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According to guidelines for the approval of pesticides, information about effects of pesticides on soil microorganisms and soil fertility are required, but the relationships of different structures of pesticides on the growth of various groups of soil microorganisms are not easily predicted. Some pesticides stimulate the growth of microorganisms, but other pesticides have depressive effects or no effects on microorganisms. For examples, carbofuran stimulated the population of Azospirillum and other anaerobic nitrogen fixers in flooded and non-flooded soil, but butachlor reduced the population of Azospirillum and aerobic nitrogen fixers in non-flooded soil. Diuron and chlorotoluron showed no difference between treated and nontreated soil, and linuron showed a strong difference. Phosphorus(P)-contains herbicides glyphosate and insecticide methamidophos stimulated soil microbial growth, but other P-containing insecticide fenamiphos was detrimental to nitrification bacteria. Therefore, the following review presents some data of research carried out during the last 20 years. The effects of twenty-one pesticides on the soil microorganisms associated with nutrient and cycling processes are presented in section 1, and the applications of denaturing gradient gel electrophoresis (DGGE) for studying microbial diversity are discussed in section 2.
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Effect of pesticides on soil microbial community
Chi-Chu Loa
a Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taiwan, ROC
Online publication date: 27 May 2010
To cite this Article Lo, Chi-Chu(2010) 'Effect of pesticides on soil microbial community', Journal of Environmental Science
and Health, Part B, 45: 5, 348 — 359
To link to this Article: DOI: 10.1080/03601231003799804
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Journal of Environmental Science and Health Part B (2010) 45, 348–359
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ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/03601231003799804
Effect of pesticides on soil microbial community
Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taiwan, ROC
According to guidelines for the approval of pesticides, information about effects of pesticides on soil microorganisms and soil fertility
are required, but the relationships of different structures of pesticides on the growth of various groups of soil microorganisms are not
easily predicted. Some pesticides stimulate the growth of microorganisms, but other pesticides have depressive effects or no effects on
microorganisms. For examples, carbofuran stimulated the population of Azospirillum and other anaerobic nitrogen fixers in flooded
and non-flooded soil, but butachlor reduced the population of Azospirillum and aerobic nitrogen fixers in non-flooded soil. Diuron
and chlorotoluron showed no difference between treated and nontreated soil, and linuron showed a strong difference. Phosphorus(P)-
contains herbicides glyphosate and insecticide methamidophos stimulated soil microbial growth, but other P-containing insecticide
fenamiphos was detrimental to nitrification bacteria. Therefore, the following review presents some data of research carried out during
the last 20 years. The effects of twenty-one pesticides on the soil microorganisms associated with nutrient and cycling processes are
presented in section 1, and the applications of denaturing gradient gel electrophoresis (DGGE) for studying microbial diversity are
Keywords: Fungicides; herbicides; insecticides; PCR-DGGE.
According to the guidelines for the approval of pesticides,
effects of pesticides on soil microorganisms and soil fertil-
ity should be determined. The fertility of soil depends not
only on the textures of soil but also on the biological ability
within it. The microbial diversity may have been changed
following pesticide use, and such changes may affect soil fer-
tility. Soil microorganisms therefore play important roles in
the soil fertility. The use of pesticides to protect crops may
alter the soil biological ability either by direct or indirect
action, but the knowledge of soil microbial ability to de-
grade pesticides and the influence of pesticides on microbial
diversity in soil are still limited.
To understand the effect of pesticides on soil microflora
and their beneficial activities is an important part of the
pesticide’s risk assessment. It is not easy to predict the
relationship between the chemical structure of pesticide
and its effect on the various groups of soil microorganisms.
Certain pesticides stimulate the growth of microorganisms,
but other pesticides have depressive effects or no effects on
microorganisms when applied at normal rates.
Microbiological analyses on the microbial communities
based on standard plate count methods lack precision, be-
Address correspondence to Chi-Chu Lo, Taiwan Agricultural
Chemicals and Toxic Substances Research Institute, Council of
Agriculture, 11, Kuang-Ming Road, Wufong, Taichung County,
Taiwan, ROC; E-mail:
cause approximately 1 % of the soil bacteria can be cul-
tured, and many bacteria and fungi can not be cultured in
the laboratory. Use of molecular methods to study soil mi-
crobial communities provides good alternatives,[1] but there
are some limitations on the molecular methods.
The following review presents some data of research car-
ried out during the last 20 years. The effects of 21 pesticides
on the soil microorganisms are presented in the first section
(Table 1), and the applications of denaturing gradient gel
electrophoresis (DGGE) for studying microbial diversity
are discussed in the second section (Table 2).
Effects of pesticides on soil microorganisms
Several pesticides (11 herbicide, 9 insecticides, and 5 fungi-
cides) representing different structures, mode of actions,
and applications were selected for comparison (Table 1).
Jena et al.,[2] had studied the influence of butachlor (N-
Butoxymethyl-2-chloro-2’,6’-diethylacetanilide) (Fig. 1)
applied either singly or in combination with carbaryl (1-
naphthyl methylcarbamate) (Fig. 2) on nitrogenase activity
and carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-
7-yl methylcarbamate) (Fig. 2) on the populations of
heterotrophic and nitrogen-fixing bacteria Azotobacter,
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Pesticides and soil microorganisms 349
Table 1. Effect of pesticides on soil microbial communities.
Pesticides/soil source Result Ref.
Butachlor (H), Carbary (I),
Carbofuran (I)/
rice-growing tracts
Carbary (10 µg/g) had almost no effect on nitrogenase under submerged conditions. Butachlor (20 µg/g)
and carbofuran (2 µg/g) reduced the population of Azospirillum and anerobic nitrogen fixers in a
non-flooded alluvial soil. Carbofuran (4 µg/g) stimulated the population of Azospirillum and other
anaerobic nitrogen fixers in both the soils.
Diflubenzuron (I)/maize
Diflubenzuron (100500 µg/g) had a stimulatory effect on dinitrogen fixation bacteria (Azotobacter
vinelandii) in soil.
Methylpyrimifos (I),
Chlorpyrifos (I)/maize
Methylpyrimifos (100300 µg/g) or chlorpyrifos (10300 µg/g) significantly decreased aerobic dinitrogen
fixing bacteria, however, fungal populations and denitrifying bacteria were not affected.
Metsulfuron methyl (H),
chlorsulfuron (H),
thifensulfuron methyl
(H)/Agricultural soil
Reduction of the growth of fluorescent psendomonads (77 strains). [6]
Diuron (H), linuron (H),
Similarity dendrograms based on 16S ribosomal ribonucleic acid (rRNA)-denaturing gel gradient
electophoresis (DGGE) band patterns showed that the microbial community structures of the treated and
nontreated soils were significantly different.
(F)/research plots
Fenpropimorph inhabited the growth of active fungi during the first 10 days. The number of total calculable
bacteria was significantly lowered by fenpropimorph at day 17 and stimulated at day 56. The diversity of
total bacterial DNA measured by DGGE was unaffected by fenpropimorph treatment.
Propanil (H), prometryne
(H)/soil cultivated with
sugar beet
Propanil did not affect soil bacteria significantly as indicated by DGGE and amplified rDNA restriction
analysis (ARDRA). Prometryne persisted in soil longer than propanil.
Iprodione (F)/strawberry
DGGE profiles showed that the soil bacterial community is more complex at 30Cthanat15
C. At 30C
andwith50µg/g iprodione treatment, the amounts of soil bacterial communities increased quickly but
can not be reduced to the original status after incubation for 23 days.
Glyphosate (H)/ponderosa
pine plantations
Glyphosate produces a non-specific, short-term stimulation of bacteria at a high concentration (100×field
Methamidophos (I)/soil
without agrochemical use
Methamidophos at 0.031g/pot/week and 0.31g/pot/week (total addition 3.14g/pot and 31.4g/pot,
respectively) significantly decrease microbial biomass by 4183 % compared with the control. The colony
forming unit (CFU) numbers of methamidophos metabolized bacteria in treated soil also increased
significantly by 86.1 % (0.031g/pot/week) and 88.9 % (0.31g/pot/week) compared with that of control.
