3.7 5.

Article

Changes in Bacterial and Fungal

Community of Soil under Treatment
of Pesticides

Rostislav Streletskii, Angelika Astaykina, George Krasnov and Victor Gorbatov

Special Issue
Pesticides Application and Remediation from the Environment
Edited by
Dr. Pankaj Bhatt

https://doi.org/10.3390/agronomy12010124
 agronomy
Article
Changes in Bacterial and Fungal Community of Soil under
Treatment of Pesticides
Rostislav Streletskii 1, *, Angelika Astaykina 1 , George Krasnov 1,2 and Victor Gorbatov 3

1 Soil Science Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia;
[email protected] (A.A.); [email protected] (G.K.)
2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
3 LLC Centre for Ecopesticides Research, 119234 Moscow, Russia; [email protected]
* Correspondence: [email protected]

Abstract: Experiments were carried out in soil microcosms with the treatment of pesticide formulations—
imidacloprid, benomyl, and metribuzin in single and tenfold application rates. For additional stimulation
of microorganisms, a starch–mineral mixture was added to some variants. For all samples, high-
throughput sequencing on the Illumina MiSeq platform of the V4 (16S rRNA) and ITS1 (18S rRNA)
fragments was carried out. As a result, it was possible to establish the characteristic changes in the
structure of the soil fungal and bacterial communities under pesticides application. The application of
pesticides was accompanied by dramatic shifts in alfa-diversity of the fungal community. The phylum
Basidiomycota was likely to be involved in the degradation of pesticides. The changes in the relative
abundance of the genera Terrabacter, Kitasatospora, Streptomyces, Sphingomonas, Apiotrichum, Solicoccozyma,
Gamsia, and Humicola can be proposed as an indicator of pesticide contamination. It is suggested to use
these markers for large-scale assessment of the effect of pesticides on soil microbial communities instead
of classical integral methods, including within the framework of state registration of pesticides. It is
also recommended to research the effect of pesticides on the soil microbiome during artificially initiated



successions using the additional source of carbon.


Citation: Streletskii, R.; Astaykina, Keywords: pesticide; bacteria; fungi; NGS; soil microbial community
A.; Krasnov, G.; Gorbatov, V. Changes
in Bacterial and Fungal Community
of Soil under Treatment of Pesticides.
Agronomy 2022, 12, 124. https:// 1. Introduction
doi.org/10.3390/agronomy12010124
The study of the effect of pesticides on the soil microbial community is an important
Academic Editor: Pankaj Bhatt issue, which, however, is associated with methodological difficulties, since the assessment
Received: 14 December 2021
is aimed at an extremely complex system. Until recently, methods were not available
Accepted: 2 January 2022
to capture the full microbial diversity of the soil. From the middle of the 20th century,
Published: 5 January 2022
the studies on the possible harm of pesticides to soil microbial community (Bacteria and
Fungi) have been relying on the use of integral indicators such as soil respiration and
Publisher’s Note: MDPI stays neutral
nitrification, as well as microorganism seeding on solid media. Any effects have been
with regard to jurisdictional claims in
observed only at very high concentrations and have been difficult to explain. For example,
published maps and institutional affil-
DDT (insecticide) applied at a concentration of 0.1% [1] has not affected nitrogen fixers,
iations.
nitrifiers, ammonifiers, and sulfur-oxidizing bacteria; however, it has led to an increase
in the total amount of microorganisms. Aldrin (insecticide) has also led to an increase in
the number of bacteria, enhanced soil respiration, and either stimulated or suppressed
Copyright: © 2022 by the authors.
nitrification and ammonification depending on the type of soil [2]. An assessment of
Licensee MDPI, Basel, Switzerland. the influence of 29 pesticides on respiration and nitrification has shown both positive
This article is an open access article and negative effects depending on the pesticide and the time of exposure [3]. All effects
distributed under the terms and have been observed at concentrations well above field application rates. The herbicides
conditions of the Creative Commons metribuzin and glyphosate are capable of suppressing carbon dioxide emissions from soils
Attribution (CC BY) license (https:// with low carbon content when applied at a level of 100 mg/kg [4]. A study of the effect of
creativecommons.org/licenses/by/ pyrethroid insecticides on nitrification, respiration, and dehydrogenase activity has shown
4.0/). that pesticides did not have a statistically significant long-term effect [5]. For the fungicide

Agronomy 2022, 12, 124. https://doi.org/10.3390/agronomy12010124 https://www.mdpi.com/journal/agronomy

Agronomy 2022, 12, 124 2 of 23

benomyl, which is also used in our study, no significant effect on microbiota was found by
the method of seeding and measuring soil respiration [6]. A laboratory experiment with
a set of pesticides designed to simulate comprehensive plant protection has shown the
likelihood of synergistic effects. To assess the state of the microbial community, researchers
also actively use indicators of enzymatic activity as well as dynamics of microbial biomass
in terms of carbon emission [7]. The most significant effects were exerted by preparations
containing the fungicides captafol and triadimefon, but only after the third treatment.
Often, individual phyla of microorganisms can be stimulated by pesticides, for example,
the insecticides hexachlorocyclohexane and forate at the recommended application rate had
positive effects on microorganisms involved in the nitrogen and carbon cycles [8]. Positive
effects on bacteria and fungi in the rice rhizosphere as a result of insecticide application
have been documented in a field experiment using the plate method and counting Colony
Forming Units (CFUs) [9].
It is also possible to study the effect of pesticides on microorganisms in pure cul-
tures. For example, the herbicides glyphosate and hexazinone have been found to have
a significant negative effect on ectomycorrhizal fungi in experiments in a liquid nutrient
medium [10]. However, it is not clear to what extent these results relate to soil; moreover,
only a small fraction of soil microorganisms can be efficiently grown in vitro. The study
of the effects of herbicides using BIOLOG methodology has not shown a significantly
higher sensitivity of this method compared to the classical methods for assessing the
mineralization of carbon and nitrogen [11].
At present, studies using classical methods are still ongoing. For example, the most
popular fungicides, carbendazim and tebuconazole, have been shown to exert no effect on
soil respiration and enzyme activity; the effects manifest themselves only at a concentration
of 100 mg/kg [12]. Imidacloprid (insecticide) has been shown to inhibit soil respiration
and enzymatic activity even when applied at a single application rate in loamy sand [13].
Respiration and enzymatic activity of tropical soils are less susceptible to the influence of
imidacloprid; at a single application rate, changes are weakly expressed [14].
The use of molecular genetic methods has made it possible to dramatically increase
the volume and quality of the data. For instance, the use of genetic fingerprinting methods
to separate the products of amplifications of the 16S rRNA region from total soil DNA
has revealed significant changes in the structure of the bacterial community under the
influence of herbicides [11,15]. An analysis of phylotypes based on the 16S rRNA genes of
the total soil DNA has shown the effect of methylparathion (insecticide) on the soil micro-
bial community that is manifested in the replacement of phylotypes [16]. As the results
of amplification of the V4 (16S rRNA) region followed by TGGE and band sequencing
show, carbendazim (fungicide) reduces the microbial diversity expressed as the Shannon in-
dex [17]. The fungicides penconazole and cyprodinil have a temporary effect on nitrification
in vineyard litters, but the changes in the abundance of archaea and ammonia-oxidizing
microorganisms caused by them persist for a much longer time; it should be noted that
ammonia-oxidizing bacteria are more sensitive to these fungicides [18]. The faster recovery
of nitrification can be explained by the excess microbial functions in the soil. Structural
changes have been identified using PCR-DGGE with DNA and cDNA fragments. RNA-
based analysis was found to be more informative. Ammonia-oxidizing Archaea (AOA)
and Bacteria (AOB) and are likely to be suitable target groups for monitoring the effects of
pesticides on the soil microbiota [19]. An analysis of fatty acid profile (PLFA) has shown
that imidacloprid has a temporary effect on the structure and abundance of fungal and
bacterial communities in the soil when applied at the rate of application. These data have
also been confirmed by DGGE [20]. The use of pyrosequencing has allowed identifying
changes in the structure of the soil microbiome under the influence of neonicotinoids,
in particular, concerning some families of Proteobacteria and Actinobacteria, which are
involved in the biodegradation of neonicotinoids [21].
The use of high throughput sequencing (NGS) and Taxon XOR analysis (Taxon Ex-
clusive Or) to assess the state of the soil microbial community under the influence of
Agronomy 2022, 12, 124 3 of 23

organochlorine pesticides (OCPs) (insecticides) has made it possible to determine that

