SOIL MICROBIOTA UNDER THE CONDITIONS OF AN OPEN FIELD EXPERIMENT OF FERTILIZING SOIL BY ENERGY WILLOW

The aim of the work was to study the soil agrochemical indices, soil microbiocoenosis, in case of growing of energy cultures and based on the mineralization coefficient, to make a conclusion on the speed of mineralization processes in the soils under study. In conditions of continuous field experiment (2011– 2016), the dynamics of soil microbial associations was studied for willow ( Salix triandra  Salix viminalis ‘Inger’) cultivation with application of experimental fertilizers of different types. In the research fertilizers there were used: sulfuric urea, municipal biocompost, municipal sewage sludge compost, rhyolite tuff and willow ash. The soil microbiotic communities analysis was conducted by the method of serial dilutions of soil suspension with the use of differentially diagnostic nutrient media: meat-peptone agar, starch-ammonium agar, Ashby medium, potato agar, Czapek Dox medium, starvation agar, Ploskirev medium. The direction of the microbiological processes in the soils was determined. According to the results, it was established that the most promising for the purpose of improving the metabilic activity of the soil (in the growth of energy willow) is a municipal sewage sludge compost and a municipal biocompost. In case of the use of municipal sewage sludge compost, the number of intestinal bacteria, ammonifiers, micromycetes and actinomycetes was doubled as compared with the control. In case of the use of municipal biocompost, the levels of microscopic fungi and cellulolytic bacteria doubled, and those of intestinal bacteria and pedotrophs tripled as compared with the control. While calculating the mineralization/immobilization index, it was shown that the most significant deviation from the control plot was found in the rhyolite tuff treated soil — a decrease by 6 times, and in case of willow ash by 2.3 times, which proved the inhibition of mineralization of the organic substances in the soil.

To date there have been known about 20 species of fast-growing plants that may be grown to obtain high amounts of plant biomass -e.g. eucalypt, poplar, willow, silver grass, giant reed, and others. The harvested biomass is used for the production of heat and electric energy, and may be used as primary product to produce such biofuel in the form of granules and bricks [1][2][3][4][5][6].
In the EU countries (Sweden, Denmark, United Kingdom), the use of energy cultures is especially popular. In Ukraine, plantations of energy willow are grown in Kyiv, Donetsk, Ivano-Frankivsk, Volyn, Lviv, Ternopil and Rivne oblasts (regions). Transcarpathia (Zakarpatska oblast) also has a more than ten-year-long experience of growing energy willow (Salix sp.).
This culture is used not only as an energy source but with the aim of bio-or phytoremediation. Willow may efficiently grow on the soils with heightened level of lead and cadmium [7], for instance along the roads or near industrial facilities. Its wood may be used as biofuel [3]. At the same time, willow is a very fast-growing plant, which fact allows it to be used for phytoremediation. What is more, various willow species are considered as promising plants for Ukraine's urban landscaping [8]. Earlier we have analyzed changes in the composition of the soil microbial coenosis occurring when growing The aim of the work was to study the soil agrochemical indices, soil microbiocoenosis, in case of growing of energy cultures and based on the mineralization coefficient, to make a conclusion on the speed of mineralization processes in the soils under study. In conditions of continuous field experiment (2011-2016), the dynamics of soil microbial associations was studied for willow (Salix triandra  Salix viminalis 'Inger') cultivation with application of experimental fertilizers of different types. In the research fertilizers there were used: sulfuric urea, municipal biocompost, municipal sewage sludge compost, rhyolite tuff and willow ash. The soil microbiotic communities analysis was conducted by the method of serial dilutions of soil suspension with the use of differentially diagnostic nutrient media: meat-peptone agar, starch-ammonium agar, Ashby medium, potato agar, Czapek Dox medium, starvation agar, Ploskirev medium. The direction of the microbiological processes in the soils was determined.
According to the results, it was established that the most promising for the purpose of improving the metabilic activity of the soil (in the growth of energy willow) is a municipal sewage sludge compost and a municipal biocompost. In case of the use of municipal sewage sludge compost, the number of intestinal bacteria, ammonifiers, micromycetes and actinomycetes was doubled as compared with the control. In case of the use of municipal biocompost, the levels of microscopic fungi and cellulolytic bacteria doubled, and those of intestinal bacteria and pedotrophs tripled as compared with the control. While calculating the mineralization/immobilization index, it was shown that the most significant deviation from the control plot was found in the rhyolite tuff treated soil -a decrease by 6 times, and in case of willow ash by 2.3 times, which proved the inhibition of mineralization of the organic substances in the soil. energy willow, compared to the soils of the meadow plot [9,10]. There have been many reports claiming that energy willow rapidly exhausts the stocks of soil nutrients [11], which is why the aim of our research has been to study the soil agrochemical indices and soil microbiocoenosis, in case of growing of energy cultures; to compare the obtained data with the control (a meadow ecosystem); and based on the mineralization coefficient, to make a conclusion on the speed of mineralization processes in the soils under study. • Control (without any fertilization since 2011).
Willow ash (WA) -300 kg/ha dry weight with 1% moisture content (produced at the University of Nyíregyháza with burning of willow shoots without leaves). 600 kg/ha WA was applied to the soil during 2011 and 2013, respectively.
Above fertilizers and additives were immediately rotated to upper 0-25 cm layer of the soil. The basic physical and chemical characteristics (plant nutrient content) of municipal biocompost, municipal sewage sludge compost, rhyolite tuff and willow ash were described in our previous studies [12][13][14].
The soil sampling for study soil microbiota was done by August 8, 2016. The upper 5 cm layer of the soil was removed, and then sampling was performed with the help of a drill rod from 10-15 cm soil depth. Mixed average samples were taken combining 5 soil subsamples per plot, which were taken from the neighbourhood of willow plants, located in centre of the given experimental plot. From every treatment 2 independent average samples were taken from 2 parallel plots, receiving the same treatments. All soil samples were put in snap-lockable plastic bags, and were transported immediately to laboratory in Uzhgorod in a cooler box.
The soil microbial coenosis analysis was conducted with the use of differentially diagnostic nutrient media by the method of serial dilutions of soil suspension. The ammonifying bacteria were propagated on meat-peptone agar (MPA); the actinomycetes and mycobacteria -on starch-ammonium agar (SAA); the oligotrophs -on Ashby medium; the myxobacteria -on potato agar; the micromycetes -on Czapek Dox medium; the оligonitrophils -on starvation agar; the Azotobacter -on Ashby medium using the method of soil lumps fouling; the enteric bacteria -on Ploskirev medium [15]. The results were evaluated by the number of colony-forming units per 1 g of absolutely dry soil (CFU/g) [15].
The soil was selected from each plot of the experiment in two replicates (parallel treatments). All microbiological tests were conducted with 3 replications.
The direction of the microbiological processes in the soils was determined after [16]. The mineralization/immobilization index (MII) was calculated as the ratio of the number of microorganisms grown on starchand-ammonia agar and meat-infusion agar, correspondingly. The pedotrophic index (PI) was calculated as the ration of microorganisms grown on meat-infusion agar and so il agar.
Statistical analysis of experimental data was conducted with MS Excel 10.0 software using analysis of a variance (ANOVA) followed by treatment comparison using Tukey's test. The Tukey Test is a post-hoc test based on the studentized range distribution. The test compares all possible pairs of means. The Tukey test is invoked when you need to determine if the interaction among three or more variables is mutually statistically significant. Honestly significant difference is shown when the pairwise difference between two means exceeds the value. Hereinafter the letters «a» etc. indicate statistically significant differences.