(I)/contaminated soil
(more than 20 years)
In the polluted soil, the dominant phylotype included two members of γ-porteobacteria, which were closely
relatedtothePseudomonas stutzeri (similarity 99 %) and Pseudomonas putida (similarity 99 %).
Azoxystrobin (F),
chlorothalonil (F),
(F)/conventional farm
and organic management
None of the fungicides affected bacterial community structure (16S rRNA-PCR DGGE), but ciliate
protozoan Arcuospathidium sp. (similarity 97 %), or Bresslaua vorax (similarity 99 %), was affected by
chlorothalonil. Flagellate protozoan Paraflabellula hoguae (similarity 87 %) was affected by azoxystrobin,
and ascomycete fungus Cladosporium tenuissimum (similarity 78 %) was affected by tebuconazole (18S
Isoproturon (H)/farm 16S rRNA-PCR DGGE showed that degradation in sub-soil between 40–50 and 70–80 cm depths was
associated with proliferation of Sphingomonas spp.
Methamidophos (I)/black
The potential influences of methamidophos (50, 150, and 250 mg/kg) on soil fungal community in black
soil were assessed by plate count, 28S rDNA-PCR-DGGE, and clone library analysis. High
concentrations of methamidophos (250 mg/kg) could significantly stimulate fungal populations. DGGE
fingerprinting patterns showed a significant difference between the responses of culturable and total fungi
communities under the stress of methamidophos.
Butachlor (H)/paddy soil DGGE-unweighted pair-group method using arithmetic averages (UPGMA) dendrograms showed the
diazotrophic divergences ranged from 33 % to 64 % throughout rice growth stages. For general bacterial
communities, the diversities ranged from 28 % to 52 %.
Fenamiphos (I)/soils Not toxic to dehydrogenase or urease activities but likely to be detrimental to the nitrification in the soil. [13]
F: fungicide;
H: herbicide;
I: insecticide.
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350 Lo
Table 2. Studies of the microbial community structure by denaturing gradient gel electrophoresis (DGGE) techniques.
Environment/16S or
18S rRNA region Result Ref.
Microbial mat from
DGGE specific for the V3 region of 16S rRNA (341fGC/534r, 40GC) of sulfate–reducing bacteria was
analyzed, and up to 10 distinguishable hands were present. It is possible to identify constituents which
represent only 1 % of the total population.
Soil with known
A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed for the
detection of actinomycetes in the environment with polymerase chain reaction (PCR), temperature
gradient gel eletrophoresis (TGGE), and DGGE. Most actinomycetes investigated could be separated by
TGGE and DGGE, with both techniques giving similar results.
Microbial diversity in
sludge from biological
phosphorus removal
Up to 11 DGGE bands were observed, and comparative phylogenetic analysis of the partial 16S rRNA
sequences suggested that organisms are most closely related to Magentospirillum magenetotactium,
Legionella pneumophila and other three novel groups of organisms in the gamma subclass of the
Soil (16 soils)/ V6-V8/16S Soil DNA recovered from the different soils by the direct extraction method ranged from 835 µg/g dry
soil, whereas 5 µg/g dry soil was obtained with an indirect method. The quantification of the
Enterobacter cloacae and Arthrobacter sp. targets in the soil DNA background suggested that minimally
detectable specific genome members were on the order of 5 ×106g1dry soil or higher (13×104
genomes per reaction), and 0.5–1.5 % of the total microscopically detectable bacterial community may be
detectable via community based PCR-DGGE.
Diagnostic tool for
detecting microbial in
The system is a promising tool for the analysis of infectious agents in culture-negative clinical samples. It
enables not only the detection of well-known pathogens, but also hitherto unknown pathogens.
Microbial transformation
of dieldrin in
DGGE fingerprint revealed that four microbials evolved in dieldrin-amended cultures, but not in the
dieldrin-free cultures. Partial sequence of 16S rDNA for these four organisms exhibited 94–99 %
similarity to those of genera Clostridium,Acidaminobacter and an uncultured bacterial group.
Methanogens in different
UASB granules/V3/16S
DGGE fingerprints obtained from the methanogen reference cultures of Methanosaeta concilii,
Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina mazeii and Methanobacterium
formicicum were compared to the DGGE profiles of the Archaea in the different granules. The positions
of the DGGE bands did not correspond well to the known species were sequenced.
Soil from transgenic
papaya field/V3/16S
PCR-DGGE profiles showed that there were some differences on the bacterial diversity between planting
transgenic papaya and non-transgenic papaya at the beginning of planting, and the difference decreased
after six months.
Thauera spp in an
industrial wastewater
treatment plant/V3/16S
AThauera-specific nested-PCR DGGE was developed, and this DGGE fingerprinting technology has the
potential to be a profiling tool for monitoring structural shifts of Thauera spp. in industrial waste water
treatment plant.
Nonylphenol (NP) in
Sulfate-reducing bacteria, methanogen, and eubacteria are involved in the degradation of NP. A portion of
band P from the DGGE band profiles matches the genomic sequence of Clostridium aminovalericum
(similarity, 99 %). It was the consistently dominant bacterium in the mangrove sediments under the
various treatments.
Assess species diversity of
arbuscular mycorrhizal
fungi/V3-V4 and
Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that
the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different
Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9
region of this marker could be used for reliable identification of all recognized species within this genus.
Soil after fumigation/
Chloropicrin treatment changed DGGE profiles drastically and reduced the diversity index Hin both bulk
soil and rhizosphere soil 2 months after fumigation. In contrast, 1,3-dichloropropene did not reduce the
diversity index significantly after 2 months.
Fungal diversity in
agricultural soils/V1-V2
and V7-V8/18S
Several primers were tested for PCR and DGGE: (1) NS1/GCFung, (2) FF390/FR1(N)-GC, (3)
NS1/FR1(N)-GC (for single PCR) and (4) NS1/EF3 for the first PCR and NS1/FR1(N)-GC for the
second PCR (for nested PCR). The efficacy of PCR amplification using primer set 1 and the first PCR of
primer set 4 were better than those using primer sets 2 and 3. In DGGE analysis, the PCR products of
primer sets 3 and 4 showed the highest diversity indices. However, these primer sets had drawbacks, there
were several non-specific aggregates, and reproducibility was poor of the DGGE profiles.
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Pesticides and soil microorganisms 351
Fig. 1. Structures of herbicides.
Azospirillum, and anaerobic nitrogen fixers in two tropical
paddy soils.
Application of the herbicide butachlor or the insecti-
cide carbofuran at 2 µgg
1reduced the population of
Azospirillum and anaerobic nitrogen fixers in a non-flooded
sandy-loam alluvial soil (pH 6.2). In contrast, butachlor
stimulated the population of anaerobic nitrogen fixers in a
non-flooded acid sulphate saline Pokkali soil (pH 4.2). An
increased level of carbofuran (4 µgg
1) would stimulate the
population of Azospirillum and anaerobic nitrogen fixers in
both soils. In Pokkali soil the combination of carbofuran
and butachlor inhibited the population of Azospirillum,
while carbofuran alone at 4 µgg
1was stimulatory. Car-
baryl, an insecticide commonly used in rice, had almost
no effect on nitrogenase under submerged conditions while
activity was stimulated at 10 µgg
1under non-flooded con-
dition. Their results suggest that under tropical conditions
the effects of pesticides on nitrogen fixation and nitrogen-
fixing populations depend upon pesticide concentration,
water regime (flooded or non-flooded) and soil type.
Butachlor is a pre-emergence herbicide for the control of
weeds of transplanted rice in paddy fields in Taiwan. Chen
et al.[3] studied the composition of culture-independent mi-
crobial communities and the change of nitrogenase activi-
Fig. 2. Structures of insecticides.
ties under the application of butachlor in paddy soil. Their
results showed that changes among the bacterial commu-
nity after 100 days of incubation could be discovered by
UPGMA Unweighted Pair-Group Method Using Arith-
metic averages (UPGMA) dendrograms, and nitrogen-
fixation (expressed as the amount of acetylene reduction)
was suppressed after butachlor application but was aug-
mented after 37 days in both upper and lower layer soils.
