Agriculture, Ecosystems and Environment 235 (2016) 289–305
Contents lists available at ScienceDirect
Agriculture, Ecosystems and Environment
journal homepage: www.elsevier.com/locate/agee
Review
Do genetically modified plants affect adversely on soil microbial
communities?
Zheng-jun Guana,b , Shun-bao Luc , Yan-lin Huod , Zheng-Ping Guane , Biao Liuf , Wei Weib,*
a
Department of Life Sciences, Yuncheng University, Yuncheng, Shanxi 044000, China
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
College of Life Sciences, Jiangxi Normal University, Nanchang 330022, China
d
Centre of Science Experiment, Yuncheng University, Yuncheng, Shanxi 044000, China
e
College of Food Science, Shanxi Normal University, Linfen, 041000, China
f
Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection of China, Nanjing, Jiangsu 210042, China
b
c
A R T I C L E I N F O
Article history:
Received 15 March 2016
Received in revised form 27 October 2016
Accepted 29 October 2016
Available online xxx
Keywords:
Ecological risk
GM plant
Molecular technique
Non-target effect
Soil microorganism
A B S T R A C T
With the increase in the number of commercial applications and larger cultivation areas of genetically
modified (GM) plants, their biosafety for soil microorganisms has become a controversial issue. The
effects on the diversity and abundance of soil microorganisms are important components of evaluation of
the biosafety risks of GM plants. So far, no definite conclusions have been drawn about whether GM
plants can negatively affect soil microorganisms. In this review, we discuss the advances that have been
made in recent years in the research into the effects of GM plants on soil microbial communities. It has
been argued that foreign gene products that are released from the residue of GM plants into soil by root
exudation may affect soil microbial communities. Moreover, foreign genes may change the genetic and
functional properties of soil microorganisms via horizontal transfer. The advantages and disadvantages of
various detection technologies—from classical culture-dependent methods to modern molecular
protocols—are reviewed here. To accurately and comprehensively evaluate the effects of GM plants on
microorganisms, we discuss the factors that should be considered in the assessment of risks of GM plants
for soil microorganisms (e.g., foreign proteins, marker genes, plant varieties, and environmental factors),
as well as the problems and prospects related to biosafety assessment platforms for GM plants.
ã 2016 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mechanisms underlying the effects of GM plants on soil microorganisms . . .
Effects of foreign gene products on soil microorganisms . . . . . . . . . . . .
2.1.
Effects of horizontal gene transfer (HGT) on soil microorganisms . . . .
2.2.
Detection of the effect of GM plants on soil microbial communities . . . . . . . .
Traditional culture and observation methods . . . . . . . . . . . . . . . . . . . . .
3.1.
The biolog microplate technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
The modern biomarker approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Modern molecular biological methods . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Advances in the research on the effects of GM plants on soil microorganisms
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
290
290
290
290
291
291
291
292
292
292
Abbreviations: AMV, alfalfa mosaic virus; ARDRA, amplified ribosomal DNA restriction analysis; ARISA, automated ribosomal intergenic spacer analysis; Bt, Bacillus
thuringiensis; CaMV, cauliflower mosaic virus; CFUs, colony-forming units; CLPP, community-level physiological profile; DGGE, denaturing gradient gel electrophoresis;
FISH, fluorescence in situ hybridization; GM, genetically modified; GFP, green fluorescent protein; NGS, next generation sequencing; PRSV, papaya ringspot virus; PLFAs,
phospholipid fatty acids; RT-PCR, real-time PCR; RFLP, restriction fragment length polymorphism; RISA, ribosomal intergenic spacer analysis; T-RFLP, terminal restriction
fragment length polymorphism.
* Corresponding author at: 20 Nanxincun, Xiangshan, Beijing 100093, China.
E-mail address: [email protected] (W. Wei).
http://dx.doi.org/10.1016/j.agee.2016.10.026
0167-8809/ã 2016 Elsevier B.V. All rights reserved.
290
5.
6.
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
4.1.
The impact of insect-resistant GM plants on soil microbial communities . . . . . . . . .
The impact of herbicide-resistant GM plants on soil microbial communities . . . . . . .
4.2.
The influence of disease-resistant GM plants on soil microbial communities . . . . . .
4.3.
The impact of quality-relevant GM plants on soil microbial communities . . . . . . . . .
4.4.
The influence of GM plants with other traits on soil microbial communities . . . . . .
4.5.
The impact of GM plant with stacked resistant traits on soil microbial communities
4.6.
Factors considered in the assessment of risks of GM plants for soil microorganisms . . . . . .
Persistence of GM products in soil microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
5.2.
Effects of marker genes on soil microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of plant varieties on soil microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
The effect of environmental factors on soil microorganisms . . . . . . . . . . . . . . . . . . . .
5.4.
5.5.
Effects of differential management measures on soil microorganisms . . . . . . . . . . . .
Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Microorganisms, an important component of soil, play a major
role in ecosystem processes such as substance transformation and
energy exchange. Moreover, the abundance and diversity of
microbial communities are important indicators of dynamic
changes in soil ecosystems (Powell et al., 2015; van der Heijden
et al., 2008a, 2008b, 2008c). Soil microorganisms are the link
between soil and plants, integrating these two systems. In the
soil, because the rhizosphere is a special ecological environment,
the abundance and diversity of soil microorganisms in the
rhizosphere are far higher than those in non-rhizosphere soils
(Filion, 2008). Plant–microorganism interactions in the rhizosphere soil are a major factor in the regulation of plant growth
and development. Variations in root exudate composition and
abundance may alter soil microbial biodiversity and activity as
well as have different effects on harmful or beneficial microbes
(Icoz and Stotzky, 2008).
The foreign genes in genetically modified (GM) plants express
new traits that can increase the commercial value of their specific
functions and applications (Halford and Shewry, 2000; Wolfenbarger and Phifer, 2000). The success of GM plants highlights the
progress that researchers have made in transforming nature;
however, with the increased numbers of commercial applications
and larger cultivation areas, the environmental safety of GM plants
has been receiving increasing attention (Andow and Zwahlen,
2006). Since the commercialization of GM plants, many scientists
have assessed their ecological risks (Icoz and Stotzky, 2008; Kos
et al., 2009; Liu, 2010; Velkov et al., 2005 Velkov et al., 2005).
Studies have shown that the physiological and metabolic changes
in GM plants and the release of their foreign expression products
(e.g., Bt protein) into the soil ecosystem might impact soil
microbial diversity (Hannula et al., 2014; Liu et al., 2005; Sanahuja
et al., 2011; Sanchis, 2010).
The effects on the diversity and abundance of soil microbial
communities are important factors in the evaluation of the
biosafety risks of GM plants (Kolseth et al., 2015; Turrini et al.,
2015; Wolfenbarger and Phifer, 2000). On the basis of related
previous reviews, in this paper, we focus on the recent advances
in the understanding of the effects of GM plants on soil
microbial communities and in the applied researches and
detection technologies in related to analysis those effects.
Factors to be considered in the assessment of risks of GM plants
for soil microorganisms are proposed, and the problems and
prospects of a biosafety assessment platform for GM plants are
discussed.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
292
294
295
298
299
299
299
299
299
300
300
300
301
301
301
2. Mechanisms underlying the effects of GM plants on soil
microorganisms
GM plant residues in soils are present in two forms—foreign
genes and their products—that may influence soil microbial
communities through the following two pathways.
2.1. Effects of foreign gene products on soil microorganisms
In a farmland ecosystem, the roots of GM plants inevitably
interact with microbes in the soil. Foreign and native proteins (e.g.,
new proteins produced after induction of changes in a catabolic
pathway) released from GM plants into the soil via root exudation
may have effects on soil microbial communities (Giovannetti et al.,
2005). Due to the activities of GM proteins and promoters, foreign
genes in GM plants can directly influence soil microbial
communities, for example, by changing certain functional microorganism groups or soil biodiversity. The expression of insecticidal
crystal proteins in plants is a representative case of accumulation
and persistence of GM products in the soil. Studies have shown that
after entering soils, Bt protein rapidly binds to soil clay and humic
acid. Bound Bt protein retains its insecticidal activity for 234 days
and is not easily broken down by soil microorganisms (Zwahlen
et al., 2003). This activity of Bt toxin is retained and sometimes
enhanced by adsorption and binding to clay (Tapp and Stotzky,
1995). Small amounts of Bt toxin, which are similar to the
commercial microbial Bt formulations, produced in GM plants may
persist in soil for several weeks or months (Strain and Lydy, 2015).
Persistent low levels of Bt toxin may affect some non-target
organisms and repeated use of Bt-producing plants may cause
toxin accumulation (Palm et al., 1996).
2.2. Effects of horizontal gene transfer (HGT) on soil microorganisms
Foreign genes can be integrated into the genomes of soil
microorganisms via horizontal transfer, resulting in changes in the
genetic and functional properties of soil microorganisms. The
likelihood of gene transfer from transgenic plants to microorganisms is dependent on the transgene copy number and on the
presence of homologous sequences for recombination (Demanèche et al., 2011). Soil contains an active gene bank comprising
extracellular DNA, from which some microorganisms can obtain
necessary foreign DNA under conditions of environmental stress.
After entering the soil gene bank, foreign DNA may become a part
of the gene transfer chain, for example via transformation,
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
291
Fig. 1. The number of studies on detection methods used for the effects of GM plants on soil microbial communities since 2004.
conjugation, or transduction (de Vries and Wackernagel, 2005;
Brigulla and Wackernagel, 2010). Some reports have suggested that
the cauliflower mosaic virus (CaMV) 35S promoter, a component of
GM constructs in more than 80% of GM plants, may lead to
environmental risks when horizontal transfer occurs (Hull et al.,
2000; Lee et al., 2010). In contrast, Kim et al. (2010b) found that
horizontal gene transfer does not take place between GM potatoes
and soil microorganisms in potato fields. Lee et al. (2011a, 2015)
also assessed the possible horizontal transfer of GM grass DNA into
soil microorganisms and did not detect GM genes in the
rhizosphere soils of GM and non-GM Zoysia grasses. Demanèche
et al. (2008) did not detect any cellular or molecular evidence (by
means of a sensitivity-based hybridization method) that the
antibiotic resistance blaTEM116 gene from a transgenic Bt176 corn
plant is transferred to soil bacteria. Some reviews on various GM
plants or transgenic plants indicated that the occurrence of HGT
from GM plants to soil microorganisms is less prevalent in
comparison with natural transformation rates in most cases, and
those authors even argued that potential HGT from transgenic
plants to prokaryotes is not expected to have negative effects on
human or animal health and the environment (Brigulla and
Wackernagel, 2010; Keese, 2008).
3. Detection of the effect of GM plants on soil microbial
communities
With developments in modern biotechnology, methods for
detection of soil microbial communities now include techniques
ranging from simple macroscopic examination to whole-microecosystem microbe-population analysis (Fig. 1). To avoid the
limitations inherent in the analysis of microorganisms (e.g., a high
proportion of unculturable microorganisms, their minute size, and
population effects), a combination of different methods may be
used. The current most common detection methods are presented
below.
technologies such as fluorescence in situ hybridization (FISH),
micro-autoradiography combined with FISH, and atomic force
microscopy have been widely applied to the field of microbiology
(Moter and Göbel, 2000). Despite their many disadvantages,
traditional methods may be combined with modern technologies
to gain a deeper understanding of the state of soil microorganisms
affected by GM plants and thus should not be discounted
(Chaudhry et al., 2012; Lee et al., 2011a; Sohn et al., 2015; Travis
et al., 2007). Currently, the detection and identification of microbes
still depend mainly on the traditional methods (Fig. 2).
3.2. The biolog microplate technique
The Biolog microplate culture technique can be used to
characterize bacterial communities. Samples are inoculated into
Biolog gram-negative microplates to generate sole-carbon-source
utilization patterns—that is, the so-called metabolic fingerprints—
of bacterial communities (Garland and Mills, 1991). This method
has been developed to assess community-level physiological
profiles (CLPPs) in order to analyze diversity of the carbon
substrate catabolism of microbial communities. This approach has
been increasingly applied to evaluation of the effects of GM plants
(e.g., maize, rice, wheat, and soy) on rhizosphere community
structure (Liphadzi et al., 2005; Lupwayi et al., 2007; Mulder et al.,
2007; Wei et al., 2012). Although this technique cannot be used to
analyze the activity of all community members, the method has
several advantages (over traditional culture-based methods), for
3.1. Traditional culture and observation methods
Some soil microorganisms can be directly examined using
classic culture-dependent methods (Hu et al., 2013; Wang et al.,
2013); however, soil microorganisms are diverse and cultivable
microbes account for less than 10% of all soil microorganisms.
Therefore, the cultivation-dependent total colony-forming units
(CFUs) reflect changes only in a small subset of soil microbes, this
drawback limits the use of this approach. With the rapid
developments in modern microscopy, several advanced
Fig. 2. The number of studies on the effects of various GM plants on soil
microorganisms since 2004.
292
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
example, automated data collection, simultaneous evaluation of 95
carbon sources, and rapid comparison of environmental samples
(Haack et al., 1995). Furthermore, this method centers on
functional characteristics of the community, complementing
taxonomic structural analysis such as the isolation and characterization of bacterial strains (Liu et al., 2015; Stefanowicz, 2006).
Nevertheless, Biolog microplates have some limitations such as
those of artificial culture, the effects of culture conditions on
empirical results, and the accuracy of the results (Garland and
Mills, 1991).
3.3. The modern biomarker approach
Soil microorganisms mostly comprise bacteria and fungi.
Characteristic compounds (e.g., quinones and fatty acids) in their
cell membrane or cell wall may serve as useful biomarkers for
analysis of microbial communities. Phospholipid fatty acids
(PLFAs) are essential components of every living cell and are
considered the markers of a viable microbial community because
they are not found in storage products or in dead cells. Once cells
die, the phosphate groups are quickly hydrolyzed (Zelles, 1999).
Because culturing is not required, the output of this method
directly reflects the original structure of the soil microbial
community. Determination of PLFA patterns of soil organisms
has become a commonly used approach to characterization of
microbial community structure (Frostegård et al., 2011). PLFAs can
be analyzed by multivariate procedures to determine statistically
significant changes in soil microbial-community structure and in
relative abundance levels in soils under different management
regimens. PLFA analyses are quantitative and have relatively high
throughput, these advantages make PLFA biomarkers well suitable
for field studies of ecosystem management and for tracking of the
microbial communities of GM plants (Chun et al., 2011; Griffiths
et al., 2005; Lahl et al., 2012; Wu et al., 2009; Gao et al., 2015). The
following are limitations of the PLFA biomarker approach: this
method is suitable only for living microbes, and different species of
microbes may produce overlapping maps. Other protocols are now
necessary to complement the PLFA method in the analysis of
diversity of soil microbes.
3.4. Modern molecular biological methods
Molecular techniques are highly sensitive to even small changes
in the structure of microbial communities and can detect minor
variation in bacterial composition. Accordingly, molecular techniques are used to explore the community diversity of unculturable
microorganisms and to identify novel microorganisms without
cultivation. In particular, DNA extraction from soil samples is
widely used in the studies on soil microbial populations. The
common methods include DNA chips, single-strand conformation
polymorphism (SSCP), restriction fragment length polymorphism
(RFLP), amplified ribosomal DNA restriction analysis (ARDRA),
automated ribosomal intergenic spacer analysis (ARISA), denaturing/temperature gradient gel electrophoresis (DGGE/TGGE), and
metagenomic library-based protocols. Among them, the DGGE
technology has been mainly used for assessment of the effects of
GM plants on soil microorganisms in the last decade.