Results and Discussion
In Table 1  Actinomycetes. The number of bacteria using mineral forms of nitrogen was growing if MSSC, or MBC were used. A considerable decrease in the number of bacteria using mineral forms of nitrogen was found when RT was used. The number of these bacteria in plots to be growing with the use SU and WA did not differ from the control plot.
Pedotrophs. The number of pedotrophs was observed to be growing in the soil of plot to be growing if SU top-dressing, and if MBC and RT were used.
Myxobacteria. No significant myxobacteria changes were found in the soils of the experimental plots, however it is worth noting their lowering only in the soil of to be growing if WA was applied.
Oligonitrophils. The number of oligonitrophils was observed to be decreasing with the use of MSSC, WA, and RT.
Celulosolytic organisms. The number of cellulosolytic microorganisms was found to decline in case of the use of MSSC, and RT.
Scientific literature is aware of such regularities. The stimulating activity of municipal sewage upon the content of most bacterial and fungal groups was also proved experimentally by the example of grey-brown podzolic soils, during both the first and the second year of the experiment [17]. When secondary-treated municipal sewage was added to the soil with willows, the microbial biomass, microbial breathing and fermentative activity were observed to grow [18].
In case of the use of MBC, rise in the levels of intestinal bacteria, microscopic fungi, actinomycetes, pedotrophs and cellulosolytic bacteria was found (Table).
The calculated coefficients and indices reflected the elements of the morphofunctional structure of microbial coenoses, and revealed the direction of the soil processes. The mineralization/immobilization index (MII) shows the intensity of the processes of mineralization and assimilation of nitrogen compounds, and characterizes the level of intensity of mobilization processes in soil. The pedotrophic index characterizes the level of assimilation of soil organic substances by microorganisms and thus the functionality of the structure of the soil microbial coenosis. Low pedotrophic indices were evidence of the inhibition of mineralization of the organic substances. Increase of the pedotrophic index testified to the growing transformation intensity of the organic substances [19].
In case of well-balanced ratio between the processes of synthesis and decomposition of organic substances, the values of mineralization indices approximated one. These were the trends observed in case of the control plot. In that case, the mineralization/ immobilization index equaled to 1.12 (Fig. 1).
Similar values were observed in the treatments MBC and MSSC (1.08 and 1.  Based on the detailed analysis of the mineralization indices, we observed that the variant with addition of MBC was the closest to the control. The obtained results proved concordant with other authors' data. Introduction of compost into soil with energy willow plantations was observed to change the direction of environmental links, and increase the integrity and resistance of the "soilmicroorganisms -plant" system [20].
T hus, the most indicative changes in the soil microbiota against the control plot were found in case of the use of municipal sewage sludge compost: rise in the number of intestinal bacteria, ammonifiers, micromycetes and actinomycetes, and fall in the number of oligonitrophils and cellulosolytic microorganisms.
While calculating the mineralization/ immobilization index, it was shown that the most significant deviation from the control plot was found in variant of rhyolite tuff -a decrease by 6 times, and in case of willow ash in 2.3 times, which proves the inhibition of mineralization of the organic substances in the soil.
In all treatments of the experiment, the number of free-living nitrogen-fixing microorganisms (Azotobacter) was found to be equal to 100%.
Calculation of the pedotrophic index showed growth of humus formation speed in the soils of sulfuric urea top-dressing and municipal biocompost, while in plot of municipal sewage sludge compost the pedotrophic index was found to be decreasing significantly.