The diversities in bacterial communities ranged from 28 %
to 52 %, and the divergences became higher with the cultiva-
tion period.[2] The authors concluded that the application
of butachlor imposed a significant variation on microbial
community shift.
Dinoterb and metamitron
Engelen et al.[4] had studied the patterns generated by am-
plified ribosomal sequences to investigate the suitability of
these approaches for the analysis of the impacts of herbicide
applications on the function and structure of soil bac-
terial communities. Herbogil (dinoterb, 2-tert-butyl-4,6-
dinitrophenol, Fig. 1), mineral oil Oleo (paraffin oil used
as an additive to herbicides), and Goltix (metamitron, 4-
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352 Lo
Fig. 1) were tested. Effects on metabolic sum parameters
were determined by monitoring substrate-induced respira-
tion (SIR), dehydrogenase activity, and carbon and nitro-
gen mineralization. These three conventional ecotoxicolog-
ical testing procedures are required in pesticide registration.
Inhibition of biomass-related activities and stimulation
of nitrogen mineralization were the most significant effects
caused by the application of Herbogil. The application of
Goltix resulted in smaller effects and the additive Oleo was
the least-active compound even though Goltix and Oleo
were used at a higher dosage (10 times higher). The au-
thors found that the application of Herbogil inhibited many
catabolic pathways. The authors also suggested that data
of partial 16S rDNA amplification products in tempera-
ture gradient gel electrophoresis (TGGE) analysis could
provide details about changes in the diversity of functions
and structural composition of the bacterial communities.[4]
Diuron, linuron, and chlorotoluron
El Fantroussi et al.[5] studied the effect of three
phenyl urea herbicides [diuron [3-(3,4-dichlorophenyl)-1,1-
dimethylurea, Fig. 1], linuron [3-(3,4-dichlorophenyl)-1-
methoxy-1-methylurea, Fig. 1], and chlorotoluron [3-(3-
chloro-p-tolyl)-1,1-dimethylurea], Fig. 1] on soil microbial
communities in soil samples with a 10-year history of treat-
DGGE was applied for the analysis of 16S rRNA genes
(16S rDNA), and similarity dendrograms showed that the
microbial community structures of the herbicide-treated
and nontreated soils were significantly different. The bacte-
rial diversity decreased in soils treated with urea herbicides,
and the sequence of several DGGE fragments showed that
the most affected species in the soils treated with diuron
and linuron were associated with an uncultivated bacterial
group. BIOLOG data showed that the functional abilities
of the soil microbial communities were altered by the ap-
plication of the herbicides.
Enrichment cultures of the different soils in medium
with the urea herbicides as the sole carbon and nitrogen
source showed that there was no difference between treated
and nontreated soil in the rate of transformation of diuron
and chlorotoluron, but there was a strong difference in the
case of linuron. In linuron-treated soil, linuron disappeared
completely after 1 week, whereas no significant transforma-
tion was observed in cultures inoculated with nontreated
soil even after 4 weeks. The authors concluded that both
the structure and metabolic potential of soil microbial com-
munities were clearly affected by a long-term application
of urea herbicides.[5]
Metsulfuron methyl, chlorsulfuron and thifensulfuron
The sulfonylurea herbicides are highly selective, safe
herbicides, and the major mode of action is interfering the
enzyme acetolactate synthase (ALS) on sensitive weeds.
The commercial interests of fluorescent pseudsmonals are
their ability to act as antagonists against plant pathogens.
Boldt and Jacobsen[6] studied the effect of sulfonylurea
herbicides on a total of 77 strains of genetically differ-
ent fluorescent Pseudomonas strains. Test strains were
incubated in various concentrations of the sulfonylurea
herbicides metsulfuron methyl [methyl 2-(4-methoxy-6-
chlorsulfuron [1-(2-chlorophenylsulfonyl)-3-(4-methoxy-
6-methyl-1,3,5-triazin-2-yl) urea] and thifensulfuron
methyl [methyl 3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl-
carbamoylsulfamoyl) thiophene-2-carboxylate] (Fig. 1).
The authors found that the herbicides resulted in a re-
duction of the growth of the fluorescent pseudomonads.
Metsulfuron methyl had a reducing effect on the growth of
76 out of the 77 isolated strains, and was toxic to 15 strains
at 5 ppm (19.5 %). Chlorsulfuron was found to be less toxic
in low concentrations at 5 ppm but toxic in concentrations
at 30 ppm. Thifensulfuron methyl was toxic to a minority
of the strains, only 10 % (seven strains) of the 77 isolated
strains were affected at 300 ppm. The authors suggested
that the difference of bacterial sensitivity to sulfonylurea
herbicides might be due to the different genetic ALS, The
authors concluded that the toxic effects of the sulfonylurea
herbicides could be neutralized when the strains were grown
in the presence of an excess amount o the three amino acids,
because ALS is involved in the synthesis of the branched
amino acids valine, leuctine and isoleucine.
Isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea]
(Fig. 1) is a member of the phenylurea group of herbicides,
and is frequently detected as contaminants of ground water
and surface fresh water in Europe. Bending et al.[7] studied
the factors controlling change in the biodegradation rate
of isoproturon with soil depth in a field with sandy loam
soil. Soil was sampled at five depths between 0–10 and
70–80 cm.
The degradation rate of isoproturon declined progres-
sively down the soil profile, and decreasing degradation
rates with soil depth were associated with an increase in
the length of the lag phase prior to exponential degrada-
tion, suggesting the time required for adaptation within
communities controlled degradation rates. But differences
in degradation rate between soil depths were not associated
with the starting population size of catabolic organisms, or
the number of catabolic organisms proliferating following
100 % degradation. Sequences from the bands in DGGE
showed that degradation in sub-soil between 40–50 and 70–
80 cm depths was associated with proliferation of the same
strains of Sphingomonas spp (known to contain multiple
heterogeneous 16S rRNA gene copies).[8]
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Pesticides and soil microorganisms 353
Glyphosate [N-(phosphonomethyl)glycine] (Fig. 1) is
one of the most commonly used herbicides worldwide.
Glyphosate inhibits protein synthesis via the shikimic acid
pathway in bacteria, fungi, and plant. Commercial for-
mulations contained polyoxyethylene fallow amine, which
is toxic to bacteria and protozoa. Glyphosate is a P-
containing amino acid that can be used as a sole P, C, or N
source for microbial growth, thus, Ratcliff et al.[9] studied
the effect of glyphosate in forest soil microbial communi-
Glyphosate applied at the recommended field rate (5 kg
a.i. ha1) to a clay loam and a sandy loam forest soil re-
sulted in few changes in microbial community structure.
Total and culturable bacteria, fungal hyphal length, bacte-
rial:fungal biomass, carbon utilization profiles (BIOLOG),
and bacterial and fungal phospholipid fatty acids (PLFA)
were all unaffected at Days 1, 3, 7, or 30 after application of
a commercial formulation (Roundup
R). In contrast, a high
concentration of glyphosate (100 ×field rate), simulating
an undiluted chemical spill, altered the bacterial commu-
nity substantially in both soils. Increases in total bacteria,
culturable bacteria, and bacterial:fungal biomass was fast
after application. Culturable bacteria increased from abut
1 % of the total population in untreated soil to 25 % at the
high concentration by day 7, indicating enrichment effect
on bacterial growth.
Community composition in both soils shifted from
fungal dominance to an equal ratio of bacteria to fungi.
Functional diversity of culturable bacteria, estimated
by C substrate utilization, also increased at the high
glyphosate concentration, particularly in the clay loam
soil. Only minor changes in bacterial PLFA resulted after
the 100×field rate application at Day 3. Changes in
fungal properties (hyphae, propagules, PLFA biomarkers)
were few and transient. The authors concluded that the
commercial formulation of glyphosate has a friendly effect
on microbial community structure when applied at the
recommended field rate, or even 100-fold field rate.