Furthermore, commercially available genome-wide DNA microarrays were used to examine Bacillus subtilis- and Streptomyces
coelicor-related bacteria in Bt maize rhizobacterial communities,
suggesting that this sensitive method may serve as a useful tool for
molecular monitoring of rhizobacterial communities (Val et al.,
2009). Using a metagenomic approach, which combines molecular
screening and pyrosequencing methods, Demanèche et al. (2009)
screened 77,000 clones from a soil metagenomic library,
with subsequent identification and characterization of nine
denitrification gene clusters. Various investigators have monitored
the cumulative effects of GM plant cultivation by high-throughput
DNA pyrosequencing of bacterial DNA coding for the 16S rRNA
hypervariable V6 region from rhizobacterial communities
(Barriuso et al., 2012; Liang et al., 2014; Sohn et al., 2015). These
modern biological methods do not require microbial culturing,
thus facilitating the analysis of total microbial diversity. On the
other hand, the detected microbial communities are usually
limited to colonies with significant environmental advantages, and
the same band may represent multiple microbial species in DNA
molecular genetic maps (Mardis, 2008).
In addition, application of the next-generation sequencing
(NGS) technology to soil biodiversity has led to an increasing
number of metabarcoding surveys on biodiversity variation in
relation to various factors such as soil type, climate, and land use
(Nielsen and Wall, 2013; Ranjard et al., 2013). DNA metabarcoding
is a molecular approach by which each operational taxonomic
units can be identified through a specific sequence of DNA
(barcode) (Orgiazzi et al., 2015). Owing to the relative ease of
practical application together with the continuous reduction in
time and costs involved in the use of NGS platforms (and the
development of new bioinformatics pipelines to analyze large
amounts of data on below-ground diversity), metabarcoding may
open up distinct opportunities for research on the spatial
distribution of soil biodiversity and can be applied to DNA from
any environment or organism, thereby gaining increasing prominence in biodiversity studies (Schmidt et al., 2013; Yang et al.,
2013). This approach is already commonly used to characterize soil
microbial communities, and its application is now being extended
to other soil organisms, i.e., meso- and macro-fauna (Fahner et al.,
2016). Nevertheless, the use of this method for the risk assessment
of GM plants has not yet been reported. With extended studies and
better understanding of the utilization of this approach, metabarcoding can help to evaluate the effects of GM plants on soil
microorganisms. Finally, metabarcoding may identify the differences in soil microbes caused by the release of GM plants in a more
precise manner and provide a sensitive methodology for risk
assessment.
4. Advances in the research on the effects of GM plants on soil
microorganisms
Analysis of the process of genetic transformation leading to
environmental and ecological risks is a relatively complicated and
systematic scientific problem. Because of the interference from
various biotic and abiotic factors, no definite consensus conclusions about whether GM plants can affect soil microorganisms
have been drawn to date. A number of studies assessing the effects
of GM plants on soil microorganisms have been conducted in the
recent years and yielded significantly different results.
4.1. The impact of insect-resistant GM plants on soil microbial
communities
Bt crops have been commercialized for more than 20 years. Bt
toxin-related proteins have been expressed in GM plants to confer
pest resistance. Bt GM crops have been overwhelmingly successful
and beneficial with respect to increasing yields and reducing
chemical pesticide use. Nonetheless, their application has evoked
some criticism, particularly regarding the ecological risks Bt GM
crops for a soil ecosystem (Sanchis, 2010; Cheeke, 2012). Therefore,
GM Bt plants are commonly selected as experimental systems in
studies pertaining to the assessment of the effects of GM plants on
non-target soil microorganisms (Table 1). To date, most studies on
the impact of GM Bt plants have shown no effects on soil
microorganisms (Fig. 2). The structures of bacterial communities
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
293
Table 1
The impact of insect-resistant GM plants on soil microbial communities.
GM
plant
Foreign
gene
Trial
conditions
monitoring
period
(year)
Foreign
Protein
Methods
Effects of GM plants on soil microbial community
References
maize
Bt
field
1
PLFA; CLPP
rice
cry1Ab
field
1
Cry1Ab;
Cry1F
Cry1Ab
CFUs
Blackwood and
Buyer (2004)
Wu et al. (2004)
maize
cry3Bb
field
2
Cry3Bb
T-RFLP
Significantly reduced the presence of eukaryotic
PLFA biomarker
Some occasional significant effects (eg. higher total aerobic bacterial
counts,lower population of actinomycetes) on microbial community
No deleterious effects on soil bacterial
maize
field
1
Significant positive effects on the rhizosphere communities
DGGE; Biolog
No effects on bacterial diversity
maize
cry1Ab
Cry1Ab
DGGE
A significantly lower level of mycorrhizal colonization
maize
cry1Ab
greenhouse; 1
field
greenhouse; 1
field
field
2
Cry1Ab;
PAT
Bt
CLCP; ARISA
maize
cry1Ab;
bar; Bla
Bt
Cry1Ab
CLPP; PLFA
No effects on soil microbial community structure
maize
cry1Ab
field
1
Cry1Ab
SSCP
Minor effects on diversity of rhizobacteria
cotton
cry1Ac
field
1
Cry1Ac
CFUs
cotton
cry1Ab
field
1
Cry1Ab
CLPP
Significant negative differences in the numbers of the three
functional bacteria
No effects on the functional diversity of microbial communities
maize
cry1Ab
glasshouse
1
Cry1Ab
maize
cry1Ab
field
1
Cry1Ab
CLPP;
PLFA
Biolog
spruce
cry1Ab
field
1
Cry1Ab
ARDRA
maize
cry1Ab
field
2
Cry1Ab
CLPP; PLFA
tobacco
mti-2
field
1
Pls
DGGE
maize
cry1Ab
field
2
Bt
CFUs
cotton
cry1Ac;
cry2Ab
field
1
Cry1Ac;
Cry2Ab
maize
cry1Ab;
cry3Bb
cry1Ab
field
4
field
2
Cry1Ab;
Cry3Bb
Cry1Ab
ARDRA; RISA;
BOX-PCR; ERICPCR
DGGE
DGGE; T-RFLP
Brassica
rapa
rice
maize
cry1AC
field
1
Cry 1AC
DGGE
cry1Ab
cry
greenhouse
field
1
1
Cry1Ab
Cry
cotton
field
5
rice
cotton
cry1A;
cpti
cry1Ab
cry
field
field
2
1
Cry1A;
CpTI
Cry1Ab
Cry
PLFA
Genome-Wide
Microarrays
CFUs
maize
rice
Bt
cry1Ab
field
field
1
1
Bt
cry1Ab
DGGE
T-RFLP
maize
cotton
Bt
cry1Ac;
CpTI
field
field
1
3
PCR-DGGE
CFUs;
MPN
maize
cry3Bb
field
1
Bt
Cry1Ac;
CpTI
protein
Cry3Bb
rice
rice
Bt
Bt
field
field
1
1
Bt
Bt
Biolog; DGGE
TGGE
maize
cry1Ab
field
4
Cry1Ab
maize
cry1Ab
field
1
Cry1Ab
brinjal
cry1Ac
field
2
Cry1Ac
cotton
Bt
field
1
Bt
454pyrosequencing
454pyrosequencing;
T-RFLP
QT-PCR;
RFLP
CFUs
rice
Bt
field
1
Bt
maize
Bt
field
2
Bt
rice
T-RFLP
CFUs; T-RFLP
T-RFLP
DGGE;
RT-PCR
Biolog; DGGE
Devare et al.
(2004)
Brusetti et al.
(2004)
Fang et al.
(2005)
Castaldini et al.
(2005)
Griffiths et al.
(2005)
Baumgarte and
Tebbe (2005)
Rui et al. (2005)
Shen et al.
(2006)
No effects on the microbial and communities
Griffiths et al.
(2006)
Significantly increased microbial consumption of carbohydrates
Mulder et al.
(2006)
All diversity indices of rhizobacteria significantly increased
LeBlanc et al.
(2007)
No effects on the microbial community structure
Griffiths et al.
(2007b)
No significant effects on the dominant members of the bacterial
Riglietti et al.
community
(2008)
No effects on culturable aerobic bacteria, actinomycetes, and fungi Oliveira et al.
(2008)
No effects on diversity richness of PPFMs
Balachandar
et al. (2008)
No consistent statistically significant differences in the numbers of
different groups of microorganisms
No measurable adverse effects on microbial community
composition
No significant effects on the genetic composition of rhizosphere
bacterial communities
No significant effects on the microbial community composition
No effects on rhizobacteria
Icoz et al. (2008)
Liu et al. (2008)
Jung et al. (2008)
Wu et al. (2009)
Val et al. (2009)
No significant effects on the numbers of different functional bacteria Hu et al. (2009)
groups
No significant effects bacterial and fungal community compositions Lu et al. (2010a)
No adverse effects on the diversity of the microbial communities. Kapur et al.
(2010)
No apparent impacts on bacterial and fungal communities
Tan et al. (2010)
Significant negative effects on fungal community composition; no Lu et al. (2010b)
effects on bacterial community composition
No adverse effects on the arbuscular mycorrhizal fungal community Tan et al. (2011)
No significant effects on the number of bacteria, fungi, azotobacter, Li et al. (2011)
and the diversity indices of microorganisms
No significant effects on bacterial decomposer communities; minor
effect on fungal decomposer communities
No significant effects on the diversity of microbial communities
No significant effects on the richness, evenness and community
structure of soil microorganisms
No significant effects on bacterial community
No significant effects on AM fungal communities;
Significantly lower abundance of bacterial structure
Xue et al. (2011)
Wei et al. (2012)
Fang et al. (2012)
Barriuso et al.
(2012)
Verbruggen
et al. (2012)
Singh et al.
(2013a)
Transient and not persistent negative effects on numbers of bacteria, Hu et al. (2013)
actinomycetes and fungi
Significant negative effects on methanogenic archaeal and
Han et al. (2013)
methanotrophic bacterial community abundance and diversity.
Significantly higher bacterial and fungal microbial catabolic abilities Velasco et al.
(2013)
294
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
Table 1 (Continued)
GM
plant
Foreign
gene
Trial
conditions
monitoring
period
(year)
Foreign
Protein
Methods
Effects of GM plants on soil microbial community
brinjal
cry1Ac
field
1
Cry1Ac
RT-PCR;
maize
Bt
field
1
Bt
CFUs; DGGE
cotton
cry1Ac
field
1
Cry1Ac
CFUs
maize
cry1Ab
field
1
Cry1Ab
Biolog; CLPP
maize
Bt
field
1
Bt
maize
Bt
field
1
Bt
454pyrosequencing
Biolog
rice
field
4
maize
cry1Ac;
cpti
Bt
field
1
Cry1Ac;
Cpti
Cry1Ab
T-RFLP
brinjal
cry1Ac
field
1
Cry1Ac
clone library
maize
cry1Ab
field
2
Cry1Ab
cotton
cry1Ac;
CpTI
cry1Ac
cry1Ab;
cry1F
cry1Ac
field
2
field
field
1
1
field
3
CrylAc;
CpTI
Cry1Ab
Cry1Ab;
Cry1F
Cry1Ac
T-RFLP; clone
library
CFUs
cry1Ac
field
1
Cry1Ac
Negative effects on the actinomycetes population size and diversity Singh et al.
(2013b)
No effects on the abundances of total bacteria
Cotta et al.
(2013)
No effects on microbial population and microbial diversity indices Velmourougane
and Sahu, 2013)
No effects on bacterial functional diversity
Lupwayi and
Blackshaw
(2013)
No significant effects on fungal diversity and community structure Kuramae et al.
(2013)
No significant effects on bacterial functional community
Bumunang et al.
(2013)
No significant effects on the diversity of bacterial and fungal
Song et al.
community
(2014)
No significant effects on AMF communities
Zeng et al.
(2014)
Minor effect on the fungal community
Singh et al.
(2014)
No significant effects on bacterial communities composition
Ondrei9
cková
et al. (2014)
No apparent impact on microorganism populations
Zhang et al.
(2014)
Significant differences in methanogenic community composition
Zhu et al. (2014)
Minor positive effects on abundances of ammonia-oxidizing
Cotta et al.
bacterial and archaeal communities
(2015)
No effects on microbial communities
Zhang et al.
(2015)
no direct effects on microbial community
Liu et al. (2015)
rice
maize
cotton
oilseed
rape
DGGE; RT-PCR
clone library
DGGE; QT-PCR
DGGE
CFUs
References
Note: arbuscular mycorrhizal fungi (AMF); automated ribosomal intergenic spacer analysis (ARISA); colony forming units (CFUs); community level catabolic profiling (CLCP);
community-level physiological profile (CLPP); denaturing gradient gel electrophoresis (DGGE); phospholipid fatty acid (PLFA); pink-pigmented facultative methylotrophs
(PPFMs); serine protease inhibitors (PIs); temperature gradient gel electrophoresis (TGGE); terminal restriction fragment length polymorphism (T-RFLP); 3-tube most
probable number method (MPN).
inhabiting the GM (Cry1Ab) maize rhizosphere are not different
from those of non-GM maize cultivars, as revealed by polymerase
chain reaction (PCR)-amplified 16S rRNA gene SSCPs (Baumgarte
and Tebbe, 2005). Subsequent research addressed on soil microbial
and faunal community responses to Bt maize in a glasshouse by
PLFA analysis and CLPP, and no significant effects of Bt proteins on
soil microbial populations were detected (Griffiths et al., 2006). In
addition, by means of differential carbon substrate utilization
profiling and DNA fingerprinting approaches such as ARDRA, RISA,
and ERIC-PCR, researchers showed that the diversity of pinkpigmented facultative methylotrophs present in the rhizoplane
does not differ between Bt cotton and non-Bt cotton (Balachandar
et al., 2008; Zhang et al., 2015).
On the other hand, some studies have shown that Bt GM plants
have significant effects on soil microbial communities. Castaldini
et al. (2005) assessed the effects of Bt corn on rhizospheric
eubacterial communities in greenhouse experiments by DGGE
analyses of 16S rRNA genes. The differences between Bt and non-Bt
corn plants were analyzed in rhizospheric eubacterial communities, culturable rhizospheric heterotrophic bacteria, and mycorrhizal colonies, and it was found that Bt corn plants have a
significantly lower level of rhizospheric mycorrhizal colonization.
The impact of transformation of rice with Cry1Ab on the soil
microbial community composition in paddy fields was determined
by terminal restriction fragment length polymorphism (T-RFLP),
and the results showed that Bt rice roots have significant negative
effects on fungal community composition at the early stage of root
decomposition (Lu et al., 2010b). DGGE and real-time (RT)-PCR
analysis revealed that GM Bt rice significantly reduces the
abundance and diversity of methanogenic archaea and methanotrophic bacterial communities (Han et al., 2013). Furthermore, a
time-course field experiment was conducted for 2 years to
evaluate the potential impact of GM Bt maize on rhizosphere
microorganisms by Biolog microplate analyses. Marked positive
effects of Bt maize were observed in rhizosphere microbial
community structure, according to bacteria- and phylum-specific
PCR-DGGE and PCR cloning approaches (Velasco et al., 2013). GM
brinjal with the Cry1Ac gene has a minor effect on the fungal
community according to phylogenetic analysis of ITS rRNA clones
(Singh et al., 2014). RNA-stable isotope probing combined with
clone library analyses suggested that insertion of the cry1Ab gene
into the rice genome has a potential to modify composition of the
methanogenic community in the rice rhizosphere (Zhu et al.,
2014).
4.2. The impact of herbicide-resistant GM plants on soil microbial
communities
In the past decade, studies on the effects of herbicide-resistant
GM plants on soil microbial communities have become prevalent
and comprehensive(Kremer, 2014) (Table 2 & Fig. 2). For example,
the community structures and bacterial activities of the rhizospheric microbes from GM Basta-tolerant oilseed rape were found
to be positively affected by genetic modifications (Sessitsch et al.,
2005). These effects, however, are small compared with the
influence of the plant growth stage according to some studies.
