Response of Soil Microflora to Pesticides - Pesticides: Evaluation of Environmental Pollution - page 247

8.6.1 PCR-DGGE Profiling
One of the most popular methods based on DNA profiling is denaturing gradient gel elec-
trophoresis (DGGE), which enables rapid and reproducible separation of PCR-amplified
16S rDNA fragments in a mixed PCR. DNA fragments of the same length but different
nucleotide sequences are separated based on the difference in the mobilities of the mole-
cules in polyacrylamide gels with a linear denaturant gradient (Muyzer and Smalla 1998).
Differences in the electrophoretic profiles between the samples reflect the differences and
abundance in a community composition (Kent and Triplett 2002).
Changes in the microbial diversity as a result of herbicide application was found by Hua
et al. (2009), who studied the impact of different dosages (0-80 mg/kg soil) of naprop-
amide on the microbial community structure. Napropamide apparently increased the
number of bands that are considered to represent the dominant microbial populations,
on days 7 and 14 after application. The cluster analysis of 16S rDNA showed that simi-
larities among the different banding patterns obtained for control and pesticide-treated
samples were more than 90% on day 7; however, these similarities decreased to 40% over
the next week. These results suggested that some particular bacteria may be adapted
to the pesticide applied and dominated in the DGGE patterns obtained. Seghers et al.
(2003) used PCR-DGGE profiling to study the chronic effect of the herbicides atrazine
and metolachlor on the community structure in the soil of a maize monoculture. For the
extended analysis, they used the primers for both entire bacterial community and specific
group of bacteria such as Acidobacterium , the actinomycetes, the ammonium oxidizers,
and the methanotrophs. This approach allowed them to observe that the physiological
groups of bacteria differed in their reaction to pesticide application. The biggest differ-
ences between the control and the pesticide-treated soils were found in the case of metha-
notrophs. In their DGGE profiles from herbicide-treated soil, some bands were absent or
much weaker when compared with the patterns obtained for the control soil. The altera-
tions in the methanotrophic community observed at the first sampling time were also
seen in the next year. However, cluster analysis showed that the long-term use of atrazine
and metolachlor did not negatively affect the community structures of the tested micro-
bial groups (Seghers et al. 2003).
Similarly, bensulfuron-methyl applied at the field-recommended and 10-fold field-
recommended dosages did not affect the bacterial community over 8 weeks in a model
paddy soil. PCR-DGGE analysis revealed the same banding patterns in all pesticide-
treated and control soils on all sampling days (Saeki and Toyota 2004). By contrast, Ros
et al. (2006) analyzing PCR-DGGE banding patterns obtained for the soils treated with
atrazine found that regardless of the sampling time, herbicides at concentrations of 10, 100,
and 1000 mg/kg soil affected the bacterial community, compared with the control and at a
dosage of 1 mg/kg soil. This effect was observed even on day 45 when atrazine had been
degraded. The significant changes in both abundance and diversity of ammonia oxidizers
group under atrazine and other herbicide (dicamba, fluometuron, metolachlor, and sulfen-
trazone) exposure over an experimental period were also observed by Chang et al. (2001).
A response of the microbial community to another herbicide was studied by Chen et al.
(2009). Application of butachlor at 1- and 10-fold field-recommended dosages shifted the
nitrogen-fixation bacterial community and the effect was related to the stages of rice.
Changes among this bacterial group were much higher than those observed among the
total bacterial community in soils, which suggested that the diazotrophic community
might be more susceptible to the addition of butachlor as compared with other groups of
microorganisms.