some species are absent in contaminated soils and their absence can serve as a marker
of contamination [22]. Other species, on the contrary, are characteristic of soils contami-
nated with OCPs and are probably involved in the bioremediation of pollution. Escherichia
and Mortierella are the main genera were found inside the contaminated soil. The low
abundance of Nitrospirae species and the disappearance of Glomeromycota in contaminated
soil indicate that the use of OCPs leads to serious toxicological consequences. Sometimes
during the joint application of pesticides, certain smoothing of the effects is possible. For
example, soil microbial activity and functional diversity based on NGS data have shown
a tendency to suppression after carbendazim (fungicide) application and a tendency to
suppression–restoration–stimulation when treated with chlortetracycline (antibiotic) and
carbendazim simultaneously [23]. At the same time, repeated treatments with carben-
dazim do not lead to an increase in the resistance of the microbial community to it. The
genera of bacteria most sensitive to carbendazim are Bacillus, Pseudomonas, Arthrobacter,
Mycobacterium, Streptococcus, Burkholderia, and Corynebacterium.
High throughput sequencing of the 16s rRNA has shown that the number of OTU
significantly decreased in the samples of soils treated with imidacloprid in comparison
with the control samples and amounting to 31,173 and 21,909. The genus Gemmata has
completely disappeared in the soil treated with imidacloprid, whereas the species belonging
to the genus Prevotella (class Phycisphaerae) have been identified in treated samples [24].
Approximately 10 years ago, a consensus began to emerge regarding the assessment
of methods for determining the effects of pesticides on the soil microbial community. The
researchers came to realize that integral methods are not informative enough and that there
is a need for the use of DNA sequencing [25], as well as for the combined application of
classical and molecular methods. At the same time, the need to use new tools to assess the
effect of pesticides on soil microorganisms to satisfy the corresponding state regulations was
also discussed [26]. Currently, according to the Organization for Economic Co-operation
and Development (OECD) and EU rules, only nitrogen and carbon mineralization tests are
used to assess the effect of pesticides on soil microorganisms in framework of pesticides
registration [27–29]. Moreover, although scientific research has advanced significantly in
recent years, nevertheless, in the field of government regulation in the OECD countries,
there are no new standards.
A multilevel approach for assessing the effect of pesticides on microbial communities
has also been proposed; it includes the analysis of fatty acids and potential enzymatic
activity, as well as qPCR for individual phylogenetic groups [30].
A separate direction of research is the assessment of enzymatic activity by determining
the expression of the corresponding genes, which is probably more informative than the
direct determination of enzyme activity [31]. In addition, specific enzymes associated with
the destruction of herbicides have been proposed to be used as biomarkers of the effect
of the latter on the soil microbiome [32]. The genes encoding these enzymes are located
inside of the plasmids, who allows them to actively spread among bacteria. However, this
method does not provide any information about the ongoing changes in the structure of
the soil microbiome.
In our opinion, an insufficient number of informative studies aimed at the identifica-
tion of the most sensitive components of the microbiome has yet been carried out using
high-throughput sequencing allowing us to assess changes in the structure of the bacterial
community in response to pesticide treatment. To detect these components, it is important
to assess the effects of pesticides both individually and in the combined application. At the
first stage, it is preferable to carry out these assessments in laboratory conditions, where
the problem of sample homogeneity is not so acute.
Our task is to identify the most sensitive taxa to the introduction of pesticides. These
taxa can be used as universal indicators of pesticide contaminations in a soil.
Agronomy 2022, 12, 124 4 of 23

2. Materials and Methods

2.1. Chemical Substances
The experiment used formulations produced in local factories containing metribuzin
(an herbicide, CAS № 21087-64-9), imidacloprid (an insecticide, CAS № 138261-41-3), and
benomyl (a fungicide, CAS № 17804-35-2). Benomyl converts very quickly into the main
metabolite of carbendazim (CAS № 10605-21-7) in the soil. The main characteristics of
these pesticides are listed in Table 1. Metribuzin has a low sorption capacity (Koc = 38)
and a rather high solubility (~10.7 g/L), which determines its bioavailability. Imidacloprid
and carbendazim (the main metabolite of benomyl), due to their high persistence in soil
(DT50 > 60 to 120 days), can act on soil microorganisms for a long time. These three
pesticides can be used together in real field conditions, for example, on potatoes.

Table 1. Physical/Chemical Properties of pesticides.

Solubility—In Octanol–Water
Active Chemical Vapor Pressure at
Molecular Weight Water at 20 ◦ C Partition Coefficient
Substances Formula 20 ◦ C (mPa)
(mg/L) at pH 7, 20 ◦ C, log P
Metribuzin C8 H14 N4 OS 214.29 10,700 1.75 0.121
Imidacloprid C9 H10 ClN5 O2 255.66 610 0.57 4.0 × 10−7
Benomyl C14 H18 N4 O3 290.32 2 1.4 0.005
Carbendazim C9 H9 N3 O2 191.21 8 1.48 0.09

2.2. Soil and Experimental Design

Umbric Albeluvisols (IUSS Working Group WRB, 2014) soil was air-dried and sieved
through a 1 mm sieve. The main properties of the soil are presented in Table 2.

Table 2. Main soil properties.

Parameter Value
Organic matter (%)
1.5 ± 0.04
0–20 cm
pHwater 5.6 ± 0.2
Texture class (USDA)
Silty loam
0–25 cm
Soil bulk density, g/cm3 1.2 ± 0.1

Firstly, 100 g of soil was mixed in a mortar with 20 mL of water (control variants)
or with 20 mL of water supplemented with pesticide formulations or their mixture. The
application rate for the herbicide was 1.4 L/ha (0.98 kg/ha metribuzin), for the insecticide
0.1 L/ha (0.02 kg/ha imidacloprid), and for the fungicide 3 kg/ha (1.5 kg/ha benomyl).
There were also experimental variants with a 10-fold rate of application. This was necessary
to simulate a “worst-case” scenario where the concentration of pesticides was locally
overdosed (long-term pesticide application) or an overestimated rate was applied.
After that, the soil samples were placed in 250 mL glass jars. Umbric Albeluvisols soil
is rather poor (Corg = 1.5%); therefore, microbiological processes go on in it rather slowly,
which can complicate monitoring the state of the microbial community. Because of this, we
decided to include in the experiment variants with the initiation of succession due to the
addition of an additional carbon source. Preliminary experiments with measuring carbon
dioxide emissions showed that starch in an amount of 5 g/kg of air-dry soil was best suited
for this purpose. Water-soluble cellulose, glucose, sucrose were also tested. Along with
the starch, we also introduced mineral salts into the soil in order to avoid NPK limitation.
Moreover, K2 HPO4 and (NH4 )2 SO4 were introduced in an amount of 1 g/kg. The design of
the experiment was as follows in Table 3. Glass jars were placed in the thermostat at 20 ◦ C.
Agronomy 2022, 12, 124 5 of 23

Table 3. Experimental design.

Individual Application of
Mix of Pesticides Application
Experiment Pesticides
Option Substrate Sampling Time, Substrate Sampling Time,
Induction Days Induction Days
control yes 14 yes 7, 14, 28, 56
control no 14 no 7, 14, 28, 56
1-fold yes 14 yes 7, 14, 28, 56
1-fold no 14 no 7, 14, 28, 56
10-fold yes 14 yes 7, 14, 28, 56
10-fold no 14 no 7, 14, 28, 56

2.3. Sampling and DNA Extraction

Soil samples were extracted with a micro-drill from microcosms, so as to collect the
soil layer completely to the very bottom. Then the soil samples collected in this way were
placed in 1.5 mL test tubes and frozen.
Isolation of total DNA was carried out from 0.5 g samples of soil using the FastDNA®
SPIN Kit for Soil (MP Biomedicals, Irvine, CA, USA).

2.4. Assessment of Pesticide’s Residual Quantities

The analytical standards of active substances manufactured by Dr. Ehrenstorfer GmbH
(Augsburg, Germany) was used for quantitative determination. Benomyl was not deter-
mined because it is extremely unstable [33] and almost immediately turns into carbendazim
in soil. Absolute calibration with analytical standards was used for quantitation. The corre-
lation coefficient was 0.999.
Residual quantities were measured using an Agilent 1100 series HPLC (Agilent Tech-
nologies, Santa Clara, CA, USA) system with DAD. Wavelength: carbendazim—280 nm,
metribuzin—290 nm, imidacloprid—270 nm. Chromatographic column was Phenomenex
Synergy Polar-RP 80A C18 150 × 4.6 mm, 4 µm. Eluents are water (A) and acetonitrile (B)
with the addition of acetic acid (0.1%) in a gradient mode (0 min—20% «B»; 12 min—80%
«B»). Extraction of pesticides was carried out from 5 g of soil with a mixture of acetonitrile
and water (95:5) for 15 min in 50 mL centrifuge flacks on a vibration platform at 2400 rpm
with an amplitude of 4 mm, followed by sonication in an ultrasonic bath for 5 min at
160 kHz. To increase the efficiency of extraction on the platform, three metal balls with a
diameter of 5 mm were added to each tube. Successively the extracts were centrifuged for
5 min at 13,400× g, the supernatant was additionally filtered through a filter with a pore
diameter of 0.45 µm. The extraction was performed twice and the extracts were combined
and subjected to evaporation until dry on a rotary evaporator at 40 ◦ C. The extract was
dissolved with 1 mL of acetonitrile, treated for 30 s in an ultrasonic bath, and transferred
into chromatographic vials. The analysis was carried out for three independent samples.
The soils samples were analyzed with the addition of analytical standards of active
ingredients at the level of 0.01 and 0.1 mg/kg to determine the metrological characteristics
of the method. The efficiency of extraction was for metribuzin—93 ± 1%, carbendazim—
90 ± 1%, imidacloprid—91 ± 1%.
Moreover, some samples were analyzed using an Agilent 1200 series HPLC with a
quadrupole time-of-flight mass spectrometric detector (6520 Accurate-Mass Q-TOF LC/MS,
ionization source electrospray (Agilent Technologies, Santa Clara, CA, USA), column
Phenomenex Hydro-RP C18 4 µm 4.6 × 100 mm, the mobile phase was water and methanol
with the addition of formic acid (10 mM), the volume of the injected sample is 5 µL to
search for metabolites.