Glufosinate ammonium-tolerant GM maize T25 cultivars grown
under glasshouse conditions negatively altered soil microbial
community structures, as measured using ester-linked fatty acids
(ELFA) (Griffiths et al., 2008). Lee et al. (2011a) studied the effects of
field-grown GM Zoysia grass (tolerant to the herbicide Basta) on
bacterial community structure by means of culture-independent
approaches and constructing 16S rDNA clone libraries. The
bacterial diversity of the GM clone library was found to be
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
295
Table 2
The effects of herbicide-resistant GM plants on soil microbial communities.
GM plant
Foreign gene
Trial
conditions
monitoring
period
(year)
Foreign Protein
Methods Effects of GM plants on soil microbial community
oilseed rape
Pat gene
field
1
Basta
DGGE
soy; maize
glyphosateresistant gene
field
3
glyphosate resistance
SIR;
Biolog
maize
glufosinateresistance gene
field
1
glufosinate resistance
CLPP;
ELFA
soybean
glyphosateresistant gene
field
1
glyphosate-resistance
FAMEs
wheatcanolawheatpea
maize
glyphosateresistant gene
field
4
glyphosate resistance
Biolog
glasshouse
1
glufosinate-ammonium
tolerance
ELFA
rape; maize
glufosinateammonium
tolerant gene
Basta gene
field
4
glufosinate resistance
PLFA
maize
EPSPS gene
field
1
glyphosate resistance
maize
bar gene
field
1
glufosinate resistance
QT-PCR;
T-RFLP
CFUs
Zoysia grass
bar gene
field
1
rice
PPO gene
field
2
soybean
ahas gene
field
3
soybean
EPSPS gene
greenhouse 1
tolerance to the herbicide CFUs;
Basta
clone
library
resistance to PPOT-RFLP
inhibiting herbicides
resistance to herbicides of DGGE
the imidazolinone group
glyphosate resistance
T-RFLP
maize
pat gene
field
1
Zoysia grass
bar gene
field
1
phosphinothricin-Nacetyltransferase
glufosinate resistance
DGGE;
QT-PCR
CFUs;
clone
library
References
Minor positive effects on bacteria community
Sessitsch
et al.
(2005)
No effects on soil microbial communities
Liphadzi
et al.
(2005)
Minor negative effects on abundance and diversity Griffiths
of bacteria
et al.
(2007b)
Small and transient negative effects on microbial Weaver
community
et al.
(2007)
Small, and inconsistent negative effects on
Lupwayi
microbial community
et al.
(2007)
Significant negative effects on soil microbial
community structure
Griffiths
et al.
(2008)
No effects on microbial biomass
Ernst et al.
(2008)
No effects on abundance and community structure Hart et al.
of denitrifying bacteria and fungi
(2009)
No effects on bacteria and fungi
Tothova
et al.
(2010)
Considerable effects on bacterial community
Lee et al.
structure
(2011a)
No effects on the diversity indices and community Chun et al.
composition of bacterial and fungal communities (2012)
No quantitative effects on microbial communities Souza et al.
(2013)
No effects on that bacterial diversity
Arango
et al.
(2014)
Minor positive effects on abundances of ammonia- Cotta et al.
oxidizing bacterial and archaeal communities
(2014)
No significant difference in the abundance of total Lee et al.
cultivable bacteria
(2015)
Note: ester-linked fatty acids (ELFA); fatty acid methyl esters (FAMEs); protoporphyrin oxidase (PPO); substrate-induced respiration (SIR).
significantly lower than that of the non-GM library, suggesting that
alterations in microbial-community composition are associated
with GM Zoysia grass.
Glyphosate tolerance is a common modification to corn,
soybeans, and canola (Widmer, 2007). Some studies have
documented only minor or transient effects. Liphadzi et al.
(2005) reported that glyphosate treatment on glyphosate-resistant
soybean and corn does not affect the structure of soil bacteria
communities. Weaver et al. showed that glyphosate-resistant
soybean did not significantly change soil microbial communities in
terms of structure, function, or activity (Weaver et al., 2007).
Lupwayi et al. (2007) found that microbial communities grown via
wheat-corn/wheat-pea rotation are relatively insensitive to
cropping practices, and glyphosate-resistant crops cause only
small and inconsistent negative changes in microbial communities. Using quantitative PCR to measure microbial abundance and
T-RFLP to analyze community structure, Hart et al. (2009) showed
that glyphosate-resistant GM corn does not affect rhizosphere
fungal communities, and that seasonality is a significant determinant of fungal abundance and their diversity. In addition, the
expression of PPO or AHAS herbicide resistance gene does not affect
soil microbial communities(Chun et al., 2012; Souza et al., 2013).
Nonetheless, negative effects of glyphosate-tolerant GM soybean
on soil microorganisms have been reported (Kremer and Means,
2009; Zobiole et al., 2011). Empirical data of many years need to be
accumulated to clarify whether negative effects occur in the long
run.
4.3. The influence of disease-resistant GM plants on soil microbial
communities
The expression of GM proteins with antimicrobial activity (e.g.,
chitinases, glucanases, and lysozymes) has been explored for many
years as a means to improve resistance to plant diseases. Still,
plants that express broad-spectrum and disease-suppressive traits
have the potential to have undesirable effects on non-target and
potentially beneficial microbial communities (Table 3 and Fig. 2).
Lysozyme has a negative impact on the stability of a peptidoglycan
that is more prevalent in the cell wall of gram-positive than gramnegative bacteria. Rasche et al. (2006) carried out a greenhouse
experiment to analyze the possible effect of GM potatoes
expressing antibacterial compounds (attacin/cecropin and T4
lysozyme) on rhizosphere bacterial communities by 16S rRNAbased T-RFLP analysis. T4 lysozyme had a stronger negative effect
on bacterial communities. The influence of the genetic modification was found to be both transient and weak, in comparison with
other factors (such as soil type, plant genotype, and vegetation
stage). Using PCR-DGGE fingerprints, van Overbeek and Van
296
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
Table 3
The influence of disease-resistant GM plant on soil microbial communities.
GM plant
Foreign gene
potato
gene expressing Magainin II field
1
production
gene expressing attacin/
greenhouse 1
cecropin and T4 lysozyme
potato
Trial
conditions
monitoring
period
(year)
Foreign Protein
Methods
Effects of GM plants on soil microbial
community
References
Magainin II
production
attacin/cecropin;
T4 lysozyme
CFUs
Significantly positive effects on abundance of
rhizobacteria
Minor negative effects on diversity of
rhizobacteria
O’Callaghan
et al. (2005)
Rasche et al.
(2006)
T-RFLP;
clone
libraries
CFUs
a replicase mutant gene of
PRSV
watermelon CGMMV-resistant gene
field
1
field
1
papaya
a replicase mutant gene of
the PRSV
field
1
replicase mutant
protein
potato
1
tobacco
field
a gene resistant to
kanamycin and a gene for
T4 lysozyme
gene expressing a synthetic field
magainin
aadA gene
field
potato
T4 lysozyme gene
field
1
resistance to
kanamycin and T4
lysozyme
antimicrobial
DGGE
peptide magainin
ARISA
resistance to
spectinomycin
and streptomycin
T4 lysozyme
DGGE
tomato
gene expressing beta-1,3glucanase and chitinase
greenhouse 1
papaya
potato
1
1
replicase mutant
protein
CGMMV-resistant
T-RFLP
CFUs; TRFLP;
DGGE;
ARDRA
Py-FIMS
expression of
beta-1,3glucanase and
chitinase
resistance to
CGMMV
CFUs; RTPCR
watermelon gene resistant to CGMMV
field
1
poplar
field
1
b-glucuronidase
ITS
field
1
DGGE
watermelon gene resistant to CGMMV
field
1
white
endochitinase gene
spruce
chili pepper a viral coat protein gene
greenhouse 1
late blightresistant
CGMMV
resistance
endochitinase
field
1
resistance to CMV
Nicotiana
tabacum
field
1
degrades QS
signals (AttM)
PLFA; TRFLP
DGGE
field
1
AMV coat protein
greenhouse 1
resistance to
fungal pathogens
resistance to
kanamycin and
neomycin
balsam pear
chitinase;
potatoes
alfalfa
white
spruce
maize
gene expressing betaglucuronidase
nifH gene
gene expressing the
lactonase AttM that
degrades QS signals (AttM)
AMVcp-s gene coding for
AMV coat protein
endochitinase gene
field
3
tobacco
neomycin
phosphotransferase gene
(nptII)
McChit1 gene
field
1
wheat
WYMV-Nib8 gene
field
2
American
chestnut
gene encoding an oxalate
oxidase
greenhouse 1
T-RFLP
PLFA
RT-PCR
control the wheat
yellow mosaic
virus disease
blight-resistant
CFUs;
Biolog
PCR
RT-PCR
CFUs
DGGE
RFLP
Significantly increased total number of CFUs Wei et al.
of bacteria, actinomycetes, and fungi
(2006)
No effects on bacterial communities
Park et al.
(2006)
Significantly positive effects on the total
Hsieh and Pan
number of CFUs of bacteria, actinomycetes, (2006)
and fungi
No effects on microbial activity
Melnitchouck
et al. (2006)
Significantly greater population sizes and
diversity of bacterial and fungal populations
Transient and positive effect on bacterial
community structure tended to disappear
after 96 h of incubation
No significant effects on total bacterial,
actinobacterial and Pseudomonas
communities
No significant effects on arbuscular
mycorrhizal fungi
O’Callaghan
et al. (2008)
Brusetti et al.
(2008)
Van Overbeek
and Van Elsas
(2008)
Girlanda et al.
(2008)
No significant effects on the diversity of
bacteria and fungi community
Yi et al.
(2009);
Stefani et al.
(2009)
No significant effects on ectomycorrhizal
Stefani et al.
(EM) fungi
(2009)
No significant effects on the structure of the Zadorina et al.
microbial associations
(2009)
No significant adverse effects on bacterial
Yi and Kim
and fungal relative abundance
(2010)
No effects on fungal biomass
Stefani et al.
(2010)
Significant positive effects on microbial
Chun et al.
community(bacterial and fungal)
(2011)
No significant effects on bacterial
D’Angelopopulations
Picard et al.
(2011)
Significantly negative effects on densities of Faragová et al.
rhizospheric bacteria
(2011)
No effects on the fungal phylogenetic
Lamarche
community structure
et al. (2011)
No significant effects on kanamycin-resistant Ma et al.
(Km(R)) and neomycin-resistant (Nm(R)) soil (2011)
bacterial populations
Wang et al.
No significant effects on the number of
rhizospheric fungi and the ratio of fungi to (2013)
bacteria (F/B)
No adverse impact on microbial community Wu et al.
diversity
(2014)
No significant differences in beneficial fungi D’Amico et al.
(2015)
HNote: alfalfa mosaic virus (AMV); amplified ribosomal DNA restriction analysis (ARDRA); cucumber green mottle mosaic virus (CGMMV); cucumber mosaic virus (CMV);
internal transcribed spacer (ITS); papaya ringspot virus (PRSV); pyrolysis-field ionization mass spectrometry (Py-FIMS); terminal restriction fragment length polymorphism
(T-RFLP).
Elsasalso (2008) also found the absence of significant effects of
plant genotypes (both GM potatoes and non-GM cultivars) on soil
bacterial communities and that the plant growth stage overrides
any effect of the plant genotype on bacterial communities.
Heterogeneous chitinases have been introduced into many
plant species with the aim of increasing the resistance to fungal
diseases (Singh et al., 2006). Because the cell wall components
targeted are typical of most of filamentous fungi, overproduction of
these enzymes may also affect ecologically important non-target
native fungi. Pasonen et al. (2009) studied the effects of sugar beet
chitinase IV expression on the degree of ectomycorrhizal
colonization and the structure of fungal communities in a GM
silver birch field trial and found that one GM line differs
significantly from the non-GM controls with respect to fungal
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
community structure. In contrast, using CFU and RT-PCR analyses,
research groups demonstrated that constitutive expression of
chitinase has no significant effect on indigenous nonpathogenic
fungi associated with GM plants (e.g., tomato, white spruce, and
tobacco) (Girlanda et al., 2008; Lamarche et al., 2011; Wang et al.,
2013).
In addition, some resistance genes related to by the viral coat
protein—e.g., genes for resistance to papaya ringspot virus (PRSV),
297
cucumber green mottle mosaic virus (CGMMV), alfalfa mosaic
virus (AMV), and wheat yellow mosaic virus (WYMV) —have been
introduced into many GM crops. A viral coat protein is
constitutively expressed in all parts of virus-resistant plants,
producing large quantities of a GM protein that have a potential for
off-target effects on rhizosphere soil microorganisms. Studies
showed that significant differences in the total number of bacterial
CFUs and actinomycete and fungi cells exist between soils where
Table 4
The impact of quality relevent GM plants on soil microbial communities.
GM plant
Foreign gene
Trial
conditions
monitoring
period
(year)
potato
gene altering starch
composition
gene Ov from Japanese
quail
field
1
field
1
alfalfa
Foreign Protein
Methods
Effects of GM plants on soil microbial References
community
altered starch composition
DGGE
methionine-rich protein
ovalbumin
CFUs
Significantly enhanced the relative
abundance of rhizobacteria
Significantly positve effects on
abundance and diversity of aerobic
bacteria
Minor and positive effects on
abundance and diversity of
rhizobacteria
Minor and positive effects on diversity
of rhizobacteria
Minor effects on Microbial
community composition
Milling et al.
(2005)
Faragova et al.
(2005)
citrange
gene altering
Phytohormone balance
field
1
Phytohormone
balance
CFUs
citrange
rolABC genes
field
2
growth habit change
aspen
1
altered stem lignin
1
Saccharose
Fructosyltransferase
and Fructan
Fructosyltransferase
T-RFLP
No effects on bacterial community
Saha et al.
(2007)
aspen
greenhouse
gene encoding
coniferaldehyde 5hydroxylase
field
Saccharose
Fructosyltransferase
gene; Fructan
Fructosyltransferase
gene
polyphenol oxidase gene greenhouse
Biolog; ARDRA;
DGGE
NLFA; PLFA
1
polyphenol oxidase
clone library
tobacco
Lcbhl-2gene
field
1
higher photosynthetic ARDRA; DGGE
activities
Oliver et al.
(2008)
Andreote et al.
(2008)
potatoes
fructan-producing gene
field
3
fructan productiom
Minor and positive effects on the
bacterial and fungal populations
Significantly positive effects on the
diversity of the culturable microbial
community
No effects on microbial community
cherry
field
1
eucalyptus
a phytochrome A rice
gene
Lhcb1-2 gene from pea
greenhouse
1
Arabidopsis
thaliana
gene producing an
field
exogenous glucosinolate
alteried tight
perception
higher photosynthetic ARDRA; DGGE
capacity
glucosinolate
DGGE
eucalyptus
heterologous DNA from
the pea Lhcb1-2 gene
fused to the
nptII resistance marker
gene
granule-bound starch
synthase gene
gene amylopectinaccumulating
potato
potato
potato
potato
potato
potato
poplar
soybean
rice
gene amylopectinaccumulating
gene cyanophycin
producing
gene amylopectin
accumulating
Tannins gene
gene expressing
Arabidopsis
cystathionine
c-synthase
four synthetic genes
1
greenhouse
1
increase of
photosynthetic
capacity
RT-PCR;
DGGE
field
1
T-RFLP
greenhouse;
field
1
Inhibition of amylose
production
amylopectin
greenhouse
1
starch metabolism
field
3
cyanophycin
field
3
amylopectin
field
1
field
1
tannins
(proanthocyanidins)
methionine
field
1
b-carotene
field
1
tomato
Gene expressing
cytokinin
ath-miR399d gene
field
1
cellular
cytokinin
phosphorus uptake
soybean
AtDCGS gene
field
2
high methionine
poplar
T-RFLP;
clone library
CFUs
No effects on the number of bacteria
PLFA
clone library
Abbate et al.
