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Section 10. Agriculture
Zhurlov Oleg Sergeevich, Institute for Cellular and Intracellular Symbiosis, Ural Branch Russian Academy of Sciences,
Orenburg, Russia E-mail: [email protected] Grudinin Dmitriy Aleksandrovich, Institute of Steppe, Ural Branch Russian Academy of Sciences,
Orenburg, Russia E-mail: [email protected] Mushinskiy Alexandr Alekseevich, Institute for Cellular and Intracellular Symbiosis, Ural Branch Russian Academy of Sciences,
Orenburg, Russia E-mail: [email protected]
Yakovlev Ilya Gennadievich, Institute of Steppe, Ural Branch Russian Academy of Sciences,
Orenburg, Russia E-mail: [email protected]
METHODOLOGICAL APPROACHES TO MONITORING MICROBIAL COMMUNITIES OF AGRICULTURAL LANDSCAPE OF THE STEPPE ZONE
Abstract: The article analyzes the methodological features of sampling soil for agrochemical, microbiological and metagenomic analysis. The possibility of integrating soil sampling methods on the basis method for sampling soil from equidistant points with GPS positioning is discussed, as a single methodical technique, most accurately reflecting the features of the distribution of physical and chemical properties and microbial communities of soil. Standardization of soil sampling methods for physicochemical, microbiological and metagenomic research is necessary for the construction of a GIS "Microbial communities of agricultural landscape".
Keywords: agroecology, bacterial communities, agricultural landscape, geographic information system (GIS).
Introduction
Methodological development of issues of increasing soil fertility, the effectiveness of "self-restoration" and re-cultivation of agricultural landscape, are an actual task of agroecology.
Microorganisms of the soil are the main destructors of organic matter and promote the transfer of nutrients in form that is accessible to plants. They have a dominant role in the processes of mineralization and humification of plant residues in the soil.
One of the main tasks contributing to the increase of soil fertility is the conservation and effective regulation of the qualitative composition of microbial communities of agricultural landscape. Microbial succession, under the influence of external and internal environmental factors, is the main reason for the change in the composition of microbial communities of agricultural landscape and natural landscapes.
Therefore, the development of a system of geoin-formation mapping of microbial communities will help control their changes under the influence of biotic and abiotic factors.
Development of methodological foundations ofphy-logenetic mapping of microbial communities of soils will contribute to the effectiveness of recultivation of agricultural lands and restoration of natural landscapes.
Monitoring the physicochemical parameters of soil
The lack of unified requirements for soil sampling for agrochemical, microbiological and metagenomic analysis does not allow analyzing the totality of soil «health» indicators. Today, for the sampling of soil, accredited organizations use several techniques [1, 2, 3, 4]. Often, the territory under investigation is not homogeneous. Features of microrelief and soil mosaic affect the distribution ofvegetation and microbial communities of soil [5].
The methods used aver average the physicochemical parameters of soil samples taken in different parts of the field, smoothing out this heterogeneity. This approach is dictated by the "old" labor-consuming methods of research and analysis of a typical "dominant" in the study of the physical and chemical properties of the soil, which are extrapolated over the entire area of the investigated soil region. Modern methods of soil analysis are more economical, both from the point of view of the use of time, the amount of material taken, and the reagents used.
Paradoxically, until now there is no method that would meet the requirements of sampling automation for a number of physical and chemical properties of the soil and showed physicochemical parameters in a certain sector of the field.
The standardization of soil sampling techniques and their integration into a single methodical technique will allow in the future carrying out complex monitoring studies of the chemical composition and microbial
communities of soils. The most popular method of sampling in soil science and agrochemistry is the «konvert» method [6].
However, it does not take into account the sterility of the soil sample, it is impossible to determine the thickness of the layer of the soil horizon, an average sample (total sample) is selected. According to the results of agrochemical and microbiological analysis, it is impossible to compare the actual distribution of microbial communities depending on the agrochemical indicators of the soil.
Moreover, the features of soil sampling of the agricultural landscape and natural landscapes associated with the formation of monitoring sites are not suitable for soils in urban areas. The presence of communications, anthropogenically-modified horizons makes it impossible to form stationary sites in urban areas.
Therefore, it is necessary to develop a single method for sampling soil for physicochemical, microbiological and metagenomic analysis.
Monitoring of microbial communities
An important factor for constructing a geoinforma-tion system for phylogenetic mapping of microbial communities will be the effectiveness of soil sampling.
