Polish Journal of Ecology
Pol. J. Ecol. (2014) 62: 67–76
Regular research paper
Katarína ONDREIČKOVÁ1*, Daniel MIHÁLIK1, 2, Andrej FICEK3,
Martina HUDCOVICOVÁ1, Ján KRAIC1, 4, Hana DRAHOVSKÁ3
Plant Production Research Centre, Plant Production Research Institute, Bratislavská cesta 122,
SK-921 68 Piešťany, Slovak Republic,
*e-mail: [email protected] (corresponding author)
2
Institute of High Mountain Biology, University of Žilina, Tatranská Javorina 7,
SK-059 56 Tatranská Javorina, Slovak Republic
3
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava,
Mlynská dolina B-2, SK-842 15 Bratislava, Slovak Republic
4
Department of Biotechnology, University of SS. Cyril and Methodius in Trnava, Nám. J. Herdu 2,
SK-917 01 Trnava, Slovak Republic
1
IMPACT OF GENETICALLY MODIFIED MAIZE ON THE GENETIC
DIVERSITY OF RHIZOSPHERE BACTERIA:
A TWO-YEAR STUDY IN SLOVAKIA
ABSTRACT: Nowadays, genetically modi-
fied plants are cultivated in many countries and
it is important to consider their safety for surrounding environment. So, the environmental
risk assessments of genetically modified plants are
evaluated. This assessment consists of an objective
evaluation of risk and involves generating; collecting and assessing of information on a GM plant
with the aim to determine its impact on human
or animal health and the environment relative to
non-genetically modified organisms. One of the
numerous methods used to investigate the impact
of GM plants on the environment is the Terminal
Restriction Fragment Length Polymorphism. This
method was used for comparison of genetic variation in populations of bacteria isolated from rhizosphere of genetically modified maize MON810
carrying the gene cry1Ab and genetically nonmodified maize. Rhizosphere samples were collected in Slovakia during two years (2008, 2009)
in July and September and 16S rRNA gene was
amplified from metagenomic DNA using universal eubacterial primers. Differences in the number
of terminal restriction fragments between control
and GM maize hybrids were not detected. Additionally, variation within bacterial communities
composition from rhizosphere of MON810 and
non-GM hybrids was not observed, nevertheless
negligible differences in composition of bacterial
community were observed between two sampling
periods (July and September). These changes were
observed in non-GM as well as in GM maize hybrids and reflected effects of environment and
conditions, no influence of genetic modification.
The 16S rDNA clone library creation from rhizosphere sample of MON810 maize followed by
DNA sequencing revealed that the Proteobacteria
were major group of bacteria and Actinobacteria,
Firmicutes, and Chloroflexi were less represented.
This study did not confirm any changes in the soil
ecosystem, which would have been larger than
normal variations caused by external conditions.
KEY WORDS: bacterial diversity, MON810,
rhizosphere, Slovakia, transgenic maize, T-RFLP
1. INTRODUCTION
The genetically modified (GM) maize
containing transformation event MON810
(called Bt-maize) is actually permitted for
cultivation in several countries out of the
Europe (USA., Canada, Argentina, Brazil, Japan, South Africa, Uruguay, the Philippines).
The Commission of the European Communities decided to place MON810 on the EUmarket in the year 1998. Its cultivation is still
68
Katarína Ondreičková et al.
concentrated mainly in Spain and only very
small areas were sown in the past in other
EU-countries. In addition to Spain only Portugal, the Czech Republic, Romania, and Slovakia cultivated MON810 in the year 2012.
The MON810 maize expresses the Cry1Ab protein encoded by gene cry1Ab introduced into plant genome from the soil bacteria Bacillus thuringiensis (Bt). This protein
is expressing constitutively in plant tissues
and hydrolyses in the midgut of larvae of
susceptible insects. Produced polypeptide
Bt-toxins are deadly for target larvae so provide self-protection of plant against certain
lepidopteran insect pests including the European corn borer (Ostrinia nubilalis Hübner)
and pink borers (Sesamia spp.). The soil microbiota is non-target object of this protein;
nevertheless protein is deposited into soil by
pollen grains released after flowering and
by decomposed plant residues after harvest
(D one g an et al. 1997). The subsequent tillage embeds plant residues into deeper soil
layers leading to interactions of alien protein
with a large number of soil microorganisms.