Methylpyrimifos and chlorpyrifos
Methylpyrimifos [2-Diethylamino-6-methylpyrimidine-4-
yl dimethyl phosphorothionate] and chlorpyrifos [O,O-
diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate]
(Fig. 2) are two widely used organophosphorus insecticides
in agriculture at concentrations of 3 to 15 kg ha1for pest
control. The effects of methylpyrimifos and chlorpyrifos
applied at normal pest control value up to thirty times on
soil microflora were conducted in an agricultural loam.[10]
The authors found that methylpyrimifos at concentrations
of 100 to 300 µgg
1, or chlorpyrifos at concentrations
from 10 to 300 µgg
1significantly decreased aerobic
dinitrogen fixing bacteria and dinitrogen fixation.[10]
Nitrifying bacteria decreased at concentrations of 200 and
300 µgg
1of methylpyrimifos.[10] Total number of bacteria
decreased at the concentration of 10 to 300 µgg
chlorpyrifos. Fungal populations and denitrifying bacteria
were not affected as a consequence of the addition of the
organophosphorus insecticides to the agricultural soil.
Diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)
urea] (Fig. 2) is a widely used insecticide in agriculture and
forestry. Martinez-Toledo et al.[11] studied the effects of 100,
200, 300, 400, and 500 µg diflubenzuron per gram of soil
in nonsterile soil incubated under aerobic conditions, and
in sterilized soil.[11] Both soils were inoculated with Azoto-
bacter vinelandii, and the authors found that the presence
of 100 to 500 µgg
1had a stimulatory effect on dinitrogen
fixation in both nonsterile and sterile soil.
Methamidophos [(RS)-(O,S -dimethyl phosphoramidoth-
ioate)] (Fig. 2) is a widely used insecticide in China. Wang
et al.[12] had studied the effect of methamidophos on micro-
bial diversity in soil by using soil microbial biomass analy-
sis and community level physiological profiles (CLPPs).[12]
They found that both a low and a high level of methami-
dophos application (CS2, 0.031 g pot1week1, and CS3,
0.31 g pot1week1) and urea application (CS4, 0.5 g
pot1week1) significantly decreased microbial biomass C
(Cmic) by 41–83 % compared with the control (CS1). The
respiration activity of treated soils was significantly higher
than the control. The soil total of N and P (Ntol and Ptol)
were significantly increased, and the Corg Ntol 1and Corg
Ptol1values were decreased after application of methami-
dophos. Substrate richness, Shannon and Simpson indices
of microbial communities under chemical stresses, were in-
creased significantly. The CFU (colony-forming unit) num-
bers of methamidophos metabolized bacteria in CS2 and
CS3 increased significantly by 86.1 % and 188.9 % com-
pared with that of CS1. They concluded that methami-
dophos could reduce microbial biomass and enhance func-
tional diversities of soil microbial communities, and some
species of soil bacteria may be enriched by the addition of
Fenamiphos [(RS)-(ethyl 4-methylthio-m-tolyl isopropy-
lphosphoramidate)] (Fig. 2) is an organophosphorus
insecticide and nematicide widely used in agriculture to
control soil pests, particularly nematodes. For effective
control, it should be incorporated into soil in the zones of
root growth. Caceres et al.[13] conducted research on the
effects of fenamiphos on important soil microbial activities
such as dehydrogenase, urease and potential nitrification
in four soils from Australia and Ecuador.
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354 Lo
Dehydrogenase is an intracellular enzyme belonging to
oxido-reductase, present in all microorganisms and is usu-
ally used as a measure of total microbial activity in soils.
Urease and potential nitrification are involved in nitrogen
metabolism, and usually used to evaluate the toxicity from
pesticides and other agricultural chemicals.
The authors founded that fenamiphos was not toxic to
dehydrogenase and urease up to 100 mg kg1soil. Potential
nitrification was found to be highly sensitive to fenamiphos
with a significant inhibition recorded at 10 mg kg1soil.
The nitrification activity in soils was decreased with an
increase in fenamiphos concentration, and the calculated
EC50 values for nitrification in all the tested soils ranged
between 19 and 56 mg fenamiphos kg1dry soil. There-
fore, the authors suggested that fenamiphos is likely to be
detrimental to nitrification at field application rate.
Methylparathion [ O,O-dimethyl O-4-nitro-phenyl phos-
phorothioate] (Fig. 2) is organophosphate insecticide,
which is extensively used despite its high toxicity. Zhang
et al.[14] had studied the long-term effects of methyl-
parathion contamination on the diversity of soil microbial
community by a culture-independent approach using small
subunit ribosomal RNA (SSU rRNA) gene-based cloning.
Microbial DNA extracted from both the control soil sample
and methylparathion contaminated soil sample was sub-
jected to PCR amplification with primers specific for bac-
terial 16S rRNA gene sequences. Phylogenetic analysis of
the sequences of the dominant phylotypes showed that the
bacterial communities changed noticeably. In the control
soil, the dominant bacterial groups included some novel
bacterial division, the bacillus genus, and α-proteobacteria,
while in methylparathion contaminated soil, the dominant
phylotypes were replaced by a member of the flexibactera-
cytophaga-bacteroides division and two members of the
γ-proteobacteria subdivision.
Diazinon, Profenophos
Diazinon [O,O-diethyl O-2-isopropyl-6-methylpyrimidin-
4-yl phosphorothioate] and profenophos [(RS)-(O-4-
bromo-2-chlorophenyl O-ethyl S-propyl phosphoroth-
ioate)] (Fig. 2) are two widely used organophosphorus
insecticides in agriculture. Gomez et al.[15] studied the
influences of 10, 50, 100, 200 and 300 µgmL
1of pro-
fenophos and diazinon in cells of Azospirillum brasilense
grown in chemically defined medium and dialyzed-soil
medium. The authors found that diazinon did not affect
microbial growth, levels of ATP, dinitrogen fixation and
production of vitamins of A. brasilense in either chemically
defined medium or dialyzed-soil medium. Profenophos
significantly reduced dinitrogen fixation, intracellular
levels of ATP, production of pantothenic acid, thiamine,
niacin and growth in cells grown in chemically defined
medium. These negative effects were not significant in cells
grown in dialyzed-soil medium. The authors concluded
Fig. 3. Structures of fungicides and related compounds.
that diazinon and profenofos at concentrations from 10 to
300 µgmL
1were not detrimental to A. brasilense.
Fenpropimorph [cis-4-[(RS)-3-(4-tert-butylphenyl)-2-me-
thylpropyl]-2,6-dimethylmorpholine] (Fig. 3) is a widely
used fungicide for the control of the pathogens Erysiphe
graminis (powdery mildew) and Puccinia recondita (cereal
rusts) in Denmark. Bjornlund et al.[16] studied the effect
of fenpropimorph on the microbial number on litterbags
containing freshly harvested wheat roots with adhering rhi-
zosphere soil over a 4-month period.
Rhizosphere soils were treated with different concentra-
tions of fenpropimorph (0, 1, 10 and 100 mg kg1soil).
They found that bacteria [colony forming units (CFUs)]
and protozoa [most probable number (MPN)] followed
a similar pattern: a sudden decrease at the onset of the
study period, then followed by a fast recovery and in-
crease. Maximum mean values were 7.5 ×107bacteria
g1root litter by day 7, and 3 ×106protozoa g1root
litter by day 20, respectively. The fungi were unaffected by
fenpropimorph until day 20. Fluorescein diacetate-active
(FDA-active) hyphae were significantly inhibited in all
fenpropimorph treated plots after Day 20. Fungal CFUs
on fenpropimorph-containing agar showed a selection to-
wards more fenpropimorph tolerant fungi in the 100 mg
kg1fenpropimorph treated plots. The authors suggested
that long-erm effects of fenpropimorph on the activity of
saprotrophic fungi at high dose fenpropimorph treatment
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Pesticides and soil microorganisms 355
can induce the selection for fenpropimorph-tolerant fungal
Thirup et al.[17] had conducted the study on the effects of
the fungicide fenpropimorph (in the formulation Corbel)
on primary decomposer organisms in soil over a 56-day
period. Fenpropimorph was added to the soil in a recom-
mended dose. The soil fenpropimorph degraded with the
formation of concomitant fenpropimorphic acid (Fig. 3).
