Identification of Microbial Populations Present in Agricultural and Nonarable Soils in the Talas Valley, Northern Kyrgyzstan, in Autumn

Soil bacterial and fungal communities were investigated in relation to soil type and farm management practices after vegetation harvesting in autumn. Soils from felds cultivated with Phaseolus vulgaris (bean) and Pyrus comminus (pear) and nonarable, natural areas were studied. Microbial diversity was analysed using cultivation-dependent methods (isolation of pure cultures) and cultivation-independent methods (direct extraction of DNA from soil, followed by PCR amplifcation of the 16S rRNA and 18S rRNA genes). Te use of cultivation-dependent methods revealed that there were no diferences in the biodiversity of the soil bacterial and fungal communities between felds cultivated with bean plants and pear trees. However, the use of cultivation-independent methods showed that there were clear soil and crop type-specifc efects on the composition of the soil bacterial and fungal communities. Te density of the bacterial population was two times higher in northern mountain-valley serozem (NMVS) soil samples than in light chestnut (LC) soil samples. In contrast, the densities of the fungal communities were almost equal in the studied soil types. Te density of the actinomycetes community was almost two times higher in LC soil than in NMVS soil under bean plants. Te Shannon index values showed that the bacterial biodiversity in the NMVS soil samples was greater than that in the LC soil samples. Soils under fallow appeared to have diverse bacterial communities that mainly consisted of local autochthonous microfora and a small amount of zymogenic microfora (since fresh plant residue does not enter the soil). Te Shannon index results revealed two interesting facts: (1) the soil bacterial community was highly diverse in soils that supported bean plants and (2) the soil fungal biodiversity was high under pear trees in both soil types.


Introduction
Intensive farming with an annual application of fertilizers, the use of plant-protection chemicals, and various types of tillage and erosion all cause losses of the total soil carbon reserve, leading to an increase in the release of gases into the atmosphere, namely, N 2 O and CO 2 [1][2][3][4][5][6][7].
Interestingly, almost 95% of the total soil biomass consists of microorganisms, and they play essential roles in soil formation, converting nutrients in nature from plant litter, maintaining the entire terrestrial ecosystem, and protecting the environment [8].Hence, microorganisms are an indispensable part of living soil and essential for soil health.Te soil microbial communities are diverse, and their diversity and abundance are primarily infuenced by altitude and ecological and geological factors, such as the vegetation type, temperature, and soil pH [9][10][11].Climate is another factor that directly afects soil microorganisms, and global warming has been identifed as a potential environmental problem in Central Asia, particularly in Kyrgyzstan [12].
While some studies have revealed that soil type afects the microbial community structure far more than plant species, it is known that the exudates of various plants difer signifcantly in chemical composition [13,14].Others have also observed that both soil and plants could afect the community structure of soil microbes [15][16][17][18][19].
Te level of microbial biodiversity in the soil determines the functioning of the ecosystem; for example, a decrease in species diversity can afect the processes that control the nitrogen and carbon cycles.It is also worth noting that the composition of soil bacterial and fungal communities is dynamic, subject to strong fuctuations within short periods (e.g., months) during the growing season, and undergoes temporary changes that are dependent on tillage and crop rotation [20][21][22].
It has been reported that the rooting depth of perennial crops and the composition of the litter create a sensitive and dynamic microbial system that cycles plant residue [23].Any change in the soil microbial biomass is regarded as an early indicator of change in the organic carbon cycle and soil organic matter [24,25].Studies have shown that the greater the biodiversity of soil microbes, the larger the plant biomass; that is, there is evidence of a relationship between the richness of bacteria and the stability of the plant biomass, especially the richness of fungi for plant biomass production [26].Crop types (i.e., annual vs. perennial) and farming practices (i.e., the addition of fertilizers and manures) have also been shown to impact the dynamics of the soil microbial community and its biomass [27][28][29][30][31]. Plant species in longterm cultivation are the dominant driving force in the composition of soil microbial diversity [32][33][34].Other studies have indicated that continuous cultivation of different agricultural plants has diferent efects on bacterial communities in diferent soil complexes (e.g., the rhizosphere soil and bulk soil) [35][36][37].In addition, long-term use of high doses of nitrogen, phosphorus, and potassium (NPK) fertilizers reduces the richness of soil bacterial species [38,39].
Te aim of this study was to gain a better understanding of how land management, long-term cultivation, plant species, and soil types afect the soil microbial community and its biomass in the autumn after a harvest.More specifcally, the main goal was to identify and compare the microbial communities present in long-term cultivated soils under two diferent plants and in uncultivated soils in the Talas region, North Kyrgyzstan.
Te fndings will support our understanding of the impact of land use on soil microbial communities and will be useful for predicting the future cycling of carbon and other nutrients in the region.

Short Description of the Study Area.
Te Talas region is located in northwest Kyrgyzstan.Te area has a midtemperate continental climate, with warm summers and cold winters.Te annual average temperature is 6-8 °C.Te yearly precipitation is about 320 mm, and it generally occurs during the crop-growing season (late spring).Te region is considered favourable for cultivating industrial crops; in recent years, farmers have grown beans in a monocultural manner and have obtained high yields.Commercial varieties of dwarf apple and pear trees are also cultivated.Te beans and fruit trees are the most representative economic crops of this region; the seeds, fresh fruits, and products of these crops are exported to European countries.

Sampling Sites.
Te soil microbial communities present in natural and crop areas in the Talas region were analysed in autumn, after harvest in the crop areas, and after the growth of natural vegetation in the noncrop areas (Figures 1 and 2).
Nonarable soil samples, including soil samples under bean and pear trees, were collected in mid-September.Te underground and aboveground organs of beans were still semidecomposing in the soil; pear trees still had green leaves, but the litter of yellow leaves predominated under the crown.Fluctuations in night and day temperatures were very noticeable; daytime air temperatures ranged from 15.0 to 19.00 °C during the sampling period.Soil temperature ranged from 7.9 to 15.0 °C (Table 1).