Many factors affect accumulation of Cry1Ab
protein in soil as amount of transgene and
content of relevant protein in plant tissues,
protein persistence and resistance to degradation, chemical and physical parameters of
soil as well as environmental conditions (L iu
et al. 2005). Bt-maize releases the Cry1Ab
protein into soil also by root exudates (S a x e na et al. 1999, D u n f i el d and G e r m i d a
2004). According to S a xe na et al. (2002)
the Cry1Ab protein adsorbed on the surfaceactive particles of soil retains insecticidal
activity at least 180 days. On the other side,
no extraordinary stability in soil and no persisting immunoreactive Cry1Ab protein was
detected in any soil shortly before the next
seeding over the last third of nine years of Btmaize planting (Gr ub e r et al. 2012). However, any changes in chemical composition
of root exudates can beneficially or deleteriously change soil microenvironment and
modify biological diversity and activity of
microorganisms resident in soil, especially in
rhizosphere (Ly nch 1994). Different experiments about direct effects of Cry proteins
from Bt-crops on soil microbial communities reviewed Ic oz and Stot z ky (2008) and
reported none, minor or significant impacts.
Soil microbial communities are relevant and
sensitive tools for monitoring of different interactions between soil and cultivated crops,
farming practices, fertilizers, pesticides, environmental changes. Particularly rhizosphere
bacteria exposed to plant root exudates are
appropriate for plant-soil relationships analyses including impact studies of GM crops
cultivation.
The total number of bacteria per gram of
dry soil is ca. 1.5 × 1010 but only 0.1–0.5 %
can be cultivated on traditional cultivation
media (Tors v i k et al. 1990). Therefore the
cultivation independent techniques based on
analysis of metagenomic DNA isolated from
soil (Hi rs ch et al. 2010) provide more effective tools for relationships studies between
microorganisms and soil, plants, biotic and
abiotic factors. An effective approach for bacterial studies by metagenomic DNA analysis
is technique using small subunit ribosomal RNA sequences (Av an iss- Ag haj an i
et al. 1994, 1996) later named as terminal
restriction fragment length polymorphism
(T-RFLP) (L iu et al. 1997).
Genetically modified plants, including
maize, play an important role in agriculture
and economy in many countries. In spite of
their advantages also their impacts to environment must be considered. An essential
is also to examine whether cultivation of
GM plants leads generally to changes in microbial diversity in their rhizosphere. Such
study has not been yet conducted in Slovakia over seven years of MON810 cultivation. Therefore aims of this study were to:
i) compare genetic variation and differences
between bacterial communities in rhizosphere of Bt-maize containing MON810
transformation event and non-GM hybrid
and ii) characterize bacteria by creating of
16S rDNA clone library from rhizosphere
of MON810 maize.
2. STUDY AREA
Experimental fields were located at the
Research Station in Borovce (Fig. 1A) of Plant
Production Research Center Piešťany (GPS
48°34’38’’ N and 17°43’45’’ E) in Slovakia.
Soil and climatic conditions of the workplace:
loamy luvic chernozem with pH 5.2, mean
annual temperature 9.2°C (for vegetation
Impact of transgenic maize on rhizosphere bacteria
69
Table 1. Genetically modified MON810 (GM) and conventional (C) maize hybrids and their codes in
the experiments.
Rhizosphere samples
Sampling
Hybrids of conventional maize
Hybrids of MON 810 maize
date
DKC4442YG
MEB483BT
TPA422-H
DKC3511
DKC3511
July
GM1J
GM2J
GM3J
C1J
C2J
September
GM1S
GM2S
GM3S
C1S
C2S
Fig. 1. Location of field trials in the Western Slovakia (black point) (A). Positions of rhizosphere collecting sites within field plots (crosses) during both years 2008 and 2009 (B). Positioning of maize hybrids in
plots in 2008: plot No.1 – C1, No.2 – GM1, No.3 – GM2, No.4 – GM3, No.5 – C2; and in 2009: plot No.1
– C1, No.2 – GM3, No.3 – GM1, No.4 – GM2, No.5 – C2 (codes of the samples are according to Table 1).
