Bioindication of desalination in grassed soil by microbiome diversity

. Vast areas of Western Siberia (about 40%) are occupied by saline soils. Solonetzic agricultural lands are mainly used for growing perennial grasses - phytomeliorants. To increase the yield of fodder crops on solonetz lands, specialists of the Siberian Research Institute of Fodder Crops in the 80s of the last century developed phytomeliorative crop rotations. The article considers the effect of grassing phytomeliorative crop rotations with a mixture of brome (Bromus inermis Leyss.) and alfalfa (Medicago varia Mart.) on the microflora of meadow solonetzes (hydromorphic) (Gleyic Solonetz Albic) (for 13 years). It is shown that post-rotational grassing led to a significant desalinization of the upper soil horizon and a decrease in its alkalinity, as well as to an improvement in the water-air regime of solonetzes. Long-term cultivation of fodder crop rotations and subsequent grassing formed a specific soil microbiome, characterized by taxonomic diversity of microorganisms and a greater proportion of copiotrophs in the dominant phyla, which indirectly indicates an increase in the carbon content and nitrogen available to plants in the phytomeliorated solonetz.


Introduction
The conditions of the soil environment play a priority ecological role in the life and diversity of soil microorganisms, which is clearly manifested in intrazonal soils with clearly defined limiting factors.In saline soils, they are the alkaline reaction of the environment, the accumulation of water-soluble salts (sodium in solonetzes is more than 15% of the total exchangeable cations), high density, and low water permeability [1].All this forms a specific microbial cenosis and severely limits plant productivity.The agricultural use of such soils is associated with their transformation with the help of gypsum or long-term reclamation with grasses.These techniques desalinate solonetz, transform its biological properties [2][3] and ultimately increase the mass of herbs by several times.Long-term use of phytomeliorants is more economically preferable than a single application of gypsum.On the solonetzes of the Baraba lowland of Siberia, whose area is 3686 thousand hectares, it is recommended to cultivate salt-tolerant ameliorant grasses in fodder crop rotations.This allows you to combine the positive effect of the impact on the soil of the crop itself with the impact of mechanical processing (agrobiological method of reclamation).At relatively low salt concentrations on saline soil, grassing with legumes and cereal grasses is recommended [4].This approach is possible not only on intrazonal soils, but also on secondarily saline arable lands, which today number 950 million hectares [5].
The influence of the nature of vegetation on the community of microorganisms in the root zone (rhizosphere) has been studied in a large number of studies.The microbiome (cultivated and non-cultivated forms of microorganisms) has been most fully characterized with the advent of high throughput sequencing and metagenomics [6][7][8].It has been shown that changes in the composition of the rhizosphere microbiome are mainly associated with the type of vegetation, and the soil factor is the next most influential factor [9][10].The microbiome has been studied under monocots and dicots [11].Under the latter, an increased representation in the microbiocenosis was noted in the phylums Actinobacteria (family Streptomicetaceae) and Proteobacteria (Pseudomonadaceae) and under monocots, in Bacteroidetes and Rhizobiales [12].
In the Baraba lowland of Western Siberia, the influence of crop rotations with perennial awnless brome (monocot) and biennial sweet clover (dicot) on solonetzes has been studied in dynamics for more than 30 years.The direction of transformation of the microflora of solonetzes in the first 20 years of cultivation of fodder crop rotations is shown in the work of T. G. Lomova, L. N. Korobova [13].Further changes in the microbial cenosis under the influence of crop rotations are reflected in the works of L. N. Korobova et al. [14] and V. S. Riksen [15].But the question of what happens to the soil microflora of the solonetz transformed by crop rotations after abandoning its mechanical loosening, where the part of the crop rotation area was grassed that is only ameliorant grasses left the impact on the soil, remained completely unexplored.
The purpose of this work is to show the changes in the microflora of the shallow solonetz and the salt content that have occurred over 13 years of grassing the soil with a grass mixture of brome and alfalfa after different fodder crop rotations.