(2005)
Bradley et al.
(2007)
Becker et al.
(2008)
Cirvilleri et al.
(2008)
Andreote et al.
(2009a)
Bressan et al.
(2009)
No effects on density of bacterial
community
Significantly negative effects on
bacterial, archaea and fungal
community structures
Significantly positive effects on
Andreote et al.
rhizobiales-related
(2009b)
alphaproteobacteria and the diversity
of methylobacterium
No effects on fungal abundance and
fungal community composition
RT-PCR
No effects on microbial community
composition and total bacterial and
fungal abundances
PLFA
No effects on microbial rhizosphere
community structure
PLFA
No effects on microbial structural
diversity of microorganisms
T-RFLP
No effects on function of the fungal
communities
DGGE
No effects on diversity of bacterial and
fungi communities
pyrosequencing No statistically significant difference
in bacterial community structure
DGGE; clone
library
PLFA
Cirvilleri et al.
(2005)
Hannula et al.
(2010)
Gschwendtner
et al. (2010)
Gschwendtner
et al. (2011)
Lahl et al.
(2012)
Hannula et al.
(2012)
Winder et al.
(2013)
Liang et al.
(2014)
No effects on bacterial communities
Li et al. (2014)
Negative effects on microbial
community
Transient adverse effects on microbial
community and diversity
No effects on AM fungal communities
Nam et al.
(2014)
Gao et al.
(2015)
Note: granule bound starch synthase gene (gbss); neutral lipid fatty acids (NLFA); starch branching enzyme (SBE).
298
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
GM papaya resistant to the PRSV and non-GM lines are planted
(Wei et al., 2006). A significant positive change was observed
between CMV-resistant wild-type and GM peppers with regard to
their bacterial composition, according to the T-RFLP profile (Chun
et al., 2011). Statistically significant negative effects on the
rhizospheric bacterial density were detected for GM alfalfa
carrying an introduced AMV coat protein gene by means of the
Biolog system (Faragová et al., 2011).
4.4. The impact of quality-relevant GM plants on soil microbial
communities
Genetic engineering of plants has also been used to improve the
quality and quantity of crop production. Despite the great potential
of this technology for improving agricultural yields and quality,
major concerns about the ecological effects of GM crops on soil
ecosystem functions have been voiced, especially regarding the
key functional groups of non-target species (Birch et al., 2007)
(Table 4). Using DGGE, T-RFLP, and PLFA analyses, some researchers
showed that GM potatoes with altered starch composition or
amylopectin accumulation have little or no effect on the diversity
of soil and rhizosphere bacteria and fungi in a greenhouse
experiment and field trial (Gschwendtner et al., 2010; Hannula
et al., 2012, 2014; Milling et al., 2005). Other researchers assessed
the effects of culture-independent GM eucalyptus-carrying pea
Lhcb1 and Lhcb12 genes, which increase its photosynthetic
capacity-on the diversity of Rhizobiales-related Alphaproteobacteria and Methylobacterium in the rhizosphere and rhizoplane
(Andreote et al., 2009b). The results showed significantly positive
effects of the GM plant on the bacterial communities of
rhizospheres according to RT-PCR and PCR-DGGE analyses.
Transgenic ath-miR399d tomatoes have enhanced phosphorus
uptake and accumulation in shoots and have the potential to
improve phosphorus utilization in agricultural soils. On the other
hand, these tomatoes have transient adverse effects on the
microbial community and diversity in rhizosphere soil
(Gao et al., 2015).
Table 5
The effects of GM plants with other traits on soil microbial communities.
GM plant
Foreign gene
Trial
conditions
potato
DREB1A gene
greenhouse 1
potato
NDPK2 gene
field
1
rice
TPSP gene
field
3
rice
ABC-TPSP gene
field
1
tobacco
MCM6 gene
greenhouse 1
salinity-tolerant CFUs; CLPP
rice
PDH45 gene
field
1
field
1
overexpression
of PDH45 gene
salt tolerance
CFUs
field
1
HSA production
CFUs; MPN
field
1
field
2
CFUs; CLPP; DGGE Significantly increased microbial community
biomass and metabolic activity
454No influences on the biodiversity of fungal
pyrosequencing
communities
field
1
Nitroreductase
overexpression
suppressed
activity
of antisense
CAD
N-AHLS
CFUs; DGGE; PLFA
No effects on bacterial communities
gene producing
opine
gene
overexpressing
ferritin
gene
tobacco
overexpressing
ferritin
gene
tobacco
overexpressing
ferritin
eucalyptus codA gene
field
1
ARDRA; CFUs
Significantly positive effects on rhizobacteria
field
1
opine
production
Ferritin
overexpression
CFUs
Significantly positive effects on bacterial density
field
1
Ferritin
overexpression
ARISA; PCR-RFLP
Significantly positive effects on bacterial and
pseudomonad communities
field
1
Ferritin
overexpression
CFUs; RAPD
No effects on the abundance of rhizobacteria;
Robin et al.
significant effects on the diversity of rhizobacteria (2007)
field
4
salt tolerance
CFUs
rice
AhSTS1 gene
field
1
resveratrol
Oguchi et al.
(2014)
Sohn et al.
(2015)
rice
SUV3 gene
field
1
salt tolerance
CFUs; DGGE;
Pyrosequencing;
CLPP
CFUs
No significant differences in the number of soil
microbes
No significant effects on soil microbes
No significant effects on microorganism
biodiversity
Sahoo et al.
(2015)
eucalyptus choline oxidase
(codA) gene
gene producing
tobacco
HSA
tobacco
bacterial nitroreductase gene
poplar
gene encoding
antisense CAD
tobacco
N-AHLS gene
birdsfoot;
trefoil
tobacco
monitoring
Foreign Protein
period (year)
environmental
stress tolerance
environmental
stress tolerance
stress and
drought
resistance
ABC-TPSP
Method
Effects of GM plants on soil microbial community
References
RISA
No effects on microbial diversity
PCR; AFLP
No impacts on the communities of soil
microorganisms
No significant impacts on the communities of
microorganisms
Mimura et al.
(2008)
Kim et al.
(2010b)
Kim et al.
(2010a)
multiplex PCR;
AFLP
T-RFLP; RT-PCR
CFUs
No effects on diversity indices of bacterial and
fungal communities
No (or only minor) effects on diversity of culturable
bacterial communities
No effects on variety of bacteria
No significant effects on rhizosphere microbe
community
No effects on abundance of rhizobacteria
Lee et al.
(2011b)
Chaudhry et al.
(2012)
Sahoo and
Tuteja (2013)
Yu et al. (2013)
Sabharwal
et al. (2007)
Travis et al.
(2007)
Danielsen et al.
(2012)
D’AngeloPicard et al.
(2004)
Oger et al.
(2004)
Robin et al.
(2006b)
Robin et al.
(2006a)
Note: amplified fragment length polymorphism (AFLP); colony-forming units (CFUs); community level physiological profiling (CLPP); minichromosome maintenance (MCM);
nucleoside diphosphate kinase (NDPK); pea DNA helicase (PDH); random amplification polymorphism DNA(RAPD); 3-tube most probable number method (MPN); trehalose6-phosphate synthase and phosphatase (TPSP).
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
4.5. The influence of GM plants with other traits on soil microbial
communities
In recent years, the effect of GM plants-engineered for stress
tolerance, salinity tolerance, phytoremediation, or root exudationon soil microbial communities has been evaluated in numerous
studies (Table 5). Lee et al. (2011b) assessed the temporal dynamics
of bacterial and fungal communities in a soil ecosystem supporting
GM rice expressing a fusion of trehalose-6-phosphate synthase
and phosphatase by T-RFLP analysis and real-time quantitative
PCR. GM rice did not affect soil bacterial and fungal community
structures as compared to non-GM rice. The impact of salinity
stress-tolerant mini-chromosome maintenance complex subunit 6
overexpression in GM tobacco was examined on the functional
diversity of rhizosphere microbial communities in the presence or
absence of salt stress (Chaudhry et al., 2012). The diversity of
cultivable bacterial communities showed no significant changes or
only minor alterations in the rhizosphere soil of tobacco plants
owing to the presence of transgenes.
Environmental contamination with recalcitrant toxic chemicals
poses a serious and widespread problem with respect to the
functional capacity of soils. It was found that GM tobacco plants
overexpressing a bacterial nitroreductase gene can detoxify soil
that is contaminated by the highly explosive 2,4,6-trinitrotoluene
and significantly increase microbial community biomass and
metabolic activity in the rhizospheres of the GM plants, as
compared to those of wild-type plants (Travis et al., 2007). This
finding revealed that GM plants engineered for the phytoremediation of organic pollutants can increase the functional and
genetic diversity of the rhizosphere bacterial community in acutely
polluted soil. Other studies on GM plants with altered root
exudates have also been conducted for risk assessment, and
showed an obvious impact on rhizobacterial diversity and
abundance. A GM tobacco overexpressing ferritin (P6) was shown
to accumulate more iron than the wild type does, thus leading to
reduced iron availability in the rhizosphere and alterations in the
pseudomonad community (Robin et al., 2007). Suppression of
cinnamyl alcohol dehydrogenase (CAD), the final enzyme in the
lignin monomer biosynthesis, yields lignin with altered structure.
No influence on fungal root or soil communities was detected by
the 454 pyrosequencing technology for GM poplar lines with
suppressed CAD activity (Danielsen et al., 2012). Although while
large-scale growth of GM lignin-modified trees may fully meet the
requirements of commercial applications to bioenergy, the
potential ecological influence must also be considered.
4.6. The impact of GM plant with stacked resistant traits on soil
microbial communities
GM plants with stacked traits (such as resistance gene
compounds, insect-resistant also herbicide-resistant) are an
important and growing in popularity type of GM crops. Nonetheless, there are few research reports evaluating the effects of a GM
plant with stacked traits on soil microbial communities. For
example, compared with its non-transgenic counterpart, transgenic Cry1Ab + Bar maize (Zea mays) 176 cultivated in a
greenhouse, according to ARISA and DGGE analyses, showed
differences in the rhizosphere bacterial communities at different
plant ages and a significantly lower level of mycorrhizal colonization (Brusetti et al., 2004; Castaldini et al., 2005). GM potatoes
(containing the attacin E gene and cecropin B gene) expressing
antibacterial compounds showed a differentiation in the activity
rates and structures of the associated rhizosphere bacterial
communities in a greenhouse experiment (Rasche et al., 2006).
Nevertheless, no consistent significant differences were observed
in the numbers of different groups of functional bacteria between
299
the rhizosphere soil of transgenic Cry1A + CpTI cotton lines (such as
SGK321 and Zhong-41) and that of with non-transgenic cotton
counterparts in the field during multi-year cultivation (Hu et al.,
2009; Li et al., 2011; Zhang et al., 2014). The cultivation of
transgenic cry1Ac/CpTI rice has no significant effects on composition and abundance of bacterial and fungal communities in paddy
soil in the short term (Song et al., 2014).
5. Factors considered in the assessment of risks of GM plants for
soil microorganisms
The rapid development of genetic biotechnology and bioengineering and the practical application of GM plants have brought
about huge economic benefits to society but also risks to soil
microorganisms. How should the unexpected effects of GM plants
on soil microorganisms be assessed scientifically and precisely?
The first basic consideration should be given to the factors
influencing soil microbial populations (Filion, 2008). The impact of
GM plants has been analyzed in a growing number of studies, in
which changes in soil microorganisms have been assessed. To
accurately and comprehensively evaluate the effects of GM plants
on soil microorganisms, we suggest that the following factors be
considered when implementing cultivation strategies.
5.1. Persistence of GM products in soil microorganisms
Foreign proteins expressed in GM plants may alter the
abundance and diversity of soil microorganisms in a variety of
ways. The goal of genetic engineering is to produce specific target
compounds; however, some newly developed compounds may
affect non-target soil microbes to some extent. The residual period
of the GM products in soil is an important factor that affects soil
microbes (Filion, 2008). For example, these proteins may be
introduced into the rhizosphere through root exudation, or the
plant may release some non-proteinaceous substances as a result
of alteration of some metabolic pathways (Raubuch et al., 2007). A
3-year study was conducted to determine the abundance,
persistence, and movements of the Cry1Ab/1Ac protein released
from Bt rice, and those authors concluded that Bt rice can release
detectable amounts of Cry1Ab/1Ac protein into the soil and field
water during the growth period, but that the Bt protein does not
move into adjacent paddies along with the irrigation water and
does not persist in soil for more than 2 months (Wang et al., 2013).
The Cry1Ab protein can persist in an aquatic microcosm for 2
months (Strain and Lydy, 2015). Bt proteins from transgenic crops
may enter soil ecosystems mainly through root exudates (Icoz and
Stotzky, 2008) and can end up in aquatic ecosystems through plant
residues (Liu et al., 2016). In addition, the increasing numbers of
species and foreign genes may make the effects more complex. For
instance, the application of stacked resistance of GM plants
increases the complexity of assessment of the effects of GM plants
on soil microbes (Brusetti et al., 2004; Hu et al., 2009; Song et al.,
2014).
5.2. Effects of marker genes on soil microorganisms
Marker genes have been widely used to confirm the success or
failure of gene transformation in GM plants, but, these marker
genes may affect soil microorganisms. Currently, nptII from
Escherichia coli, which confers neomycin phosphotransferase
resistance to kanamycin and neomycin, is widely applied in
plants. LeBlanc et al. (2007) analyzed the rhizosphere-inhabiting
microbial communities of GM white spruce by biolistic transformation with CryIA(b), uidA (b-glucuronidase), and nptII genes.
Statistically significant differenceS were observed among the
microbial communities inhabiting the rhizospheres of trees
300
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
carrying all three transgenes, those carrying uidA and nptII only,
and control trees. The potential for HGT of antibiotic resistance
markers from GM plants to bacteria of environmental importance
was also assessed. An Arabidopsis thaliana ABC transporter,
Atwbc19, which confers kanamycin resistance on GM plants, does
not provide resistance in E. coli (Burris et al., 2008).
Green fluorescent protein (GFP) is one of the most widely
studied and exploited proteins in biochemistry and has many
applications as a marker, especially in plant transformation
systems. GFP can be a helpful indicator in environmental risk
assessment, in particular, for tracing the transfer of the gfp gene
and its product in nature. Compared to a wild-type control, a fieldreleased transplastomic tobacco (Nicotiana tabacum) with a gfp
transgene has no significant effect on the microbial population
during the entire plant growth cycle including seedling, vegetative,
flowering, and senescing stages (Lv et al., 2014). In contrast, the
developmental stages have a greater effect than the plant types do
(GFP-transformed and wild-type), suggesting that the small gfp
molecule does not burden the host plant and that the chloroplast
transformation with GFP marker genes has no significant effect on
soil microbial communities; such genes may serve as a promising
platform for future plant biotechnologies. Therefore, whether
marker genes are safe for human beings and microbes is debatable
and warrants further study.
should be considered in a reasonably designed experiment. It was
argued that the plant growth stage and year have the strongest
effect on both diversity and function of the fungal communities,
whereas the GM trait under study is the least explanatory factor
(Hannula et al., 2012). The effects of a cultivar and soil type were
intermediate in that study; however, these changes were detected
only in a single soil type, growth stage, or year. A 2-year study was
performed on a Cry1Ac-transformed brinjal crop (Bt) and its nearisogenic untransformed counterpart (non-Bt) to elucidate whether
GM crops alter rhizospheric bacterial community structure (Singh
et al., 2013a). By means of the high-throughput DNA pyrosequencing technology, rhizobacterial communities of Bt maize during a 4year cultivation period were monitored, and variations that were
detected in the rhizobacterial community structure were possibly
due to climatic factors rather than the presence of the Bt gene
(Barriuso et al., 2012). Although the abundance of bacterial 16S
rRNA gene copies was lower in soils associated with brinjal Bt,
Cry1Ac-expressing Bt brinjal overall had a smaller impact as
compared to that caused by seasonal changes. In another study,
clear-cut differences were observed in these rhizosphere communities between soil types and periods of the year in which the
maize was cultivated (Cotta et al., 2013, 2014).