15.5°C), mean annual rainfall 625 mm (for
vegetation 358 mm), and altitude 167 m. The
production area is transient maize-sugar beet
farming.
3. MATERIALS AND METHODS
3.1. Rhizosphere sampling
Rhizosphere samples were collected in
July and September 2008 and 2009, always
from the same sites. There were cultivated
three hybrids of GM maize MON810 – DKC4442YG, MEB483BT, and TPA422-H and
the conventional maize DKC3511 as control.
Simplified names of the samples of maize hybrids are shown in Table 1. Maize was planted
in field plots 3 m × 200 m and from each plot
four separate rhizosphere samples were collected (Fig. 1B). MON810 plants were taken
out from soil, soil residues from roots were
gently removed and rhizosphere soil was
scraped from roots with sterile scalpel and
stored at 4°C.
3.2 T-RFLP analysis
Each rhizosphere sample was homogenized and metagenomic DNA was extracted
by the PowerSoil DNA Isolation Kit (MoBio
Laboratories, Inc.) according to the manufacturer’s protocol. Concentration and purity of
isolated DNA were pre measured by Nanodrop
1000 Spectrophotometer (Thermo Scientific)
and samples were diluted to the same final
concentration (25 ng/μl). DNA amplification
was carried out in 50 µl reaction mixture containing FailSafeTM PCR PreMix Selection Kit
(Epicentre Biotechnologies); 0.10 μM of both
primers and 1 µl of DNA extracted from soil.
Bacterial universal primers for the 16S rRNA
gene 8F (AGAGTTTGATCCTGGCTCAG,
FAM labelled) (Edwards et al. 1989) and 926R
(CCGTCAATTCCTTTRAGTTT) (Muy z e r
et al. 1995) were used. PCR was performed
using the program: 3 min at 95°C, 35 cycles of
30 s at 94°C, 30 s at 47°C, 1 min at 72°C and
final polymerization 10 min at 72°C in GeneAmp PCR System 9700 (Applied Biosystems). Triplicate reactions were pooled, and
Katarína Ondreičková et al.
70
PCR products were purified by the PCR Purification & Agarose Gel Extraction Combo
Kit (E. coli s.r.o.). Purified PCR products were
digested separately with restriction enzymes
CfoI, MspI and RsaI (Roche) in digestion
mixture with total volume of 20 µl containing 10 U of restriction enzyme, 2 µl of 10 ×
relevant buffer, and 10 µl of purified PCR mix
and incubated for 3 h at 37°C. Digested samples were purified by purification kit mentioned above. Terminal restriction fragments
(T-RFs) were separated by electrophoresis in
ABI Prism 3100 Avant apparatus (Applied
Biosystems) with LIZ 600 internal standard.
Electrophoretograms were analyzed by GeneMapper 3.5 (Applied Biosystems). Only fragments between 70 bp and 600 bp were used
for evaluation.
3.3. Clone library and sequencing
Bacterial clone library was created from
metagenomic DNA isolated from the rhizosphere of MON810 maize (MEB483BT) collected on September 2009, extracted, and amplified as described above. Amplicons were
ligated into the pGEM-T Easy vector (Promega) and transformed into E. coli TOP10F’
(Invitrogen) according to the manufacturer’s
instructions. Plasmids from one hundred
independent colonies (labelled 1-100) were
isolated by GeneJET Plasmid Miniprep kit
(Fermentas). Variation of cloned inserts was
assessed by T-RFLP according to the procedure described, but all three enzymes in
a single reaction were used for the cleavage.