Fenpropimorph inhibited the growth of active fungi dur-
ing the first 10 days. The number of total culturable bac-
teria was also significantly lowered by fenpropimorph at
day 17 and stimulated at day 56. Culturable Pseudomonas
and actinomycetes were not affected. The diversity of to-
tal bacterial DNA measured by DGGE was unaffected by
fenpropimorph treatment. The authors suggested an effect
of fenpropimorph on soil fertility was possible.
Iprodione [3-(3,5-dichlorophenyl)-N-isopropyl-2,4-dioxo-
imidazolidine-1-carboxamide] (Fig. 3) has been introduced
to control a variety of crop disease in strawberry, tobacco,
and pear in Taiwan, Wang et al.[18] studied the effect of
iprodione on the soil bacterial communities by treating
two kinds of soils with different concentrations of iprodi-
one. The 16S rDNA-DGGE profiles showed that the bac-
terial communities were changed and recovered rapidly to
the original status when incubated at a lower temperature
(15C) with a lower iprodione concentration (5 µgg
1). At
the same temperature but with more iprodione (50 µgg
added, the soil bacterial community increased slowly and
regained the original status slowly. When incubated at the
higher temperature (30C), the soil bacterial community
was more complex than that at the lower temperature. The
amounts of soil bacterial communities increased quickly
at 30C with 50 µgg
1iprodione added, but the com-
munities could not be returned to the original status after
incubation for 23 days. The authors concluded that the re-
sponse of the soil bacterial community to the iprodione was
faster at higher temperature than that at lower temperature,
and the bacterial communities changed when iprodione of
50 µgg
1was added.
Azoxystrobin, tebuconazole, and chlorothalonil
Bending et al.[19] studied the impacts of the fungicides (at
the maximum recommended doses) azoxystrobin [methyl
3-methoxyacrylate] (5 mg kg1), tebuconazole [(RS)-1-p-
thyl)pentan-3-ol] (5 mg kg1) and chlorothalonil (tetra-
chloroisophthalonitrile) (10 mg kg1) (Fig. 3) on different
soil microbial properties. The fungicides selected repre-
sented a range of solubilities and capacities to sorbs to
soil organic matter, and different modes of action. Tebu-
conazole inhibits ergosterol biosynthesis, chlorothalonil
inhibits conjugation of thiols, and chlorothalonil blocks
mitochondrial respiration.
Degradation of all fungicides was fastest in the high OM
biomass1soil, with tebuconazole the most persistent com-
pound. Chlorothalonil was the most readily degraded. Pes-
ticide sorption distribution coefficient (Kd) did not differ
significantly between the soils. Chlorothalonil had the high-
est Kd(97.3), followed by azoxystrobin (13.9) and tebu-
conazole (12.4). None of the fungicides affected microbial
biomass in either soil. All fungicides significantly reduced
dehydrogenase activity to varying extents in the low OM
biomass1soil, but not in the high OM biomass1soil.
Bacterial PCR DGGE (16S rDNA) indicated that none of
the fungicides affected bacterial community structure, but
results from 18S rRNA PCR-DGGE revealed that a small
number of eukaryote bands were absent in certain fungi-
cide treatments, and sequencing data suggested these bands
represented protozoa and fungi. The authors suggested
chlorothalonil generally had greater and more prolonged
impacts on soil microbial properties than azoxystrobin or
tebuconazole, and further work to develop methods for
using microbial community profiling and measurement of
narrow niche functions for the assessment of pesticide im-
pact is recommended.
Magarey and Bull [20] studied the effect of using the fungi-
cide mancozeb [manganese ethylenebis (dithiocarbamate)
(polymeric) complex with zinc salt] (Fig. 3) to control fun-
gal root pathogens in sugarcane soils. They found that to-
tal fungi, actinomycetes and Pseduomonas bacteria were
significantly reduced but total bacterial populations were
increased.[20] Pandey and Singh found that the stimulation
of bacteria from the application of mancozeb probably re-
sulted from reduced competition for nutrients following the
reduction in the fungal and actinomycete populations.[21]
Nematicides and insecticides are unlikely to have a di-
rect impact on soil microbial populations, but their ac-
tivity may temporarily perturb the functioning of the soil
food web resulting in short term fluctuations in microbial
Studying microbial community structures by DGGE
General limitations in studying pesticides effects on the soil
microorganisms based on the counts and DGGE
Torsvik et al.[22] estimated that in 1 g of soil there are 4000
different bacterial “genomic units” based on DNA-DNA
reassociation. It has also been estimated that about 5000
bacterial species have been described.[23,24] Approximately
1 % of the soil bacterial population can be cultured by stan-
dard laboratory practices[25] and an estimated 1,500,000
species of fungi exist in the world.
There are many papers in the literature focusing on the ef-
fects of pesticides on specific groups of soil microorganisms.
Most of the work on the effects of pesticides on microor-
ganisms is based on cultivation-dependent methods. Due
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356 Lo
to the limitations of the culture-dependent methods, the
uses of molecular techniques are increasing. Techniques Bi-
olog GN, include terminal restriction fragment length poly-
morphisms (T-RFLP), amplified rDNA restriction analy-
sis (ARDRA), regional integrated science and assessment
(RISA), and DGGE. Biolog GN is a rapid method, but
inoculums density, incubation time, and the source of envi-
ronments are critical, thus the method has some drawbacks.
ARDRA and RFLP techniques start with DNA digestion
by restriction enzymes, then the digested DNA fragments
are separated on gels. However, some genus of microor-
ganisms could produce several bands, thus reducing the
resolution. RISA is used to amplify the region between the
16S and 23S rRNA genes,then the PCR-amplified products
are separated by gel, and bands are excised for sequencing.
But the RISA technique is limited by the database, because
the number of space sequences in the database was far
more less than the database of 16S rDNA. Therefore, the
DGGE method is the most widely used for studying micro-
bial communities in different environmental samples. The
PCR products produced by the amplified 16S rRNA gene
or 18S rRNA gene can be separated on gels, and the visible
bands can be excised, sequenced, and compared with the
Band information obtained by DGGE would be var-
ied by the conditions that are applied to the DGGE sys-
tem. Conditions include the PCR reactions, length of PCR
products produced, location of the rRNA region selected,
percentage acrylamide used for the gel, temperature used
in crossing denaturant concentrations, and electrophoresis
volt-hours applied. Thus, this review is focused on the con-
ditions of PCR-DGGE method, and the effects pesticides
on bacterial and fungal diversities are also discussed.
DGE fingerprinting of microbial communities
Muyzer et al.[26] first applied denaturing gel electrophoresis
(DGE) techniques for the analysis of whole bacterial com-
munities. Denaturing gel electrophoresis allows the sepa-
ration of small polymerase chain reaction products, com-
monly up to 500 bp. The separation of the DNA fragments
is achieved as a function of their different G+Ccontent
and distribution. Consequently, the fingerprinting pattern
is built according to the melting behavior of the sequences
along a linear denaturing gradient.[27] Such a gradient is
obtained using either denaturing chemicals for denaturing
gradient gel electrophoresis (DGGE) or heat for temper-
ature gradient gel electrophoresis (TGGE) and temporal
temperature gradient electrophoresis (TTGE). Denaturing
gradient gel electrophoresis (DGGE) is the most commonly
used method among the culture-independent fingerprinting
techniques (Table 2).
The DGE techniques were applied using 16S rDNA frag-
ments to the analysis of bacterial communities in numerous
habitats[2830] and aquatic environments.[3134] The addi-
tion of a 30-to 40-bp GC clamp to one of the polymerase
chain reaction (PCR) primers is to insure the fragment
of DNA will remain partially double-stranded in the low-
est melting domain.[27] The GC clamp PCR fragments ob-
tained from PCR reaction are then separated in a denatur-
ing gradient polyacrylamide gel based on their differential
denaturation (melting).