Characteristics of the Studied Soil Types. According to
Mamytov [40,41], the Talas region contains three types of soil: northern mountain-valley serozem (NMVS; low carbonate) soil, light chestnut (LC) soil, and dark chestnut soil.Cultivation has been practiced in some parts of the region for many years; however, other parts continue to support natural vegetation while being used for agricultural purposes, and livestock graze here during the spring and summer months.
(1) Northern Mountain-Valley Serozem (NMVS) Soil.NMVS soil is confned to the middle and lower parts of the Kyrgyz and Talas ridges' foothill plume within the absolute heights of 600-900 m.Te mechanical composition of NMVS soil varies from skeletal sandy to cartilaginous silt loam.Te humus content in the upper horizons fuctuates between 1.5 and 2.5%.Te nitrogen content is 0.10-0.18%.NMVS soil is classifed as low carbonate in terms of the amount of CO 2 .Te low carbonate content is associated with the hydrothermal features of the regime of these types of grey soil and the nature of the source rocks.Te pH of the studied NMVS soil samples ranged from 6.53 to 7.80.A chemical analysis performed in the Agrochemical Laboratory in Bishkek in 2020-2021 showed that these soils contained the following compounds: mobile phosphorus compounds at 26.0 mg/kg, exchangeable potassium compounds at 184 mg/kg, and nitrate nitrogen at 31.6 mg/kg.
(2) Light Chestnut (LC) Soil.LC soil is the standard soil type found on the Talas foothill trails, from 1,000 to 1,500 m.Regarding temperature, the belt of LC soil in Northern Kyrgyzstan is located in moderately warm areas.Te temperature is above 10 °C at 2,700 m and is 20 °C at 1,800−2,200 m, respectively.
Atmospheric precipitation is 300-400 mm per year.In terms of mechanical composition, LC soil is predominantly a medium to heavy loam.Te proportion of particles <0.01 mm is usually 33-55%.Silt particles (<0.001 mm) make up approximately 7.4-17.6% of the soil.Tere are also light loamy cartilaginous varieties of LC soil.Te LC soil located on the foothill plains is highly susceptible to erosion processes.Te carbonate content of LC soil varies considerably, ranging from carbonate-free to highly carbonate, and tends to increase with depth.Te CO 2 content in the arable 2 Applied and Environmental Soil Science horizon ranges from 0 to 8.2% (average of 2.7%) and in the subsoil from 0 to 11% (average of 4.6%).LC soil is slightly alkaline to alkaline, with a pH of 7.2-8.6.Te humus content varies widely depending on the degree of erosion, thickness, and texture.In the arable horizon, it ranges from 0.7 to 4.4% (average of 2.3%), and in the subarable horizon, it ranges from 0.5 to 2.3% (average of 1.5%).Te total nitrogen content ranges from 0.10 to 0.30%, and the ratio of carbon to

Isolation of Microorganisms from the Soil Using
Cultivation-Dependent Approaches.From each collected soil sample, 10 g was taken and ground in a sterile porcelain mortar for 5 min in aseptic conditions.After grinding, the soil sample was washed with sterile water.Ten, 1 g of each sample was added to 10 mL of Luria-Bertani broth in a 125 mL fask, and the mixture was bufered with sodium acetate (0.25 M, pH 6.8).Te broth was incubated in a shaker at 200 rpm for 4 h at 28 °C.After incubation, 1 mL aliquots were spread on nutrient agar (NA) plates and incubated at 27 °C for 7-10 days to obtain bacterial colonies [42,43].Te following media were typically used for the growth and qualitative and quantitative characterisation of the soil microfora: Czapek's medium and potato dextrose agar (PDA) for fungal species (incubated at 25 °C for 10-12 days), Ashby's medium for oligonitrophilic microorganisms (namely, acetobacter bacteria), starch-ammonia medium (SAA) for actinomycetes (incubated for 15 days), and meatpeptone agar (MPA), starvation agar, and soil agar for bacterial species.Colonies were continually subcultured on new agar plates until pure cultures were obtained, and they were stored at 4 °C until further identifcation was performed.
Te phenotypic and biochemical characteristics of the bacterial isolates were determined with reference to Bergey's Manual of Determinative Bacteriology [44] and classifed according to Bergey's Manual of Systematics of Archaea and Bacteria [45].Te morphological characteristics of the fungal isolates were identifed using the Principles of Fungal Taxonomy and Illustrated Genera of Imperfect Fungi handbooks and the Mycology Online website [46][47][48].
Images of bacterial cells and fungal mycelia were obtained using a microscope camera (Motic Images Plus, Version 2.0ML, Quick Start Guide, 163 Series Compound Biological Microscope).

Isolation of Microorganisms from the Soil Using
Cultivation-Independent Methods  45 sec at the primer-specifc annealing temperature, and an elongation step at 68 °C for an amplicon-specifc elongation time, followed by a 5 min fnal elongation step at 68 °C.PCR product size was controlled by 1, 5% agarose gel electrophoresis.Raw sequence data were combined into a single consensus sequence for each marker using version 6 of the MEGA software package [50].

Determination of the Number of Microbial Colony-
Forming Units (CFU) per Gram of Soil.Under aseptic conditions, 1 g of soil was moistened to a paste state and ground with a rubber pestle for 5 min.To determine the hyphae length of the fungi, 10 g of soil was mixed with 90 mL of water, and an RT-2 tissue microgrinder was used for 5 min at 5,000 rpm.After preliminary dispersion by grinding, the soil suspensions were diluted (from 1 : 2 to 1 : 100,000, depending on the group of microorganisms considered) and plated.Soil bacterial counts were estimated at 10 4 dilutions and fungal population counts at 10 2 dilutions.
Te most accurate estimates are obtained when 50-200 colonies of bacteria and actinomycetes and 30-50 colonies of fungi developed on a plate.Te seeded plates were incubated at 27-28 °C for bacterial and actinomycete growth and at 21-23 °C for fungal growth.Counting of the bacterial colonies that grew on NA in Petri dishes was performed after 7 days for the r-strategies group, after 10 days for the Kstrategies group, and after 15 days for the actinomycetes.Fungal colonies were counted after 10-12 days of growth on PDA.Colonies were usually counted using a magnifying glass without uncovering the Petri dish.Each counted colony was marked with a dot on the outer surface of the dish bottom using a glass marker for convenience.After determining the number of colonies on all parallel plates, the average number of colonies per plate was calculated.