2.5. Amplification and DNA Sequencing

Amplification of the variable region V4 (16S rRNA) was carried out in one round
using forward and reverse primers 515F (5′ -GTGCCAGCMGCCGCGGTAA-3′ ) and 806R
Agronomy 2022, 12, 124 6 of 23

(5′ -GGACTACHVGGGTWTCTAAT-3′ ) [34] with dual index sample multiplexing. These

primers are specific to both bacteria and archaea. Amplification of the variable region ITS1
(18S rRNA) was carried out using the following primers BITS (5′ -CTACCTGCGGARGGAT
CA-3′ ) and B58S3 (5′ -GAGATCCRTTGYTRAAAGTT-3′ ) [33]. The PCR products were
purified using the Cleanup Mini Kit (Evrogen, Moscow, Russia) for DNA isolation. The
concentration of the obtained libraries of 16S rRNA and ITS1 in solution was measured
on a Qubit® fluorometer (Invitrogen, Waltham, MA, USA) using a Quant-iT™ dsDNA
High-Sensitivity Assay Kit. The purified amplicons were mixed in an equimolar way in ac-
cordance with the concentrations obtained, and the quality of the resulting library prepared
for sequencing was assessed with agarose gel electrophoresis. Further samples preparation
and sequencing of the pooled samples were carried out using a MiSeq sequencer (Illumina,
San Diego, CA, USA) and the MiSeq Reagent Kit v2 (500 cycles).

2.6. Sequencing Data Processing

Bioinformatics analysis was carried in R version 3.6.3 (R Core Team, 2018) using the
package DADA2 (Divisive Amplicon Denoising Algorithm). For fungal 18S rRNA gene
sequencing, forward and reverse reads have been pre-merged at the initial step using
MeFiT tool because of high diversity of the amplicon lengths. The derived RSV (Ribosomal
Sequencing Variants) lengths were about 253 bp (with minimal variability) for bacterial 16S
V4 fragments and 140 to 343 for fungal ITS1 (18S rRNA) regions.
In contrast to Operation Taxonomic Units (OTUs), the current analysis, based on RSVs
(also referred to as Amplicon Sequence Variants (ASVs) or Exact Sequence Variants (ESVs)),
does not imply merging of closely related amplicon variants (<3% differences) into a single
consensus sequence (i.e., OTU), and, therefore, can distinguish single-nucleotide differences
between species [35].
For taxonomic annotation of the derived RSV sequences, we also used the DECIPHER
Bioconductor package supplied with SILVA v132 reference database [36] and the UNITE
ITS database v. 8.0 [37]. RSVs annotated as chloroplasts, mitochondria, cercozoa, etc., have
been removed. Raw data loaded in NCBI (BioProject ID PRJNA723173). The derived read
counts data were normalized between the samples using the number of reads annotated at
the Kingdom level.
The remaining analysis was also conducted in R environment (version 3.6.3). To
assess alpha diversity, we calculated Shannon, Simpson, Chao1, and ACE indexes using
fossil 0.4.0 and vegan 2.5–6 packages. When calculating these indices, the read counts
data were rarefied to match the sample with the minimum number of reads. For beta
diversity analysis, we used Bray–Curtis and Jaccard metrics. The remaining analyses
and visualization were performed using phyloseq 1.30.0, plotly 4.9.2.2, phytools 0.7–47,
pheatmap 1.0.12, ggplot2 3.3.3 packages. To visualize the differences between samples,
metrical and non-metrical multidimensional scaling (MDS) was applied. The association
analysis between period time and taxon abundances was performed using Spearman
and Pearson correlations. For non-paired comparison of two groups, we used the Mann–
Whitney U test, and for paired comparisons, we applied the Wilcoxon test. FDR was
calculated using Benjamini–Hochberg adjustment for the derived p-values.

3. Results
3.1. Analysis of Pesticides Residual Amounts
Pesticides residues were analyzed on days 0, 7, 14, 28, and 56 [38]. The DT50 of the
three pesticides were calculated using the first order single phase kinetic equation [39],
(Table 4).
Agronomy 2022, 12, 124 7 of 23

Table 4. Half-life (t1/2) of active substances with standard deviation, day (n = 3).

Benomyl
Imidacloprid Metribuzin
(Carbendazim)
1-fold
86 ± 4 104 ± 7 41 ± 1
−SMM
10-fold
52 ± 5 57 ± 3 20 ± 0.3
−SMM
1-fold
78 ± 7 102 ± 8 37 ± 1
+SMM
10-fold
50 ± 2 42 ± 2 20 ± 1
+SMM

At a tenfold application rate, the pesticides were decomposed much faster, which was
probably due to that they were not adsorbed and remained available to microorganisms.
The addition of starch did not affect the rate of decomposition of the pesticides.
On the 56th day of the experiment, the residual amounts of pesticides were benomyl
(by carbendazim)—1.57 mg/kg at a single (1×) dose rate and 12.51 mg/kg at a 10-fold
(10×) dose rate, which corresponds to 63 and 50% of the first application; imidacloprid—
0.057 mg/kg at 1× dose rate and 0.44 mg/kg at 10× dose rate, which corresponds to 69
and 53% of the application level; metribuzin—0.52 mg/kg at 1× dose rate and 3.65 mg/kg
at 10× dose rate, which corresponds to 45 and 31% of the application level.

3.2. Alpha Diversity of Soil Prokaryotic and Fungal Communities

The study showed that pesticides have practically no effect on the alpha diversity
of the soil bacterial community, in both situations, with the addition of a starch–mineral
mixture (SMM) and without SMM (Table 5). There was a very small decrease in the Shannon
and Chao1 indexes (by 10–15%), but it was not statistically significant (p = 0.1 . . . 0.2).

Table 5. Indices of alpha diversity of the bacterial community at the genus level in all variants of the
experiment.

Treatment, and Bacteria −SMM Bacteria +SMM

Sampling Time, Days Shannon Chao1 Shannon Chao1
Control 7 d 3.17 237.5 2.98 178.8
Mix 1–7 d 2.98 224.6 2.04 98.0
Mix 10–7 d 3.35 229.1 2.24 147.8
control 14 d 3.19 164.5 2.45 140.1
mix 1–14 d 2.99 180.1 2.67 159.0
mix 10–14 d 3.12 152.4 2.28 136.6
control 28 d 3.36 169.3 2.97 195.0
mix 1–28 d 3.16 136.8 2.58 145.4
mix 10–28 d 3.31 178.5 2.61 214.3
control 56 d 3.63 172.6 3.08 222.6
mix 1–56 d 3.38 135.7 3.03 194.6
mix 10–56 d 3.40 162.0 2.39 207.9
control 14 d 3.15 170.2 3.10 180.1
imidacloprid 1–14 d 2.90 119.5 2.98 143.4
imidacloprid 10–14 d - - 2.96 145.3
Benomyl 1–14 d 3.05 100.0 2.80 151.6
benomyl 10–14 d 2.93 111.1 2.52 141.4
metribuzin 1–14 d 2.91 103.0 3.10 177.6
metribuzin 10–14 d - - 2.97 149.0

However, the application of the SMM itself resulted in a decrease in alpha diversity—at
genus level, and the average Shannon index decreased from 3.1 to 2.04 (p-value < 0.001 for
Agronomy 2022, 12, 124 8 of 23

Welch’s t-test, Mann–Whitney U test, paired Wilcoxon test) (Table 5). The same observation
was made at RSV level too (p < 0.01 for all these tests).
By the 56th day, the alpha diversity in both variants (with and without SMM) of the
experiment increased according to the Shannon index, but decreased according to the
Chao index. Most likely, this indicates that the composition of the bacterial community
became more balanced over time (more taxa began to have a significant proportion in the
bacterial community), but many minor taxa that were originally present in the soil die
with time. This trend was observed both for control samples and for samples treated with
pesticides. At the same time, in general, temporary succession made a significantly greater
contribution to the structure of bacterial communities than treatment with pesticides; the
addition of SMM produced an even greater effect. It should be noted that alpha diversity
was more affected by pesticides when we added the starch–mineral mixture. However,
these observations are only trends; no statistically significant differences were noticed here.
Surprisingly, we also noticed a strong positive correlation between the rarefied Chao1 and
ACE, Jackknife indices and initial read counts derived for samples.
The analysis of alpha diversity indices for soil fungal communities showed that the
addition of SMM led to a decrease in diversity at genus level. For instance, at the genus
level, the average value of the Shannon index decreases from 2.07 to 1.15 (p < 0.001 for
Welch’s, Mann–Whitney and Wilcoxon tests) (Table 6). The same is applied for Chao1 index
(decrease from 52 to 33 at genus level; p < 0.01 for all listed tests) (Table 6). These differences
were also observed at RSV level and were also statistically significant.

Table 6. Indices of the alpha diversity of the fungal community at the genus level in all variants of
the experiment.

