FEMS Microbiology Ecology 38 (2001) 1^9
www.fems-microbiology.org
Diversity of bacterial communities in the rhizosphere and root
interior of ¢eld-grown genetically modi¢ed Brassica napus
Kari E. Dun¢eld, James J. Germida *
Department of Soil Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada S7N5A8
Received 14 May 2001; received in revised form 7 August 2001; accepted 7 August 2001
First published online 27 September 2001
Abstract
Plant roots significantly affect microbial diversity in soil, but little is known on how genetically modified plants influence soil microbial
communities. We conducted a 2-year field study to assess the effects of herbicide-tolerant genetically modified canola (oilseed rape, Brassica
sp.) on microbial biodiversity in the rhizosphere. During the 1998 and 1999 field seasons, four genetically modified and four conventional
canola varieties were grown at four different field locations across Saskatchewan, Canada. The rhizosphere and root interior microbial
communities were characterized through fatty acid methyl ester analysis and community level physiological profiles. Principal component
analysis indicated that the root interior and rhizosphere bacterial community associated with the genetically modified variety Quest
(Brassica napus) was different from conventional varieties Excel (B. napus) and Fairview (Brassica rapa), based on both fatty acid
composition and carbon substrate utilization. In addition, all root-associated microbial communities associated with genetically modified
canola varieties had significantly higher levels of 10:02OH, 12:02OH, 12:03OH, a15:0, 15:1g5c, cy17:0, 18:3g6,9,12c, 19:0g8c and Sum in
Feature 3, suggesting alterations in the composition of the microbial community associated with plants. This study indicates that the
composition and functional diversity and the microbial community were influenced by plant variety. ß 2001 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Bacterial diversity; Rhizosphere ; Root interior; Genetically modi¢ed plant; Brassica spp.
1. Introduction
The recognition that microbial species and their interaction with soil in£uence a number of ecosystem processes
has led researchers to examine soil microbial biodiversity
[1]. Soil microbial communities vary depending on soil
physical and chemical properties, type and amount of
plant cover and climate [2]. It is well known that plants
in£uence the biodiversity of bacteria in soils. Through the
release of compounds such as amino acids, sugars and
growth factors in plant root exudates, microbial activity
and growth are stimulated [3]. Because bacteria respond
di¡erently to these compounds, di¡erences in the composition of root exudates can in£uence the types of bacteria
present in the rhizosphere community [4]. Although all
plants show a rhizosphere e¡ect, the plant species can
* Corresponding author. Tel. : +1 (306) 966-6836;
Fax: +1 (306) 966-6881.
E-mail address : [email protected] (J.J. Germida).
in£uence the types of bacteria that are present in the rhizosphere [5,6].
Recently, genetically modi¢ed canola (oilseed rape;
Brassica sp.), tolerant to non-selective broad-spectrum
herbicides such as glyphosate or glufosinate ammonium,
have received environmental and nutritional food and feed
safety clearances by Agriculture and Agri-Food Canada
and Health Canada [7]. These varieties provide an economic and agronomic bene¢t to farmers because they allow superior weed control with the use of a minimal number of herbicides. However, there are concerns regarding
the e¡ects of genetically modi¢ed plants on soil and rhizosphere microbial communities [8]. For example, Donegan
et al. [9] suggest that unintentional changes in plant characteristics resulting from genetic modi¢cation may impact
on soil and plant biota. Consequences of these changes
include decreases in plant decomposition rates, and in
soil carbon and nitrogen levels that could a¡ect soil fertility [8]. In addition, the root-associated communities of
genetically modi¢ed plants may be signi¢cantly di¡erent
from non-genetically modi¢ed plants [10,11]. Di Giovanni
et al. [12] found di¡erences in carbon substrate utilization
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 6 7 - 2
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K.E. Dun¢eld, J.J. Germida / FEMS Microbiology Ecology 38 (2001) 1^9
patterns between rhizosphere bacterial communities of parental and transgenic alfalfa plants grown in a growth
chamber.