Primer choice of 16S rRNA region and 18S rRNA region
The most commonly employed target for PCR-DGGE is
the ribosomal DNA, but the PCR itself may be a source
of bias in molecular study. Targeting different 16S variable
regions may lead to different results in species composition
of the same sample,[35] therefore, the choice of the primers
for PCR-DGGE is important. Analysis of the amplified
variable V3 region and V6-V8 region of the 16S rDNA is
usually selected to differentiate and identify bacteria than
other regions of the 16S rDNA (Table 2), although some
other regions have also been used in different environmen-
tal samples. For examples, Ferris et al.[36] used V9 region
to analyze populations inhabiting a hot spring microbial
mat community. Klijn et al.[37] used V1 and V3 regions to
identify the mesophilic lactic acid bacteria. Walter et al.[38]
used V2-V3 region to detect and identify the gastrointesti-
nal Lactobacillus species, and Leong et al.[39] used V7-V9
region to detect bacteria found in cerebrospinal fluid.
To differentiate and identify eukaryote communities, the
analysis of the amplified variable the 18S rDNA is usually
selected,[40] such as V3-V4 region and V9 region,[41] V1-V2
region and V7-V8 region.[4243]
Optimization of PCR-DGGE
Denaturing gradients can be performed with the DCode
Universal Mutation Detection System (Bio-Rad). PCR
samples are loaded onto 6 or 8 % polyacrylamide gels con-
taining a denaturing gradient ranging from 30 to 70 % or
45 to 65 % (where 100 % denaturant corresponds to 7 M
urea and 40 % (V/V) formamide).
Electrophoresis can be performed at constant voltage
70V, 100V, and 200V for 15h, 16h, and 5hr, respectively.
Temperature can be maintained at a constant temperature
of 60–65 C. After electrophoresis, the gels can be stained
with SYBR
RGreen I, and photographed. The banding
patterns can be analyzed by the NT-SYS program (Exeter
Software, NY) by using the unweighted pair group with the
mathematical averages method (UPGMA).
Cocolin et al.[44] found that the 40 to 60 % denatu-
rant gradient allowed differentiation of members of the
Micrococcaceae, while the 30 to 50 % denaturant gradi-
ent allowed differentiation of all the Lactobacillus spp.
tested. They found that the presence of up to 10 distin-
guishable bands in DGGE separation pattern, which were
most likely derived from as many different species consti-
tuting these populations. However, our experience showed
that the number of distinguishable bands in DGGE (V3
region, 341Fgc/534r, 234bp) is not always equal to the
number of species. A similar result was also reported by
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Pesticides and soil microorganisms 357
Sekiguchi et al.[45] They found that the excised DNA from
the DGGE (V3 region, 357FGC/518R, 202bp) gel showed
a single band, but the library consisted of several different
sequences phylogenetically.[45] Therefore, the assumption
that one band equals one genome is not always valid, and
the excision and sequencing of the bands is required to
confirm the meanings of the bands.
DGGE could usually separate sequences differing by a
single base pair, but multiple sequence differences were
not so easily resolved. Jackson et al.[46] found that two
sequences that differ by 2 base pairs showed identical mi-
gration in DGGE gels and could not be separated in a
mixed sample. This limitation should be considered when
using DGGE to examine natural bacterial communities.
Denaturing gradient in the DGGE system is also an
important factor. Several denaturing gradients have been
used, for example, 15–55 %[26] , 20–70 %[47], 30–50 %[38] ,30–
70 %[48], 35–50 %[49] , 35–80 %[36], 40–60 %[50] , 40–70 %[31],
45–70 %[51]. In our experience, use of small gradients (20–
45 %) reduced the clarity of bands, and the improvement
on clarity is limited when high gradients (30–80 %) was
used. Therefore, denaturant gradient of 40–70 % in DGGE
system is used in our laboratory.
The quality of DNA extracts from environment is very
important when DNA extracts are used as templates for
the PCR-DGGE, because other components in the extracts
may act as inhibitors. Therefore, the DNA extraction pro-
cedure needs to be verified in order to achieve the right
degree of purity for PCR reaction. We have compared four
soil DNA extraction methods in six soils of different type
and pH, and Zhou’s soil DNA extraction method plus QI-
Aquick gel is recommended in our laboratory for DGGE
to monitor the microbial diversity in soil.[52]
The presence of multiple copies of the 16S rDNA gene
in a single species is also a problem in the study of bacterial
community diversity on the basis of 16S rRNA genes using
DGGE. A single species with multiple rRNA copies can
display multiple bands in a DGGE profile which overesti-
mates the community diversity.[53]
The effects of pesticides on soil microbial communities are
important for the approval of pesticides, but the relation-
ships between pesticide structure and soil microorganisms
associated with soil fertility are not easily predicted.
Diflubenzuron can stimulate the activity of nitrogen
fixation bacteria in soil, but methylpyrimitos decreases the
activity of nitrogen fixation bacteria. Investigation on the
microbial populations are obtained by culture-dependent
methods, but many microorganisms are uncultivable,
therefore, the uses of molecular methods are important.
PCR-DGGE is the most widely used molecular technique
compared with other molecular techniques, such as Biolog
GN, amplified fragment length polymorphism (AFLP),
ARDRA, and RISA. Although the analysis of 16S rDNA
fragments by DGGE has great value in environmental
microbiology, it still has limitations. Many factors could
affect the performance of PCR-DGGE, such as extracted
DNA quality, template concentration, range of denaturing
gradient, % acrylamide gel, electrophoresis condition
(voltage, temperature, time), and different copies of 16S
rRNA gene in one strain. Also at least 1 in 20 16S rRNA
sequence records is likely to be corrupt.[8] Therefore, to
study the effect of chemicals (pesticides, food components,
industrial waste water) on microbial community in differ-
ent environments, it would be wise to know the benefit and
the limitation of PCR-DGGE.
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... This eventually may alter the uptake of nutrient(s) for example increased abundance of nitrogen fixing microbes in rhizosphere of soybeans has been documented in response to eCO 2 helping to meet increased demand of nitrogen by the plant . On the contrary, microbial communities in the agricultural system may strongly respond to other variables such as nitrogen fertilizers affecting production of root exudates as documented in wheat (Usyskin-Tonne et al., 2021) and maize (Zhu et al., 2016), while crop diversification (Maiti et al., 2011), tillage (Miller et al., 1995) and application of different pesticides could affect the microbial diversity in the rhizosphere (Lo, 2010;Jin et al., 2013). Hence, impact of climate change factors on the soil microbial diversity, composition and activity in agricultural system have to be analysed differently in comparison to the studies on the natural ecosystems ( Fig. 1). ...
Crop improvement programs are facing serious challenges to sustain food and more importantly nutritional demand from global population, due to changing climate. Major staple cereals provide the caloric requirement for the global population, but are poor sources of micronutrients. Besides nutritional dilution due to the yield oriented breeding, and unprecedented increase in atmospheric CO2, altered composition of beneficial microbes in the rhizosphere is seen as a plausible reason driving low nutrient accumulation in cereals. A complex network of signalling between plant and microbes in the rhizosphere reveals extensive links between environment, microbes and crop nutrition. Despite established roles of rhizospheric microbes on crop nutrient dynamics, limited knowledge is available on the impact of climate change on rhizosphere biology and the subsequent modulation of crop nutrition. Here, we emphasize the potential role of microbes to help sustain the global nutritional demand, achieved through the development of microbiome responsive nutrient rich staple crops. To succeed in this goal, dynamics of rhizosphere biology altered by climate change factors (CO2, temperature, precipitation), farming practices and soil properties needs to be untangled and a robust research pathway established to enhance crop nutrition with microbial biofortification to ensure a sustainable route to achieve global nutritional security.
... Repeated application of pesticides has been reported to significantly lower the soil microbial biomass, mainly the fungal populations, and has been reported to increase certain bacterial populations (Smith et al., 2000;Pal et al., 2006;Singh et al., 2015). Molecular techniques like DGGE were also to determine the effect of specific pesticides on the soil microbial community (Lo, 2010). They found that carbofuran stimulated the population of Azospirillum and anaerobic nitrogen fixers in flooded and non-flooded soil. ...
