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Study of Biological Properties of Effective Pesticides Destructor Pseudomonas Putida P2

A promising approach for remediation of contaminated soils is the use of biological products based on microorganisms-destructors. Of particular interest are the strains that can decompose several types of pollutants. Potential strains-destructors should be not only effective but also adapted for certain soil and climatic conditions. In addition, when introducing a significant amount of biological agents into the ecosystem, the lack of toxicological and pathogenic effects on plants, animals, and humans is particularly important. The object of the study is the destructor strain of sim-triazine herbicide of prometrin Pseudomonas putida P2. The study explores the ability of strain to use various pesticides as the only carbon source, the effect of bacterial culture on seed germination, biotesting of strain on toxicity using earthworms. Besides, the study presents the phytopathogenic factors and enzymes causing bacteria invasiveness into animals and humans. The determination of dehydrogenase activity of prometrin destructor strain shows that this strain can also use active ingredients of pesticide malathion, HCH, and 4,4-DDT as only sole carbon source. It is also established that the studied strain Pseudomonas putida P2 increases the germination of seeds of the variety Saratovskaya 70 and carrots of the variety Samson and does not possess pathogenicity factors. The strain biotesting on the earthworms revealed no acute toxicity of the culture. The analysis of data obtained from the model pollution shows that the introduction of the destructor of Prometrium Pseudomonas putida P2 into the soil can create favorable conditions for vital activity and reproduction of earthworms.


Introduction
One of the urgent tasks of modern biotechnology is the creation of biologicals based on xenobiotic strains-destructors isolated from native microflora to solve a set of tasks related to rehabilitation of contaminated soils as a result of increasing man-made stress [1--3]. Currently in Russia and abroad there are many products produced for cleaning the environment. They are mainly aimed at oil and oil products decomposition [4]. However, pesticides relate to significant global polluter of the Earth. Pesticides are widely used throughout the world and are constantly released into the environment. Pesticides can be accumulated in water, soil, plants, in living organisms and can even be transmitted through trophic chains, many of them have carcinogenic, mutagenic properties and cause disturbances in the vital activity of organisms. Soils mostly degrade due to the intensive use of pesticides violations the norms and rules for their use, which leads to their accumulation in soils. The disposal sites for unused or prohibited chemicals are also of particular danger [6,7]. Natural processes of soil self-purification are unable to cope with such volumes of pollution. A promising approach for remediation of contaminated soils is the use of biological products based on microorganisms [8,9]. The use of biological products can reduce labor costs by 3--5 times and energy consumption by 50--100 %. It can increase the efficiency of cleaning 5 to 10 times. Due to the selective adaptation, the bacteria are able to multiply at high concentrations of pollutants and use the latter as sources of food and energy [10]. Today, it is an economically viable strategy and an environmentally friendly technology for the remediation of polluted ecotops [11].
Potential strains-destructors should not only be effective, but also adapted for certain soil and climatic conditions. In addition, when introducing a significant amount of biological agents into the ecosystem, their lack of toxicological and pathogenic effects on plants, animals and humans is particularly important. The absence of antagonistic relations between the introduced species and the autochthonous microbiological community is also very important. In this regard, the selection of strains-destructors for the use as a basis of biological product is a very crucial stage. In this regard, we studied the factors of pathogenicity and toxigenicity of strain-destructor of pesticide.