Treatment and Sampling Fungi −SMM Fungi +SMM

Time, Days Shannon Chao1 Shannon Chao1
control 7 d 2.60 63.0 1.43 20.0
mix 1–7 d 1.58 31.0 1.80 36.0
mix 10–7 d 2.05 59.0 1.80 77.8
control 14 d 1.68 46.0 0.33 15.0
mix 1–14 d 2.55 59.0 0.87 28.5
mix 10–14 d 2.08 66.3 1.88 81.8
control 28 d 1.98 60.0 0.20 16.0
mix 1–28 d 2.43 52.0 1.44 30.0
mix 10–28 d 2.12 50.0 1.58 43.5
control 56 d 2.47 58.0 0.35 13.0
mix 1–56 d 1.55 38.0 0.51 15.3
mix 10–56 d 2.00 47.3 1.23 13.0
control 14 d 2.30 53.0 0.98 30.0
benomyl 1–14 d 1.55 40.0 0.68 19.0
benomyl 10–14 d 2.12 68.3 2.22 59.8

At the same time, in the variant with SMM, treatment with pesticides led to increase
in diversity from 0.20 to 1.88 according to the Shannon index compared to the control
variants (Table 6). The application of pesticides (at 10× concentrations; both benomyl and
a mixture of three pesticides containing benomyl) was accompanied by dramatic shifts in
the composition of the fungal community, the elimination of dominant species, and, as a
consequence, a striking increase in the proportion of many other taxa and the resulting
increase in diversity according to the Shannon index. Generally, the SMM was causing
an avalanche-like restructuring of the entire fungi community. For example, Humicola,
which was actively growing on starch and accounted for 60% of the total fungal community
(without SMM—only 5%), was up to 300 times reduced under treatment with benomyl. The
effect of pesticides (partially, benomyl) was less noticeable in the variants without SMM.
Agronomy 2022, 12, 124 9 of 23

3.3. Taxonomic Structure of Soil Bacterial and Fungal Communities

The addition of SMM led to a change in dominants and a restructuring of the bacterial
community. For the control, the dominants were the phyla Proteobacteria, Actinobacteria
and Firmicutes (Figure 1) and the genera Thermomonas, Sphingomonas, and Pseudoarhtrobacter
(Figure 2).

Figure 1. Bacteria’s phyla in all variants of the experiment without SMM (A) and with SMM (B).

After the application of SMM, the representation of Actinobacteria sharply increased

while the representation of Proteobacteria decreased (Figure 1).
The genus Streptomyces sharply increased in the variant with SMM, while genus
Thermomonas decreased in the variant with SMM (Figure 2).
Agronomy 2022, 12, 124 10 of 23

Figure 2. Bacteria’s genus in all variants of the experiments without SMM (A) and with SMM (B).
Figure 2. Bacteria’s genus in all variants of the experiments without SMM (A) and with SMM (B).

The observed changes were expected since the proportion of bacteria actively decom-
posing complex substrates increased. The application of pesticides led to a change in the
dominants of the bacterial community both in the control and in the soil supplemented
with SMM.
A pairwise comparison using the Wilcoxon test showed an impact of the addition of
pesticides on the abundance of several bacterial phyla (p ≤ 0.05). The pesticide application
with SMM led to a reduction in the relative abundance≤ of bacterial phyla Myxococcota,
Bacteroidetes, Gemmatimonadetes, Proteobacteria. The relative abundance of Acidobac-
teria, Chloroflexi, and Planctomycetes was also decreased in the variant without SMM.
The phylum Actinobacteria increased both in with and without SMM addition, which was
associated with the high hydrolytic activity of this bacteria (Table 7).
Treatment with pesticides (at 1× and 10× concentrations) affected 29 genera in the
variant without SMM and 28 genera in the variant with SMM; among them, Catenulispora,
Sphingomonas, Terrabacter, Haliangium, Bradyrhizobium, Burkholderia, and Mycobacterium were
affected by pesticides in both variants (Table 8).
Agronomy 2022, 12, 124 11 of 23

Table 7. Pairwise comparison of abundances of various bacterial phyla between control and treated soil samples.

Log2(Fold Change)
Reads Mean Wilcoxon
Phylum (Aver- Mix1_ Mix10_ Mix1_ Mix10_ Mix1_ Mix10_ Mix1_ Mix10_ Imi1_ Imi10_ Ben1_ Ben10_ Met1_ Met10_
LogFC p CTRL CTRL 7 CTRL CTRL CTRL CTRL CTRL CTRL CTRL CTRL CTRL14 CTRL CTRL CTRL
age)
7d d 14 d 14 d 28 d 28 d 56 d 56 d 14 d 14 d d 14 d 14 d 14 d
Without SMM
Acidobacteria 908 −0.54 0.0009 −0.65 0.05 −0.98 −0.41 −0.42 0.15 −0.06 −0.40 −0.96 −0.22 −0.75 −0.85 −0.93 −0.69
Actinobacteria 5970 0.14 0.002 0.41 0.72 −0.08 0.35 0.08 0.11 0.18 0.13 −0.05 0.11 0.26 0.10 0.09 −0.09
Bacteroidetes 380 −0.27 0.0009 −0.69 −0.31 0.01 −0.10 −0.13 0.08 −0.29 −0.29 −0.57 −1.24 −0.04 −0.26 −0.26 −0.39
Chloroflexi 407 −0.59 0.0009 −0.41 0.20 −0.89 −0.39 −0.31 −0.03 −0.09 −0.35 −1.03 −0.12 −1.17 −1.46 −1.11 −1.51
Myxococcota 270 −0.38 0.0006 −0.07 0.16 −0.18 −0.46 −0.34 −0.58 −0.90 −0.75 −0.43 −1.79 −0.18 −0.24 −0.11 −0.52
Planctomycetes 201 −1.03 0.004 −0.83 0.45 −1.35 −0.63 −1.44 −0.49 0.39 −0.68 −1.38 −0.50 −1.21 −1.82 −1.80 −2.61
Verrucomicrobia 297 −0.33 0.09 −0.61 0.48 −1.31 −0.33 −0.63 −0.01 0.82 −0.20 −1.33 0.33 −1.05 −1.07 −1.10 −1.23
With SMM
Actinobacteria 21,392 0.22 0.0009 0.50 0.32 −0.03 −0.01 0.02 0.15 0.05 0.31 0.28 0.26 0.28 0.46 0.16 0.34
Bacteroidetes 423 −0.82 0.01 −2.32 −1.03 −0.82 −1.74 −1.19 −1.82 0.42 −0.34 −0.66 −0.33 −0.61 −0.32 −0.18 −1.14
Gemmatimonadetes 276 −0.23 0.04 −0.70 −0.23 0.16 −0.28 0.29 −0.64 0.05 −0.82 −0.11 −0.37 −0.10 −0.57 −0.02 0.00
Myxococcota 209 −0.36 0.02 −1.03 −0.23 0.47 0.16 0.02 0.06 −0.27 −1.06 −0.68 −0.38 −0.32 −1.00 −0.38 −0.37
Proteobacteria 6242 −0.51 0.0002 −1.76 −1.36 0.04 −0.31 −0.30 −0.75 −0.07 −0.97 −0.44 −0.33 −0.38 −0.69 −0.22 −0.73
Explanation: Mix—the mixture of three pesticides; 1—the recommended application rate; 10—the ten-fold application rate; CTRL—the control; 7, 14, 28, 56 days—sampling time; Imi,
Ben, Met—imidacloprid, benomyl, metribuzin; SMM—the starch–mineral mixture. Abundances of microbial phyla with positive scores are marked with red color, abundances with
negative scores—with blue color. The degree of difference is shown by the brightness of the color.
Agronomy 2022, 12, 124 12 of 23

Table 8. Pairwise comparison of abundances of various bacterial genera between control and treated soil sample.

Log2(Fold Change)

Met10_CTRL 14 d
Ben10_CTRL 14 d
Mix10_CTRL 14d

Mix10_CTRL 28d

Mix10_CTRL 56d

Imi10_CTRL 14d
Mix10_CTRL 7 d

Mix1_ CTRL 28d

Met1_CTRL 14 d
Mix1_CTRL 14d

Mix1_CTRL 56d

Ben1_CTRL14 d
Imi1_CTRL 14d
Mix1_CTRL 7d
Reads Mean Wilcoxon
Genus LogFC
(Average) p

Without SMM
Actinoplanes 31 −0.22 0.05 −0.39 −0.57 0.15 −0.30 −0.01 0.12 0.00 −0.08 −0.06 −0.70 −0.68
Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium 28 −0.72 0.02 0.06 0.03 −0.20 −0.03 0.22 −1.02 −1.37 −0.65 −3.45 −1.43 −1.31 −1.28
Arenimonas 40 −0.58 0.0001 −0.55 −0.59 −0.47 −0.93 −0.68 −1.34 −0.26 −0.49 −0.42 −0.52 −1.35 −0.40 −0.14 −0.75
Bradyrhizobium 383 −0.19 0.004 −0.03 0.17 −0.28 −0.21 −0.31 −0.23 −0.50 −0.40 0.07 −0.19 −0.19 −0.15 0.00 −0.28
Bryobacter 102 −0.17 0.01 −0.32 −0.07 −0.17 −0.18 −0.23 −0.06 −0.21 0.05 0.31 −0.42 −0.61 −0.19 −0.17 −0.09
Burkholderia–Caballeronia–Paraburkholderia 185 0.20 0.009 −0.11 0.05 0.03 0.16 0.41 0.31 −0.14 0.51 0.04 0.49 0.43 0.41 0.07 0.13
Candidatus Solibacter 20 −0.59 0.03 −0.52 −0.58 −0.51 −0.81 −0.64 −0.46
Catenulispora 43 0.45 0.0007 0.52 0.50 0.23 0.08 0.55 0.39 0.38 0.24 −0.29 0.42 1.33 0.90 0.68
Cohnella 20 −0.45 0.03 0.48 −0.71 −0.59 −1.07 −0.61 −0.32 −0.41 −0.40 −0.39 −0.15
Ellin6067 126 −0.30 0.02 −0.44 −0.60 −0.50 −0.64 −0.14 −0.23 −0.45 −0.75 0.26 0.36 −0.38 0.08 −0.27 −0.04
Gaiella 50 −0.31 0.003 −0.04 0.16 −0.03 −0.35 −0.16 −0.17 −0.74 −0.77 −0.02 −2.82 −0.21 −2.82 −0.25 −0.33
Haliangium 132 −0.40 0.002 −0.20 −0.10 −0.10 −0.61 −0.12 −0.93 −0.90 −0.97 −0.36 −4.08 −0.40 −0.13 0.24 −0.23
Hyphomicrobium 126 −0.36 0.02 −0.44 −0.21 −0.05 −0.04 −0.29 −0.55 −1.16 −0.53 0.40 −3.92 −0.20 −0.45 −0.84 0.68
Janthinobacterium 19 −0.81 0.03 −1.47 −1.25 −0.30 −0.55 −0.86 −0.42
Kitasatospora 464 1.03 0.0001 1.11 1.73 1.20 1.35 0.63 0.70 1.09 1.03 0.93 1.03 1.40 0.90 1.01 0.63
Marmoricola 45 0.91 0.01 0.64 1.12 1.30 0.89 0.07 −0.94 1.12 1.36 1.30 1.23 0.54
Massilia 953 −0.12 0.02 −0.59 −0.61 −0.04 −0.26 0.01 −0.09 −0.21 0.03 −0.34 0.00 −0.20 0.07 −0.12 0.07
Mycobacterium 570 −0.22 0.001 −0.13 0.12 0.02 −0.11 −0.34 −0.21 −0.45 −0.32 −0.21 −0.43 −0.01 −0.28 −0.12 −0.58
Occallatibacter 39 −0.73 0.005 −0.43 0.13 −1.57 −0.49 −0.72 −0.17 −0.59 −0.62 −1.03 0.70 −1.73 −1.07 −0.61 −1.74
Agronomy 2022, 12, 124 13 of 23

