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Section 2. Biology
Zhurlov Oleg Sergeevich, Institute for Cellular and Intracellular Symbiosis, Ural Branch Russian Academy of Sciences, Orenburg, Russia E-mail: jurlov1968@mail.ru Grudinin Dmitriy Aleksandrovich, Institute of Steppe, Ural Branch Russian Academy of Sciences, Orenburg, Russia E-mail: grudininda@yandex.ru Mushinskiy Alexandr Alekseevich, Institute for Cellular and Intracellular Symbiosis, Ural Branch Russian Academy of Sciences, Orenburg, Russia E-mail: SAN2127@yandex.ru Yakovlev Ilya Gennadievich, Institute of Steppe, Ural Branch Russian Academy of Sciences, Orenburg, Russia E-mail: russo-turisto01@yandex.ru
COMPARATIVE ANALYSIS OF PROKARYOTIC COMMUNITIES ASSOCIATED WITH CONVENTIONAL CROPLANDS AND FALLOW LANDS
Abstract: A comparative analysis of the phylogenetic composition of microbial communities of croplands and long-term fallow lands showed their similarity in terms of the dominant groups ofbacteria and differences minor taxa. The composition of the microbial communities of conventional croplands was characterized by a high proportion of Planctomycetes and the absence of representatives of Chloroflexi and Caldithrix, which dominated of prokaryotic communities of long-term fallow lands.
In croplands, the dominant position in the microbial communities was occupied by representatives of actinomy-cetes - Micromonosporaceae, Nocardioidaceae, Conexibacteraceae and Solirubrobacteraceae. Soil microbial communities of long-term fallow lands were dominated by bacteria-cellulozolitics (Chitinophagaceae) and bacteria dominating in the soils of urbanized areas with features of the granulometric composition of the soil (Rubrobacteraceae, Caldi-thrixaceae, Chthoniobacteraceae).
Keywords: soil fertility, microbial communities, fallow lands.
Introduction erts, wastelands, wetlands and ravines. According to the cal-
One of the global problems of world agriculture is the culations of UN experts, nearly 2 billion hectares of land are limited availability of land. Humanity in its history irrevoca- subject to anthropogenic degradation. Soil salinity is result bly lost more fertile land than their swings around the world, of irrigation, erosion (wind, water) and desertification soil as
m
aking the productivity of conventional croplands into des- result of climate change.
The contemporary soil cover of the Orenburg region, according to the soil-geographical regionalization of the country's territory [1, 2], is mainly represented by southern Chernozem (21.1%), conventional Chernozem (15.5%) and dark chestnut soils (10.4%). Under crops of crops conventional Chernozem and southern Chernozem, the most rich in humus soils, are used.
However, the low thickness of the humus horizon and the heavy granulometric composition of soils contribute to the deterioration in water penetration and retention, soil compaction, aeration reduction and slowing of microbiological processes (ammonification and nitrification).
The change in the granulometric composition of soil particles is associated with a change in prokaryotic communities of the soil. So, microorganisms of the soil (more than 250 ^m) contain bacteria belonging to the filaments Actinobateria, Bac-teroidetes, Verucomicrobia and Proteobacteria, and in microaggregates (50-250 ^m), representatives of Rubrobacteriales and Chloroflexi predominate [3, 4].
In addition, the humus (horizon A) is the main habitat of the soil microbial community. Reducing humus layer thickness influences the microbial community composition and promotes the conversion of agricultural land in low productivity with plowed field dominance oligotrophic microorganisms. A long period of self-restoration of the surface, humus horizon of degraded soils, does not facilitate the return of such lands to a crop rotation.
Finally, the fertility of the soil depends on the state of the humus horizon, which determines the biological activity of the soil. Each hectare of infertile soil accounts for 2.5-3 tons of microbial biomass, and for highly-fertile soil - up to 16 tons. The number of microorganisms per gram of soil can vary from 1-3 x 106 to 20-25 x 109 [5].
Today, the use of soil-saving technologies in agriculture (No-Till), helps to reduce soil moisture losses and reduce degradation of croplands with wind and water erosion, but can contribute to its salinization and compaction (reduces its oxygen saturation) and the accumulation of phytopathogenic microorganisms.
The aim of the work was a comparative analysis of the qualitative composition of the bacterial communities of croplands and long-term fallow lands.