Materials and methods
The studies were carried out in the Chanovsky district of the Novosibirsk region at the station of SibNII Kormov (Siberian Scientific Research Institute of Fodder Crops) of the Siberian Federal Scientific Centre of Agrobiotechnologies of the Russian Academy of Sciences (55.389º N, 78.927º E).
Research objects: bacterial complex of solonetzes, shallow solonetz of the virgin soil (meadow hydromorphic solonetz; WRB, 2006, Gleyic Solonetz Albic.)[16], high-columnar medium-sodium soda-sulfate type of salinity, heavy granulometric composition, shallow solonetz, grassed with brome and alfalfa for 13 years; the grass mixture was sown after 20 years of cultivation of crop rotations.The chemical characteristics of the solonetz modified by grass sowing are given in the work of N. V. Elizarov et al. (2019) [2].
The microflora was studied in the following variations: • Virgin soil.
• Grassing after crop rotation with sweet clover.
Crop rotations in the solonetz station were represented by 6 fields.Crop rotation with sweet clover: 1st field -sweet clover of the 1st year of life with a cover crop of Sudan grass; 2nd field -sweet clover of the 2nd year of life; 3rd field -oats for grain haylage; 4-6th fields -repetition of 1-3rd.Crop rotation with awnless brome: 1st field -millet; 2nd field -awnless brome with millet cover crop; 3rd-6th fields -awnless brome.In crop rotations and on grassing, yellow sweet clover of the Alsheevsky variety, awnless brome of the SibNIISKhoz (Siberian Scientific Research Institute of Agriculture) 189 variety, and blue-hybrid alfalfa of the Omskaya 8893 variety were sown.
Soil tillage in crop rotations -layer-by-layer milling to a depth of 8-10 cm or disking with heavy harrows once per rotation, and then non-moldboard tillage with SibIME (Siberian Scientific Research Institute of Mechanization and Electrification of Agriculture) racks up to 30-35 cm.There is no tillage on the grassing.Soil samples were taken in the first decade of August in 2016, 2018-2020 from a layer of 0-20 cm (0-15 in virgin soil) on odd working plots of the station with an area of 200 m2 in 10 repetitions from each working plot along a diagonal transect.For classical microbiological investigations, one mixed sample was made and four mixed samples were made for metagenomic analysis in 2020.
The total salt content in the soil was determined using a KL-S-1 conductometer by the specific electrical conductivity of soil paste (SEC) diluted with water in a ratio of 1:5.The taxonomic affiliation of bacteria was revealed in the samples of the 2020 year at the ICBFM RAS at the "Genomics" Collective Use Center (Novosibirsk) by high-throughput sequencing of the V3-V4 region of the 16S rRNA gene.Total DNA was isolated from the samples using the DNeasy PowerSoil Kit (Qiagen).Samples were sequenced on a MiSeq instrument (Illumina, USA), OTE sequences were assigned to taxa using SINTAX [17].The repetition of the analysis of soil samples is four times.
The statistical significance of differences in taxa representation and diversity indices (analyzed by Usearch v11.0.667) by variations was proved using the Mann-Whitney U-test.