Table 8. Cont.

Log2(Fold Change)

Met10_CTRL 14 d
Ben10_CTRL 14 d
Mix10_CTRL 14d

Mix10_CTRL 28d

Mix10_CTRL 56d

Imi10_CTRL 14d
Mix10_CTRL 7 d

Mix1_ CTRL 28d

Met1_CTRL 14 d
Mix1_CTRL 14d

Mix1_CTRL 56d

Ben1_CTRL14 d
Imi1_CTRL 14d
Mix1_CTRL 7d
Reads Mean Wilcoxon
Genus LogFC
(Average) p

Paenisporosarcina 393 0.35 0.002 0.54 1.31 0.51 0.61 0.19 0.61 −0.22 0.20 0.40 −0.08 0.54 0.27 0.23 0.04
Phycicoccus 154 0.39 0.01 0.67 0.97 −0.40 0.34 0.32 0.74 0.72 0.14 −0.38 1.38 0.47 0.03 0.14 0.30
Ramlibacter 289 0.25 0.02 −0.51 −0.34 −0.01 0.02 0.52 0.11 0.51 0.10 0.11 0.42 0.41 0.34 0.50 0.50
Sphingomonas 2893 0.16 0.001 0.10 0.00 0.17 0.27 0.01 0.02 −0.09 0.09 0.30 0.28 0.13 0.46 0.36 0.26
Streptacidiphilus 55 0.65 0.0005 0.30 0.79 0.26 0.54 0.79 0.25 0.69 0.96 0.83 1.22 0.84 0.47
Streptomyces 325 0.74 0.0001 1.33 1.69 0.34 0.91 0.53 0.76 0.13 0.58 0.65 0.55 1.14 0.75 0.72 0.77
Terrabacter 592 0.62 0.0009 0.67 0.92 −0.34 1.12 0.51 1.20 1.03 0.58 −0.10 1.09 0.31 0.35 0.19 0.54
Terracidiphilus 48 −0.52 0.05 −0.77 −0.17 −0.96 0.08 −0.28 0.53 0.61 −0.20 −1.38 −3.53 −0.32 −1.22 −3.53 0.06
Thermomonas 6779 0.12 0.05 0.19 −0.33 0.29 0.00 0.16 0.06 0.35 0.42 0.20 −0.12 −0.02 0.09 0.06 0.20
Xylophilus 31 0.17 0.01 0.27 0.30 0.43 0.06 0.12 −0.06 0.19 0.29 −0.02 0.13
With SMM
Aminobacter 58 −0.33 0.05 −1.39 −0.87 −0.10 −0.25 0.73 −0.45 0.16 0.01 −0.48 −0.47 −0.28 −0.01
−0.46 −0.81
Arachidicoccus 17 −2.78 0.008 −0.08 −1.06 −4.13 −3.34 −1.71 −3.34 −3.34
−3.34 −3.34
Bradyrhizobium 318 −0.28 0.02 −0.78 −0.50 0.63 −0.14 0.10 −0.89 0.07 −0.88 −0.23 −0.17 −0.09 −0.60
−0.15 −0.25
Bryobacter 32 −0.50 0.01 −0.90 −1.03 0.62 −0.45 0.24 −0.43 −0.26 −1.53 −0.91 −0.07 −0.65 −1.90
−0.22 −0.08
Burkholderia–Caballeronia–Paraburkholderia 109 −0.31 0.02 −0.74 −0.79 0.26 −0.61 −0.19 −1.00 0.07 −1.72 0.02 −0.24 0.18 −0.05
−0.08 −0.44
Catenulispora 25 −0.55 0.04 0.84 −0.01 −1.46 −0.34 −0.59 −0.72
−0.44 −1.17
Conexibacter 56 −0.29 0.03 −0.24 −0.23 −0.12 −0.56 −0.39 −0.40 −0.58 −1.30 −0.28 0.03 −0.13 0.26−0.64 0.51
Devosia 56 −0.55 0.01 −1.02 −0.99 0.08 −0.21 −1.29 −0.73 0.59 −0.84 −0.59 −0.32 −0.26 −0.24
−0.26 −1.23
Dyella 862 −1.35 0.0001 −4.42 −4.79 −0.08 −1.56 −2.15 −2.79 −0.92 −3.78 −0.51 −0.10 −0.28 0.00−0.67 −0.76
Edaphobacter 15 0.51 0.03 1.78 0.41 0.17 0.06 0.310.36
Frateuria 504 −1.31 0.001 −6.47 −6.47 −0.03 −1.47 −2.01 −1.96 −0.48 −3.50 −0.74 −0.43 −0.41 −0.72
−0.41 −1.34
Gemmatimonas 191 −0.21 0.05 −0.91 −0.21 0.24 −0.27 0.36 −0.54 −0.03 −0.70 −0.11 −0.43 −0.03 −0.50
−0.07 0.04
Hyphomicrobium 93 −0.46 0.02 −1.06 −0.88 0.28 −0.11 0.52 −0.22 −0.62 −1.00 −0.95 −0.32 −0.39 0.17−1.07 −0.24
Janibacter 30 0.84 0.02 3.37 −0.05 −0.31 0.76 0.51 1.97 0.86 0.86 0.98
Jatrophihabitans 63 −0.93 0.002 −3.74 −0.59 0.18 −3.61 −1.04 −0.53 −2.12 −0.54 0.08 −0.72 −0.43 −0.02 −0.71
Kribbella 246 −0.97 0.02 −2.32 −3.47 −0.99 −0.62 −6.59 −1.41 0.90 −2.41 −0.67 −0.10 0.10 0.08 −0.38 −0.90
Leifsonia 171 0.22 0.02 0.19 0.54 0.24 0.88 0.18 0.20 0.21 0.96 0.08 0.22 0.00 −0.16 0.29 −0.46
Luteimonas 33 −0.48 0.006 −1.12 −0.09 0.12 −0.60 −1.31 −0.84 −1.98 −0.55 0.06 −0.40 −0.34 0.13 −0.18
Agronomy 2022, 12, 124 14 of 23

Table 8. Cont.

Log2(Fold Change)

Met10_CTRL 14 d
Ben10_CTRL 14 d
Mix10_CTRL 14d

Mix10_CTRL 28d

Mix10_CTRL 56d

Imi10_CTRL 14d
Mix10_CTRL 7 d

Mix1_ CTRL 28d

Met1_CTRL 14 d
Mix1_CTRL 14d

Mix1_CTRL 56d

Ben1_CTRL14 d
Imi1_CTRL 14d
Mix1_CTRL 7d
Reads Mean Wilcoxon
Genus LogFC
(Average) p