Determination of the Presence of Nitrogen-Fixing Bacteria
(Azotobacter) in the Soil Samples.Te soil lumps method was used to detect Azotobacter bacteria in the soil samples.Te soil was moistened with sterile tap water until it reached a pasty state.Te lumps were laid out in regular rows (according to a stencil) with a microbiological loop or needle, 30 in each Petri dish, on Ashby's medium.Tree Petri dishes were used in parallel in triplicate for each soil sample.Te dishes were placed in a thermostat-controlled humid chamber.After 4-6 days, the number of soil lumps overgrown with mucous colonies of Azotobacter was determined.Azotobacter colonies are usually slimy with a doughy consistency, white opaque, and the formation of a brownish pigment is observed with age, appearing on the 4th-6th day of incubation.In terms of their morphology, the cells resemble short, thick rods or cocci, and are often connected in pairs and are surrounded by a capsule.Azotobacter colonies should be distinguished from lipomycete yeasts, which form slimy, spreading colonies with a glossy sheen and appear on the 14th-16th day of incubation.

Statistical Analysis.
Te Shannon index was used to determine the complete species composition of the bacterial communities, including the abundant rare species, present at the studied sites [51].Te Shannon-Weaver diversity index is based on communication theory.Te Shannon function H ′ measures the uncertainty, corresponds to the entropy concept, and is calculated using the following equation: where pi � ni/N (the share of the i-species in the biotope), ni � the number of the i-species (specimen), N � the total number of microbial species, ln � the natural logarithm, Σ is the Greek symbol that means "sum," ln � natural log, and p � the proportion of the entire community made up of species I. Te higher the value of H ′ , the higher the species diversity in a particular community.Te lower the value of H ′ , the lower the diversity.A value of H ′ � 0 indicates a community with only one species.Te obtained data were statistically processed using SPSS version 25 software (IBM, USA).

Density of Microbial Communities in the Studied Soils.
Te studied areas mainly consisted of two soil types: NMVS and LC soil, according to the descriptions of Mamytov [40,41].Te studied sites also included arable land that supports bean and orchard plots, as well as three natural sites.Table 2 shows the density of bacterial, actinomycete, and fungal cells found in soil samples collected from the various study sites.
When the densities of the bacterial populations in the diferent soil (NMVS vs. LC) and land (plant cover vs. bulk soil) types were compared, it was found that the greatest proportion of CFU was present in soil from the TAL-88 Cholponbai site.At this site, which has NMVS soil and bulk soil without vegetation, the number of CFU/g of soil reached 3.9 × 10 6 ± 0.84.Another two sites, TAL-99 Berdike (LC soil, arable land with beans) and TAL-97 Cholponbai (NMVS soil, bulk soil without vegetation), were also found to contain a signifcant number of bacterial cells/g of soil, at 1.2 × 10 6 ± 0.72 and 1.03 × 10 6 ± 0.93, respectively.Te TAL-77 Boo-Terek village site (NMVS soil, land cultivated 6 Applied and Environmental Soil Science Applied and Environmental Soil Science with dwarf pear trees) was also characterised by a signifcant bacterial content (1.2 × 10 6 ± 0.78 bacterial cells/g of soil).Te remaining sites were found to contain low levels of bacterial cells, numbering only a few thousand cells per g of soil (Figure 3).Te studied soils were characterised by a lower content of microscopic fungi than bacteria and actinomycetes.Te sites most densely populated with fungal cells were TAL-70 (LC soil, cultivated with beans), TAL-102 (LC soil, fruit nursery, and pear seedlings), TAL-77 (NMVS soil, cultivated with dwarf pear trees), TAL-44 (NMVS soil, cultivated with beans), and TAL-30 (NMVS soil, cultivated with beans).Te rest of the sites were characterised by lower fungal cell content (Figure 4).
Te actinomycete content of the soils was found to be lower than that of all bacteria.Te most signifcant densities of actinomycete spores were found at the following sites: TAL-70 (LC soil, cultivated with beans), TAL-78 (NMVS soil, cultivated with beans), and TAL-41 (NMVS soil, cultivated with pear trees).Moderate actinomycete cell densities were found at TAL-44 (NMVS soil, cultivated with beans), TAL-86 (LC soil, cultivated with beans), and TAL-88 (NMVS soil, bulk soil) (Figure 5).
Te quantity of free-living, nitrogen-fxing Azotobacter species in the soil samples was determined by overgrowing soil lumps with colonies of these bacteria (Figure 1S).Applying this simple method across several replicates made it possible to compare the presence and enrichment of Azotobacter species in the studied sites and soil types, which serves as an indirect bioindicator of soil fertility.Only two sites (TAL-107 and TAL-97) were found to have low levels of nitrogen-fxing bacteria, both below 50%.Te remaining sites were enriched with nitrogen-fxing bacteria, by up to 100%.
Azotobacter levels were low at sites with LC soil and without vegetation.Te soil at these sites does not contain enough phosphorus and other elements; that is, its fertility is low (Figure 6).

Microbial Composition of the Studied Soils Determined
Using Cultivation-Dependent Approaches.Te bacterial diversity of the NMVS and LC soils was represented by two phyla: Firmicutes and Proteobacteria (Figure 7(a)).Firmicutes species made up < 80% of the bacteria in the NMVS soil samples and 70% of the bacteria in the LC soil samples.Such compositions were observed in samples from almost all sites and were found to not depend on the plant cover or land use practice during the autumn period (Figure 2S).
In terms of the bacterial genera present in the soil samples, an abundance of spore-forming bacteria from the Bacillus genus (80-85%) was found in NMVS soil samples.Non-spore-forming species from the Pseudomonas (5.0%), Enterobacter (5.0%), and Staphylococcus (5.0%) genera were also detected, as well as autochthonous microfora, such as Nocardia species (5.0%) (Figure 7(b)).Te proportions of the pseudomonads and autochthonous microfora were higher in the LC soil samples than in the NMVS soil samples.
Te species diversity of the soil fungi difered slightly between the two studied soil types.Representatives of nine known genera were found in the NMVS soil samples, including common saprophytic species from the Aspergillus and Penicillium genera involved in the decomposition of plant residue and pathogenic Fusarium species.Tis was expected because the soil samples were from sites used for the monoculture of beans (Figures 3S-10S).
In the LC soil samples, fungi from fve genera were detected.Tese soil samples were dominated by species from the Fusarium and Penicillium genera (Figures 8(a) and 8(b)).