Results and discussion
A metagenomic investigation of the microbiome showed that solonetz phytomelioration (including 20-year cultivation of fodder crop rotations and subsequent thirteen-year grassing with brome and alfalfa) affected bacterial biodiversity differently in different taxa (table 1).The diversity of phyla in the phytomeliorated solonetz increased: on the grassing after crop rotation with brome by 18%, and after crop rotation with sweet clover by 11.1%.In the rest of the taxa, the changes in the grassing were not unidirectional and were more associated with the type of grasses cultivated earlier in the crop rotation than with the influence of the grass mixture sown later.Thus, in the "grassing after crop rotation with sweet clover" variation, 361 bacterial genera from 197 families were revealed, while in the virgin soil there were 9.1% less of them.When grassed after a crop rotation with brome, the generic composition of bacteria turned out to be 8.5% less representative than in virgin soil, and they belonged to 181 families.Among the Bacteria domain, 14 phyla dominated in all variations: Actinobacteria, Acidobacteria, Proteobacteria, bacteria highlighted as unc_Bacteria, Verrucomicrobia, Gemmatimonadetes, Bacteroidetes, and others.Their share in the virgin and grass-transformed solonetz differed for each of the four repetitions of the studied variations.In virgin lands, microorganisms of the phylum Actinobacteria turned out to be the most common, the representation of which was 30.3% (table 2).The second were prokaryotes from the phylum Acidobacteria, accounting for up to 25.1% of the microbiome, the third were Proteobacteria (13.2%).Their share presence was 1.9 times lower than that of Acidobacteria and 2.3 times lower than that of Actinobacteria.Such a distribution with a predominance of Actinobacteria seems to be characteristic of the microbiomes of saline soils, which is confirmed by N. Fierer et al. [18] and T. P. Makhalanyane et al. [19].
In the microbiome, 5 phyla of the 6 most significant dominants of the grassed areas (i.e., 83.4%) had a percentage that was statistically different from the virgin soil (table 2).The phylum unc_Bacteria was stably the same both in the virgin soil and in each variation of grassing.
The phyla Bacteroidetes, Chloroflexi, and Candidatus Saccharibacteria, ranked 8-10, were less representative than those considered in table 2. In microbiomes, they accounted for 1.5-4.4%,but each of them also had comparable shares in virgin and phytomeliorated soils.
An analysis of the composition and abundance of smaller taxa included in phyla made it possible to reveal the nature of changes in the solonetz that occurred under sown grasses.It was found that the number of genomic sequences from the Gp 6 and Gp 4 groups of the most numerous Acidobacteria phylum increased significantly in the transformed soil.In the "grassing after crop rotation with sweet clover" variation they totaled 22.8%, and in the "grassing after crop rotation with brome" -29.5%.In the virgin soil, these Acidobacteria were much less: 1.9 and 2.4 times, respectively.Researchers of the bacterial domain often associate the abundance of Acidobacteria of the Gp 6 and Gp 4 groups with the pH of the soil solution [20][21].The proportion of these Acidobacteria is higher in soils with low acidity (pH).Therefore, we can assume that the phytomeliorative effect of grass sowing on shallow solonetz manifested itself in a decrease in soil alkalinity.It should be noted that desalinization of the upper soil horizon also occurred in the grassed solonetz.For 13 years of grassing and 20 years of cultivation of phytomeliorative crop rotations, the amount of salts, according to the indications of specific electrical conductivity (SEC), decreased in it: by 6.8 times in the "grassing after crop rotation with sweet clover" variation and by 7.1 times in the "grassing after crop rotation with brome" variation.
Long-term phytomelioration has changed the water-air regime of the investigated soils.An indicator of this fact was the abundance of the bacterial type Gemmatimonadetes.It has been noted in the literature that their representation in the microbial community is always higher under conditions of moisture deficiency [22].In the solonetz transformed by phytomeliorants, Gemmatimonadetes became less: in soil with grassing after crop rotation with sweet clover by 1.9 times, under crop rotation with brome by 2.3 times.Therefore, it can be considered that, against the background of long-term phytomelioration, there was a change in the hydrothermal properties of soils, which influenced their biotic component.
Long-term cultivation of grasses in crop rotation and subsequent grassing were accompanied by additional input of plant mass into the soil, and this increased the content of carbon and nitrogen in the soil.As a result, the abundance of the phylum of Proteobacteria increased in the transformed solonetz, especially the classes Betaproteobacteria (by 1.6-2 times compared to virgin soil), Gammaproteobacteria (by 1.9-2.1 times), and Deltaproteobacteria (by 2.9-3.2 times compared to virgin soil).These are copiotrophic microorganisms with high metabolic activity.Functionally, they are always associated with improved soil fertility [23].
An improvement in soil fertility in the transformed soil is also evidenced by a decrease in the percentage of oligotrophs among the first six dominant phyla.Their representation in the microbial community of the solonetz decreased in the grassed area to 48.9 and 51.8% against 61.2% in virgin soil.Oligotrophic Actinobacteria, Acidobacteria, and Gemmatimonadetes [24] have been displaced in the soil by representatives of Proteobacteria, which prefer a nutrient-rich habitat.
The predominance of oligotrophic flora in the microbial community of the virgin lands was also noted by us using the methods of classical microbiology in 2016, 2018-2020.The coefficient of oligotrophy of the virgin solonetz calculated on the basis of these data turned out to be, on average, 4.4-6.7 times higher than the soil transformed with grasses (table 3).At the same time, in the grassed soil, the mineralization coefficient increased by 1.4-1.6 times, which indicates an increased nitrogen content under sown grasses.All of the above indicates functional changes in the community of microorganisms under the influence of phytomelioration.

Conclusion
Long-term cultivation of fodder crop rotations and their subsequent grassing has become the main ecological factor in the transformation of the microbial community of virgin shallow solonetz and its properties.The agrobiological impact and grass sowing of brome with alfalfa led to the desalinization of the upper horizon of the saline soil and the formation of a specific soil metagenome.It differs by an increase in the share of copiotrophs in the dominant phyla of the bacterial domain and a decrease in the representation of oligotrophs, which indirectly indicates an increase in the content of carbon and nitrogen in the phytomeliorated solonetz.Based on the data on the taxonomic structure of the microbiome, positive changes in the water-air regime of the solonetz transformed by grasses and a decrease in its alkalinity were revealed.The revealed microbiological features of the grassed shallow solonetz contribute to new knowledge about how long post-crop changes persist under grasses in the absence of mechanical tillage.

Table 1 .
Taxonomic representation in microbiomes of virgin and phytomeliorated shallow solonetz.

Table 2 .
Percentage representation of dominant bacterial phyla in virgin and grass-transformed shallow solonetz a .
a Note.Different letters denote the mean values of the variations, which differ significantly from each other at p < 0.05.