5.5. Effects of differential management measures on soil
microorganisms
5.3. Effects of plant varieties on soil microorganisms
Different plant genotypes and cultivars of the same plant
species affect soil microorganisms differentially. Therefore, testing
whether GM plant genotypes have stronger effects than plant
varieties on soil microorganisms may serve as a reference method
for assessing whether GM plants significantly affect soil microbes.
For example, molecular techniques based on 16S (bacteria) and 18S
(fungi) rDNA were used to study the potential effects of bacterial
and fungal diversity on the rhizosphere and soil of a GM potato line
(SIBU S1, whose starch composition was modified by the RNA
antisense technology), as compared to the non-GM parental
cultivar (SIBU) and another non-GM cultivar (SOLANA) (Milling
et al., 2005). No significant differences were found between the
two cultivars and the GM line; however, obvious effects of the
SOLANA cultivar on the structure of the Pseudomonas community,
compared to SIBU, were detected. Effects of a GM amylopectinaccumulating potato cultivar, the parental isoline, and four
unrelated cultivars on rhizosphere fungi were compared and
differences were detected among the cultivars, the amylopectinaccumulating potato line, and its parental variety (Hannula et al.,
2012). Thus, the differences between non-GM parental cultivars
and other conventional cultivars should be taken into account and
may help to more objectively assess the safety of the soil
environment in the presence of GM plants. The effects of six
potato (Solanum tuberosum) cultivars (including one GM line) on
the bacterial communities in field soil were studied recently.
Positive effects of the potato rhizosphere on bacterial abundance
_
lu et al., 2013). Neverthelevels were observed in 2 years (Inceo
g
less, the community structures in the potato rhizosphere compartments were mainly affected by the growth stage of the plants and,
to a lesser extent, by the plant cultivar type.
5.4. The effect of environmental factors on soil microorganisms
Environmental factors mainly include the season, weather, soil
type, and geographical location, all of which are important drivers
of changes in the soil microbial community (Marschner et al.,
2004). To accurately evaluate the effects of GM plants on soil
microorganisms, both natural changes in these factors in farmland
ecosystems and variation in space and time during sampling
Farmland management measures include farming methods as
well as the use of fertilizers, pesticides, and herbicides, and
different rotation systems have strong effects on soil microorganisms (Höflich et al., 1999). When assessing the effects of GM
plants under field conditions, the changes in these factors should
be taken into account. For example, the cultivation of GM Bt crops
may help to reduce the application of certain insecticides. In this
case, however, the effect of the pesticide change on soil microorganisms should be assessed simultaneously. Soil microbial
functional diversity was evaluated in glyphosate-resistant wheat–canola rotations under a low-disturbance direct seeding regimen
and conventional tillage in field experiments (Lupwayi et al.,
2007). Depending on tillage, glyphosate-resistant crop frequency
affected the functional diversity of rhizosphere soil bacteria in only
two of 20 site-years. Shifts in the structures of bacterial
communities, related to glyphosate-resistant crop frequency, were
detected, but they were uncommon and inconsistent. Tillage
affected the functional diversity of soil bacteria in the rhizosphere
in 3 site-years, but the effects were not consistent. In addition, the
impact of the AHAS transgene and herbicides associated with
soybean crops showed that microbial-community evaluations
were sensitive and valid for monitoring of different technologies
and agricultural management methods (Souza et al., 2013).
Like GM crops, agricultural practice changes resulting from GM
crops have the potential to change soil microorganisms. For
example, agricultural practice models of the GM Roundup Ready1
(GM RR) crop mainly showed no-till and heavy herbicide use
(Ammann, 2005; Klümper and Qaim, 2014), and thus may affect
the soil microorganisms (Liphadzi et al., 2005). It is often argued
that GM RR soybean is environmentally sustainable because it
enables the use of no-till, a farming method that avoids ploughing
with the aim of conserving soil. Some experts, however, assumed
that these agricultural practice changes caused serious ecological
and agronomic problems, including the spread of glyphosateresistant weeds, erosion of soils, loss of soil fertility and nutrients,
and loss of species and biodiversity (such as the diversity of soil
microorganisms) (Bolliger et al., 2006; Antoniou et al., 2010). Some
studies have shown that, the expansion of GM soybean monoculture results in intensification of agriculture on a massive scale, a
decline in soil fertility, and an increase in soil erosion, rendering
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
some soils unusable (Altieri and Pengue, 2005; Pline-Srnic, 2005;
Robert and Krishna, 2004).
The long-term effects of GM plants on soil microorganisms
compared to non-GM parental counterparts and closely related
species, as well as plants carrying marker genes should be
monitored simultaneously for more than a 2-year period (Blackword, 2004; Filion, 2008). Furthermore, in each field trial, various
factors should be taken into consideration, such as different soil
types, management styles, seasons, and different growth stages.
Research has shown that plant growth stage and year have the
strongest effect on the rhizosphere soil microbial community
(Cotta et al., 2014; Liang et al., 2015; Zhang et al., 2015). In addition,
in terms of technology, various quantification and monitoring
methods should be applied to the overall assessment of relevant
effects, when appropriate.
6. Conclusion and perspectives
Soil microorganisms play essential roles in agricultural
production systems. With the increase in cultivation of various
GM plants (e.g., insect-resistant or disease-resistant plants), soil
microorganisms will be exposed to risks that may eventually
adversely affect agricultural production systems. The introduction
of foreign genes into GM plants may affect species diversity and
amounts of root exudates, alter micro-ecological environments in
the soil, and can strongly influence the soil ecosystem functions.
Therefore, the core effect of growing GM plants on a soil is the
change in soil ecosystem function, especially the effects on the
related microbial communities. Some investigators believe that no
inherent connection exists between the diversity and function, and
the abundance of the species was found to not affect soil function
substantially (Bardgett, 2002). There is no direct evidence showing
that soil microbial diversity is related to soil ecosystem function.
That is, a decrease in microbial diversity does not necessarily
worsen soil ecosystem function, but the change in certain key
species may matter (Tilman et al., 2001). On the other hand, Bender
et al. (2016) synthesized the potential of soil organisms to enhance
ecosystem service delivery and demonstrated that soil biodiversity
promotes multiple ecosystem functions simultaneously (i.e.,
ecosystem multifunctionality). Current techniques and methods
may not be effective enough to obtain the relevant evidence, or the
available data may be inadequate to draw such conclusions. For
example, soil ecosystem function may have correlations only with
a few microbial species or show functional redundancy (Loreau
et al., 2001). Nevertheless, the species abundance and diversity of
soil microbes comprise an important reference parameter in the
system of safety evaluation for GM plants and may represent a key
element of the biosafety assessment of animal and plant diversity.
Many Studies on the effects of GM plants on soil microorganisms have been carried out, and obvious differences among
these studies have been noted. These studies, however, were
conducted on different GM plants, and in different environments,
and the resulting data were analyzed with different methods
characterized by specific detection thresholds. For example, a
DGGE, T-RFLP or SSCP profile may reveal the diversity manifesting
as 25–40 most abundant microorganisms, whereas a 454 or
Illumina-based approach easily detects easily 2000–5000 different
organisms in a rhizosphere. Thus, assuming that the same number
of replicates are analyzed, the latter methods have a much higher
probability to detect significant differences between a GM plant
and the comparator but also between non-GM varieties or
different plant growth stages. Furthermore, most of research
conclusions were obtained on the basis of a 1-year cultivation
period. In view of various factors, it is not enough to evaluate the
ecological risk of GM plants in a short trial timeframe. Furthermore, more stacked traits of GM plants are expected to be
301
developed in future agricultural practice. Multiple transgenic
products and other secondarily metabolized components can be
released into soil and affect the soil microbial communities. Other
agricultural practice changes, e.g. application of an herbicide in the
case of herbicide-resistant plant stacked with other traits, may also
influence the soil microbes. Therefore, the effects of GM plants
with stacked traits on soil microorganisms may be more
complicated in comparison with single-trait crops in future
biosafety assessments.
Because of the limitations of current studies, further research at a
more specific level is necessary to assess the correlation among the
components and functions of soil microbial communities, the
response of soil microbial structure and function to natural
fluctuations in the soil system (e.g., the season, climate, rotation,
and pesticide use), the extent of the influence of GM plants on soil
microbes, and the limits of these various risks. The limitations of
current research methods are another important reason for the
discrepant conclusions in various studies. On the basis of the findings
presented above and existing standardized evaluation protocols
(Birch and Wheatley, 2005), the discussion and analyses as well as an
assessment of safety assessment of GM plants for soil microbes
should involve a better evaluation method and theoretical system for
analysis of soil microecology, with objective and scientific verification. The combined application of traditional culture methods,
modern biochemistry, and molecular biological technologies should
probably be able to circumvent the drawbacks of each method and
help to fully exploit the complementary advantages. Especially, the
development and improvement of NGS technologies (such as 454pyrosequencing and Illumina MiSeq sequencing) and DNA metabarcoding will help to better evaluate the effects of GM plants on soil
microorganisms. In addition, for GM plants of different species, a case
study should be conducted by combining long-term fixed plot tests.
Risk assessment of the safety of GM plants is a long term and difficult
task. A better understanding of the advantages and disadvantages of
cultivation of GM plants can be obtained over time and mostly
depends on the progress of technologies and knowledge in the
scientific community.
Funding
This work was funded by two projects of the National Natural
Science Foundation of China (31200422, 31360136), two projects of
the China’s Postdoctoral Science Foundation (2012M520455,
2013T60193), a project of the Commonwealth Scientific Programme of Environmental Protection (201409060), a project of the
National Special Program for the R&D of Transgenic Crops in China
(2016ZX08012005) and a Yuncheng University Doctor Scientific
research project (YQ-2014022).
References
Abbate, C., Ascher, J., Pietramellara, G., Ambrosoli, R., Gennari, M., 2005. Analysis of
bacterial communities in the rhizosphere of transgenic rolABC citrange Troyer:
preliminary studies. Fresen. Environ. Bull. 10, 867–872.
Altieri, M.A., Pengue, W.A., 2005. Roundup Ready Soybean in Latin America: A
Machine of Hunger, Deforestation and Socio-ecological Devastation. RAP-AL,
Uruguay. http://webs.chasque.net/rapaluy1/transgenicos/Prensa/
Roundupready.html.
Ammann, K., 2005. Effects of biotechnology on biodiversity: herbicide-tolerant and
insect-resistant GM crops. Trends Biotechnol. 23, 388–394.
Andow, D.A., Zwahlen, C., 2006. Assessing environmental risks of transgenic plants.
Ecol. Lett. 2, 196–214.
Andreote, F.D., Mendes, R., Dini-Andreote, F., Rossetto, P.B., Labate, C.A., PizziraniKleiner, A.A., van Elsas, J.D., Azevedo, J.L., Araújo, W.L., 2008. Transgenic tobacco
revealing altered bacterial diversity in the rhizosphere during early plant
development. Antonie Van Leeuwenhoek 4, 415–424.
Andreote, F.D., Carneiro, R.T., Salles, J.F., Marcon, J., Labate, C.A., Azevedo, J.L., Araújo,
W.L., 2009a. Culture-independent assessment of Rhizobiales-related
Alphaproteobacteria and the diversity of Methylobacterium in the rhizosphere
and rhizoplane of transgenic eucalyptus. Microbial. Ecol. 1, 82–93.
302
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
Andreote, F.D., Rossetto, P.B., Mendes, R., Avila, L.A., Labate, C.A., Pizzirani-Kleiner, A.
A., Azevedo, J.L., Araújo, W.L., 2009b. Bacterial community in the rhizosphere
and rhizoplane of wild type and transgenic eucalyptus. World J. Microb. Biot. 6,
1065–1073.
Antoniou, M., Brack, P., Carrasco, A., Fagan, J., Habib, M., Kageyama, P., Leifert, C.,
Nodari, R.O., Pengue, O., 2010. GM soy: sustainable? responsible?. Summary of
key findings . http://agris.fao.org/agris-search/search.do?
recordID=GB2013201601.
Arango, L., Buddrus-Schiemann, K., Opelt, K., Lueders, T., Haesler, F., Schmid, M.,
Ernst, D., Hartmann, A., 2014. Effects of glyphosate on the bacterial community
associated with roots of transgenic Roundup Ready1 soybean. Eur. J. Soil Biol.
63, 41–48.
Balachandar, D., Raja, P., Nirmala, K., Rithyl, T., Sundaram, S., 2008. Impact of
transgenic Bt-cotton on the diversity of pink-pigmented facultative
methylotrophs. World J. Microb. Biot. 10, 2087–2095.
Bardgett, R.D., 2002. Causes and consequences of biological diversity in soil. Zoology
4, 367–375.
Barriuso, J., Valverde, J.R., Mellado, R.P., 2012. Effect of Cry1Ab protein on
rhizobacterial communities of Bt-maize over a four-year cultivation period.
PLoS One 4, e35481.
Baumgarte, S., Tebbe, C.C., 2005. Field studies on the environmental fate of the
Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on
bacterial communities in the maize rhizosphere. Mol. Ecol. 8, 2539–2551.
Becker, R., Behrendt, U., Hommel, B., Kropf, S., Ulrich, A., 2008. Effects of transgenic
fructan- producing potatoes on the community structure of rhizosphere and
phyllosphere bacteria. FEMS Microbiol. Ecol. 2, 411–425.
Bender, S.F., Wagg, C., van der Heijden, M.G., 2016. An underground revolution:
biodiversity and soil ecological engineering for agricultural sustainability.
Trends Ecol. Evol. 31, 440–452.
Birch, A., Wheatley, R., 2005. GM pest-resistant crops: assessing environmental
impacts on non-target organisms. Issues Environ. Sci. Technol. 31.
Birch, A.N.E., Griffiths, B.S., Caul, S., Thompson, J., Heckmann, L.H., Krogh, P.H.,
Cortet, J., 2007. The role of laboratory, glasshouse and field scale experiments in
understanding the interactions between genetically modified crops and soil
ecosystems: a review of the ECOGEN project. Pedobiologia 3, 251–260.
Blackwood, C.B., Buyer, J.S., 2004. Soil microbial communities associated with Bt
and non-Bt corn in three soils. J. Environ Qual. 3, 832–836.
Bolliger, A., Magid, J., Carneiro, J., Amado, T., Neto, F.S., de Fatima dos Santos Ribeiro,
M., Calegari, A., Ralisch, R., de Neergaard, A., 2006. Taking stock of the Brazilian
zero-till revolution: a review of landmark research and farmers' practice. Adv.
Agron. 91, 49–111.
Bradley, K.L., Hancock, J.E., Giardina, C.P., Pregitzer, K.S., 2007. Soil microbial
community responses to altered lignin biosynthesis in Populus tremuloides
vary among three distinct soils. Plant Soil 1–2, 185–201.
Bressan, M., Roncato, M.-A., Bellvert, F., Comte, G., el Zahar Haichar, F., Achouak, W.,
Berge, O., 2009. Exogenous glucosinolate produced by Arabidopsis thaliana has
an impact on microbes in the rhizosphere and plant roots. ISME J. 11, 1243–1257.
Brigulla, M., Wackernagel, W., 2010. Molecular aspects of gene transfer and foreign
DNA acquisition in prokaryotes with regard to safety issues. Appl. Microbiol.