Presence of Actinomycetes in the Studied Soils.
Te actinomycetes are a group of oligotrophic microorganisms that give rise to slow-growing colonies on the surface of a starch-ammonia medium.Actinomyces colonies derived from the studied soils were classifed into fve sections according to their aerial and substrate mycelium morphology and colour [52].In both soils, representatives of the Albus section dominated, reaching 33.0 ± 0.71% to 35.0 ± 0.41%.
Cinereus-type species were the second most prevalent, accounting for 20.0 ± 0.12% to 25.5 ± 0.31%.Tey were more abundant in the LC soil samples than in the NMVS soil samples.Representatives of the Roseus section were also prominent, reaching 19.0 ± 0.72% and 20.0 ± 0.23% in the NMVS and LC soil samples, respectively.Species from the Azureus and Helvolo-Flavus sections were found in medium and low proportions.
A rich biodiversity of actinomycetes was found at the TAL-44 site in soil-supporting beans (soil pH � 7.27), the TAL-102 site in soil-supporting pear trees, and the TAL-107 site in soil-supporting natural vegetation.Te proportions of the actinomycetes belonging to each of the fve sections found in the two soil types are shown in Figures 9(a) and 9(b), respectively.

Microbial Composition of the Studied Soils Determined
Using Cultivation-Independent Methods

Bacterial Diversity in NMVS Soil
Samples.When the bacterial composition of the two types of soils located under diferent vegetation was examined via direct analysis of the 16S rRNA gene, rich bacterial biodiversity was found; such biodiversity was not detected when classical cultivation on a nutrient medium was performed.Applying this approach (i.e., examining genes) to the analysis of soil bacteria made it possible to determine the presence and level of bacterial biodiversity in the diferent soil types and under diferent vegetation types.For example, the 16S rRNA analysis revealed that Bacillus species prevailed in NMVS soil samples from felds with a monoculture of beans, namely, Bacillus subtilis (including B. subtilis ssp.subtilis), Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, and Bacillus pumilus (Figure 10).Among the identifed species were industrially important species, such as B. atrophaeus and B. amyloliquefaciens.B. amyloliquefaciens is a root-colonising biocontrol bacterium used to fght some plant root pathogens in agriculture [53].Te second most predominant group consisted of species from the Brevibacterium genus (order: Actinomycetales).Tese bacteria inhabit insects and decaying organic matter and can metabolise carbon and nitrogen [54].
Te same Bacillus species were found at the sites where pear trees were grown, including B. atrophaeus, B. pumilus, B. amyloliquefaciens, and B. tequilensis (Figure 11).In other words, the soil bacterial communities in the soils supporting pear trees were similar in composition to those in the soils supporting bean plants.Regardless of the vegetation type, the soil type and pH may afect the levels of the diferent bacterial species.For example, more dead organic residue enters the ground in autumn.
While spore-forming bacteria were also dominant at sites without vegetation, the species difered from those found in the soil under bean plants and pear trees.Specifcally, the Bacillus species were diferent at sites with NMVS soil without vegetation from those at sites with cultivated soil.In addition, classical ammonifers were found, such as Bacillus mycoides, Bacillus megaterium, Bacillus cereus, Bacillus albus, and Bacillus wiedmannii ( with the optimum around 30 °C, and produce a broad set of enzymes that support the modifcation of organic matter and substances in soil.B. megaterium strains are considered halophiles and grow in a saline (2-10% NaCl) environment.Tus, these bacteria are adapted to diferent soil compositions [53].Spore-forming bacteria are credited with playing the leading role in soil formation processes due to their ability to metabolise organic residue that enters the soil.In most of the studied sites, Bacillus species were found to dominate.
Non-spore-forming bacterial species from the Proteobacteria phylum and from a range of genera were detected; in some locations, they were found to be abundant (Figure 13).In soils under bean plants, Ochrobactrum species were dominant.According to other authors, these bacteria are found widely in the environment, including soil, plants, and animals [55].Non-spore-forming Alcaligenes species were also frequently found.Alcaligenes faecalis has been identifed as an active ammonifer due to its ability to decompose urea to ammonia and has adapted to alkaline environments, including soil [56].
In addition, pathogenic and opportunistic bacteria were detected in the soil under bean plants, such as Stenotrophomonas spp.and Agrobacterium species.

Bacterial Diversity in LC Soil
Samples.At the sites with LC soil, representatives of the Proteobacteria phylum prevailed over those of the Firmicutes phylum.It was found that most of the Proteobacteria species were diferent from those found at the sites with NMVS soil.A wide range of aerobic heterotrophic bacterial genera were represented, such as Achromobacter, Alcaligenes, and Aminobacter, as well as the  Applied and Environmental Soil Science Gammaproteobacteria and Betaproteobacteria classes.Most of the bacterial species were from the Achromobacter genus (Figure 14(a)).At the sites with LC soil, most of the Firmicutes species also difered from those found at the sites with NMVS soil (Figure 14(b)).
As the results show, LC soil under bean plants was dominated by bacteria from the Alcaligenaceae family and the Achromobacter genus.Tese bacteria are heterotrophic, rod-shaped, Gram-negative and are isolated from water, soil, humans, and other animals.Tey decompose many organic substances, including polychlorinated biphenyls, using them as sole carbon and energy sources.In addition, Betaproteobacteria and Gammaproteobacteria species were found to be widely distributed and abundant in the soil at these sites.
When analysing the biodiversity of the bacterial communities according to the Shannon index, it was found that there was greater biodiversity at the sites with NMVS soil than at those with LC soil.NMVS soil samples collected from under bean plants had a higher sensitivity biodiversity index value (6.21) than those collected from under pear trees (3.9).Even in fallow soil, signifcant biodiversity was detected (Figure 15).