Siciliano et al. [10] found that the carbon utilization
patterns and fatty acid methyl ester (FAME) pro¢les of
the microbial community associated with the roots of the
¢eld-grown genetically modi¢ed canola variety, Quest
(Brassica napus), were signi¢cantly di¡erent from the pro¢les of communities associated with two non-genetically
modi¢ed varieties, Excel (B. napus) and Parkland (Brassica
rapa). However, they only evaluated one transgenic variety
and were unable to predict whether the observed changes
were due to the transgenic nature of the plants. Here, we
describe a 2-year ¢eld study, which compared the root
interior and rhizosphere communities of eight canola varieties grown at four geographically distinct ¢eld sites
across Saskatchewan. We included four herbicide-tolerant
genetically modi¢ed canola varieties. Community level
physiological pro¢ling (CLPP) was used to assess the functional diversity of the microbial community by determining the ability of the microorganisms extracted from the
root system to use a variety of carbon substrates [10,13].
In addition, the composition of fatty acids present in the
rhizosphere and roots of plants was used to analyze community structure using FAME analysis [10,14]. The purpose of this study was to validate ¢ndings reported by
Siciliano et al. [10].
2. Materials and methods
2.1. Experimental design
This study was a part of a larger multiple ¢eld site,
multiple ¢eld year study where eight canola varieties
were seeded at six ¢eld locations in Saskatchewan, Canada, over three ¢eld years (1998, 1999 and 2000). This study
examined four of these ¢eld locations; ¢eld characteristics
are described in Table 1. The purpose of this experiment
was to validate ¢ndings reported in Siciliano et al. [10].
Their 1997 ¢eld sites were at Denholm and Watrous, Saskatchewan. In 1998, we examined soil communities from
plants grown at Eyebrow and Melfort, the closest sites
available to those of the 1997 study. In 1999, we returned
to the Denholm and Watrous locations. Eight commercially available canola varieties (four genetically modi¢ed
and four conventional ; Table 2) were seeded at each site in
a replicated (n = 4) randomized complete block design.
2.2. Sample processing
Field sites and plants were sampled on various dates
when plants were at the £owering stage of growth (universal growth stage: 6.5 [15]). The root material and soil were
collected from each replicate as described by Siciliano et
al. [10]. Brie£y, plants were removed from soil with a
trowel, placed in a plastic bag, transported to the laboratory and processed within 24 h of removal from the
ground. The shoots were removed with a scalpel and the
roots with the soil adhering were sieved (5 mm) for 5 min.
A 0.6-g portion of root with adhering soil and a 5-g portion of rhizosphere soil were placed individually into
screw-cap test tubes and stored (320³C) for FAME analysis.
The number of colony forming units (CFU) per gram of
root was determined by placing a portion (5 g) of roots
with adhering soil into a 1-l Erlenmeyer £ask containing
495 ml of phosphate-bu¡ered saline (PBS ; 1.2 g l31
Na2 HPO4 , 0.18 g l31 NaH2 PO4 , 8.5 g l31 NaCl) and shaking on a rotary shaker (200 rpm) at 22³C for 20 min.
Dilutions (1:10) of this solution were made in sterile
PBS and 0.1 ml of the appropriate dilutions was spread
plated onto 1/10 Trypticase soy agar (TSA; 3 g l31 Trypticase soy broth (TSB), 15 g l31 agar) plates. Plates were
incubated at 28³C for 72 h and colonies counted. The 1034
dilution was saved for CLPP.
To extract bacteria from the root interior for plate
counts and CLPP, roots were removed from the 1-l Erlenmeyer £ask and transferred to a 500-ml Erlenmeyer £ask
containing 200 ml of NaClO (1.05% v/v) in sterile water,
and placed on a rotary shaker (200 rpm) at 22³C for 10
min. Roots were rinsed four times with 200 ml of sterile
PBS, and 0.1 ml of the ¢nal wash was diluted in 9.9 ml of
1/10 TSB to check for contamination [16]. The roots were
then chopped into 6 1-mm sections and triturated with a
sterile mortar and pestle. The roots were diluted with sterile PBS (1:10) and inoculated onto 1/10 TSA plates for
enumeration. The 1033 dilution was saved for CLPP.
Table 1
Selected soil characteristics of Saskatchewan ¢eld sites
Field site name and location
Soil name and type
FAO soil classi¢cation
Conductivity
(mS cm31 )a
pHa
Eyebrow 50³48PN, 106³09PW
Melfort 52³52PN, 104³37PW
Denholm 52³39PN, 108³01PW
Watrous 51³40PN, 105³28PW
Weyburn Sandy Loam
Oxbow Loam
Oxbow Silty Loam
Weyburn Silty Clay Loam
Kastanozem
Chernozem
Chernozem
Kastanozem
0.1
0.1
0.4
0.4
6.1
6.2
6.2
6.6
a
1:2 soil:water dilution.