Clones representing T-RFLP specific profiles were sequenced and analyzed by the
Chromas 2.33 (Technelysium Pty Ltd). DNA
sequences of clones were deposited into the
GenBank database (accessions KC693737KC693751 and KF638285-638289). The Basic Local Alignment Search Tool (BLAST)
of the National Center for Biotechnology Information (NCBI) was used for searching of
homologous sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/)
and the NEBcutter V2.0 (Vincze et al. 2003)
was used for in silico restriction cleavage of
sequenced clones.
3.4. Statistical analysis
Results were statistically evaluated by
Fisher’s least significant difference (LSD)
procedure at the 95% confidence level. Bacterial communities in different samples were
compared by the presence/absence of specific
T-RFs. T-RF peaks were classified as present
(1) or absent (0) in each sample and the binary system was used for Principal Component
Analysis (PCA) using the statistical software
Statgraphics Centurion XV.II. Dendrogram
based on DNA sequences was constructed
by the UPGMA method using the program
CLUSTAL 2.1.
4. RESULTS
4.1. Genetic variation in rhizosphere
bacteria of GM and non-GM maize
Rhizosphere samples were collected from
three hybrids of Bt maize MON810 and one
conventional hybrid in four sampling dates
(July 2008 and 2009, September 2008 and
2009), and bacterial 16S rRNA genes were
amplified and analyzed by T-RFLP. T-RFLP
analysis yielded between 22 and 65 T-RFs
in individual community profiles which had
intensities of at least 50 fluorescence units
using CfoI enzyme, from 13 to 73 T-RFs using MspI and from 16 to 34 T-RFs using RsaI
restriction enzyme. Nevertheless, statistically
significant differences between samples were
not confirmed (LSD, P> 0.05). It was possible
to detect the lengths of T-RFs, which were
located in all samples from each sampling
period. More of these ubiquitous T-RFs were
detected in the year 2008 in comparison to
Table 2. Ubiquitous T-RFs in bp detected in both years after CfoI, MspI and RsaI digestion.
Enzyme
CfoI
MspI
RsaI
Year 2008
86, 88, 102, 134,
175, 190, 337
71, 72, 77, 83, 86, 107,
116, 122, 133, 135, 143,
145, 153, 155, 163, 183
70, 76, 87, 96
Year 2009
75, 76, 86, 206, 432
135, 137, 143, 145, 147
70, 100, 103, 112, 123
Impact of transgenic maize on rhizosphere bacteria
71
Table 3. Table of sequenced clones compared with 16S rDNA sequences of bacteria with the highest
similarity.
Clone
No.
1
2
4
5
6
9
15
16
18
19
20
29
30
33
41
52
53
56
57
63
GenBank
accession no.
NR_041585.1
NR_041994.1
NR_041798.1
NR_040834.1
NR_043037.1
NR_044306.1
NR_043184.1
NR_040936.1
NR_044284.1
NR_042502.1
NR_024776.1
NR_043411.1
NR_042003.1
NR_044095.1
NR_025673.1
NR_043615.1
NR_042368.1
NR_037121.1
NR_025374.1
NR_043934.1
The closest genera
Rhizobacter dauci
Bosea thiooxidans
Ferrimicrobium acidiphilum
Porphyrobacter tepidarius
Bradyrhizobium pachyrhizi
Microlunatus aurantiacus
Ochrobactrum cytisi
Rhodoplanes serenus
Ammonifex thiophilus
Massilia aurea
Thermaerobacter nagasakiensis
Chloroflexus aurantiacus
Pseudonocardia hydrocarbonoxydans
Filomicrobium insigne
Schlegelella thermodepolymerans
Variovorax dokdonensis
Modestobacter versicolor
Blastochloris sulfoviridis
Methylobacterium fujisawaense
Azospirillum zeae
Similarity
(%)
96
94
85
91
91
93
98
96
83
95
83
81
94
93
85
98
96
93
86
84
Phylogenetic group
Gammaproteobacteria
Alphaproteobacteria
Actinobacteria
Alphaproteobacteria
Alphaproteobacteria
Actinobacteria
Alphaproteobacteria
Alphaproteobacteria
Firmicutes
Betaproteobacteria
Firmicutes
Chloroflexi
Actinobacteria
Alphaproteobacteria
Betaproteobacteria
Betaproteobacteria
Actinobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Fig. 2. T-RFLP profiles from rhizosphere sample of GM maize in 2008 generated after CfoI, MspI and RsaI
cleavage. Ubiquitous T-RFs detected in this year in all samples are marked with the respective sizes in bp.