Biodiversity of Streptomyces Species in NMVS Soil
Samples.Actinomycetes are an essential part of soil microfora.Teir ability to adapt to various conditions allows them to metabolise a broad group of substances as energy    sources.Streptomyces is the largest genus of the Actinobacteria phylum; over 500 species of Streptomyce bacteria have been described, and most are found in soil and decaying vegetation [57].
In this study, 16S rRNA gene analysis revealed that there was a rich biodiversity of Streptomyces species in the NMVS soil under bean crops, especially at site TAL-44 (Figure 16).Streptomyces species are typically found in such soil habitats, and various decomposing organic substances dominate at the sites mentioned above.Te presence of decaying vegetation during autumn is a favourable ecological niche for Streptomyces species.In addition, antibiotic producers were detected, such as Streptomyces griseus, Streptomyces venezuelae, Streptomyces anulatus, Streptomyces noisiness, Streptomyces omiyaensis, and Streptomyces avidinii.
In the same soil type under pear tree seedlings, the Streptomyces biodiversity was less than in the soil under bean plants.Also, even though antibiotic-producing species and heterotrophic species were found, the species composition of the detected Streptomyces difered (Figure 17(a)).It was also found that the Streptomyces biodiversity was diferent at sites with LC soil under pear seedlings than at sites with NMVS soil.However, some species found under bean plants in NMVS soil were also found (Figure 17(b)).
In soil without vegetation (LC soil), completely diferent Streptomyces species were found (Figure 18).
Te obtained results indicate that the soil and vegetation type infuence the quantitative and qualitative composition of the Streptomyces population.Among the species found, a large proportion were antibiotic-producing species and species that simultaneously participated in soil-forming processes to decompose organic residue.All these organisms grow in a wide pH range (from 5 to 11) and produce diferent colour pigments when they grow as colonies.
Te Shannon index values were calculated to determine the biodiversity of the Streptomyces species in the soil samples.NMVS soil under bean plants had a higher value (9.2) than LC soil under the same crop (4.21).When the biodiversity of these microorganisms in the soil under pear trees was assessed, it was found that NMVS soil had a higher index (4.44)than LC soil (4.25).In soils without vegetation, the biodiversity index of Streptomyces was slightly higher in NMVS soil (1.2) than in LC soil (1.0) (Figure 19).

Biodiversity of Fungi in NMVS Soil Samples.
Microscopic fungi contribute to essential processes in soil, particularly the decomposition of plant and animal debris.PCR amplifcation of internal partial sequences of the spacer ITS region of 18S rRNA allowed us to determine the biodiversity of the soil fungal communities at the studied sites.
At sites with NMVS soil under bean plants, the biodiversity of the soil fungi difered from that at sites with LC soil.In soil under bean plants, the dominant species were from the genus Fusarium (division: Ascomycota, family: Nectriaceae), and these were found at almost every site.Despite their predominant role in natural soils as saprophytes, many strains of Fusarium oxysporium are plant pathogens, especially in agricultural land.Geotrichum candidum (division: Ascomycota, family: Dipodascaceae) was also found in abundance in such soil under bean plants.Tis species is widespread in soils around the world and is found 14 Applied and Environmental Soil Science on all continents.Also, at some sites with bean crops, Myrothecium verrucaria and Albifmbria verrucaria (division: Ascomycota, family: Stachybotryaceae) were found.Tese are widespread in soil and plant material and are pathogenic species and saprophytes capable of decomposing cellulose [58] (Figure 20).Te same type of mycofora, consisting of only F. oxysporium, was found in most of the NMVS soil samples (Figure 21(a)).F. oxysporium is a ubiquitous soil dweller; it can exist as a saprophyte and decompose lignin and other complex carbohydrates in plant residue [59].Te lack of fungal biodiversity in soils without vegetation may be associated with a lack of plant litter, and fungal species better adapted to such an environment are found in these areas.Te soils under pear trees were found to be dominated by fungal species that are specifcally associated with fruits, such as Talaromyces species (division: Ascomycota, family: Trichocomaceae) (Figure 21(b)).

Biodiversity of Fungi in LC Soil Samples.
At almost all the studied sites and in both types of soils, fungi from the Fusarium genus dominated.In cultivated areas, pathogenic species from this genus predominated, followed by pathogenic species from the Alternaria genus.Compared to uncultivated sites, sites cultivated with beans were found to have greater amounts of fungal species from the Fusarium and Alternaria genera.Te results indicate that monoculture farming over a long period may result in the accumulation of phytopathogenic organisms, including fungi, in the soil (Figure 22).At sites with LC soil under pear tree seedlings, fungal species from the Cladosporium genus (division: Ascomycota, family: Davidiellaceae) dominated.Usually, these fungi are widespread indoors and outdoors and found on living and dead plants.Among them are plant pathogens, fungal parasites, and saprophytes [60].
In addition, species from the genus Talaromyces (division: Ascomycota, family: Trichocomaceae) accounted for a signifcant share of the soil fungi found in samples collected from under pear tree seedlings (Figure 23).
In LC soil without vegetation, only Penicillium species were found in abundance (Figure 24).
It is well known that Penicillium is one of the most widespread genera of fungi in the world, with representatives found in a wide variety of places: in soil, on plants, in the air, indoors, on food, and in the sea.Species of this genus are saprotrophs and weak plant parasites.However, only a few species have been recorded in the rhizosphere soil [61].
When the biodiversity of the soil fungi was analysed, it was found that the Shannon index was higher for sites with LC soil under pear trees (4.188) than for sites with NMVS soil under the same crop (3.9).For soils under bean plants, the Shannon index was higher for sites with LC soil (4.059) than for sites with NMVS soil (3.86).In terms of the bulk soil samples, sites with LC soil had a higher index (2.889)than those with NMVS soil (0.964) (Figure 25).
Tus, based on the obtained results, it can be suggested that, among all the abiotic and biotic factors that afect the formation and function of the individual groups of microorganisms in the plant rhizosphere, the vegetation type has the most signifcant impact (≤60%).Tis is followed by soil pH and soil type, which have a ≤30% impact on the biodiversity of microorganisms in the soil.Monoculture farming has a minor infuence (approximately 10%) on the biodiversity of the soil microbial species (Figure 26).