Biot. 4, 1027–1041.
Brusetti, L., Francia, P., Bertolini, C., Pagliuca, A., Borin, S., Sorlini, C., Abruzzese, A.,
Sacchi, G., Viti, C., Giovannetti, L., 2004. Bacterial communities associated with
the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic
counterpart. Plant Soil 1 (-2), 11–21.
Brusetti, L., Rizzi, A., Abruzzese, A., Sacchi, G.A., Ragg, E., Bazzicalupo, M., Sorlini, C.,
Daffonchio, D., 2008. Effects of rhizodeposition of non-transgenic and
transplastomic tobaccos on the soil bacterial community. Environ. Biosaf. Res. 1,
11–24.
Bumunang, E.W., Babalola, O.O., Barros, E., 2013. Bacterial community profiling in
the rhizosphere of field grown GM and non-GM maize. J. Pure Appl. Microbiol. 7,
157–164.
Burris, K., Mentewab, A., Ripp, S., Stewart, C.N., 2008. An Arabidopsis thaliana ABC
transporter that confers kanamycin resistance in transgenic plants does not
endow resistance to Escherichia coli. Microb. Biotechnol. 2, 191–195.
Castaldini, M., Turrini, A., Sbrana, C., Benedetti, A., Marchionni, M., Mocali, S.,
Fabiani, A., Landi, S., Santomassimo, F., Pietrangeli, B., 2005. Impact of Bt corn on
rhizospheric and soil eubacterial communities and on beneficial mycorrhizal
symbiosis in experimental microcosms. Appl. Environ. Microb. 11, 6719–6729.
Chaudhry, V., Dang, H.Q., Tran, N.Q., Mishra, A., Chauhan, P.S., Gill, S.S., Nautiyal, C.S.,
Tuteja, N., 2012. Impact of salinity-tolerant MCM6 transgenic tobacco on soil
enzymatic activities and the functional diversity of rhizosphere microbial
communities. Res. Microbiol. 8, 511–517.
Cheeke, T.E., 2012. Effects of the cultivation of genetically modified Bt crops on
nontarget soil organisms. In: Cheeke, T.E., Coleman, D.C., Wall, D.H. (Eds.),
Microbial Ecology in Sustainable Agroecosystems. CRC Press, Boca Raton, pp.
153–227.
Chun, Y.J., Kim, D.Y., Kim, H.-J., Park, K.W., Jeong, S.-C., Park, S., Lee, B., Harn, C.H.,
Kim, H.M., Kim, C.-G., 2011. Do transgenic chili pepper plants producing viral
coat protein affect the structure of a soil microbial community? Appl. Soil Ecol.
51, 130–138.
Chun, Y.J., Kim, H.-J., Park, K.W., Jeong, S.-C., Lee, B., Back, K., Kim, H.M., Kim, C.-G.,
2012. Two-year field study shows little evidence that PPO-transgenic rice
affects the structure of soil microbial communities. Biol. Fertil. Soils 4, 453–461.
Cirvilleri, G., Spina, S., Scuderi, G., Gentile, A., Catara, A., 2005. Characterization of
antagonistic root-associated fluorescent pseudomonads of transgenic and nontransgenic citrange troyer plants. J. Plant Pathol. 87, 179–186.
Cirvilleri, G., Spina, S., Iacona, C., Catara, A., Muleo, R., 2008. Study of rhizosphere
and phyllosphere bacterial community and resistance to bacterial canker in
genetically engineered phytochrome A cherry plants. J. Plant Physiol. 10, 1107–
1119.
Cotta, S.R., Dias, A.C.F., Marriel, I.E., Gomes, E.A., van Elsas, J.D., Seldin, L., 2013.
Temporal dynamics of microbial communities in the rhizosphere of two
genetically modified (GM) maize hybrids in tropical agrosystems. Antonie Van
Leeuwenhoek 3, 589–601.
Cotta, S.R., Dias, A.C.F., Marriel, I.E., Andreote, F.D., Seldin, L., van Elsas, J.D., 2014.
Different effects of transgenic maize and nontransgenic maize on nitrogentransforming archaea and bacteria in tropical soils. Appl. Environ. Microbiol. 80,
6437–6445.
Cotta, S.R., Dias, A.C.F., Seldin, L., Andreote, F.D., van Elsas, J.D., 2015. The diversity
and abundance of hytase genes (b-roeller hytases) in bacterial communities of
the maize rhizoshere. Lett. Microbiol. 62, 264–268.
D’Amico, K.M., Horton, T.R., Maynard, C.A., Stehman, S.V., Oakes, A.D., Powell, W.A.,
2015. Comparisons of ectomycorrhizal colonization of transgenic American
chestnut with those of the wild type, a conventionally bred hybrid, and related
Fagaceae species. Appl. Environ. Microbiol. 81, 100–108.
D'Angelo-Picard, C., Faure, D., Carlier, A., Uroz, S., Raffoux, A., Fray, R., Dessaux, Y.,
2004. Bacterial populations in the rhizosphere of tobacco plants producing the
quorum-sensing signals hexanoyl-homoserine lactone and 3-oxo-hexanoylhomoserine lactone. FEMS Microbiol. Ecol. 1, 19–29.
D’Angelo-Picard, C., Chapelle, E., Ratet, P., Faure, D., Dessaux, Y., 2011. Transgenic
plants expressing the quorum quenching lactonase AttM do not significantly
alter root-associated bacterial populations. Res. Microbiol. 9, 951–958.
Danielsen, L., Thürmer, A., Meinicke, P., Buee, M., Morin, E., Martin, F., Pilate, G.,
Daniel, R., Polle, A., Reich, M., 2012. Fungal soil communities in a young
transgenic poplar plantation form a rich reservoir for fungal root communities.
Ecol. Evol. 8, 1935–1948.
Demanèche, S., Sanguin, H., Poté, J., Navarro, E., Bernillon, D., Mavingui, P., Wildi, W.,
Vogel, T.M., Simonet, P., 2008. Antibiotic-resistant soil bacteria in transgenic
plant fields. Proc. Natl. Acad. Sci. U. S. A. 10, 3957–3962.
Demanèche, S., Philippot, L., David, M.M., Navarro, E., Vogel, T.M., Simonet, P., 2009.
Characterization of denitrification gene clusters of soil bacteria via a
metagenomic approach. Appl. Environ. Microb. 2, 534–537.
Demanèche, S., Monier, J.M., Dugat-Bony, E., Simonet, P., 2011. Exploration of
horizontal gene transfer between transplastomic tobacco and plant-associated
bacteria. FEMS Microbiol. Ecol. 1, 129–136.
de Vries, J., Wackernagel, W., 2005. Microbial horizontal gene transfer and the DNA
release from transgenic crop plants. Plant Soil 1–2, 91–104.
Devare, M., Jones, C., Thies, J., 2004. Effect of Cry3Bb transgenic corn and tefluthrin
on the soil microbial community. J. Environ. Qual. 3, 837–843.
Ernst, D., Rosenbrock-Krestel, H., Kirchhof, G., Bieber, E., Giunaschwili, N., Muller, R.,
Fischbeck, G., Wagner, T., Sandermann, H., Hartmann, A., 2008. Molecular
Investigations of the soil, rhizosphere and transgenic glufosinate-resistant rape
and maize plants in combination with herbicide (Basta (R)) application under
field conditions. Zeitschrift für Naturforschung C 63 (11–12), 864–872.
Fahner, N.A., Shokralla, S., Baird, D.J., Hajibabaei, M., 2016. Large-scale monitoring of
plants through environmental DNA metabarcoding of soil: recovery, resolution,
and annotation of four DNA markers. PLoS One 11 (6), e0157505.
Fang, M., Kremer, R.J., Motavalli, P.P., Davis, G., 2005. Bacterial diversity in
rhizospheres of nontransgenic and transgenic corn. Appl. Environ. Microb. 7,
4132–4136.
Fang, H., Dong, B., Yan, H., Tang, F., Wang, B., Yu, Y., 2012. Effect of vegetation of
transgenic Bt rice lines and their straw amendment on soil enzymes,
respiration, functional diversity and community structure of soil
microorganisms under field conditions. J. Environ. Sci. 7, 1259–1270.
Faragová, N., Gottwaldová, K., Faragó, J., 2011. Effect of transgenic alfalfa plants with
introduced gene for Alfalfa Mosaic Virus coat protein on rhizosphere microbial
community composition and physiological profile. Biologia (Bratisl.) 5, 768–
777.
Faragova, N., Farago, J., Drabekova, J., 2005. Evaluation of abundance of aerobic
bacteria in the rhizosphere of transgenic and non-transgenic alfalfa lines. Folia
Microbiol. 6, 509–514.
Filion, M., 2008. Do transgenic plants affect rhizobacteria populations? Microb.
Biotechnol. 6, 463–475.
Frostegård, Å., Tunlid, A., Bååth, E., 2011. Use and misuse of PLFA measurements in
soils. Soil Biol. Biochem. 8, 1621–1625.
Gao, N., Shen, W., Lin, X., Shi, W., 2015. Influence of transgenic ath-miR399d tomato
lines on microbial community and diversity in rhizosphere soil. Soil Sci. Plant
Nutr. 61, 259–268.
Garland, J.L., Mills, A.L., 1991. Classification and characterization of heterotrophic
microbial communities on the basis of patterns of community-level solecarbon-source utilization. Appl. Environ. Microb. 8, 2351–2359.
Giovannetti, M., Sbrana, C., Turrini, A., 2005. The impact of genetically modified
crops on soil microbial communities. Rivista di Biologia/Biology Forum 393–
418.
Girlanda, M., Bianciotto, V., Cappellazzo, G.A., Casieri, L., Bergero, R., Martino, E.,
Luppi, A.M., Perotto, S., 2008. Interactions between engineered tomato plants
expressing antifungal enzymes and nontarget fungi in the rhizosphere and
phyllosphere. FEMS Microbiol. Lett. 1, 9–18.
Griffiths, B., Caul, S., Thompson, J., Birch, A., Scrimgeour, C., Andersen, M.N., Cortet, J.,
Messean, A., Sausse, C., Lacroix, B., 2005. A comparison of soil microbial
community structure: protozoa and nematodes in field plots of conventional
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
and genetically modified maize expressing the Bacillus thuringiens is CryIAb
toxin. Plant Soil 1–2, 135–146.
Griffiths, B.S., Caul, S., Thompson, J., Birch, A.N.E., Scrimgeour, C., Cortet, J., Foggo, A.,
Hackett, C.A., Krogh, P.H., 2006. Soil microbial and faunal community responses
to maize and insecticide in two soils. J. Environ. Qual. 3, 734–741.
Griffiths, B.S., Heckmann, L.H., Caul, S., Thompson, J., Scrimgeour, C., Krogh, P.H.,
2007b. Varietal effects of eight paired lines of transgenic Bt maize and nearisogenic non-Bt maize on soil microbial and nematode community structure.
Plant Biotechnol. J. 1, 60–68.
Griffiths, B., Caul, S., Thompson, J., Hackett, C., Cortet, J., Pernin, C., Krogh, P.H., 2008.
Soil microbial and faunal responses to herbicide tolerant maize and herbicide in
two soils. Plant Soil 1–2, 93–103.
Gschwendtner, S., Reichmann, M., Müller, M., Radl, V., Munch, J.C., Schloter, M., 2010.
Effects of genetically modified amylopectin-accumulating potato plants on the
abundance of beneficial and pathogenic microorganisms in the rhizosphere.
Plant Soil 1–2, 413–422.
Gschwendtner, S., Esperschütz, J., Buegger, F., Reichmann, M., Müller, M., Munch, J.
C., Schloter, M., 2011. Effects of genetically modified starch metabolism in potato
plants on photosynthate fluxes into the rhizosphere and on microbial degraders
of root exudates. FEMS Microbiol. Ecol. 3, 564–575.
Höflich, G., Tauschke, M., Kühn, G., Werner, K., Frielinghaus, M., Höhn, W., 1999.
Influence of long-term conservation tillage on soil and rhizosphere
microorganisms. Biol. Fertil. Soils 1, 81–86.
Haack, S.K., Garchow, H., Klug, M.J., Forney, L.J., 1995. Analysis of factors affecting the
accuracy, reproducibility, and interpretation of microbial community carbon
source utilization patterns. Appl. Environ. Microb. 4, 1458–1468.
Halford, N.G., Shewry, P.R., 2000. Genetically modified crops: methodology,
benefits, regulation and public concerns. Br. Med. Bull. 1, 62–73.
Han, C., Zhong, W., Shen, W., Cai, Z., Liu, B., 2013. Transgenic Bt rice has adverse
impacts on CH4 flux and rhizospheric methanogenic archaeal and
methanotrophic bacterial communities. Plant Soil 1 (-2), 297–316.
Hannula, S.E., De Boer, W., Van Veen, J., 2010. In situ dynamics of soil fungal
communities under different genotypes of potato: including a genetically
modified cultivar. Soil Biol. Biochem. 12, 2211–2223.
Hannula, S.E., De Boer, W., Van Veen, J., 2012. A 3-year study reveals that plant
growth stage, season and field site affect soil fungal communities while cultivar
and GM-trait have minor effects. PLoS One 4, e33819.
Hannula, S.E., De Boer, W., Van Veen, J., 2014. Do genetic modifications in crops
affect soil fungi? A review. Biol. Fertil. Soils 50, 433–446.
Hart, M.M., Powell, J.R., Gulden, R.H., Dunfield, K.E., Peter Pauls, K., Swanton, C.J.,
Klironomos, J.N., Antunes, P.M., Koch, A.M., Trevors, J.T., 2009. Separating the
effect of crop from herbicide on soil microbial communities in glyphosateresistant corn. Pedobiologia 4, 253–262.
Hsieh, Y.-T., Pan, T.-M., 2006. Influence of planting papaya ringspot virus resistant
transgenic papaya on soil microbial biodiversity. J. Agric. Food Chem. 1, 130–137.
Hu, H.-Y., Liu, X.-X., Zhao, Z.-W., Sun, J.-G., Zhang, Q.-W., Liu, X.-Z., Yu, Y., 2009.
Effects of repeated cultivation of transgenic Bt cotton on functional bacterial
populations in rhizosphere soil. World J. Microb. Biotechnol. 3, 357–366.
Hu, H., Xie, M., Yu, Y., Zhang, Q., 2013. Transgenic Bt cotton tissues have no apparent
impact on soil microorganisms. Plant Soil Environ. 8, 366–371.
Hull, R., Covey, S., Dale, P., 2000. Genetically modified plants and the 35S promoter:
assessing the risks and enhancing the debate. Microb. Ecol. Health Dis. 1, 1–5.
_
lu, Ö., van Overbeek, L.S., Salles, J.F., van Elsas, J.D., 2013. Normal operating
Inceo
g
range of bacterial communities in soil used for potato cropping. Appl. Environ.
Microb. 4, 1160–1170.
Icoz, I., Stotzky, G., 2008. Fate and effects of insect-resistant Bt crops in soil
ecosystems. Soil Biol. Biochem. 3, 559–586.
Icoz, I., Saxena, D., Andow, D., Zwahlen, C., Stotzky, G., 2008. Microbial populations
and enzyme activities in soil in situ under transgenic corn expressing cry
proteins from Bacillus thuringiensis. J. Environ. Qual. 37, 647–662.
Jung, S., Park, S., Kim, D., Kim, S.B., 2008. Denaturing gradient gel electrophoresis
analysis of bacterial community profiles in the rhizosphere of cry1AC-carrying
Brassica rapa subsp. pekinensis. J. Microbiol. 1, 12–15.
Kapur, M., Bhatia, R., Pandey, G., Pandey, J., Paul, D., Jain, R.K., 2010. A case study for
assessment of microbial community dynamics in genetically modified Bt cotton
crop fields. Curr. Microbiol. 2, 118–124.