Materials and Methods Site description
Soil samples were collected on the territory of Perevo-lotsky district of the Orenburg region, on a plot of croplands and long-term fallow lands. The climate of the region is moderately continental, the snow cover is thin, in summer frequent dry winds. The average annual amount of precipitation is 250-300 mm. The soil profile corresponds to the chernozem to the southern low-humified low-moisture light loam.
Isolation of DNA
The samples for the isolation of total DNA were samples of frozen soil (0.5 g each). DNA from the samples was isolated using NucleoSpin®Soil (Macherey-Nagel, GmbH & Co. KG (Dep., IT-EDV) in accordance with the manufacturer's instructions. The purity of the isolated DNA was monitored by photometry on a NanoDrop 8000 instrument (Termo Fisher Scientific Inc., USA) and also by electrophoresis on a 1.5% agarose gel. The DNA concentration in the samples was determined on a fluorometer "Quantus" ("Promega" USA) using a set of Quanti Fluor dsDNA of the same manufacturer. DNA libraries for sequencing were created using the Illumina protocol (http://support.illumina.com/documents/documentation/ chemistry documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf), using primers to V3 and V4 regions 16S rRNA for prokaryotes (direct SD-Bact-0341-bS-17 and reverse SD-Bact-0785-aA-21). Metagenomic sequencing was performed on a MiSeq sequencer (Illumina, USA) with a set of reagents according to the manufacturer's recommendations at the collective use center for scientific equipment "Persistence of microorganisms" of the Institute of Cellular and Intracellular Symbiosis of the Ural Branch of the Russian Academy of Sciences.
Metagenome analysis
Taxonomic identification of nucleotide sequences and a comparative analysis of microbial communities were carried out using the Internet resource VAMPS (Visualization and Analysis of Microbial Population Structures) available on http://vamps.mbl.edu/. The sequence was sorted using the RDP (Ribosomal Database Project), available at http:// rdp.cme.msu.edu/.
Statistical analysis of results
The obtained data were subjected to statistical processing using variational statistics methods using MS Excel 10.0 for Windows spreadsheet processor.
Results
Sampling in arable field sown with winter wheat (Triticum aestivum L.). The humus content in the soil is 4.5 ± 0.7%, the thickness of the humus horizon is 32.0 ± 3.1 cm, pH = 8.0 ± 0.1. The content of ammonium nitrogen was (12.7 ± 1.3 mg/kg) and nitrate nitrogen (12.9 ± 2.6 mg/kg).
The 16S metagenomic analyses of the soil shows that the prokaryotic composition were classified into bacteria with 76.773 reads (99.8%), archaea with 18 reads (0.02%) and 161 reads (0.2%) were unclassified at the kingdom level, suspected to belong to the third kingdom (domain), the eucaryota.
Prokaryotic organisms belonging to 27 Phyla were detected using this 16S metagenomic tool. The top 8 of 27 phyla (with the number of reads) belonged to the Phyla Firmicutes (11.1%), Proteobacteria (27.5%), Actinobacteria (39.1%), Ver-
rucomicrobia (1.7%), Planctomycetes (1.6%) and Bacteroidetes (4.02%). At this level, 8.8% were unclassified.
A total of 56 classes of prokaryotic organisms were detected from the soil. The top 8 of 56 classes were identified to be Deltaproteobacteria (4.8%), Gammaproteobacteria (5.7%), Alphaproteobacteria (12.8%), Clostridia (7.1%), Actinobacteria (26.8%), Thermoleophilia (8.1%) and Sphingobacteriia (3.6%).
The 16S metagenomic result of the soli identified and thus classified all the procaryotic organisms present into a total of 223 families. The identities of the top 8 of the 223 families as Pseudonocardiaceae (3.9%), Conexibacteraceae (4.7%), Mi-cromonosporaceae (4.1%), Nocardioidaceae (4.1%), Sphingo-monadaceae (3.5%), Solirubrobacteraceae (3.1%) and Thermo-anaerobacteraceae (2.2%). A total of 21.2% were unclassified.
Using the 16S metagenomic tools, a total of907 prokaryotic species were identified to be present in the soil. The seven most predominant species were identified to be Solirubrobac-ter soli (2.0%), Caldithrixpalaeochoryensis (1.5%), Nocardioi-des islandensis (1.4%), Chondromycespediculatus (1.2%), Euze-bya tangerina (1.2%) Sphingomonas oligophenolica (1.1%) and Cohnella soli (1.0%). Of this 907 species, 60.2% of the species were considered unclassified.
