 16-35 caused focal burns of lamina at an initial concentration of 1 mg/ml, so in subsequent experiments they were investigated in a concentration of 0.75 mg/ml.
From the above it follows that preparations virucidal effect neutralizing virus infectivity depends on: metal-complexing agent [tin (IV) compounds are more active (I, % 65-79) as compared to germanium (IV) complexes (I, % 26-62)], the number of functional groups in ligand molecules [ The most active complexes antivirus action possible mechanisms may be the TMV direct inhibition by attaching to the structural proteins of TMV and conglomerates formation (aggregates of the virus and the modified LPS) or the degradation of viral RNA (by host RNAase at mechanical inoculation).It is possible involvement of preparations in the induction (stimulation) of plant defense mechanisms, which consequence is the increased resistance of plants to TMV and inhibition of viral reproduction (obstacle to the development of productive infection).
Thus, "TMV -ultrasensitive plant" model study of LPS and its modified preparations (1-35) antiviral activity showed high antiviral activity of preparations 7, 21, 14, 23, 20 and 26, which may be due to their composition and structural features.Such preparations are of interest as promising agents in the fight against viral diseases of plants.
In available literature we have not found the data on antiviral effect of bacteria of the genus Pseudomonas LPS, however, based on the above data it is fair to assume that the observed activity of Р. chlororaphis subsp.aureofaciens strains may be associated with non-specific stimulation of infected with TMV plants protective forces.Ключевые слова: Pseudomonas chlororaphis subsp.аureofaciens, липополисахарид, координационные соединения Ge(IV) и Sn(IV), антивирусное действие, вирус табачной мозаики.