72
Katarína Ondreičková et al.
2009 (Table 2). The intensities of the fluorescent signals of these T-RFs were different
for each sample. Ubiquitous T-RFs generated
after CfoI and RsaI cleavage had more balanced amount of fluorescent signal, but on
the other hand, T-RFs 135 bp, 143 bp and 145
bp generated by MspI cleavage showed the
strongest fluorescent signal in comparison to
other ubiquitous MspI T-RFs (Fig. 2). Only
one T-RF (86 bp) after CfoI digestion, three
T-RFs after MspI digestion (135 bp, 143 bp
and 145 bp) and one T-RF after RsaI digestion (70 bp) were present in all rhizosphere
samples in both years. These five ubiquitous
T-RFs could reflect the most commonly occurring bacteria or bacteria specific for soil in
experimental location, respectively.
Also bacteria collected from the same
either GM or non-GM maize plants and at
the same time (either on July or September)
did not grouped together. Grouping of plants
reflecting presence of cry1Ab gene, year of
cultivation, or date of sampling was not revealed. This suggests that other factors than
root exudates from Bt-maize are particularly responsible for genetic variation within
bacterial rhizosphere communities. Genetic
variation evaluated by the PCA analysis
(Fig. 3) indicated that there were no significant changes and corresponding differences
between bacterial rhizospheres of GM and
non-GM plants. Simultaneously, soil bacterial diversity in rhizosphere was influenced
by seasonal changes caused by external environmental conditions. The Bt-maize impact
on soil bacteria was not demonstrated.
4.2. Analysis of 16S rDNA clone library
Rhizosphere sample from Bt maize
MEB483BT collected in September 2009 was
elected for constructing of 16S rDNA clone
library. Twenty clones with specific T-RFLP
profiles were sequenced and compared with
16S rDNA sequences of several the closest
bacterial genera available in the GenBank database using the BLAST software (Table 3).
Absolute (i.e. 100%) identity between compared DNA sequences was not observed.
DNA sequences of clones with the highest
similarity were used to construct dendrogram (Fig. 4) where 18 clones grouped in
accordance with DNA sequence similarity
but two clones (no. 2 and 52) were classified
Table 4. Comparison of T-RF sizes obtained experimentally and in silico. Restriction enzyme that digested 16S rDNA sequences in silico as the first is shown in the fourth column.
Experimental
In silico T-RF
Clone No.
Enzyme
T-RF size (bp)
size (bp)
1
137
141
MspI
2
128
132
MspI
4
130
134
MspI
5
128
132
MspI
6
102
109
CfoI
9
52
61
CfoI
15
52
61
CfoI
16
103
110
RsaI
18
156
162
MspI
19
59
66
CfoI
20
279
286
MspI
29
76
86
CfoI
30
273
278
MspI
33
127
128
MspI
41
63
67
CfoI
52
146
152
MspI
53
273
280
MspI
56
144
152
MspI
57
52
61
CfoI
63
70
79
MspI
Impact of transgenic maize on rhizosphere bacteria
otherwise. Clones were spread throughout
the dendrogram indicating high diversity of
soil bacteria in the Bt-maize rhizosphere. The
clone no. 2 was the most similar to the Alphaproteobacteria (Table 3); nevertheless cluster
analysis allocated this clone separately from
other bacteria. The clone no. 52 assigned to
the phylogenetic group of Betaproteobacteria
(Table 3) was clustered to Alphaproteobacteria. Twelve from clones from Bt-maize rhizosphere were classify to Proteobacteria and
to Alphaproteobacteria, Betaproteobacteria,
and Gammaproteobacteria, respectively. Actinobacteria was the second most abundant
taxon where 4 clones were classified. Other
taxons were Firmicutes containing 2 and
Chloroflexi with 1 sequenced clone.