FEMSEC 1283 14-12-01
Soil texture (% by weight)
Sand Silt
Clay
57
55
19
11
15
16
22
37
28
29
59
52
K.E. Dun¢eld, J.J. Germida / FEMS Microbiology Ecology 38 (2001) 1^9
Table 2
Selected characteristics of canola varieties
3
prokaryotes, were not included in statistical analysis
[14,19].
Variety
name
Species
Herbicide tolerance
45A71
Excel
Exceed
B. napus
B. napus
B. napus
Fairview
Hyola
Innovator
B. rapa
B. napus
B. napus
Invigor
B. napus
Quest
B. napus
Imidazolonone-tolerant, mutagenesisa
None
Glufosinate ammonium-tolerant canola,
genetically modi¢ed
None
None
Glufosinate ammonium-tolerant canola,
genetically modi¢ed
Glufosinate ammonium-tolerant canola,
genetically modi¢ed
Glyphosate-tolerant, genetically modi¢ed
a
Herbicide-tolerant through mutagenesis considered a conventional variety for this study.
2.3. CLPP
The CLPP was performed as described by Siciliano and
Germida [17] with Biolog1 Gram-negative (GN2) microplates (Biolog, Inc., Hayward, CA, USA). Brie£y, 100 Wl
of the 1033 or 1034 dilutions, for root interior (i.e. endophytes) and rhizosphere samples respectively, was inoculated into each well and plates incubated at 28³C for 5
days. In 1998, Biolog wells were read visually and scored
positive or negative for growth at 24, 48, 72 and 96 h. In
1999, color development was measured as optical density
at 590 nm, with an automated microplate reader (Molecular Devices, Inc., Sunnyvale, CA, USA) and Microlog 3E
software (Biolog, Inc., Hayward, CA, USA). The average
well color development (AWCD) was calculated as described by Garland and Mills [18].
2.4. FAME analysis
FAME analysis was performed as described by Cavigelli
et al. [14] and Siciliano et al. [10]. Brie£y, 5 g of soil or
0.6 g of roots with adhering soil were mixed with 5 ml of
methanoic NaOH (15% w/v NaOH in 50% v/v methanol)
and saponi¢ed at 100³C for 30 min. Esteri¢cation of fatty
acids was performed with 10 ml of 3.25 N HCl in 46% v/v
methanol at 80³C for 10 min. The FAMEs were extracted
into 1.5 ml of 1:1 v/v methyl-tert-butyl ether-hexane, centrifuged at 110Ug for 5 min, and the top phase was transferred to a 10-cm test tube. This organic extract was
washed with 3 ml of 1.2% w/v NaOH and analyzed using
a Hewlett-Packard 5890 Series II Gas Chromatograph.
FAME peaks were automatically integrated by HewlettPackard 3365 ChemStation software and FAMEs identi¢ed using the MIDI Microbial Identi¢cation System software (Sherlock TSBA Library version 3.80; Microbial ID,
Inc., Newark, DE, USA). In order to minimize the fatty
acids derived from plant and animal sources, fatty acids
with chain lengths exceeding 20 carbons, which are generally more characteristic of eukaryotic organisms than
2.5. Statistical analysis
To standardize FAMEs, the adjusted response area of
each sample was calculated by multiplying the percentage
composition of each individual FAME by the total named
area for that chromatogram [20]. We decided to report
FAME data as an adjusted area response instead of as
percentage composition (e.g. [14]), because the use of proportional data has been shown to adversely a¡ect multivariate statistical analysis [21]. Soil and root FAME pro¢les were compared by principal component analysis
(PCA) using the correlation matrix (Minitab v. 12, Minitab, Inc., State College, PA, USA). The principal component data were analyzed using analysis of variance (ANOVA). In order to minimize the number of fatty acids that
were derived from plants, common plant fatty acids were
excluded from the root FAME pro¢les. These fatty acids
were determined by extracting fatty acids from sterile canola roots and included 12:0, 14:0, 15:0, 16:0, 16:02OH,
16:1g7c, 18:0, 18:02OH, 20:0, Sum in Features 6 and 9.
The AWCD was used as a standardized reference point
in color development [9,18]. Absorbance data (A590 ) from
microplates having AWCD of approximately 0.75 were
used for statistical analysis. PCA was done as described
above. Substrates were grouped into functional guilds as
described by Siciliano et al. [10] with the exception that
only Gram-negative guilds were used. The utilization rate
for each guild was calculated by determining the number
of positives for each guild at each successive reading time.