Marmoricola 33 −0.62 0.05 −3.44 −0.10 −0.70 −0.39 −0.45 0.02 0.37 −2.12 −0.34 −0.86
Mycobacterium 841 −0.29 0.009 −0.75 −0.08 0.19 −0.28 −0.33 −0.61 0.17 −1.16 −0.24 −0.12 −0.42 −0.47 −0.06 −0.29
Nakamurella 154 −0.42 0.02 −0.79 −0.73 0.63 −0.42 −0.70 −0.99 −0.40 −1.24 −0.04 −0.03 −0.27 −0.48 0.30 −0.32
Ochrobactrum 49 0.50 0.05 0.36 0.11 −0.80 0.81 0.01 0.69 1.04 1.57
Paenarthrobacter 62 0.44 0.05 1.47 0.89 0.55 −0.10 0.95 0.85 −0.90 0.74−0.85 0.36 0.25 −0.24 0.17 1.36
Paenisporosarcina 278 −0.28 0.01 −1.92 −1.69 0.71 −0.11 0.03 −0.64 −0.54 0.23
−0.16 −0.22 −0.09 −0.36 −0.03 −0.67
Pseudolabrys 102 −0.24 0.05 −0.97 −0.37 0.30 −0.36 0.49 −0.35 −0.22 −0.89
−0.12 −0.12 −0.10 −0.49 −0.18 −0.12
Ramlibacter 38 −0.25 0.05 −2.88 −0.19 0.54 −0.01 0.38 −0.52 −0.02 −0.32 −0.62 −0.64 −0.38 −0.14
Sphingomonas 1073 −1.08 0.0006 −2.92 −2.62 0.22 −0.40 −1.37 −2.12 0.18 −2.61 −0.52 −0.48 −0.57 −1.12 −0.38 −1.19
Thermomonas 228 −0.64 0.004 −2.54 −2.72 0.22 −0.18 0.22 −0.53 −0.16 −0.26 −0.55 −0.77 −0.81 −1.27 −0.30 −1.61
Explanation: Mix—the mixture of three pesticides; 1—the recommended application rate; 10—the ten-fold application rate; CTRL—the control; 7, 14, 28, 56 days—sampling time; Imi,
Ben, Met—imidacloprid, benomyl, metribuzin; SMM—the starch–mineral mixture; Abundances of microbial genera with positive scores are marked with red color, abundances with
negative scores—with blue color. The degree of difference is shown by the brightness of the color.
Agronomy 2022, 12, 124 15 of 23

Note that the genus Terrabacter increased while Haliangium, Bradyrhizobium, and My-
cobacterium decreased in both situations, with and without SMM addition. The genus
Sphingomonas slightly increased in the variant without SMM; after the addition of SMM, its
share decreased by about 1.5 times.
The addition of SMM led to a radical restructuring of the soil fungi community.
Mucoromycota practically disappeared while the share of Ascomycota, on the contrary,
increased (Figure 3).

Figure 3. Fungi’s phylum in all variants of the experiments without SMM (A) and with SMM (B).

In the control, the dominants were the genera Mortierella, Saitozyma, Apiotrichum,
Humicola, Solicoccozyma, Fusarium, and Trichoderma (Figure 4).
Agronomy 2022, 12, 124 16 of 23

Figure 4. Fungi’s genus in all variants of the experiments without SMM (A) and with SMM (B).

After the application of SMM, the share of Humicola in the soil significantly increased
with a sharp decline in the representation of Mortierella, Saitozyma, and Solicoccozyma.
The genus Trichoderma completely disappeared (Figure 4). The dominants of the fungal
community turned out to be susceptible to the influence of pesticides, in particular, benomyl,
especially a 10× dose rate (Figure 4).
Treatment with pesticides led to an increase in the relative abundance of Basidiomycota
with a decrease in the share of Ascomycota, which was confirmed by paired samples
Wilcoxon test (p ≤≤ 0.05; Table 9).
The results of this test have shown that the effect of pesticides on the fungal community
was significantly higher with SMM at the phylum level.
A pairwise comparison using the Wilcoxon test (p ≤ 0.05) showed that exposure to
pesticides in the variant without SMM affected the genus Apiotrichum (Table 10).
Agronomy 2022, 12, 124 17 of 23

Table 9. Paired comparison of abundances of various fungal phyla between control and treated soil samples.

Log2(Fold Change)
Reads Mean Wilcoxon
Phylum LogFC Mix1_ Mix10_ Mix1_ Mix10_ Mix1_ Mix10_ Mix1_ Mix10_ Ben1_ Ben10_
(Average) p
CTRL 7 d CTRL 7 d CTRL 14 d CTRL 14 d CTRL 28 d CTRL 28 d CTRL 56 d CTRL 56 d CTRL14 d CTRL 14 d
Without SMM
Ascomycota 11,935 −0.49 0.02 −0.73 −0.59 0.34 −0.67 0.04 −0.55 −1.05 −0.71 −0.31 −0.43
With SMM
Ascomycota 27,377 −0.46 0.004 −0.46 −1.70 −0.14 −1.16 −0.04 −0.70 −0.03 0.00 −0.11 −1.06
Basidiomycota 5978 2.77 0.004 1.67 3.12 3.22 5.66 2.53 6.12 1.90 −1.26 0.27 3.77
Mucoromycota 136 2.09 0.05 0.38 3.64 1.93 4.56 1.37 3.16 −1.94 2.05
Explanation: Mix—the mixture of three pesticides; 1—the recommended application rate; 10—the ten-fold application rate; CTRL—the control; 7, 14, 28, 56 days—sampling time;
Ben—benomyl; SMM—the starch–mineral mixture; Abundances of microbial phyla with positive scores are marked with red color, abundances with negative scores—with blue color.
The degree of difference is shown by the brightness of the color.

Table 10. Paired comparison of abundances of various fungal genera between control and treated soil samples.

Log2(Fold Change)

Ben10_CTRL 14 d
Mix10_CTRL 14d

Mix10_CTRL 28d

Mix10_CTRL 56d
Mix10_CTRL 7 d

Mix1_CTRL 14d

Mix1_CTRL 28d

Mix1_CTRL 56d

Ben1_CTRL14 d
Mix1_CTRL 7d

Reads Mean Wilcoxon

Genus LogFC
(Average) p

Without SMM
Acremonium 139 −0.87 0.002 −0.67 −0.33 −1.03 −0.06 −1.71 −0.62 −0.90 −0.81 −1.21 −1.36
Apiotrichum 6385 1.30 0.010 1.73 1.96 −0.22 1.59 −0.10 1.34 1.74 2.05 0.79 1.35
Didymella 150 −1.02 0.04 −0.81 −1.96 −2.51 −1.31 −1.19 0.36 0.44 −0.77 −4.43 0.02
Gibberella 23 −2.20 0.05 −0.11 −3.21 −3.17 −3.17 −1.33 −3.67 2.77
Plectosphaerella 1171 −0.40 0.03 −0.62 −0.89 −0.69 −0.18 −0.05 −0.23 0.11 0.48 −1.51 −0.64
Trichocladium 232 −1.29 0.05 −3.10 −2.23 1.53 0.37 0.81 −1.20 −1.95 −1.53 −2.96 −1.65
Agronomy 2022, 12, 124 18 of 23

Table 10. Cont.

Log2(Fold Change)

Ben10_CTRL 14 d
Mix10_CTRL 14d

Mix10_CTRL 28d

Mix10_CTRL 56d
Mix10_CTRL 7 d

Mix1_CTRL 14d

Mix1_CTRL 28d

Mix1_CTRL 56d

Ben1_CTRL14 d
Mix1_CTRL 7d
Reads Mean Wilcoxon
Genus LogFC
(Average) p

With SMM
Acremonium 49 3.15 0.02 3.98 4.17 2.26 3.15 2.38 3.70 −0.75 3.43
Apiotrichum 4627 3.11 0.004 1.60 3.15 3.54 6.04 2.62 6.38 2.07 −1.83 0.91 4.95
Candida 204 1.48 0.05 −0.51 0.87 2.10 4.12 1.71 3.73 0.25 −0.85 2.19
Cephalotrichum 1346 4.80 0.006 5.44 5.09 −3.53 6.09 6.55 10.74 2.95 3.50 2.92 5.89
Didymella 93 3.57 0.03 2.33 4.50 2.81 5.40 4.46 1.94
Fusicolla 82 3.61 0.02 2.86 5.17 3.48 4.04 4.73 5.50 −3.33 1.37
Gamsia 961 7.60 0.03 4.83 5.06 8.54 9.45 8.85 8.88
Holtermanniella 77 1.78 0.02 1.37 1.29 2.14 2.73 3.47 0.66 1.38
Humicola 13,471 −3.86 0.03 0.27 −5.24 −0.39 −7.64 −2.79 −8.08 −0.01 −8.96 0.77 −6.99
Metarhizium 183 1.99 0.02 1.37 0.95 0.92 2.76 3.93 0.32 4.70
Mortierella 134 2.08 0.05 0.38 3.61 1.93 4.51 1.37 3.16 −1.94 2.02
Plectosphaerella 274 1.90 0.02 2.91 −0.53 2.32 1.24 2.30 2.01 6.71 0.12 2.43
Saitozyma 828 2.05 0.01 1.74 2.83 1.95 3.96 2.08 4.35 1.20 −0.02 −0.22 2.67
Solicoccozyma 224 1.94 0.04 0.31 3.77 1.93 4.19 1.60 3.17 0.00 −1.80 2.81
Explanation: Mix—the mixture of three pesticides; 1—the recommended application rate; 10—the ten-fold application rate; CTRL—the control; 7, 14, 28, 56 days—sampling time;
Ben—benomyl; SMM—the starch–mineral mixture; Abundances of microbial genera with positive scores are marked with red color, abundances with negative scores—with blue color.
The degree of difference is shown by the brightness of the color.
Agronomy 2022, 12, 124 19 of 23

In the variant with SMM, exposure to pesticides affected the genus Solicoccozyma. The
genera Acremonium, Apiotrichum, Plectosphaerella, and Didymella were affected by pesticides
in both variants (Table 10).
At the same time, 10× dose rate of a mixture of three pesticides resulted in an increase
in the relative abundance of the fungal genus Gamsia. Thus, the effect of pesticides under
study on the fungal community was significantly higher than on the bacterial one and
was manifested already at the phylum level, affecting the taxonomic structure of the entire
community as a whole.
Multidimensional scaling (MDS) analysis performed at various taxonomic levels for
the fungal community showed that samples with 10× dose rate of pesticides (both benomyl
and a mixture) formed a distinct cluster and had consistent differences in the structure of
microbial community from other samples (control and with 1× dose rate) (Figure 5).