All twenty sequences of clones were in
silico digested using the NEBcutter V2.0
(Vincze et al. 2003) using CfoI, MspI, or
RsaI, respectively, to determine the expected
sizes of the digested fragments. Subsequently
experimentally obtained sizes of T-RFs were
compared with T-RFs identified in silico and
showed that there were differences in the TRFs size (Table 4). The experimental values
were in average shorter by 6 bp than expected
and these variations are caused by different
migration of internal standard (LIZ 600) and
FAM-labeled restriction fragments.
73
changes in the rhizosphere bacterial diversity
between GM and non-GM maize. Simultaneously the results obtained by the PCA analysis indicate that there were seasonal changes
in soil bacterial diversity in the rhizosphere
between individual maize hybrids, but these
changes are slight and are most likely caused
by external environmental conditions. The
impact of Bt maize on soil bacteria was not
demonstrated at all whereas the changes were
observed between the non-GM maize. Similar results as these were published in several
works. D oneg an et al. (1995) found that
endotoxin derived from Bacillus thuringiensis
subsp. kurstaki, produced in transgenic plants
had no direct effect on soil microorganisms
but observed effects of plant cultivars. Fang
et al. (2005) reported that bacterial diversity
in the rhizosphere of GM and non-GM maize
was more under the influence of soil structure
than GM plants. None effects of Bt-maize and
Bt-protein on the microbial community was
5. DISCUSSION
Soil microbial communities perform
complex processes which have major ecological and agricultural importance. One of the
basic functions is to maintain healthy soils by
regulating the nutrient cycle. Simultaneously
genetically modified crops, similar to any agricultural crops can influence soil processes,
due to the narrow interactions between plants
and soil ecosystems. For this reason it is necessary to determine whether the commercial
cultivation of GM crops induces changes in
biodiversity and soil processes and whether
these changes are higher than the natural
changes caused by environmental factors or
as a result of the conventional farming system.
Therefore, the impact of GM maize MON810
on the composition of bacterial communities
in the rhizosphere was studied by T-RFLP
method. The obtained result of the PCA analysis indicates that there were no significant
Fig. 3. The PCA analysis of T-RFLP profiles of
bacterial communities from rhizosphere samples
in 2008 (up) and 2009 (down) created by combination of all T-RFLP data using all three restriction enzymes (codes of samples are in the Table 1).
74
Katarína Ondreičková et al.
Fig. 4. Dendrogram created using the 16S rDNA sequences obtained from rhizosphere of MON810
maize with sequences of bacteria with the highest similarity (filled circle indicates the sequenced clones).
observed in other studies and soil type was
determined as the main factor influencing
evaluated parameters (Gr if f it hs et al. 2006).
Baumgarte and Tebbe (2005) presented that
the structure of bacterial community in the
rhizosphere of Bt-maize was less affected by
Cry1Ab toxin than by environmental factors
such as the plant growth phase and native
soil diversity. Also Bl ackwo o d and Buye r
(2004) showed that for significant differences
in soil microbial communities was not responsible Bt-maize. Soil type was designated
as the most important factor affecting internal
composition of soil communities. Pr is ch l
et al. (2012) observed that the expression of
one or more cry genes did not affected endophytic bacterial communities associated
with the GM maize hybrid MON89034 ×
MON88017, their parental lines MON89034,
MON88017, and non-transgenic near-isogenic line. Recent study of D ohr mann et al.
(2013) indicates that the rhizosphere bacterial
community of GM maize did not responded
abnormally to the presence of three insecticidal proteins in root tissues. Our results revealed minor seasonal variation in genetic diversity of rhizosphere bacteria between maize
hybrids just like Ic oz et al. (2007) who also
observed the seasonal changes in the number of microorganisms and enzyme activity,
but these seasonal differences were independent of presence of the Cry proteins in plants.