In summary, the prokaryotic composition of the soil was so diverse that 907 different species, 505 genera, 223 families, 105 orders, 56 classes, 27 phyla and 2 domains (excluding those unclassified) were identified using the 16S metage-nomic tools as compared to the results obtained using the culture-dependent techniques. It is important to note that the pie chart shows only all classifications above 3.5% abundance while the "other" category in the pie chart is the sum of all classifications with less than 3.5% abundance.
The section of the long-term deposit was characterized by the presence of a central ridge and signs of an average and weak degree of water erosion, which together with a low thickness of the humus horizon (10 cm) and a change in the agro-chemical properties of the soil, slightly alkaline pH (7.5 ± 0.1), humus content (1.4 ± 0.3%), ammonium (12.3 ± 1.2 mg/kg) and nitrate nitrogen (1.15 ± 0.18 mg / kg), indicated a decrease in soil fertility. From the surface horizon A (15 cm) soil samples were taken for metagenomic analysis of total bacterial DNA.
The 16S metagenomic analyses of the soil shows that the prokaryotic composition were classified into bacteria with 98.883 reads (99.4%), archaea with 145 reads (0.2%) and 483 reads (0.5%) were unclassified at the kingdom level, suspected to belong to the third kingdom (domain), the eucaryota.
Prokaryotic organisms belonging to 27 Phyla were detected using this 16S metagenomic tool. The top 8 of 27 phyla (with the number of reads) belonged to the Phyla Firmicutes (14.6%), Proteobacteria (26.8%), Actinobacteria
(22.5%), Verrucomicrobia (4.0%), Chloroflexi (1.2%), Caldithrix (7.3%) and Bacteroidetes (6.6%). At this level, 11.6% were unclassified.
A total of 57 classes of prokaryotic organisms were detected from the soil. The top 8 of 57 classes were identified to be Deltaproteobacteria (5.2%), Gammaproteobacteria (6.0%), Alphaproteobacteria (10.2%), Clostridia (9.8%), Actinobacteria (14.3%), Caldithrixae (7.3%) and Sphingobacteriia (6.1%).
The 16S metagenomic result of the soli identified and thus classified all the procaryotic microorganisms present into a total of 227 families. The identities of the top 8 of the 227 families as Pseudonocardiaceae (2.0%), Caldithrixaceae (7.3%), Rubrobacteraceae (4.8%), Chitinophagaceae (3.9%), Sphingomonadaceae (2.3%), Chthoniobacteraceae (2.0%) and Thermoanaerobacteraceae (3.6%). A total of 22.6% were unclassified.
Using the 16S metagenomic tools, a total of 935 pro-karyotic species were identified to be present in the soil. The seven most predominant species were identified to be Tepi-danaerobacter syntrophicus (2.7%), Caldithrix palaeochoryensis (6.5%), Megasphaera hominis (1.1%), Chondromyces pediculatus (1.0%), Euzebya tangerina (0.9%), Chthoniobacterflavus (1.9%) and Segetibacter aerophilus (0.9%). Of this 935 species, 57.22% of the species were considered unclassified.
In summary, the prokaryotic composition of the soil was so diverse that 935 different species, 523 genera, 227 families, 110 orders, 57 classes, 27 phyla were identified using the 16S metagenomic tools as compared to the results obtained using the culture-dependent techniques. It is important to note that the pie chart shows only all classifications above 3.5% abundance while the "other" category in the pie chart is the sum of all classifications with less than 3.5% abundance.
Discussion
Thus, as a result of our comparative studies of the composition of the microbial communities of the croplands and section of long-term fallow lands (self-regenerating low-productive croplands), the differences in the composition of the bacterial communities of long-term fallow lands were associated with the appearance of representatives of Chloroflexi, Caldithrix and absence of Planctomycetes in the dominant group.
In croplands, the dominant position in the microbial communities was occupied by representatives of actinomycetes -Micromonosporaceae, Nocardioidaceae, Conexibacteraceae and Solirubrobacteraceae. Microbial communities of long-term fallow lands were dominated by bacteria-cellulozolitics (Chitinophagaceae) and bacteria dominating in the soils of urbanized areas with features of granulometric composition of the soil (Rubrobacteraceae, Caldithrixaceae, Chthoniobacteraceae).
The succession of microbial communities, under the influence of stress factors of different nature, is associated with a
change in the proportion ofbacteria that are underrepresented in the microbial community [6, 7].
Acknowledgements
The reported research was funded by Russian Foundation for Basic Research and the government of the region of the Russian Federation, grant № 18-44-560005.
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