Figure 5. MDS plot representing the beta diversity of fungal community compositions (at genus level)
in soil samples (without SMM) treated with pesticides (red) and control ones (blue). Samples treated
with 10× application rate concentration form a separate cluster, distant from the other samples.

4. Discussion
The decrease in the diversity according to the Shannon index with the addition of
SMM was caused, first of all, by an increase in the proportion of hydrolytic bacteria, such as
Actinobacteria. Despite this, after the addition of SMM, a larger number of bacterial genera
Agronomy 2022, 12, 124 20 of 23

were affected by pesticide treatment. In general, the spectra of bacterial and fungal genera
reacting to pesticides, as well as the dynamics of this reaction, are significantly different
in the variants without SMM and with SMM. This is quite expected since the addition
of carbon source triggers the succession, which is visible in the microbiome structure in
the control samples. The starch led to changes in the microbiome structure that persisted
throughout the entire duration of the experiment. This substrate is rather slowly degraded
in comparison with, for example, such a universal source of carbon in microbiological
experiments as glucose [27]; at the same time, it is accessible enough to initiate a succession
in a short time. Note that the soil, for the most part, is practically “dead mass”, and only in
some zones (hotspots) is there a high activity of microorganisms [40], provided that readily
available substrates are supplied. In laboratory experiments, the soil is dried, sieved, and
homogenized, which, on the one hand, makes it homogeneous, and on the other hand,
completely destroys the hotspots. It cannot be argued that starch is an ideal substrate
for initiating succession. Moreover, additional research is needed to select the optimal
concentration. To select the optimal substrate, separate studies should be carried out using
DNA-sequencing. Stimulation of actinomycetes with starch somewhat blur the picture, but
at the same time long-term changes in the structure occur, which corresponds to our goals.
In fungi, pesticide treatment without SMM reduces Ascomycetes; with the SMM addition,
the same tendency was observed, but Basidiomycetes increase statistically significantly as
well. Basidiomycetes were likely to be involved in the degradation of pesticides, which
was confirmed by other studies, for example, where it has been shown that Basidiomycetes
can degrade organochlorine pesticides [41,42].
The increase in the genus Terrabacter can also be explained by the involvement of
these bacteria in the degradation of pesticides [43]. Kitasatospora and Streptomyces increase
their relative abundance upon treatment with pesticides. The genera of Actinobacteria are
also degraders; strains of these genera isolated from paddy soils have been shown to be
capable of degrading ipconazole [44]. It is noteworthy that although the total abundance of
Actinobacteria increases upon treatment with pesticides, no significant differences from the
control were found for the genera Kitasatospora and Streptomyces after the addition of SMM.
This is probably due to the fact that actinomycetes increase their share by actively utilizing
starch and the stimulating effect of pesticide application in relation to some genera is lost.
As noted above, polymeric substrates applied together with pesticides mask the increase in
the share of degraders due to the pesticide treatment, which has to be borne in mind when
conducting a search for pesticide-degrading strains. It probably makes sense to use lower
concentrations of substrates, as well as to use mixtures of them.
Regardless of the SMM addition, the relative abundance of bacteria of the genera
Burkholderia and Mycobacterium decreases under the influence of pesticides; as shown
previously, these genera are sensitive to carbendazim [23]. The share of Sphingomonas
slightly increases in comparison with the control variants without SMM and decreases with
the addition of SMM. The increase in the share may be associated with the involvement of
representatives of this genus in the degradation of pollutants [45]. Note that representatives
of this genus are characterized by many important properties in the soil, such as stimulation
of plant growth under stress conditions and the synthesis of phytohormones [46].
Among the representatives of the fungal community, yeast fungi of the genera Api-
otrichum and Gibberella are susceptible to the influence of pesticides without SMM addition;
in the case of SMM addition, positively affected by the pesticide application and the same
influence was observed for the genera Candida and Saitozyma. The relative representation
of the genus Apiotrichum increases upon treatment with pesticides, which is much more
pronounced with the addition of SMM. The increase in the proportion of yeast fungi is
likely to be associated with vacating the niches due to a decrease in the representation of
other species. This is more noticeable with the addition of SMM, since the decomposition
of starch results in the appearance of glucose, which is readily assimilated by copiotrophs.
Agronomy 2022, 12, 124 21 of 23

5. Conclusions
In conclusion, it is also noteworthy that the analysis of the structure of microbial
communities based on the 16S and 18S rRNA gene sequencing provides information only
about the relative proportion of different microorganisms; it should be remembered that
the absolute content of microorganisms between different samples (for example, with
SMM and without SMM) can significantly differ. Further research is needed with the
addition of carbon sources to establish optimal conditions for such experiments. The
changes in the relative abundance of the genera Terrabacter, Kitasatospora, Streptomyces,
Sphingomonas, Apiotrichum, Solicoccozyma, Gamsia, and Humicola can be proposed as an
indicator of pesticide contamination. It is proposed to use these signs for large-scale
assessment of the effect of pesticides on soil microbial communities instead of classical
integral methods, including within the framework of state registration of pesticides. It is
also proposed to conduct research on the effect of pesticides on the soil microbiome during
artificially initiated successions using the additional source of carbon.

Author Contributions: Conceptualization, A.A. and R.S.; methodology, R.S. and G.K.; software, A.A.;
validation, G.K.; formal analysis, G.K.; investigation, R.S. and A.A.; resources, R.S. and V.G.; data
curation, A.A. and G.K.; writing—original draft preparation, R.S.; writing—review and editing, R.S.
and A.A.; visualization, G.K.; supervision, V.G.; project administration, A.A.; funding acquisition,
R.S. and V.G. All authors have read and agreed to the published version of the manuscript.
Funding: The DNA sequencing was funded by a grant from the President of the Russian Federation,
grant number MK-92.2021.1.5. The conceptualization was supported by the state assignment of
Ministry of Science and Higher Education of the Russian Federation, number 117031410017-4 and
number 121040800154-8. The chromatographic analyses were performed using the equipment of
Collective Use Center, Soil Science Faculty, Lomonosov Moscow State University.
Data Availability Statement: The data sets for this study can be found in the NCBI Sequence Read
Archive, BioProject ID PRJNA723173.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Wilson, J.K.; Choudhri, R.S. Effects of DDT on Certain Microbiological Processes in the Soil. J. Econ. Èntomol. 1946, 39, 537–538.
[CrossRef]
2. Fletcher, D.W.; Bollen, W.B. The Effects of Aldrin on Soil Microorganisms and Some of Their Activities Related to Soil Fertility.
Appl. Microbiol. 1954, 2, 349–354. [CrossRef]
3. Bartha, R.; Lanzilotta, R.P.; Pramer, D. Stability and Effects of Some Pesticides in Soil. Appl. Microbiol. 1967, 15, 67–75. [CrossRef]
4. Marsh, J.A.P.; Davies, H.A.; Grossbard, E. The effect of herbicides on respiration and transformation of nitrogen in two soils I.
Metribuzin and glyphosate. Weed Res. 1977, 17, 77–82. [CrossRef]
5. Tu, C.M. Influence of five pyrethroid insecticides on microbial populations and activities in soil. Microb. Ecol. 1980, 5, 321–327.
[CrossRef]
6. Peeples, J.L. Microbial Activity in Benomyl-Treated Soils. Phytopathology 1974, 64, 857–860. [CrossRef]
7. Schuster, E.; Schröder, D. Side-effects of sequentially- and simultaneously-applied pesticides on non-target soil microorganisms:
Laboratory experiments. Soil Biol. Biochem. 1990, 22, 375–383. [CrossRef]
8. Das, A.; Mukherjee, D. Insecticidal effects on soil microorganisms and their biochemical processes related to soil fertility. World J.
Microbiol. Biotechnol. 1998, 14, 903–909. [CrossRef]
9. Das, A.; Chakravarty, A.; Sukul, P.; Mukherjee, D. Insecticides: Their effect on microorganisms and persistence in rice soil.
Microbiol. Res. 1995, 150, 187–194. [CrossRef]
10. Chakravarty, P.; Chatarpaul, L. Non-target effect of herbicides: I. effect of glyphosate and hexazinone on soil microbial activity.
Microbial population, and in-vitro growth of ectomycorrhizal fungi. Pestic. Sci. 1990, 28, 233–241. [CrossRef]
11. Engelen, B.; Meinken, K.; von Wintzingerode, F.; Heuer, H.; Malkomes, H.-P.; Backhaus, H. Monitoring Impact of a Pesticide
Treatment on Bacterial Soil Communities by Metabolic and Genetic Fingerprinting in Addition to Conventional Testing Procedures.
Appl. Environ. Microbiol. 1998, 64, 2814–2821. [CrossRef] [PubMed]
12. Wang, C.; Wang, F.; Zhang, Q.; Liang, W. Individual and combined effects of tebuconazole and carbendazim on soil microbial
activity. Eur. J. Soil Biol. 2016, 72, 6–13. [CrossRef]
13. Cycoń, M.; Piotrowska-Seget, Z. Biochemical and microbial soil functioning after application of the insecticide imidacloprid.
J. Environ. Sci. 2015, 27, 147–158. [CrossRef] [PubMed]
Agronomy 2022, 12, 124 22 of 23