Keese, P., 2008. Risks from GMOs due to horizontal gene transfer. Environ. Biosaf.
Res. 03, 123–149.
Kim, S.E., Moon, J.S., Kim, J.K., Choi, W.S., Lee, S.H., Kim, S.U., 2010a. Investigation of
possible horizontal gene transfer from transgenic rice to soil microorganisms in
paddy rice field. J. Microbiol. Biotechnol. 1, 187–192.
Kim, S.E., Moon, J.S., Kim, J.K., Yoo, R.H., Choi, W.S., Lee, E.N., Lee, S.H., Kim, S.U.,
2010b. Monitoring of possible horizontal gene transfer from transgenic potatoes
to soil microorganisms in the potato fields and the emergence of variants in
Phytophthora infestans. J. Microbiol. Biotechnol. 6, 1027–1031.
Klümper, W., Qaim, M., 2014. A meta-analysis of the impacts of genetically modified
crops. PLoS One 9, e111629.
Kolseth, A.K., D’Hertefeldt, T., Emmerich, M., Forabosco, F., Marklund, S., Cheeke, T.E.,
Hallin, S., Weih, M., 2015. Influence of genetically modified organisms on agroecosystem processes. Agric. Ecosyst. Environ. 214, 96–106.
Kos, M., van Loon, J.J., Dicke, M., Vet, L.E., 2009. Transgenic plants as vital
components of integrated pest management. Trends Biotechnol. 11, 621–627.
Kremer, R.J., Means, N.E., 2009. Glyphosate and glyphosate-resistant crop
interactions with rhizosphere microorganisms. Eur. J Agron. 3, 153–161.
Kremer, R.J., 2014. Environmental implications of herbicide resistance: soil biology
and ecology. Weed Sci. 62, 415–426.
303
Kuramae, E.E., Verbruggen, E., Hillekens, R., de Hollander, M., Röling, W.F., van der
Heijden, M.G., Kowalchuk, G.A., 2013. Tracking fungal community responses to
maize plants by DNA-and RNA-based pyrosequencing. PLoS One 7, e69973.
Lahl, K., Unger, C., Emmerling, C., Broer, I., Thiele-Bruhn, S., 2012. Response of soil
microorganisms and enzyme activities on the decomposition of transgenic
cyanophycin-producing potatoes during overwintering in soil. Eur. J. Soil Biol.
53, 1–10.
Lamarche, J., Stefani, F.O., Séguin, A., Hamelin, R.C., 2011. Impact of endochitinasetransformed white spruce on soil fungal communities under greenhouse
conditions. FEMS Microbiol. Ecol. 2, 199–208.
LeBlanc, P.M., Hamelin, R.C., Filion, M., 2007. Alteration of soil rhizosphere
communities following genetic transformation of white spruce. Appl. Environ.
Microb. 13, 4128–4134.
Lee, B., Park, J.-Y., Park, K.W., Harn, C.H., Kim, H.M., Kim, C.-G., 2010. Evaluating the
persistence of DNA from decomposing transgenic watermelon tissues in the
field. J Plant Biol. 5, 338–343.
Lee, H.Y., Bae, T.W., Eok, L.Y., 2011a. Effects of field-grown genetically modified
Zoysia grass on bacterial community structure. J. Microbiol. Biotechnol. 4, 333–
340.
Lee, S.-H., Kim, C.-G., Kang, H., 2011b. Temporal dynamics of bacterial and fungal
communities in a genetically modified (GM) rice ecosystem. Microb. Ecol. 3,
646–659.
Lee, Y.E., Lee, S.H., Ryu, G.D., Kang, H.G., Kwon, Y., Sun, H.J., Park, K.W., Lee, B., Song, I.
J., Lim, P.O., Lee, H.Y., 2015. Investigation into effects of transgenic glufosinateresistant Zoysia grasses with herbicide application on bacterial communities
under field conditions. J. Plant Biol. 58, 303–310.
Li, X., Liu, B., Cui, J., Liu, D., Ding, S., Gilna, B., Luo, J., Fang, Z., Cao, W., Han, Z., 2011. No
evidence of persistent effects of continuously planted transgenic insectresistant cotton on soil microorganisms. Plant Soil 1–2, 247–257.
Li, P., Dong, J., Yang, S., Bai, L., Wang, J., Wu, G., Wu, X., Yao, Q., Tang, X., 2014. Impact
of b-carotene transgenic rice with four synthetic genes on rhizosphere enzyme
activities and bacterial communities at different growth stages. Eur. J. Soil Biol.
65, 40–46.
Liang, J., Sun, S., Ji, J., Wu, H., Meng, F., Zhang, M., Zheng, X., Wu, C., Zhang, Z., 2014.
Comparison of the rhizosphere bacterial communities of zigongdongdou
soybean and a high-methionine transgenic line of this cultivar. PLoS One 9,
e103343.
Liang, J., Meng, F., Sun, S., Wu, C., Wu, H., Zhang, M., Zhang, H., Zheng, X., Song, X.,
Zhang, Z., 2015. Community structure of Arbuscular Mycorrhizal fungi in
rhizospheric soil of a transgenic high-methionine soybean and a near isogenic
variety. PLoS One 10, e0145001.
Liphadzi, K.B., Al-Khatib, K., Bensch, C.N., Stahlman, P.W., Dille, J.A., Todd, T., Rice, C.
W., Horak, M.J., Head, G., 2005. Soil microbial and nematode communities as
affected by glyphosate and tillage practices in a glyphosate-resistant cropping
system. Weed Sci. 4, 536–545.
Liu, B., Zeng, Q., Yan, F., Xu, H., Xu, C., 2005. Effects of transgenic plants on soil
microorganisms. Plant Soil 1–2, 1–13.
Liu, W., Hao Lu, H., Wu, W., Kun Wei, Q., Xu Chen, Y., Thies, J.E., 2008. Transgenic Bt
rice does not affect enzyme activities and microbial composition in the
rhizosphere during crop development. Soil Biol. Biochem. 2, 475–486.
Liu, Y., Li, J., Stewart Jr., C.N., Luo, Z., Xiao, N., 2015. The effects of the presence of Bttransgenic oilseed rape in wild mustard populations on the rhizosphere
nematode and microbial communities. Sci. Total Environ. 530–531, 263–270.
Liu, Y., Li, J., Luo, Z., Wang, H., Liu, F., 2016. The fate of fusion Cry1Ab/1Ac proteins
from Bt-transgenic rice in soil and water. Ecotoxicol. Environ. Safe. 124, 455–
459.
Liu, W., 2010. Do genetically modified plants impact arbuscular mycorrhizal fungi?
Ecotoxicology 2, 229–238.
Loreau, M., Naeem, S., Inchausti, P., Bengtsson, J., Grime, J., Hector, A., Hooper, D.,
Huston, M., Raffaelli, D., Schmid, B., 2001. Biodiversity and ecosystem
functioning: current knowledge and future challenges. Science 5543, 804–808.
Lu, H., Wu, W., Chen, Y., Wang, H., Devare, M., Thies, J.E., 2010a. Soil microbial
community responses to Bt transgenic rice residue decomposition in a paddy
field. J. Soil Sediment. 8, 1598–1605.
Lu, H., Wu, W., Chen, Y., Zhang, X., Devare, M., Thies, J.E., 2010b. Decomposition of Bt
transgenic rice residues and response of soil microbial community in rapeseed–
rice cropping system. Plant Soil 1–2, 279–290.
Lupwayi, N.Z., Blackshaw, R.E., 2013. Soil microbial properties in Bt (Bacillus
thuringiensis) corn cropping systems. Appl. Soil Ecol. 63, 127–133.
Lupwayi, N., Hanson, K., Harker, K., Clayton, G., Blackshaw, R., O’Donovan, J.,
Johnson, E., Gan, Y., Irvine, R., Monreal, M., 2007. Soil microbial biomass,
functional diversity and enzyme activity in glyphosate-resistant wheat-canola
rotations under low-disturbance direct seeding and conventional tillage. Soil
Biol. Biochem. 7, 1418–1427.
Lv, Y., Cai, H., Yu, J., Liu, J., Liu, Q., Guo, C., 2014. Biosafety assessment of GFP
transplastomic tobacco to rhizosphere microbial community. Ecotoxicology 4,
718–725.
Ma, B., Blackshaw, R.E., Roy, J., He, T., 2011. Investigation on gene transfer from
genetically modified corn (Zea mays L.) plants to soil bacteria. J. Environ. Sci.
Health Part B 7, 590–599.
Mardis, E.R., 2008. Next-generation DNA sequencing methods. Annu. Rev. Genom.
Hum. Genet. 9, 387–402.
Marschner, P., Crowley, D., Yang, C.H., 2004. Development of specific rhizosphere
bacterial communities in relation to plant species nutrition and soil type. Plant
Soil 1–2, 199–208.
304
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
Melnitchouck, A., Leinweber, P., Broer, I., Eckhardt, K.-U., 2006. Pyrolysis-field
ionization mass spectrometry of rhizodeposits–A new approach to identify
potential effects of genetically modified plants on soil organisms. Environ.
Biosaf. Res. 01, 37–46.
Milling, A., Smalla, K., Maidl, F.X., Schloter, M., Munch, J.C., 2005. Effects of
transgenic potatoes with an altered starch composition on the diversity of soil
and rhizosphere bacteria and fungi. Plant Soil 1–2, 23–39.
Mimura, M., Lelmen, K.E., Shimazaki, T., Kikuchi, A., Watanabe, K.N., 2008. Impact of
environmental stress-tolerant transgenic potato on genotypic diversity of
microbial communities and soil enzyme activities under stress conditions.
Microbes Environ. 3, 221–228.
Moter, A., Göbel, U.B., 2000. Fluorescence in situ hybridization (FISH) for direct
visualization of microorganisms. J. Microbiol. Methods 2, 85–112.
Mulder, C., Wouterse, M., Raubuch, M., Roelofs, W., Rutgers, M., 2006. Can transgenic
maize affect soil microbial communities? PLoS Comput. Biol. 9, e128.
Mulder, C., Wouterse, M., Rutgers, M., Posthuma, L., 2007. Transgenic maize
containing the Cry1Ab protein ephemerally enhances soil microbial
communities. AMBIO J. Hum. Environ. 4, 359–361.
Nam, K.J., Kim, D.Y., Nam, K.H., Pack, I.S., Park, J.H., Jeong, S.C., Choi, Y.I., Noh, E.W.,
Kim, C.G., 2014. Effects of transgenic poplars expressing increased levels of
cellular cytokinin on rhizosphere microbial communities. Eur. J. Soil Biol. 63,
78–82.
Nielsen, U.N., Wall, D.H., 2013. The future of soil invertebrate communities in polar
regions: different climate change responses in the Arctic and Antarctic. Ecol.
Lett. 16, 409–419.
O’Callaghan, M., Gerard, E., Waipara, N., Young, S., Glare, T., Barrell, P., Conner, A.,
2005. Microbial communities of Solanum tuberosum and magainin-producing
transgenic lines. Plant Soil 1–2, 47–56.
O’Callaghan, M., Gerard, E.M., Bell, N.L., Waipara, N.W., Aalders, L.T., Baird, D.B.,
Conner, A.J., 2008. Microbial and nematode communities associated with
potatoes genetically modified to express the antimicrobial peptide magainin
and unmodified potato cultivars. Soil Biol. Biochem. 6, 1446–1459.
Oger, P.M., Mansouri, H., Nesme, X., Dessaux, Y., 2004. Engineering root exudation of
Lotus toward the production of two novel carbon compounds leads to the
selection of distinct microbial populations in the rhizosphere. Microb. Ecol. 1,
96–103.
Oguchi, T., Kashimura, Y., Mimura, M., Yu, X., Matsunaga, E., Nanto, K., Shimada, T.,
Kikuchi, A., Watanabe, K.N., 2014. A multi-year assessment of the
environmental impact of transgenic Eucalyptus trees harboring a bacterial
choline oxidase gene on biomass, precinct vegetation and the microbial
community. Transgenic Res. 23, 767–777.
Oliveira, A.P., Pampulha, M.E., Bennett, J.P., 2008. A two-year field study with
transgenic Bacillus thuringiensis maize: effects on soil microorganisms. Sci.
Total Environ. 1, 351–357.
Oliver, K.L., Hamelin, R.C., Hintz, W.E., 2008. Effects of transgenic hybrid aspen
overexpressing polyphenol oxidase on rhizosphere diversity. Appl. Environ.
Microb. 17, 5340–5348.
Ondrei9
cková, K., Mihálik, D., Ficek, A., Hudcovicová, M., Kraic, J., Drahovská, H., 2014.
Impact of genetically modified maize on the genetic diversity of rhizosphere
bacteria: a two-year study in Slovakia. Pol. J. Ecol. 62, 67–76.
Orgiazzi, A., Dunbar, M.B., Panagos, P., de Groot, G.A., Lemanceau, P., 2015. Soil
biodiversity and DNA barcodes: opportunities and challenges. Soil Biol.
Biochem. 80, 244–250.
Palm, C., Seidler, R., Schaller, D., Donegan, K., 1996. Persistence in soil of transgenic
plant produced Bacillus thuringlensis var kurstaki d-endotoxin. Can. J.
Microbiol. 12, 1258–1262.
Park, S., Ku, Y.K., Seo, M.J., Kim, D.Y., Yeon, J.E., Lee, K.M., Jeong, S.-C., Yoon, W.K.,
Harn, C.H., Kim, H.M., 2006. Principal component analysis and discriminant
analysis (PCA–DA) for discriminating profiles of terminal restriction fragment
length polymorphism (T-RFLP) in soil bacterial communities. Soil Biol. Biochem.
8, 2344–2349.
Pasonen, H.-L., Lu, J., Niskanen, A.-M., Seppänen, S.-K., Rytkönen, A., Raunio, J.,
Pappinen, A., Kasanen, R., Timonen, S., 2009. Effects of sugar beet chitinase IV on
root-associated fungal community of transgenic silver birch in a field trial.
Planta 5, 973–983.
Pline-Srnic, W., 2005. Technical performance of some commercial glyphosate –
resistant crops. Pest Manag. Sci. 61, 225–234.
Powell, J.R., Welsh, A., Hallin, S., 2015. Microbial functional diversity enhances
predictive models linking environmental parameters to ecosystem properties.
Ecology 96, 1985–1993.
Ranjard, L., Dequiedt, S., Chemidlin, P.B.N., Thioulouse, J., Saby, N.P.A., Lelievre, M.,
Maron, P.A., Morin, F.E.R., Bispo, A., Jolivet, C., Arrouays, D., Lemanceau, P., 2013.
Turnover of soil bacterial diversity driven by wide-scale environmental
heterogeneity. Nat. Commun. 4, 1434.
Rasche, F., Hödl, V., Poll, C., Kandeler, E., Gerzabek, M.H., Van Elsas, J.D., Sessitsch, A.,
2006. Rhizosphere bacteria affected by transgenic potatoes with antibacterial
activities compared with the effects of soil, wild-type potatoes, vegetation stage
and pathogen exposure. FEMS Microbiol. Ecol. 2, 219–235.
Raubuch, M., Roose, K., Warnstorff, K., Wichern, F., Joergensen, R.G., 2007.
Respiration pattern and microbial use of field-grown transgenic Bt-maize
residues. Soil Biol. Biochem. 39, 2380–2389.
Riglietti, A., Ruggiero, P., Crecchio, C., 2008. Investigating the influence of transgenic
tobacco plants codifying a protease inhibitor on soil microbial community. Soil
Biol. Biochem. 12, 2928–2936.
Robert, M.Z., Krishna, N.R., 2004. Impact of Glyphosate on the Bradyrhizobium
japonicum symbiosis with glyphosate-resistant transgenic soybean: a mini
review. J. Environ. Qual. 33, 825–831.