Discussion
Soil microfora decompose organic substances and generate valuable forms of humus in the deep layers of the earth; hence, they play key roles in maintaining the soil's structure 16 Applied and Environmental Soil Science and fertility, as well as the growth and development of plants.
Numerous studies have shown that the quantitative criterion of soil fertility is the content of saprophytic and pathogenic bacteria and fungi in specifc proportions in one gram of soil [62,63].It has also been shown that water availability and soil moisture have a signifcant efect on the activity of soil microbes [64,65].Soil moisture levels are typically highest in spring and autumn due to snow melting and signifcant amounts of precipitation.Consequently, microorganisms that mineralise plant residue are most active during autumn rather than summer.
In the 1960s and 1970s, several studies were conducted using classical microbiological methods to examine the ecology, distribution, and quantitative and qualitative composition of soil microbes found in various types of soil in Kyrgyzstan [66][67][68][69].Te scientists focused their research on the ecological groups of microorganisms present in virgin soils but not in cultivated areas.Almost 50 years have passed     Given this background, the aim of the present study was to identify and quantify the biodiversity of the microbial communities present in two types of soils (NMVS and LC) in the Talas Valley in the autumn period.In addition, we aimed to compare the soil microbial communities found in soil that supported two diferent types of crops and in soil without vegetation.Soil samples consisting of a 5-15 cm layer were collected in mid-September (average air temperature � 14-15 °C and soil temperature � 8-10 °C) from under crops of bean plants and pear tree seedlings.Samples of bulk soil without vegetation were also collected.
Regarding the soil bacterial populations, the results show that the density was two times higher in NMVS soil than in LC soil.Te number of bacteria in a gram of LC soil reached 200,000 CFU/g, whereas in NMVS soil, the number reached 400,000 CFU/g.Moreover, bacteria were found at a high density at two sites with NMVS soil without vegetation: TAL-88 (pH 7.63) and (TAL-97) (pH 7.38) (Figure 3).We suggest that the higher bacterial content of the fallow soil compared to that of the cultivated soil is associated with the soil having an optimal pH and the presence of moisture, which support the development of autochthonous microfora, the most permanent members of the soil microbial population.Regardless of the availability of fresh plant residue, these bacteria can intensively reproduce and function.
In contrast to the bacterial communities, the fungal communities in the NMVS and LC soils were present at almost equal density.In the LC soil, high numbers of fungi were recorded under bean plants (pH 7.45) and pear trees (pH 7.36), whereas in the NMVS soil, the fungal density was higher under pear trees (pH 7.8).Te average number of fungi was noted in the acidic soils (pH 6.51) under bean plants and pear trees (Figure 4).It is usually accepted that fungal communities prefer acidic environments to neutral ones; however, in the studied soils, fungal communities were concentrated more in neutral soil than acidic soil, which indicates that the plant cover may specifcally impact the fungal density in soil, followed by the pH.It is also worth noting that when plant tissues (e.g.leaves, stems, and roots) are damaged during harvesting or when straw is added, various volatile compounds are released that can have a biocidal efect on rhizosphere microorganisms.In this case, the exudates and organic substances released by the root system of the pear tree were more attractive for the functioning of soil fungi than in leguminous plants.
Te actinomycete community density was almost two times higher in LC soil than in NMVS soil under bean plants (pH 7.46) (Figure 5).In LC soil, the microenvironment was the most suitable for this group of microorganisms; actinomycetes are oligotrophs that prefer poor organic compound content and assimilate mineral forms of nitrogen [70,71].Te rhizosphere efect on oligotrophic microorganisms, such as Streptomyces bacteria, was minimal in this study, and this fnding aligns with other research that has proved that there is no rhizosphere efect on oligotrophs [72].
In all the studied areas, the two soil types were rich in Azotobacter species, indicating that the content of mobile forms of phosphorus, calcium and other elements was sufcient.Only two plots (TAL-107 and TAL-97) with LC soil had low levels of these bacteria, suggesting that LC soil is less fertile than NMVS soil (Figure 6).Others have shown that agroforestry soils contain more Azotobacter species than cereal and grassland soils [73,74].
Te standards adopted in soil microbiology indicate that, depending on the presence of humic compounds in the soil and a sufcient amount of organic matter, the number of bacteria either decreases or increases depending on the vegetation type.Several other factors also afect the biomass of saprophytic bacteria, namely, the mechanical composition, genesis, origin, pH, and moisture of the soil, as well as the presence of nitrogen and other elements in the soil.It is also generally accepted that the greater the number of saprophytic bacteria, the more fertile the soil.Hence, soil fertility is primarily determined by the amount of benefcial microfora present.According to Zvyagintsev [42], the average number of microorganisms per gram of fertile soil is as follows: bacteria � 1 × 10 8 -10 9 , actinomycetes � 1 × 10 7 -10 8 , and fungi � 1 × 10 5 -10 6 .Tus, the soils of the Talas region can be classifed as moderately fertile.
Performing a 16S rRNA analysis made it possible to determine the bacterial diversity in relation to each soil and vegetation type found at the study sites.In NMVS soil under bean plants and pear trees, sharply diferent species of sporeforming bacteria were found; however, at some sites, similar species were recorded.At the sites with uncultivated soil, completely diferent species were found, mainly the classic ammonifying Bacillus species: B. mycoides, B. megaterium, B. cereus, B. albus and B. wiedmannii.Tis indicates that, depending on the chemical composition of the plant litter, diferent Bacillus species participate as active ammonifers in the decomposition process.Our data are consistent with those of other studies; these Gram-positive, endospore-forming bacteria inhabit the soil and plant roots and actively participate in plant litter decomposition, particularly in the degradation and transformation of lignin [53,75].