The curves obtained were found to ¢t a square root curve,
therefore the values were transformed by taking the square
root of each value and the slope of the linear regression
determined. The slope value, termed utilization rate, was
tested for homogeneity of variance and analyzed by ANOVA [22].
3. Results
3.1. Culturable microbial community
Neither canola variety nor soil type signi¢cantly a¡ected
the total CFU of rhizosphere and root interior microbial
communities. The CFU (g root)31 ranged from 1.2U107
to 6.3U107 for the rhizosphere, and from 2U105 to
1U106 for the root interior. In 1998, several 1/10 TSB
tubes appeared cloudy, indicating microbial growth and
contamination of the root interior community. In order
to ensure that only the microbes from the root interior
were examined, the corresponding Biolog plates were removed from the study. Therefore in 1998, two varieties,
Invigor and 45A71, and all varieties grown in Eyebrow
were not assessed for their root interior CLPP.
FEMSEC 1283 14-12-01
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K.E. Dun¢eld, J.J. Germida / FEMS Microbiology Ecology 38 (2001) 1^9
Fig. 1. PCA of FAMEs obtained from the rhizosphere soil of canola
cultivars grown at Eyebrow and Melfort, Saskatchewan, in 1998. Each
symbol is the average of four replicates at one ¢eld site (n = 4). Closed
symbols represent conventional varieties : triangles, Excel replicates;
squares, Fairview replicates ; diamonds, Hyola replicates ; circles, 45A71
replicates. Open symbols represent genetically modi¢ed varieties: triangles, Exceed replicates; squares, Innovator replicates; diamonds, Invigor
replicates ; circles, Quest replicates. Error bars represent the standard error of the mean. Principal components marked with a P value indicate
a signi¢cant variety e¡ect as determined by ANOVA.
3.2. FAME pro¢les of microbial communities
Signi¢cant interactions between ¢eld site and plant variety were evident, therefore the sites were analyzed individually. Principal components analysis of the rhizosphere
soils showed two clusters, and explained 58% of the variation in the data. The fatty acid compositions of rhizo-
Fig. 2. PCA of FAMEs obtained from the rhizosphere soil of canola
varieties grown at Denholm and Watrous, Saskatchewan, in 1999. Each
symbol is the average of four replicates from the two combined ¢eld
sites (n = 8). Closed symbols represent conventional varieties: triangles,
Excel replicates; squares, Fairview replicates; diamonds, Hyola replicates; circles, 45A71 replicates. Open symbols represent genetically
modi¢ed varieties: triangles, Exceed replicates; squares, Innovator replicates; diamonds, Invigor replicates; circles, Quest replicates. Error bars
represent the standard error of the mean.
Fig. 3. PCA of FAMEs obtained from the rhizosphere soil of canola
varieties grown at Denholm and Watrous, Saskatchewan, in 1999. Each
symbol is the average of four replicates from the two combined ¢eld
sites (n = 8). Closed symbols represent conventional varieties : triangles,
Excel replicates; squares, Fairview replicates. Open symbols represent
genetically modi¢ed varieties ; circles, Quest replicates. Error bars represent the standard error of the mean. Principal components marked with
a P value indicate a signi¢cant variety e¡ect as determined by ANOVA.
sphere soils at the Melfort ¢eld site were not signi¢cantly
di¡erent between plant varieties. Conversely, there were
signi¢cant di¡erences in the fatty acid composition of
the rhizosphere soils at the Eyebrow ¢eld site along the
PC1 axis (Fig. 1). At the Eyebrow site, there were di¡erences in the rhizosphere soil microbial communities of two
Fig. 4. PCA of FAMEs obtained from the root-associated communities
of canola varieties grown at Eyebrow and Melfort, Saskatchewan, in
1998. Each symbol is the average of four replicates from the two combined ¢eld sites (n = 8). Closed symbols represent conventional varieties:
triangles, Excel replicates; squares, Fairview replicates; diamonds, Hyola
replicates ; circles, 45A71 replicates. Open symbols represent genetically
modi¢ed varieties: triangles, Exceed replicates; squares, Innovator replicates; diamonds, Invigor replicates; circles, Quest replicates. Error bars
represent the standard error of the mean. Principal components marked
with a P value indicate a signi¢cant variety e¡ect as determined by ANOVA.