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Preventing degradation, facilitating restoration, and maintaining soil health is fundamental for achieving ecosystem stability and resilience. A healthy soil ecosystem is supported by favorable components in the soil that promote biological productivity and provide ecosystem services. Bio-indicators of soil health are measurable properties that define the biotic components in soil and could potentially be used as a metric in determining soil functionality over a wide range of ecological conditions. However, it has been a challenge to determine effective bio-indicators of soil health due to its temporal and spatial resolutions at ecosystem levels. The objective of this review is to compile a set of effective bio-indicators for developing a better understanding of ecosystem restoration capabilities. It addresses a set of potential bio-indicators including microbial biomass, respiration, enzymatic activity, molecular gene markers, microbial metabolic substances, and microbial community analysis that have been responsive to a wide range of ecosystem functions in agricultural soils, mine deposited soil, heavy metal contaminated soil, desert soil, radioactive polluted soil, pesticide polluted soil, and wetland soils. The importance of ecosystem restoration in the United Nations Sustainable Development Goals was also discussed. This review identifies key management strategies that can help in ecosystem restoration and maintain ecosystem stability.
... In contrast to the reduction in the nitrogen fixation, carbofuran under the trade names furadan improves the colony number of the nitrogen fixers, including Azospirillum in both flooded and non-flooded soils, and the application of butachlor in the agricultural field especially in paddy field boost up the nitrogen fixation ability and creates a difference in microbial flora (Chen et al., 2009;Lo, 2010). Triazine class of herbicide atrazine at low concentration can improve dehydrogenase activity of the bacteria, but higher concertation causes deleterious effects and finally total inhibition of the enzyme activity (Nweke et al., 2007). ...
The application of pesticides is extensively increasing in arable lands to minimize the impact of pests on crop yields. However, these unmanaged agricultural practices significantly affect the soil characteristics, and therefore the xenobiotic nature of the pesticides is a growing threat to the soil micro-fauna. Of the different soil microbes, beneficial soil microorganisms perform principal roles in biodegradation, nutrient recycling, soil structural stability, nitrogen fixation, plant growth promotion, disease bio-control, and other biochemical transformation. As a result, the adverse effect of pesticides on the soil micro-biota significantly altered the soil characteristics, which ultimately reduced the growth and yield of the crop plants. Since the effects of different pesticides (organochlorines, organophosphorus, carbamates, pyrethrin, and pyrethroids) on different microbes (bacteria, fungi, and microalgae) were varied; it is essential to know the action of pesticides on microbial population and biochemical transformation processes. The major focus of this work is to analyze the effects of pesticides on microbial growth and activity. The information from this study would help in choosing specific pesticides for specific agricultural lands. For a sustainable agricultural ecosystem, it is essential to maintain the proliferation of soil microbes, and this article elaborates on different strategies to create a sustainable agroecosystem with minimum utilization of agrochemicals, which supports the growth of beneficial soil microorganisms.
... Crop production is highly dependent on soil microbial biodiversity, as microbes are crucial for organic matter decomposition and assist with converting nutrients to plant-available forms [6]. Pesticides have major impacts on soil microbial communities, specifically by altering response with shifts in microbial biomass, ultimately having negative effects on soil health and fertility [7][8][9]. ...
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Pesticide application in horticultural crops has recently multiplied to increase crop yields and boost economic return. Consequently, the effects of pesticides on soil organisms and plant symbionts is an evolving subject of research. In this short-term study, we evaluated the effects of glyphosate (herbicide) and carbaryl (insecticide) on okra biomass and AMF root colonization in both shade house and field settings. An additional treatment, the combination of glyphosate and carbaryl, was applied in the field trial. Soil and root samples were collected three times during the experiment: 30 days after planting (before first spray, or T0), 45 days after planting (before second spray, or T1), and at full maturity (at 66 days after planting, or T2). Our results indicate that glyphosate and combined treatments were most effective in controlling weeds and produced almost 40% higher okra biomass than the control. There was a ~40% increase in AMF root colonization in glyphosate-treated plots from T0 to T1. This result was likely due to high initial soil P content, high soil temperature, and low rainfall, which aided in the rapid degradation of glyphosate in the soil. However, at T2 (second spray), high rainfall and the presence of excess glyphosate resulted in a 15% reduction in AMF root colonization when compared to T1. We found carbaryl had little to negligible effect on AMF root colonization.
... Apparently, the chemicals were broken down by the microbes or by the influence of different abiotic factors and thus the toxicity of the test insecticides reduced which in due course of time favoured the growth of microbes. Chu (2010) concluded that carbofuran stimulated the population of fungi, azospirillum and other anaerobic N-fixers in flooded and non-flooded soils. In the present investigation, chlorpyriphos increased the fungal population. ...
... Treatment of plants with pesticides can have an impact on the soil microbiome, which can be beneficial or detrimental (Lo, 2010). Phosphite is widely used to control P. cinnamomi damage in avocado orchards, and there is little data on how this might affect the soil microbiome. ...
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Plant growth and responses of the microbial profile of the rhizosphere soil and root endosphere were investigated for avocado plants infested or not infested with Phytophthora cinnamomi and the changes were compared in plants grown with various soil additives or by spraying plants with phosphite. Soil treatments were organic mulches or silica-based mineral mulch. Reduction of root growth and visible root damage was least in the infested plants treated with phosphite or mineral mulch applied to the soil. Rhizosphere soils and root endospheres were analyzed for bacterial communities using metabarcoding. Bacterial abundance and diversity were reduced in infested rhizospheres and root endospheres. The presence or absence of mineral mulch resulted in greater diversity and larger differences in rhizosphere community composition between infested and non-infested pots than any other treatment. Some rhizosphere bacterial groups, especially Actinobacteria and Proteobacteria, had significantly higher relative abundance in the presence of Phytophthora. The bacterial communities of root endospheres were lower in abundance than rhizosphere communities and not affected by soil treatments or phosphite but increased in abundance after infection with P. cinnamomi. These findings suggested that the addition of silicate-based mineral mulch protects against Phytophthora root rot, which may be partly mediated through changes in rhizosphere bacterial community composition. However, the changes to the microbiome induced by spraying plants with phosphite are different from those resulting from the application of mineral mulch to the soil.
Various pesticides are being widely used worldwide to protect crops from pest damages and enhance crop production. But the pesticide residues in agriculture fields give rise to many critical environmental and public health concerns. Among different physical, chemical, and biological remediation technologies, phytoremediation of pesticides seems to be the least harmful approach and requires relatively low investment. In this chapter, the behaviors and transport of pesticides in soil are briefly presented and discussed, followed by the mechanisms of their phytoremediation. The applications of phytoremediation for removing pesticides from soil have been reviewed with examples of the five most commonly used active ingredients of pesticides in recent years. The improved effectiveness of using transgenic plants for phytoremediation, as well as the associated concerns, is concisely highlighted. Several postphytoremediation plant biomass management strategies are also analyzed and discussed for their preference hierarchy.
Protists are the most abundant and diverse eukaryotes that inhabit virtually all soils. They are active players in soil food webs as phototrophic algae, plant and animal parasites, and microbiome predators. As predators, protists lead to modification of their prey community composition typically promoting functions linked to (plant-)pathogen suppression. The diverse functional roles of protists are further connected to cycling of carbon, nitrogen and other nutrients such as phosphorous and silicon. Thanks to their link to plant growth and their dynamic response to environmental changes, soil protists are further suggested as bioindicators of soil and plant health.