Regarding the presence of non-spore-forming bacteria, the PCR analysis showed that there were signifcant differences between the species found in the two soil types and under the diferent vegetation types.In NMVS soil under bean plants, heterotrophic Ochrobactrum species dominated, while other heterotrophic bacteria of the genus Achromobacter dominated in LC soil.According to other researchers, these bacteria are found widely in the environment, including soil, the plant rhizosphere, and root exudates; defcient molecular weight carbon substrates are signifcant drivers of this bacterial diversity [55,76].Such results indicate that diferent soil parameters (pH and humus content) and vegetation exudate compounds could impact the species composition of soil bacteria.In addition, we found pathogenic and opportunistic bacteria in the soil under bean plants, such as Stenotrophomonas and Agrobacterium species.Stenotrophomonas spp.are found mainly in close association with plants, have an essential ecological role in the nitrogen and sulphur cycles, and can engage in benefcial interactions with plants, promoting growth and protecting plants from attack [77,78].Moreover, we found widely distributed and abundant Betaproteobacteria and Gammaproteobacteria bacteria at these sites.Tese bacteria play essential roles in soil pH maintenance and elemental cycling.In particular, Betaproteobacteria and Gammaproteobacteria species are responsible for the crucial organic carbon, nitrogen, and sulphur cycling processes.Te relative abundance of Betaproteobacteria and Gammaproteobacteria species is also positively correlated to the dissolved organic carbon (DOC) concentration, which is a critical environmental parameter that shapes the bacterial community composition [79][80][81].
RNA analysis revealed that the Streptomyces species difered in composition between the soil and vegetation types.Diverse Streptomyces populations were found at sites with NMVS soil and bean crops, and diferent species were detected at the sites with pear trees and without vegetation.It is likely that the bean plant residue in the soil attracted more antibiotic-producing bacteria than the pear tree residue.Tese data provide further evidence that the composition of the plant residue produced during harvesting infuences the soil microbial community.In this case, the results suggest that the type of vegetation afects the soil Streptomyces biodiversity.Other researchers have shown that these bacteria play major roles in the cycling of organic matter, inhibit the growth of several plant pathogens in the rhizosphere and decompose complex polymer mixtures in dead plants [82].
To determine the composition of the fungal community at each site, PCR amplifcation of the ITS region of 18S rRNA was performed.It was found that pathogenic species, such as F. oxysporium and G. candidum, dominated under bean plants in NMVS soil.Other studies have shown these fungi to be widespread in decaying plant tissues and are the causative agents of sour soft rot in citrus fruits, tomatoes, carrots, and other vegetables [83].M. verrucaria and A. verrucaria were also detected in signifcant amounts in soils under bean plants.Other researchers have reported that these fungi are pathogenic and decompose cellulose [58].At sites with LC soil and bean crops, the pathogenic and saprophytic Alternaria and Fusarium genera dominated, while Cladosporium and Talaromus species prevailed under pear tree seedlings.Most of these species are soil dwellers and produce thermally stable ascospores; therefore, they are sometimes isolated from pasteurised fruit juices and fruitbased products [83,84].
Te studied bulk soil samples had relatively low fungal biodiversity, with Fusarium and Penicillium species being the main components.Tese fndings indicate that the type of vegetation and its secreted exudates infuence the species of fungi that are found in the rhizosphere microfora, which are responsible for the mineralisation of plant residue.Furthermore, compared to the uncultivated soils, the soils cultivated with beans contained Fusarium and Alternaria species.Tus, monoculture farming over an extended period leads to the accumulation of phytopathogenic organisms, including fungi, in the soil.
Te Shannon index is used as an indicator of biodiversity, and comparisons of the Shannon index values calculated for the diferent sites showed that there were clear diferences in the biodiversity of the rhizosphere microorganisms according to soil and vegetation type.For example, the bacteria analysed in this study were found to prefer NMVS soil over LC soil and soil under an annual plant (i.e., the bean plant) rather than that under a perennial fruit tree (i.e., the pear tree).Te Shannon index for Streptomyces species was higher for NMVS soil and the pear tree rhizosphere than for LC soil.In soil without vegetation, there was more Streptomyces biodiversity in NMVS soil than in LC soil.In terms of fungal biodiversity, the Shannon index for fungal species under pear trees was higher for LC soil than for NMVS soil.In LC soil, these micromycetes preferred bean plants as the vegetation cover over pear trees.
It can also be concluded that the soil microfora includes microorganisms with metabolic and substrate cycling activities that are intense yet relatively slow.Te most common members of the soil microbial population are autochthonous bacteria, which are physiologically inert species and their activity is helpful for plants as they assimilate soluble forms of nitrogen, preventing them from being washed away.
As noted in other studies, a diverse microbial community can simultaneously support multiple ecosystem functions.Species that do not participate in one specifc function at a particular time may contribute at another time or simultaneously to another process.Tus, asynchronous temporal fuctuations in the number of species and their contributions to various ecosystem functions in more diverse communities contribute to better overall ecosystem functioning over time [85][86][87].

