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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
URL: http://dx.doi.org/10.1080/03601231003799804
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Journal of Environmental Science and Health Part B (2010) 45, 348–359
Copyright C
Taylor & Francis Group, LLC
ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/03601231003799804
Effect of pesticides on soil microbial community
CHI-CHU LO
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
discussedinsection2.
Keywords: Fungicides; herbicides; insecticides; PCR-DGGE.
Introduction
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: lcc@tactri.gov.tw
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).
Herbicides
Butachlor
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.
[2]
Diflubenzuron (I)/maize
field
Diflubenzuron (100∼500 µg/g) had a stimulatory effect on dinitrogen fixation bacteria (Azotobacter
vinelandii) in soil.
[11]
Methylpyrimifos (I),
Chlorpyrifos (I)/maize
field
Methylpyrimifos (100∼300 µg/g) or chlorpyrifos (10∼300 µg/g) significantly decreased aerobic dinitrogen
fixing bacteria, however, fungal populations and denitrifying bacteria were not affected.
[10]
Metsulfuron methyl (H),
chlorsulfuron (H),
thifensulfuron methyl
(H)/Agricultural soil
Reduction of the growth of fluorescent psendomonads (77 strains). [6]
Diuron (H), linuron (H),
chlorotoluron
(H)/orchard
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.
[5]
Fenpropimorph
(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.
[17]
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.
[54]
Iprodione (F)/strawberry
orchard
DGGE profiles showed that the soil bacterial community is more complex at 30◦Cthanat15
◦C. At 30◦C
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.
[18]
Glyphosate (H)/ponderosa
pine plantations
Glyphosate produces a non-specific, short-term stimulation of bacteria at a high concentration (100×field
rate).
[9]
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 41∼83 % 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.
[12]
Methylparathion
(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 %).
[14]
Azoxystrobin (F),
chlorothalonil (F),
tebuconazole
(F)/conventional farm
and organic management
farm
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
rRNA-DGGE).
[19]
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.
[7]
Methamidophos (I)/black
soil
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.
[48]
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 %.
[3]
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
sediment/V3/16S
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.
[26]
Soil with known
actinomycetes
populations/V6-V8/16S
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.
[55]
Microbial diversity in
sludge from biological
phosphorus removal
reactor
EBPR/V6-V8/16S
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
proteobacteria.
[56]
Soil (16 soils)/ V6-V8/16S Soil DNA recovered from the different soils by the direct extraction method ranged from 8∼35 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 ×106g−1dry soil or higher (1∼3×104
genomes per reaction), and 0.5–1.5 % of the total microscopically detectable bacterial community may be
detectable via community based PCR-DGGE.
[57]
Diagnostic tool for
detecting microbial in
fection/V7-V9/16S
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.
[58]
Microbial transformation
of dieldrin in
sediment/V6-V8/16S
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.
[59]
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.
[51]
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.
[52]
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.
[60]
Nonylphenol (NP) in
mangrove
sediments/V6-V8/16S
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.
[61]
Assess species diversity of
arbuscular mycorrhizal
fungi/V3-V4 and
V9/18S
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.
[41]
Soil after fumigation/
V7-V8/18S
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.
[42]
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.
[43]
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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-
amino-4,5-dihydro-3-methyl-6-phenyl-1,2,4-triazin-5-one,
Downloaded By: [Taiwan Agric Chem & Toxic Subs Research Institute] At: 00:42 31 May 2010
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-
ment.
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
methyl
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-
methyl-1,3,5-trazine-2-yl-carbamoylsulfamoyl)benzoate],
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
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
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-
ties.
Glyphosate applied at the recommended field rate (5 kg
a.i. ha−1) 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.
Insecticides
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 ha−1for 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
−1of
chlorpyrifos. Fungal populations and denitrifying bacteria
were not affected as a consequence of the addition of the
organophosphorus insecticides to the agricultural soil.
Diflubenzuron
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
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 pot−1week−1, and CS3,
0.31 g pot−1week−1) and urea application (CS4, 0.5 g
pot−1week−1) 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
Ptol−1values 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
methamidophos.
Fenamiphos
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 kg−1soil. Potential
nitrification was found to be highly sensitive to fenamiphos
with a significant inhibition recorded at 10 mg kg−1soil.
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 kg−1dry soil. There-
fore, the authors suggested that fenamiphos is likely to be
detrimental to nitrification at field application rate.
Methylparathion
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.
Fungicides
Fenpropimorph
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 kg−1soil).
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
g−1root litter by day 7, and 3 ×106protozoa g−1root
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
kg−1fenpropimorph 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
populations.
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
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
(15◦C) with a lower iprodione concentration (5 µgg
−1). At
the same temperature but with more iprodione (50 µgg
−1)
added, the soil bacterial community increased slowly and
regained the original status slowly. When incubated at the
higher temperature (30◦C), the soil bacterial community
was more complex than that at the lower temperature. The
amounts of soil bacterial communities increased quickly
at 30◦C 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
(E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-
3-methoxyacrylate] (5 mg kg−1), tebuconazole [(RS)-1-p-
chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylme-
thyl)pentan-3-ol] (5 mg kg−1) and chlorothalonil (tetra-
chloroisophthalonitrile) (10 mg kg−1) (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
biomass−1soil, 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
biomass−1soil, but not in the high OM biomass−1soil.
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.
Mancozeb
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
populations.[21]
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
database.
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[28−30] and aquatic environments.[31−34] 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.[42−43]
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]
Conclusion
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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