Pesticides are important tools to increase agriculture productivity and to deal with domestic pests. Once they were elevated to the category of relevant items brought by modernity. However, when Rachel Carson published her famous work “Silent Spring” humans worldwide were awakened to the problems caused by excessive and inappropriate use of these compounds, in that particular case due to biomagnification. Nowadays, pesticides are still regarded as important tools to improve human lifestyle, but now pesticides are less persistent, less toxic (especially toward mammals), and special emphasis is put on reducing the toxic effects of new pesticides once they are released into the market. These new rules and regulations have greatly reduced the adverse effects of pesticides on the environment. Unfortunately, there are many commercial formulations and active ingredients that are still greatly toxic to nontarget organisms of which we know a few details on fate and toxicology. This chapter would try to fill up some of the blanks that exist in this information and give a general perspective on the topic. At the end of the chapter, we would recommend future prospective works and research that might enlighten what we already know about the fate and adverse effects of pesticides.
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A previous report of high levels of members of the domain Archaea in Antarctic coastal waters prompted us to investigate the ecology of Antarctic planktonic prokaryotes. rRNA hybridization techniques and denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial V3 region were used to study variation in Antarctic picoplankton assemblages. In Anvers Island nearshore waters during late winter to early spring, the amounts of archaeal rRNA ranged from 17.1 to 3.6% of the total picoplankton rRNA in 1996 and from 16.0 to 1.0% of the total rRNA in 1995. Offshore in the Palmer Basin, the levels of archaeal rRNA throughout the water column were higher (average, 24% of the total rRNA) during the same period in 1996. The archaeal rRNA levels in nearshore waters followed a highly seasonal pattern and markedly decreased during the austral summer at two stations. There was a significant negative correlation between archaeal rRNA levels and phytoplankton levels (as inferred from chlorophyll a concentrations) in nearshore surface waters during the early spring of 1995 and during an 8-month period in 1996 and 1997. In situ hybridization experiments revealed that 5 to 14% of DAPI (4',6-diamidino-2-phenylindole)-stained cells were archaeal, corresponding to 0.9 x 104 to 2.7 x 104 archaeal cells per ml, in late winter 1996 samples. Analysis of bacterial ribosomal DNA fragments by DGGE revealed that the assemblage composition may reflect changes in water column stability, depth, or season. The data indicate that changes in Antarctic seasons are accompanied by significant shifts in the species composition of bacterioplankton assemblages and by large decreases in the relative proportion of archaeal rRNA in the nearshore water column.
During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the Cytophagales and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities.
The microbial diversity of a deteriorated biological phosphorus removal reactor was investigated by methods not requiring direct cultivation. The reactor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effluent and showed very limited biological phosphorus removal activity. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands representing at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparative phylogenetic analysis of the partial 16S rRNA sequences suggested that one sequence type was affiliated with the alpha subclass of the Proteobacteria, one was associated with the Legionella group of the gamma subclass of the Proteobacteria, and the remaining four formed a novel group of the gamma subclass of the Proteobacteria with no close relationship to any previously described species. The novel group represented approximately 75% of the PCR-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rRNA probes for this novel group were designed and used in a whole-cell hybridization analysis to investigate the abundance of this novel group in situ,The bacteria were coccoid and 3 to 4 mu m in diameter and represented approximately 35% of the total population, suggesting a relatively close agreement with the results obtained by the PCR-based DGGE method. Further, based on electron microscopy and standard staining microscopic analysis, this novel group was able to accumulate granule inclusions, possibly consisting of polyhydroxyalkanoate, inside the cells.
A total of 77 strains of genetically different fluorescent Pseudomonas strains were isolated from an agricultural soil. In pure culture growth experiments the strains were screened for their ability to grow in various concentrations of the sulfonylurea herbicides metsulfuron methyl, chlorsulfuron and thifensulfuron methyl. We found that the presence of the herbicides resulted in a reduction of the growth of the fluorescent pseudomonads. Metsulfuron methyl was shown to be toxic to a major proportion of the strains in low concentrations. Chlorsulfuron was found to be less toxic in low concentrations but toxic in high concentrations. Thifensulfuron methyl was toxic only to a minority of the strains. Indirectly, the growth- reducing effect of the sulfonylurea herbicides was shown to be caused by an inhibition of the enzyme acetolactate synthase. The enzyme is involved in the synthesis of the branched amino acids valine, leucine and isoleucine, and we demonstrated that the toxic effects of the sulfonylurea herbicides could be neutralized when the strains were grown in the presence of an excess amount of the three amino acids.
Publisher Summary Genetic fingerprinting techniques are excellently suited to comparison of large numbers of samples. Genetic fingerprinting of microbial communities provides banding patterns or profiles that reflect the genetic diversity of the community. Denaturing gradient gel electrophoresis (DGGE) of PCRamplified gene fragments is one of the genetic fingerprinting techniques used in microbial ecology. In DGGE, similar-sized DNA fragments are separated in a gradient of DNA denaturants according to differences in sequence. A variant of DGGE, temperature gradient gel electrophoresis (TGGE), makes use of a temperature gradient to separate gene fragments. DGGE is relatively easy to perform and is especially suited to the analysis of multiple samples. Since its introduction into microbial ecology, it has been adapted in many laboratories as a convenient tool for the assessment of microbial diversity in natural samples. It has been shown by several studies that the approach is reproducible and sensitive. Other new fingerprinting techniques, such as automated T-RFLP might be more sensitive but identification of predominant community members still requires cloning and sequencing of PCR products. A potential future development in PCR-DGGE fingerprinting might be to use fluorescently labelled PCR primers, which might (1) make staining of gels unnecessary, and (2) make it possible to add intra-lane standards with a different fluorochrome, facilitating gel-to-gel comparisons.
The influence of pesticides applied either singly or in combination on soil nitrogenase and nitrogen-fixing micro-organisms was investigated. Carbaryl, an insecticide commonly used in rice, had almost no effect on nitrogenase under submerged conditions while activity was stimulated even at 10 μg g−l under non-flooded conditions. Application of the herbicide butachlor or the insecticide carbofuran (at 2 μg g−1) reduced the population of Azospirillum and anaerobic nitrogen fixers in a non-flooded alluvial soil. In contrast, butachlor stimulated the population of anaerobic nitrogen fixers in an acid sulphate saline Pokkali soil under a similar water regime. Further, an increased level of carbofuran (4 μg g−1) stimulated the population of Azospirillum and anaerobic nitrogen fixers in both the soils. This stimulatory effect occurred, though not to the same extent, in the presence of butachlor which, when applied alone, inhibited the population of Azospirillum and anaerobic nitrogen fixers in alluvial soil. In Pokkali soil the combination of carbofuran and butachlor inhibited the population of Azospirillum, while carbofuran alone at 4 μg g−1 was stimulatory. These results suggest that under tropical conditions the effects of pesticides on nitrogen fixation and nitrogen-fixing populations depend upon pesticide concentration, water regime and soil type.
Direct DNA extraction followed by 18S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used for the analysis of fungal diversity in agricultural soil ecosystems. Various PCR primer sets have been used for fungi, but few reports have compared the properties of the primers. We investigated the properties of four widely used primer sets for fungal 18S rDNA DGGE: (1) NS1/GCFung, (2) FF390/FR1(N)-GC, (3) NS1/FR1(N)-GC (for single PCR) and (4) NS1/EF3 for the first PCR and NS1/FR1(N)-GC for the second PCR (for nested PCR). Using six soil samples from upland and paddy fields in Japan, the primers were compared in terms of PCR amplification efficacy, detection and reproducibility of the DGGE banding profiles, the obtained diversity indices, and the discrimination ability of the fungal communities using DGGE. The efficacy of PCR amplification using primer set 1 and the first PCR of primer set 4 was better than that using primer sets 2 and 3. In DGGE analysis, the PCR products of primer sets 3 and 4 showed the highest diversity indices. However, these primer sets had drawbacks, namely, the presence of non-specific aggregates and poor reproducibility of the DGGE profiles. Although primer sets 1 and 2 yielded shorter sequences of similar length, the PCR products with primer set 1 showed higher diversity indices than those with primer set 2. Multidimensional scaling analysis of the DGGE profiles indicated that primer set 1 could most clearly discriminate each fungal community in the soil samples. Although each primer set had advantages and disadvantages, together our analyses indicated that primer set 1 is the most suitable for detecting fungal diversities in soil using DGGE analysis. Our results are useful for selecting primers according to the aim of a particular study.