Conclusion
Biodiversity and communities of microorganisms difer in soils with annual and perennial plants, which indicates that plant residues have a diferent chemical composition and diferent types of microorganisms are involved in their decomposition.Te composition of the plant residue produced during harvesting infuences the soil microbial community.
A diverse microbial community in the two soil types and under the diferent vegetation types has signifcant diferences between the species.Te most common members of the soil microbial population are autochthonous bacteria, which are physiologically inert species whose activity is helpful for plants as they assimilate soluble forms of nitrogen, preventing them from being washed away.
Among all the abiotic and biotic factors that afect the formation and function of the individual groups of microorganisms in the plant rhizosphere, the vegetation type has the most signifcant impact (≤60%).Tis is followed by soil pH and soil type, which have a ≤30% impact on the biodiversity of microorganisms in the soil.Monoculture farming has a minor infuence (approximately 10%) on the biodiversity of the soil microbial species.
According to the average number of microorganisms per gram of soil, the results proved that the soils of the Talas region can be classifed as moderately fertile.
In the future, such studies will expand and deepen in other northern and southern regions of the country to provide an ecological assessment of the soil fertility and its suitability for cultivating annual and perennial crops.

Figure 1 :Figure 2 :
Figure 1: Representative pictures of the studied crop areas.Field cultivated with beans with northern mountain-valley serozem (NMVS) soil (a), feld cultivated with dwarf pear trees with NMVS soil (b), feld cultivated with beans with light chestnut soil (c), and fallow ground with bulk chestnut soil (d).

Figure 3 :Figure 4 :
Figure 3: Bacterial colony-forming units (CFU) per g of soil found in light chestnut soil (a) and northern mountain-valley serozem soil (b) samples.Values are given as mean ± SD, n � 3, and are signifcantly diferent at P ≤ 0.05.

Figure 5 :Figure 6 :
Figure 5: Actinomyces colony-forming units (CFU)/per g of soil found in light chestnut soil (a) and northern mountain-valley serozem soil (b) samples.Values are given as mean ± SD, n � 3, and are signifcantly diferent at P ≤ 0.05.

Figure 7 :Figure 8 :Figure 9 :
Figure 7: Te bacterial phyla and genera found in the soil samples.Percentage of bacteria from the Firmicutes and Proteobacteria phyla in the northern mountain-valley serozem (NMVS) (1) and light chestnut (LC) (2) soil samples (a) and percentage of bacteria from fve genera in the NMVS (1) and LC (2) soil samples (b).Values are given as mean ± SD, n � 3, and are signifcantly diferent at P ⩽ 0.05.

Figure 10 :Figure 11 :
Figure10: A schematic illustration of the bacterial diversity within the Bacillus genus found in the northern mountain-valley serozem soil samples collected from sites cultivated with Phaseolus vulgaris.Te phylogenetic trees are based on the 16S rRNA sequences and were drawn using the neighbor-joining method.Tey show the relationships among the isolates and other similar sequences deposited in the NCBI GenBank.

Figure 12 :Figure 13 :
Figure12:A schematic illustration of the bacterial diversity within the Bacillus genus found in the northern mountain-valley serozem soil samples collected from the soil without vegetation.Te phylogenetic trees are based on the 16S rRNA sequences and drawn using the neighbor-joining method.Tey show the relationship among the isolates and similar other sequences deposited in the NCBI GenBank.

Figure 14 :Figure 15 :
Figure 14: A schematic illustration of bacterial diversity of Alcaligenaceae family (phylum: Proteobacteria) in light chestnut soils with Phaseolus vulgaris (a) and bacterial diversity of Bacillaceae family (phylum: Firmicutes) in light chestnut soils with dwarf pear trees (b).Te phylogenetic trees are based on the 16SrRNA sequences and are drawn using the neighbor-joining method.Tey show the relationship among the isolates and similar other sequences deposited in the NCBI GenBank.

Figure 16 :Figure 17 :
Figure 16: A schematic illustration of Streptomyces biodiversity in northern mountain-valley serozem soil samples with Phaseolus vulgaris.Te phylogenetic trees are based on the 16S rRNA sequences drawn using the neighbor-joining method.Tey show the relationship among the isolates and similar other sequences deposited in the NCBI GenBank.

Figure 18 :Figure 19 :
Figure 18: A schematic illustration of Streptomyces biodiversity in light chestnut soil without vegetation.Te phylogenetic trees are based on the 16S rRNA sequences and are drawn using the neighbor-joining method.Tey show the relationship among the isolates and similar other sequences deposited in the NCBI GenBank.

Figure 21 :
Figure 21: A schematic illustration of microscopic fungi biodiversity in northern mountain-valley serozem bulk soil (a) and with the pear trees (b).Te phylogenetic trees are based on the ITS sequences and are drawn using the neighbor-joining method.Tey show the relationship among the isolates and similar other sequences deposited in the NCBI GenBank.

Figure 20 :Figure 22 :
Figure 20: A schematic illustration of microscopic fungi biodiversity in northern mountain-valley serozem soil with Phaseolus vulgaris.Te phylogenetic trees are based on the ITS region of 18S rRNA sequences and are drawn using the neighbor-joining method.Tey show the relationship among the isolates and similar other sequences deposited in the NCBI GenBank.

71 Figure 23 :Figure 24 :
Figure 23: A schematic illustration of microscopic fungi biodiversity in light chestnut soil with pear trees.Te phylogenetic trees are based on the ITS sequences and are drawn using the neighbor-joining method.Tey show the relationship among the isolates and similar other sequences deposited in the NCBI GenBank.

Figure 25 :Figure 26 :
Figure 25: Te biodiversity of the soil fungal communities in the collected samples according to the Shannon index.Te left panel (a) shows the values obtained for northern mountain-valley serozem soil samples, and the right panel (b) shows the values obtained for light chestnut soil samples.

Table 1 :
Details of the soil samples collected from the Talas region.Te gross content of P 2 O 5 is 0.15-0.23%,and the gross content of K 2 O is 2.2-3.1%.Te abovementioned data were obtained from a chemical analysis of soil samples conducted at the Agrochemical Laboratory in Bishkek in 2020-2021.2.3.Soil Sample Collection.Samples of NMVS and LC soil were collected in autumn from a range of agricultural felds and nonarable areas, as shown in Table1.Table1also shows the soil pH and type, location, altitude, GPS parameters, vegetation type, and agricultural use data for each site.Soil samples were collected at a depth of 0-15 cm from under bean (Phaseolus vulgaris) crops and at a depth of 15-30 cm from under pear trees (Pyrus communis), also at a depth of 15-30 cm from bulk soil by scraping of the surface material with a sterile spatula and obtaining approximately 100 g of soil per sample.Te samples were packed in self-sealing bags and labelled well for future use.Five samples collected from one site were combined, homogenised, and sieved through a 2 mm flter according to cover type.Subsamples were placed on dry ice and stored at −80 °C in the laboratory until DNA was extracted.Te remainder of each soil sample was subjected to microbiological, chemical, and physical analyses.Samples were stored at 4 °C until use.

Table 2 :
Density of bacterial, actinomycete, and fungal cells found at the various study sites.