Today, in agricultural microbiology, the basic approaches to the determination of microbial communities of soils have changed. Whereas earlier "cup" methods were used to estimate the number of cultivated groups of microorganisms by seeding on selective nutrient media [4], today, with the development of modern methods of molecular genetics sequencing, approaches to soil sampling have changed [7].
This led to revolutionary changes in the study of the metagenome of microbiological communities of soils and contributed to the development of a new trend in molecular genetic studies-comparative metagenom-ics. Modeling the links between microorganisms using bioinformatic analysis methods often involves a hypothetical nature of the relationship, which does not allow evaluating the relationship between microorganisms in the microbial community.
The method of sampling for metagenomic research does not take into account the features of the landscape relief, the sampling points of the soil are empirically selected, and the soil samples are mixed with the formation of a common sample. Such studies do not take into
account seasonal changes in the qualitative composition of microbial communities associated with changes in temperature and soil moisture.
So, as a result of studies of the soil microbiome by molecular genetic methods it was found that the proportion of cultivated bacteria is less than 1% of the composition of microbial communities of soils. Uneven distribution of pH, soil moisture, vegetation cover lead to the creation of "biogeographic patterns" [8], the formation of the biotic nuclei of the ecosystem, which are sources of distribution of microbial communities. Interpretation of metagenomic data is problematic, because often the complex composition of microbial communities varies in time and space [9].
Today, as a result of high-throughput sequencing of 16S-rRNA gene libraries, the main taxa of microorganisms forming microbial soil communities are known - a-Proteobacteria, Actinobacteria, Acidobacte-ria, Verrucomicrobia, Bacteroidetes, Firmicutes, Gem-matimonadetes, Chloroflexi [10]. It was also shown that the composition of microbial communities strongly depends on two physicochemical parameters of the soil (soil moisture and pH). In arid regions, actinobacteria predominate and in water-saturated proteobacteria.
Undoubtedly, they form the main part of microbial communities of soils, but the most variable part is represented by poorly represented taxa whose qualitative composition is influenced by both internal and external environmental factors. Analysis of the composition of microbial communities of different types of soils, based on the results of microbiological and metagenomic analyzes, makes it possible to identify ecological-trophic groups of microorganisms and to carry out their further monitoring.
The method of phylogenetic mapping of microbial communities proposed by us is that soil sampling is performed at grid sites with a 25-meter cell centered at the GPS positioning point. Sampling soil is made to a depth of 20 cm sterile aluminum cylinders. Point samples are not combined and characterize the distribution of mi-crobial communities throughout the topsoil (organic and organo-mineral horizons). The systematization of metagenomic analysis analysis data with GPS positioning will allow not only to characterize microbial communities located at equidistant points, but also to determine the boundaries of their distribution. The creation of
GIS "microbial soil communities" will allow to identify phytopathogenic microorganisms and, when developing appropriate methods, to monitoring the content of harmful substances in the soil. No less important is the development of the direction associated with microorganisms-biosensors of physical and chemical properties of soils. Shifting the qualitative composition of microbial communities can be used as an indicator of the ecological state of agricultural landscape.
Hence, each point sample of the soil characterizes the state of physicochemical parameters in a certain sector of the field, and the number of sectors with similar characteristics reflects the diversity of the distribution of not only the physicochemical parameters, but also the differences in the composition of microbial communities in each sector of the field. The formation of a general sample in the selection of samples with "standard" methods of metagenomic analysis contributes to an increase in the proportion of "dominant" and a decrease in the share of "minor" taxa, which may lead to a shift in the distribution of microbial communities in the field region and does not allow analysis of the distribution of microbial communities along the gradient of physico-chemical soil indicators.
Conclusions
First, the creation of methodological bases for phy-logenetic mapping of microbial communities of soils will allow monitoring of the composition of microbial communities, the spread of phytopathogenic microorganisms and their regulation.
Secondly, based on the results of monitoring the phylogenetic composition of microbial communities of agricultural landscape, it will be possible to develop scientifically grounded recommendations on the rational use of organic and mineral fertilizers.
Thirdly, the standardization of the soil sampling methodology for agrochemical, microbiological and metagenomic analysis will allow us to compare the results of analyzes obtained by other researchers and to assess the effect of a complex of factors on the fertility of the soils of the agricultural landscape.
The reported research was funded by Russian Foundation for Basic Research and the
government of the region of the Russian Federation, grant № 17-44-560976.
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