D u n f iel d and G e r mi d a (2003) performed
two-year observations in field conditions and
pointed to seasonal changes in the composition of microbial communities for GM plant,
as well as for a conventional non-GM hybrid.
Differences in the measured parameters between rhizosphere and non-rhizosphere soils
observed Olivei ra et al. (2008). Under their
field conditions the presence of Bt-maize did
not caused changes in the microbial populations of the soil or in the activity of the microbial community.
Impact of transgenic maize on rhizosphere bacteria
6. CONCLUSIONS
The risks of genetically modified crops
for the environment, and especially for biodiversity, have been extensively assessed
worldwide but not yet in Slovakia. In this first
Slovak study no effect of genetically modified maize hybrids MON810, expressing the
Cry1Ab protein, on genetic diversity of rhizosphere bacteria was demonstrated. Only slight
changes were observed in composition of
bacterial communities collected in July and in
September resulted from environmental conditions, not from genetic event. Differences
in genetic diversity of bacterial communities
from rhizosphere of conventional and GM
plants were observed neither between experimental years (2008, 2009) nor between sampling dates (July and September). By analysis
of the 16S rDNA clone library from sample of
GM maize has been characterized Proteobacteria as a major group of rhizosphere bacteria.
Also by comparing T-RFLP analysis of clones
and in silico cleavage of sequenced clones,
there was observed a shift in the T-RF sizes.
Experimentally obtained T-RFs were in average of 6 bp shorter than expected.
Concerns regarding the possible negative effects of MON810 maize on the rhizosphere bacterial community have not been
confirmed and our findings are in agreement
with other published studies.
ACKNOWLEDGEMENTS: This work was
supported by the Slovak Research and Development
Agency under the contract No. APVV-0294-11.
7. REFERENCES
Avaniss-Ag haj ani E., Jones K., C hapman
D., Br un k C. 1994 – A molecular technique
for identification of bacteria using small subunit ribosomal RNA sequences – BioTechniques, 17: 144–149.
Avanis-Ag haj ani E., Jones K., Holtzman
A., Arons on T., Glover N., B oi an M.,
Froman S., Br un k C.F. 1996 – Molecular
technique for rapid identification of mycobacteria – J. Clin. Microbiol. 37: 98–102.
B aumgar te S., Tebb e C.C. 2005 – Field studies on the environmental fate of the Cry1Ab Bttoxin produced by transgenic maize (MON810)
and its effect on bacterial communities in the
maize rhizosphere – Mol. Ecol. 14: 2539–2551.
75
Bl ackwo o d C.B., Buyer J.S. 2004 – Soil microbial communities associated with Bt and
non-Bt corn in three soils – J. Environ. Qual.
33: 832–836.
D ohr mann A.B., Küt ing M., Jünemann
S., Jaenicke S., S ch lüter A., Tebb e
C.C. 2013 – Importance of rare taxa for bacte�rial diversity in the rhizosphere of Bt- and con�ventional maize varieties – ISME J. 7: 37–49.
D onegan K.K., Pa lm C.J., Fiel and V. J.,
Por te ous L.A., Ganio L.M., S cha l ler
D.L., Buc ao L.Q., S eid ler R .J. 1995 –
Changes in levels, species, and DNA fingerprints of soil microorganisms associated with
cotton expressing the Bacillus thuringiensis
var. kurstaki endotoxin – Appl. Soil Ecol. 2:
111–124.
D onegan K.K., S eid ler R .J., Fiel and V. J.,
S cha l ler D.L., Pa lm C.J., Ganio L.M.,
C ardwel l D.M., Steinb erger Y. 1997 –
Decomposition of genetically engineered tobacco under field conditions: Persistence of
the proteinase inhibitor I product and effects
on soil microbial respiration and protozoa,
nematode and microarthropod populations –
J. Appl. Ecol. 34: 767–777.
D unf ield K.E., G er mid a J.J. 2003 – Seasonal changes in the rhizosphere microbial communities associated with field-grown genetically modified canola (Brassica napus) – Appl.