Robin, A., Mougel, C., Siblot, S., Vansuyt, G., Mazurier, S., Lemanceau, P., 2006a. Effect
of ferritin overexpression in tobacco on the structure of bacterial and
pseudomonad communities associated with the roots. FEMS Microbiol. Ecol. 3,
492–502.
Robin, A., Vansuyt, G., Corberand, T., Briat, J.-F., Lemanceau, P., 2006b. The soil type
affects both the differential accumulation of iron between wild-type and ferritin
over-expressor tobacco plants and the sensitivity of their rhizosphere
bacterioflora to iron stress. Plant Soil 1–2, 73–81.
Robin, A., Mazurier, S., Mougel, C., Vansuyt, G., Corberand, T., Meyer, J.M.,
Lemanceau, P., 2007. Diversity of root-associated fluorescent pseudomonads as
affected by ferritin overexpression in tobacco. Environ. Microbiol. 7, 1724–1737.
Rui, Y.-K., Yi, G.-X., Zhao, J., Wang, B.-M., Li, Z.-H., Zhai, Z.-X., He, Z.-P., Li, Q.X., 2005.
Changes of Bt toxin in the rhizosphere of transgenic Bt cotton and its influence
on soil functional bacteria. World J. Microbiol. Biotechnol. 6–7, 1279–1284.
Sabharwal, N., Icoz, I., Saxena, D., Stotzky, G., 2007. Release of the recombinant
proteins, human serum albumin, b-glucuronidase, glycoprotein B from human
cytomegalovirus, and green fluorescent protein, in root exudates from
transgenic tobacco and their effects on microbes and enzymatic activities in
soil. Plant Physiol. Biochem. 6, 464–469.
Saha, N., Ulrich, A., Becker, R., Wirth, S., 2007. Assessing the changes in bacterial
diversity in rhizosphere and phyllosphere of transgenic and non-transgenic
potato plant. Plant Tissue Cult. Biotechnol. 1, 87–95.
Sahoo, R.K., Tuteja, N., 2013. Effect of salinity tolerant PDH45 transgenic rice on
physicochemical properties, enzymatic activities and microbial communities of
rhizosphere soils. Plant Signal. Behav. 8, e24950.
Sahoo, R.K., Ansari, M.W., Tuteja, R., Tuteja, N., 2015. Salt tolerant SUV3
overexpressing transgenic rice plants conserve physicochemical properties and
microbial communities of rhizosphere. Chemosphere 119, 1040–1047.
Sanahuja, G., Banakar, R., Twyman, R.M., Capell, T., Christou, P., 2011. Bacillus
thuringiensis: a century of research, development and commercial applications.
Plant Biotechnol. J. 3, 283–300.
Sanchis, V., 2010. From microbial sprays to insect-resistant transgenic plants:
history of the biospesticide Bacillus thuringiensis: a review. Agron. Sustain. Dev.
1, 217–231.
Schmidt, P.A., B_alint, M., Bandow, C., Rsombke, J., Schmitt, I., 2013. Illumina
metabarcoding of a soil fungal community. Soil Biol. Biochem. 65, 128–132.
Sessitsch, A., Gyamfi, S., Tscherko, D., Gerzabek, M.H., Kandeler, E., 2005. Activity of
microorganisms in the rhizosphere of herbicide treated and untreated
transgenic glufosinate-tolerant and wildtype oilseed rape grown in
containment. Plant Soil 1–2, 105–116.
Shen, R.F., Cai, H., Gong, W.H., 2006. Transgenic Bt cotton has no apparent effect on
enzymatic activities or functional diversity of microbial communities in
rhizosphere soil. Plant Soil 1–2, 149–159.
Singh, O.V., Ghai, S., Paul, D., Jain, R.K., 2006. Genetically modified crops: success,
safety assessment, and public concern. Appl. Microbiol. Biot. 5, 598–607.
Singh, A.K., Rai, G.K., Singh, M., Dubey, S.K., 2013a. Bacterial community structure in
the rhizosphere of a Cry1Ac Bt-brinjal crop and comparison to its nontransgenic counterpart in the tropical soil. Microb. Ecol. 4, 927–939.
Singh, A.K., Singh, M., Dubey, S.K., 2013b. Changes in Actinomycetes community
structure under the influence of Bt transgenic brinjal crop in a tropical
agroecosystem. BMC Microbiol. 1, 122.
Singh, A.K., Singh, M., Dubey, S.K., 2014. Rhizospheric fungal community structure of
a Bt brinjal and a near isogenic variety. J. Appl. Microbiol. 117, 750–765.
Sohn, S.I., Oh, Y.J., Kim, B.Y., Kweon, S.J., Cho, H.S., Ryu, T.H., 2015. Effect of genetically
modified rice producing resveratrol on the soil microbial communities. J. Kor.
Soc. Appl. Biol. Chem. 58, 795–805.
Song, Y.-N., Su, J., Chen, R., Lin, Y., Wang, F., 2014. Diversity of microbial community
in a paddy soil with cry1Ac/cpti transgenic rice. Pedosphere 3, 349–358.
Souza, R.A., Babujia, L.C., Silva, A.P., de Fátima Guimarães, M., Arias, C.A., Hungria, M.,
2013. Impact of the ahas transgene and of herbicides associated with the
soybean crop on soil microbial communities. Transgenic Res. 5, 877–892.
Stefani, F.O., Moncalvo, J.-M., Séguin, A., Bérubé, J.A., Hamelin, R.C., 2009. Impact of
an 8-year-old transgenic poplar plantation on the ectomycorrhizal fungal
community. Appl. Environ. Microb. 23, 7527–7536.
Stefani, F.O., Tanguay, P., Pelletier, G., Piché, Y., Hamelin, R.C., 2010. Impact of
endochitinase-transformed white spruce on soil fungal biomass and
ectendomycorrhizal symbiosis. Appl. Environ. Microb. 8, 2607–2614.
Stefanowicz, A., 2006. The biolog plates technique as a tool in ecological studies of
microbial communities. Pol. J. Environ. Stud. 5, 669.
Strain, K.E., Lydy, M.J., 2015. The fate and transport of the Cry1Ab protein in an
agricultural field and laboratory aquatic microcosms. Chemosphere 132, 94–
100.
Tan, F., Wang, J., Feng, Y., Chi, G., Kong, H., Qiu, H., Wei, S., 2010. Bt corn plants and
their straw have no apparent impact on soil microbial communities. Plant Soil
1–2, 349–364.
Tan, F., Wang, J., Chen, Z., Feng, Y., Chi, G., Rehman, S.U., 2011. Assessment of the
arbuscular mycorrhizal fungal community in roots and rhizosphere soils of Bt
corn and their non-Bt isolines. Soil Biol. Biochem. 12, 2473–2479.
Tapp, H., Stotzky, G., 1995. Insecticidal Activity of the Toxins from Bacillus
thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure
and Soil Clays. Appl. Environ. Microb. 5, 1786–1790.
Tilman, D., Reich, P.B., Knops, J., Wedin, D., Mielke, T., Lehman, C., 2001. Diversity and
productivity in a long-term grassland experiment. Science 5543, 843–845.
Z.- Guan et al. / Agriculture, Ecosystems and Environment 235 (2016) 289–305
Tothova, T., Sobekova, A., Holovska, K., Legath, J., Pristas, P., Javorsky, P., 2010. Natural
glufosinate resistance of soil microorganisms and GMO safety. Cent. Eur. J. Biol.
5, 656–663.
Travis, E.R., Hannink, N.K., Van Der Gast, C.J., Thompson, I.P., Rosser, S.J., Bruce, N.C.,
2007. Impact of transgenic tobacco on trinitrotoluene (TNT) contaminated soil
community. Environ. Sci. Technol. 16, 5854–5861.
Turrini, A., Sbrana, C., Giovannetti, M., 2015. Belowground environmental effects of
transgenic crops: a soil microbial perspective. Res. Microbiol. 166, 121–131.
Val, G., Marín, S., Mellado, R.P., 2009. A sensitive method to monitor Bacillus subtilis
and Streptomyces coelicor-related bacteria in maize rhizobacterial
communities: the use of genome-wide microarrays. Microb. Ecol. 1, 108–115.
van der Heijden, M.G.A., Bardgett, R.D., Van Straalen, N.M., 2008a. The unseen
majority: soil microbes as drivers of plant diversity and productivity in
terrestrial ecosystems. Ecol. Lett. 11, 296–310.
van der Heijden, M.G.A., Bardgett, R.D., Van Straalen, N.M., 2008b. The unseen
majority: soil microbes as drivers of plant diversity and productivity in
terrestrial ecosystems. Ecol. Lett. 11, 296–310.
van der Heijden, M.G.A., Bardgett, R.D., Van Straalen, N.M., 2008c. The unseen
majority: soil microbes as drivers of plant diversity and productivity in
terrestrial ecosystems. Ecol. Lett. 11, 296–310.
Van Overbeek, L., Van Elsas, J.D., 2008. Effects of plant genotype and growth stage on
the structure of bacterial communities associated with potato (Solanum
tuberosum L.). FEMS Microbiol. Ecol. 2, 283–296.
Velasco, A., Kowalchuk, G., Mañero, F., Ramos, B., Yergeau, E., García, J., 2013.
Increased microbial activity and nitrogen mineralization coupled to changes in
microbial community structure in the rhizosphere of Bt corn. Appl. Soil Ecol. 68,
46–56.
Velkov, V.V., Medvinsky, A.B., Sokolov, M.S., Marchenko, A.I., 2005. Will transgenic
plants adversely affect the environment? J. Biosci. 4, 515–548.
Velmourougane, K., Sahu, A., 2013. Impact of transgenic cottons expressing cry1Ac
on soil biological attributes. Plant Soil Environ. 3, 108–114.
Verbruggen, E., Kuramae, E.E., Hillekens, R., de Hollander, M., Kiers, E.T., Röling, W.F.,
Kowalchuk, G.A., van der Heijden, M.G., 2012. Testing potential effects of maize
expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) on
mycorrhizal fungal communities via DNA-and RNA-based pyrosequencing and
molecular fingerprinting. Appl. Environ. Microb. 20, 7384–7392.
Wang, B., Shen, H., Yang, X., Guo, T., Zhang, B., Yan, W., 2013. Effects of chitinasetransgenic (McChit1) tobacco on the rhizospheric microflora and enzyme
activities of the purple soil. Plant Soil Environ. (Czech Republic) 6, 241–246.
Weaver, M.A., Krutz, L.J., Zablotowicz, R.M., Reddy, K.N., 2007. Effects of glyphosate
on soil microbial communities and its mineralization in a Mississippi soil. Pest
Manag. Sci. 4, 388–393.
Wei, X., Zou, H., Chu, L., Liao, B., Ye, C., Lan, C., 2006. Field released transgenic papaya
affects microbial communities and enzyme activities in soil. Plant Soil 1–2, 347–
358.
Wei, M., Tan, F., Zhu, H., Cheng, K., Wu, X., Wang, J., Zhao, K., Tang, X., 2012. Impact of
Bt-transgenic rice (SHK601) on soil ecosystems in the rhizosphere during crop
development. Plant Soil Environ. 5, 217–223.
Widmer, F., 2007. Assessing effects of transgenic crops on soil microbial
communities. Green Gene Technology. Springer, Berlin Heidelberg, pp. 207–
234.
Winder, R.S., Lamarche, J., Constabel, C.P., Hamelin, R.C., 2013. The effects of hightannin leaf litter from transgenic poplars on microbial communities in
microcosm soils. Front. Microbiol. 4.
305
Wolfenbarger, L.L., Phifer, P.R., 2000. The ecological risks and benefits of genetically
engineered plants. Science 5499, 2088–2093.
Wu, W.-x., Ye, Q.-f., Min, H., Duan, X.-j., Jin, W.-m., 2004. Bt-transgenic rice straw
affects the culturable microbiota and dehydrogenase and phosphatase activities
in a flooded paddy soil. Soil Biol. Biochem. 2, 289–295.
Wu, W.X., Liu, W., Lu, H.H., Chen, Y.X., Devare, M., Thies, J., 2009. Use of 13C labeling
to assess carbon partitioning in transgenic and nontransgenic (parental) rice
and their rhizosphere soil microbial communities. FEMS Microbiol. Ecol. 1, 93–
102.
Wu, J., Yu, M., Xu, J., Du, J., Ji, F., Dong, F., Li, X., Shi, J., 2014. Impact of transgenic
wheat with wheat yellow mosaic virus resistance on microbial community
diversity and enzyme activity in rhizosphere soil. PLoS One 9, e98394.
Xue, K., Serohijos, R.C., Devare, M., Thies, J.E., 2011. Decomposition rates and
residue-colonizing microbial communities of Bacillus thuringiensis insecticidal
protein Cry3Bb-expressing (Bt) and non-Bt corn hybrids in the field. Appl
Environ Microb. 3, 839–846.
Yang, C.X., Ji, Y.Q., Wang, X.Y., Yang, C.Y., Yu, D.W., 2013. Testing three pipelines for
18S rDNA-based metabarcoding of soil faunal diversity. Sci. China-Life Sci. 56,
73–81.
Yi, H., Kim, H.-J., Kim, C.-G., Harn, C.H., Kim, H.M., Park, S., 2009. Using T-RFLP to
assess the impact on soil microbial communities by transgenic lines of
watermelon rootstock resistant to cucumber green mottle mosaic virus
(CGMMV). J. Plant Biol. 6, 577–584.
Yu, X., Kikuchi, A., Matsunaga, E., Shimada, T., Watanabe, K.N., 2013. Environmental
biosafety assessment on transgenic Eucalyptus globulus harboring the choline
oxidase (codA) gene in semi-confined condition. Plant Biotechnol. 1, 73–76.
Zadorina, E., Boulygina, E., Kolganova, T., Kuznetsov, B., Skryabin, K., 2009.
Evaluation of the effect of late blight-resistant potato plants on the structure of
bacterial associations in soil. Appl. Biochem. Micro. 2, 194–198.
Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the
characterisation of microbial communities in soil: a review. Biol. Fertil. Soils 2,
111–129.
Zeng, H., Tan, F., Zhang, Y., Feng, Y., Shu, Y., Wang, J., 2014. Effects of cultivation and
return of Bacillus thuringiensis (Bt) maize on the diversity of the arbuscular
mycorrhizal community in soils and roots of subsequently cultivated
conventional maize. Soil Biol. Biochem. 75, 254–263.
Zhang, Y.J., Xie, M., Peng, D.L., 2014. Effects of the transgenic CrylAc and CpTI insectresistant cotton SGK321 on rhizosphere soil microorganism populations in
northern China. Plant Soil Environ. 60, 285–289.
Zhang, Y.J., Xie, M., Wu, G., Peng, D.L., Yu, W.B., 2015. A 3-year field investigation of
impacts of Monsanto’s transgenic Bt-cotton NC 33 B on rhizosphere microbial
communities in northern China. Appl. Soil Ecol. 89, 18–24.
Zhu, W., Lu, H., Hill, J., Guo, X., Wang, H., Wu, W., 2014. 13C pulse-chase labeling
comparative assessment of the active methanogenic archaeal community
composition in the transgenic and nontransgenic parental rice rhizospheres.
FEMS Microbiol. Ecol. 87, 746–756.
Zobiole, L., Kremer, R., Oliveira, R., Constantin, J., 2011. Glyphosate affects microorganisms in rhizospheres of glyphosate-resistant soybeans. J. Appl. Microbiol.
1, 118–127.
Zwahlen, C., Hilbeck, A., Gugerli, P., Nentwig, W., 2003. Degradation of the Cry1Ab
protein within transgenic Bacillus thuringiensis corn tissue in the field. Mol.
Ecol. 3, 765–775.