Methods and Materials
The object of the study was a strain-destructor of the sim-triazine herbicide of prometrin Pseudomonas putida P2 isolated from soil contaminated with pesticides. The State Standard Samples (GSO) of pesticides of prometrin, malathion, cypermethrin, foundation, lambda cyhalothrin, HCH and 4,4-DDT were used as chemicals ( Table 1).
Identification of the ability of the strain to use various pesticides as the only carbon source was carried out by the presence of dehydrogenase. Since under the aerobic conditions the first stage of biodegradation of xenobiotics is oxidative metabolism reactions catalyzed by various oxidoreductases, the main of which are dehydrogenases, A 5 % aqueous solution of 2,3,5-triphenyltetrazolium chloride (TTC) was used to detect dehydrogenase activity in bacteria [12], which was added to M9 medium (com- The macerating ability determination was carried out according to Johnson. 10 ml of sterile potato broth was sown with the culture of the studied bacteria and kept for ten days in thermostat at the temperature of 28 ∘ C. Then the culture fluid was poured into a 20--30 ml cup and thin slices of potatoes, beets, and carrots were put into it. The tissue maceration was determined at 40 ∘ C. The degree of softening of the slice on tubers and root crops was checked with a fine needle at regular intervals for 6 hours. If the slice was softened, this indicated the presence of protopectinase in the culture fluid.
The maceration or the decay of the tissue of higher plants into individual cells occurs under the action of pectolytic enzymes that destroy pectin in the middle plate [13].
To identify the cellulolytic activity, the strain of microorganisms was seeded with the help of a loop``medallions'' (5--8 mm in diameter) on the surface of Glucose Salts Agar containing 0.2 % of soluble cellulose (carboxymethylcellulose), then incubated for 48 hours at 28 ∘ C. After this, the cups were poured with a 0.1 % aqueous solution of Congo red (4 ml) and kept for 20 minutes. The dye was decanted, the cups were washed with 8 % aqueous NaCl solution [14].
The study used positive and negative controls: • positive control is a strain from the collection of Bacillus simplex 422 (received from aphids), which has pronounced cellulolytic activity.
• negative control is sterile tap water without microorganism.
The result records were conducted visually. The presence of bright, unstained zones in the areas of bacterial growth or around medallions was considered a positive reaction (indicating the production of cellulolytic enzymes). A negative reaction was considered the absence of bright unstained zones in the places of bacterial growth and around the medallions.
The study of the effect of a destructor microorganism on germination was carried out using seeds of a monocot (wheat) and dicotyledonous (carrot) plants. For the study the intact, leveled by the size seeds of one reproduction year with good germination (≥ 90 %) were used. Before soaking, the seeds were disinfected with 1 % potassium permanganate solution for 10 minutes. Then they were placed into the sterile Petri dishes on filter paper 100 pieces each and poured the required amount of distilled water (control) and suspensions of the studied bacteria (experiment). The experiment was carried out in three replications at the temperature of 24 ∘ C. For 7 days, both germination and viability of seedlings had been carefully monitored. The germination was determined on day 10 by the formula adjusted for germination in control: where B is the number of seeds germinated in the experiment; C is the number of seeds germinated in the control [15].
Fibrinolysin, hemolysin and arginine dehydrolase have been studied as enzymes responsible for the invasiveness of bacteria in animals and humans. Fibrinolysin was determined in a test tube with fibrin (a blood clot washed from red blood cells) where the test culture was applied. After the incubation, the result was taken into account.
With a positive result, the clot had dissolved. Staphylococcus aureus was used as a test organism.
Hemolysins were determined by plating the test culture in a Petri dish with blood agar. The cups were incubated in a thermostat at 37 ∘ C during the day. With a positive reaction, the hemolysis zones are formed around the plaques. The Staphylococcus aureus was used as a test organism.
The determination of arginine dehydrolase activity was studied on the medium of the following composition (g/l): enzyme peptone --1 g; NaC1 --5 g; KHPO4 --0.3 g; L(+) arginine HCl --10 g; phenol red --0.01 g; agar-agar --15 g; pH = 6.9--7.0. Microorganisms with arginine dehydrolase form NH 3 from arginine under anaerobic conditions, which is accompanied by alkalization of the medium and a change in the indicator color. The medium was poured into 5 ml sterile tubes and, after cooling, the test culture was inoculated with two test tubes, two of which were coated with sterile paraffine oil [14].
The results were taken into account after 24--48 hours. In the case of a positive reaction in bacteria grown under anaerobic conditions, the color of the medium changes from yellow to red. As a test organism, the strain Pseudomonas fluorescens from the museum of the Department was used.
The determination of soil toxicity by biotesting using earthworms was carried out by the method of short-term biotesting (screening test) --up to 2 days and long-term

Exploring the ability of a strain-destructor to use carbon pesticides
Currently, the biological products based on microorganisms-destructors adapted to a specific type of pollutant are widely used. The most promising approach for remediation of contaminated soils is the use of biological products based on microorganismsdestructors with a broad spectrum of action. The composition of such biological products should include microorganisms from several types of destructors, or one, capable of destroying substances of different chemical composition.
While determining the dehydrogenase activity of strain-destructor of prometrin it was found out that this strain can also use the active ingredients of pesticides of malathion, HCH and 4,4-DDT as the only carbon source.

Study of enzymes of pathogenicity
The results of the study of the enzymatic activity of the strain-destructor are presented in Table 2.
The analysis of enzymatic activity of the strain in relation to plant tissues did not reveal neither macerating nor cellulolytic activities. The study of pathogenicity factors in animals and humans did not show the presence of arginine dehydrolase, fibrinolysin and hemolysin. Consequently, the strain-destructor is not pathogenic in relation to plants, animals, and humans.

The effect of destructor microorganism on seed germination
The studies allowed learning about the effect of strain Pseudomonas putida P2 on the seeds of wheat of the variety Saratovskaya 70 and carrots of the variety Samson for 10 days. The results obtained are presented in Table 3.

The toxicity of the strain Pseudomonas putida P2 determination using earthworms
The criterion of toxicity is the absence of such phenomenon as earthworms bury into tested soil, active crawling on the surface of the earth and attempts to crawl out of the box (avoidance test). Due to some ecological and physiological features the earthworms are in contact with soil particles, air and moisture, not only on the surface of the skin, but also inside the digestive tract. Processing a large amount of soil, they are exposed to the direct influence of inorganic and organic substances in the soil. The results are presented in Table 4. The biotesting of Pseudomonas putida P2 strain on the earthworms revealed no acute toxicity of the culture. It was found out that the mortality of Eisenia foetida on

Conclusion
The determination of dehydrogenase activity of strain-destructor of prometrin showed that this strain can also use the active ingredients of pesticides, i.e. malathion, HCH and 4,4-DDT as the only carbon source. It was also stated that the studied strain