Environ. Microbiol. 69: 7310–7318.
D unf ield K.E., G er mid a J.J. 2004 – Impact
of genetically modified crops on soil and plant
associated microbial communities – J. Environ. Qual. 33: 806–815.
E dwards U., R oga l l T., Blö cker H., Emde
M., B ött ger E.C. 1989 – Isolation and direct complete nucleotide determination of entire genes: characterization of a gene coding
for 16S ribosomal RNA – Nucleic Acids Res.
17: 7843–7853.
Fang M., Kremer R .J., Mot ava l li P.P.,
D av is G. 2005 – Bacterial diversity in rhizospheres of nontransgenic and transgenic corn
– Appl. Environ. Microbiol. 71: 4132–4136.
Gr if f it hs B.S., C au l S., Thomps on J.,
Birch A.N.E., S cr imge our C., C or tet
J., Fog go A., Hackett C.A., Krog h P.H.
2006 – Soil microbial and faunal community
responses to Bt maize and insecticide in two
soils – J. Environ. Qual. 35: 734–741.
Gr ub er H., Pau l V., Me yer H.D.M., Mü l ler
M. 2012 – Determination of insecticidal
Cry1Ab protein in soil collected in the final
growing seasons of a nine-year field trial of
Bt-maize MON810 – Transgenic Res. 21: 77–88.
Hirs ch P. R ., Mauch line a T.H., C l arka
I.M. 2010 – Culture-independent molecular
76
Katarína Ondreičková et al.
techniques for soil microbial ecology – Soil
Biol. Biochem. 42: 878–887.
Icoz I., Saxena D., Andow D., Zwa h len
C., Stotzky G. 2007 – Microbial populations and enzyme activities in soil in situ under transgenic corn expressing Cry proteins
from Bacillus thuringiensis – J. Environ. Qual.
37: 647–662.
Icoz I., Stotzky G. 2008 – Fate and effects of
insect-resistant Bt crops in soil ecosystems –
Soil Biol. Biochem. 40: 559–586.
L iu W.T., Marsh T.L., C heng H., For ne y
L.J. 1997 – Characterization of microbial diversity by determining Terminal Restriction
Fragment Length Polymorphisms of genes encoding 16S rRNA – Appl. Environ. Microbiol.
63: 4516–4522.
L iu B., Z eng Q., Yan F., Xu H., Xu C. 2005
– Effects of transgenic plants on soil microorganisms – Plant Soil, 271: 1–13.
Ly nch J. 1994 – The rhizosphere – form and
function – Appl. Soil Ecol. 1: 193–198.
Muyzer G., Teske A., Wirs en C.O. 1995
– Phylogenetic relationships of Thiomicrospira
species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments
– Arch. Microbiol. 164: 165–172.
Oliveira A.P., Pampu l ha M.E., B ennett
J.P. 2008 – A two-year field study with transgenic Bacillus thuringiensis maize: Effects on
soil microorganisms – Sci. Total Environ. 405:
351–357.
Pr is ch l M., Hack l E., Past ar M., Pfeif fer S., S essits ch A. 2012 – Genetically
modified Bt maize lines containing cry3Bb1,
cry1A105 or cry1Ab2 do not affect the structure and functioning of root-associated endophyte communities – Appl. Soil Ecology, 54:
39–48.
Saxena D., Flores S., Stotzky G. 1999 –
Transgenic plants: Insecticidal toxin in root
exudates from Bt corn – Nature, 402: 480.
Saxena D., Flores S., Stotzky G. 2002 –
Bt toxin is released in root exudates from 12
transgenic corn hybrids representing three
transformation events – Soil Biol. Biochem.
34: 133–137.
Torsv i k V., G oks øy r J., D aae F.L. 1990 –
High diversity in DNA of soil bacteria – Appl.
Environ. Microbiol. 56: 782–787.
Vincze T., Posfai J., R ob er ts R .J. 2003 –
NEBcutter: a program to cleave DNA with
restriction enzymes – Nucleic Acids Res. 31:
3688–3691.
Received after revision September 2013