14. Mahapatra, B.; Adak, T.; Patil, N.K.; Pandi G, G.P.; Gowda, G.B.; Jambhulkar, N.; Yadav, M.K.; Panneerselvam, P.; Kumar, U.;
Munda, S.; et al. Imidacloprid application changes microbial dynamics and enzymes in rice soil. Ecotoxicol. Environ. Saf. 2017,
144, 123–130. [CrossRef] [PubMed]
15. el Fantroussi, S.; Verschuere, L.; Verstraete, W.; Top, E.M. Effect of Phenylurea Herbicides on Soil Microbial Communities
Estimated by Analysis of 16S rRNA Gene Fingerprints and Community-Level Physiological Profiles. Appl. Environ. Microbiol.
1999, 65, 982–988. [CrossRef] [PubMed]
16. Zhang, R.; Jiang, J.; Gu, J.-D.; Li, S. Long term effect of methylparathion contamination on soil microbial community diversity
estimated by 16S rRNA gene cloning. Ecotoxicology 2006, 15, 523–530. [CrossRef]
17. Wang, X.; Song, M.; Gao, C.; Dong, B.; Zhang, Q.; Fang, H.; Yu, Y. Carbendazim induces a temporary change in soil bacterial
community structure. J. Environ. Sci. 2009, 21, 1679–1683. [CrossRef]
18. Puglisi, E.; Vasileiadis, S.; Demiris, K.; Bassi, D.; Karpouzas, D.G.; Capri, E.; Cocconcelli, P.S.; Trevisan, M. Impact of Fungicides
on the Diversity and Function of Non-target Ammonia-Oxidizing Microorganisms Residing in a Litter Soil Cover. Microb. Ecol.
2012, 64, 692–701. [CrossRef]
19. Karas, P.; Baguelin, C.; Pertile, G.; Papadopoulou, E.; Nikolaki, S.; Storck, V.; Ferrari, F.; Trevisan, M.; Ferrarini, A.; Fornasier, F.;
et al. Assessment of the impact of three pesticides on microbial dynamics and functions in a lab-to-field experimental approach.
Sci. Total. Environ. 2018, 637-638, 636–646. [CrossRef]
20. Cycoń, M.; Markowicz, A.; Borymski, S.; Wójcik, M.; Piotrowska-Seget, Z. Imidacloprid induces changes in the structure, genetic
diversity and catabolic activity of soil microbial communities. J. Environ. Manag. 2013, 131, 55–65. [CrossRef]
21. Zhang, P.; Ren, C.; Sun, H.; Min, L. Sorption, desorption and degradation of neonicotinoids in four agricultural soils and their
effects on soil microorganisms. Sci. Total Environ. 2018, 615, 59–69. [CrossRef]
22. Egbe, C.C.; Oyetibo, G.O.; Ilori, M.O. Ecological impact of organochlorine pesticides consortium on autochthonous microbial
community in agricultural soil. Ecotoxicol. Environ. Saf. 2021, 207, 111319. [CrossRef] [PubMed]
23. Fang, H.; Han, L.; Cui, Y.; Xue, Y.; Cai, L.; Yu, Y. Changes in soil microbial community structure and function associated with
degradation and resistance of carbendazim and chlortetracycline during repeated treatments. Sci. Total. Environ. 2016, 572,
1203–1212. [CrossRef] [PubMed]
24. Garg, N.; Bhattacherjee, A.K.; Shukla, P.K.; Singh, B. Influence of imidacloprid on bacterial community diversity of mango orchard
soil assessed through 16S rRNA sequencing-based metagenomic analysis. Environ. Monit. Assess. 2021, 193, 1–10. [CrossRef]
[PubMed]
25. Imfeld, G.; Vuilleumier, S. Measuring the effects of pesticides on bacterial communities in soil: A critical review. Eur. J. Soil Biol.
2012, 49, 22–30. [CrossRef]
26. Martin-Laurent, F.; Kandeler, E.; Petric, I.; Djuric, S.; Karpouzas, D.G. An FP7 European project for developing and evaluating
innovative tools for assessing the impact of pesticides on soil functional microbial diversity—towards new pesticide registration
regulation? Environ. Sci. Pollut. Res. 2013, 20, 1203–1205. [CrossRef] [PubMed]
27. OECD. Test No. 217: Soil Microorganisms: Carbon Transformation Test. OECD Guidelines for the Testing of Chemicals 2000.
Available online: https://www.oecd-ilibrary.org/environment/test-no-217-soil-microorganisms-carbon-transformation-test_
9789264070240-en (accessed on 1 December 2021).
28. OECD. Test No. 216: Soil Microorganisms: Nitrogen Transformation Test. OECD Guidelines for the Testing of Chemicals 2000.
Available online: https://www.oecd-ilibrary.org/environment/test-no-216-soil-microorganisms-nitrogen-transformation-test_
9789264070226-en (accessed on 1 December 2021).
29. Regulation (EU) No 283/2013 Data Requirements for Active Substances. Available online: https://eur-lex.europa.eu/legal-
content/EN/TXT/PDF/?uri=CELEX%3A32013R0283&from=EN (accessed on 15 July 2021).
30. Karpouzas, D.; Kandeler, E.; Bru, D.; Friedel, I.; Auer, Y.; Kramer, S.; Vasileiadis, S.; Petric, I.; Udikovic-Kolic, N.; Djuric, S.; et al.
A tiered assessment approach based on standardized methods to estimate the impact of nicosulfuron on the abundance and
function of the soil microbial community. Soil Biol. Biochem. 2014, 75, 282–291. [CrossRef]
31. Riah, W.; Laval, K.; Laroche-Ajzenberg, E.; Mougin, C.; Latour, X.; Trinsoutrot-Gattin, I. Effects of pesticides on soil enzymes: A
review. Environ. Chem. Lett. 2014, 12, 257–273. [CrossRef]
32. Thiour-Mauprivez, C.; Martin-Laurent, F.; Calvayrac, C.; Barthelmebs, L. Effects of herbicide on non-target microorganisms:
Towards a new class of biomarkers? Sci. Total. Environ. 2019, 684, 314–325. [CrossRef]
33. PPDB: Pesticide Properties DataBase. Available online: https://sitem.herts.ac.uk/aeru/ppdb/en/Reports/66.htm (accessed on
15 July 2021).
34. Fadrosh, D.W.; Ma, B.; Gajer, P.; Sengamalay, N.; Ott, S.; Brotman, R.M.; Ravel, J. An improved dual-indexing approach for
multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome 2014, 2, 6. [CrossRef]
35. Mukherjee, P.K.; Chandra, J.; Retuerto, M.; Sikaroodi, M.; Brown, R.E.; Jurevic, R.; Salata, R.A.; Lederman, M.M.; Gillevet, P.M.;
Ghannoum, M.A. Oral Mycobiome Analysis of HIV-Infected Patients: Identification of Pichia as an Antagonist of Opportunistic
Fungi. PLoS Pathog. 2014, 10, e1003996. [CrossRef]
36. Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Peplies, J.; Glöckner, F.O. The SILVA ribosomal RNA gene
database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013, 41, D590–D596. [CrossRef]
37. UNITE Community Full UNITE+INSD Dataset for Fungi UNITE Community 2019 (2019) 18.11.2018. Available online: https:
//unite.ut.ee/ (accessed on 1 December 2021).
Agronomy 2022, 12, 124 23 of 23

38. OECD. Test, No. 307: Aerobic and Anaerobic Transformation in Soil. OECD Guidelines for the Testing of Chemicals 2002.
Available online: https://www.oecd-ilibrary.org/environment/test-no-307-aerobic-and-anaerobic-transformation-in-soil_97
89264070509-en (accessed on 1 December 2021).
39. European Food Safety Authority. EFSA Guidance Document for evaluating laboratory and field dissipation studies to obtain
DegT50 values of active substances of plant protection products and transformation products of these active substances in soil.
EFSA J. 2014, 12.5, 3662.
40. Kuzyakov, Y.; Blagodatskaya, E. Microbial hotspots and hot moments in soil: Concept & review. Soil Biol. Biochem. 2015, 83,
184–199.
41. Suhara, H.; Adachi, A.; Kamei, I.; Maekawa, N. Degradation of chlorinated pesticide DDT by litter-decomposing basidiomycetes.
Biodegradation 2011, 22, 1075–1086. [CrossRef] [PubMed]
42. Henn, C.; Arakaki, R.M.; Monteiro, D.A.; Boscolo, M.; Da Silva, R.; Gomes, E. Degradation of the Organochlorinated Herbicide
Diuron by Rainforest Basidiomycetes. BioMed Res. Int. 2020, 2020, 5324391. [CrossRef] [PubMed]
43. Kasuga, K.; Nojiri, H.; Yamane, H.; Kodama, T.; Omori, T. Cloning and characterization of the genes involved in the degradation
of dibenzofuran by Terrabacter sp. strain DBF63. J. Ferment. Bioeng. 1997, 84, 387–399. [CrossRef]
44. Eizuka, T.; Ito, A.; Chida, T. Degradation of Ipconazole by Microorganisms Isolated from Paddy Soil. J. Pestic. Sci. 2003, 28,
200–207. [CrossRef]
45. Yu, F.B.; Shan, S.D.; Luo, L.P.; Guan, L.B.; Qin, H. Isolation and characterization of aSphingomonassp. strain F-7 degrading
fenvalerate and its use in bioremediation of contaminated soil. J. Environ. Sci. Health Part B 2013, 48, 198–207. [CrossRef]
46. Asaf, S.; Numan, M.; Khan, A.L.; Al-Harrasi, A. Sphingomonas: From diversity and genomics to functional role in environmental
remediation and plant growth. Crit. Rev. Biotechnol. 2020, 40, 138–152. [CrossRef]