FEMSEC 1283 14-12-01
K.E. Dun¢eld, J.J. Germida / FEMS Microbiology Ecology 38 (2001) 1^9
Fig. 5. Content of selected fatty acids of the root-associated microbial
communities associated with canola varieties grown at Eyebrow and
Melfort, Saskatchewan, in 1998. Closed bars represent conventional varieties ; open bars represent genetically modi¢ed varieties. GN, Gramnegative biomarker ; GP, Gram-positive biomarker ; F, fungal biomarker. Sum in Feature 3 is a mixture of fatty acids ; 12:0 ALDE?, unknown 10.928, 16:1 ISO I/14 :03OH, 14:03OH/16:1 ISO I.
transgenic varieties of B. napus (Innovator and Invigor) as
well as the conventional B. rapa variety (Fairview). Principal components analysis of FAMEs of soil from the
1999 Denholm and Watrous sites indicated that the fatty
acid composition of the rhizosphere soils associated with
the genetically modi¢ed variety Quest and the conventional variety Hyola was separated from the other plant varieties, along both the principal component 1 and 2 axes
(Fig. 2). Fatty acid analysis of the rhizosphere soil communities associated with three canola varieties, the genetically modi¢ed variety Quest, and two conventional varieties, Excel and Fairview, showed clear separation between
Quest and Excel (Fig. 3).
5
Fig. 6. PCA of FAMEs obtained from the root-associated communities
of canola varieties grown at Watrous, Saskatchewan, in 1999. Each
symbol is the average of four replicates from one ¢eld site (n = 4).
Closed symbols represent conventional varieties: triangles, Excel replicates; squares, Fairview replicates; diamonds, Hyola replicates ; circles,
45A71 replicates. Open symbols represent genetically modi¢ed varieties :
triangles, Exceed replicates ; squares, Innovator replicates ; diamonds, Invigor replicates; circles, Quest replicates. Error bars represent the standard error of the mean. Principal components marked with a P value
indicate a signi¢cant variety e¡ect as determined by ANOVA.
A ¢eld site by plant variety interaction was apparent in
the FAME analysis of the root-associated communities in
both ¢eld years. Principal components analysis of the
root-associated microbial community at the Eyebrow
and Melfort sites in 1998 separated the genetically modi¢ed plant varieties from the conventional plant varieties
along the principal component 1 axis (Fig. 4). In addition,
there were nine fatty acids associated with the root-associated communities at these sites that were signi¢cantly
higher (P 6 0.10) in root-associated microbial communities
of transgenic plants. These included 10:02OH, 12:02OH,
12:03OH, a15:0, 15:1g5c, cy17:0, 18:3g6,9,12c, 19:0g8c
and Sum in Feature 3 (Fig. 5). At the Denholm ¢eld site in
1999, there were no di¡erences in the fatty acids from the
root-associated communities (data not shown), whereas at
Table 3
Substrate utilization rates of GN functional groups for the rhizosphere soil-associated microbial communities from canola varieties grown at Eyebrow
and Melfort in 1998
Variety
Excel
Fairview
Hyola
45A71
Quest
Innovator
Invigor
Exceed
P value
a
b
Substrate utilization rate ((no. wells)1=2 day31 )
Amine/polymers
Carbohydrates
Carboxylic acids
Amino acids
Miscellaneous
0.71
0.68
0.66
0.73
0.71
0.69
0.67
0.71
NSb
1.21aba
1.19ab
1.34bc
1.47c
1.43c
1.16a
1.22ab
1.41c
0.21
1.14
1.21
1.27
1.28
1.25
1.18
1.10
1.18
NS
1.02a
1.09b
1.17d
1.14cd
1.14cd
1.03a
0.99a
1.10bc
0.02
0.82bc
0.88cd
0.87cd
0.93d
0.95d
0.76ab
0.73a
0.91d
0.14
Means in each column followed by the same letter are not signi¢cantly di¡erent at the P value indicated.
No signi¢cant di¡erences between varieties.
FEMSEC 1283 14-12-01
6
K.E. Dun¢eld, J.J. Germida / FEMS Microbiology Ecology 38 (2001) 1^9
Fig. 7. PCA of CLPP obtained from the rhizosphere soil-associated microbial communities of canola (A) and from the root interior-associated
microbial communities of canola (B), grown at Denholm and Watrous,
Saskatchewan, in 1999. Each symbol is the average of four replicates
from the two combined ¢eld sites (n = 8). Closed symbols represent conventional varieties : triangles, Excel replicates; squares, Fairview replicates; diamonds, Hyola replicates ; circles, 45A71 replicates. Open symbols represent genetically modi¢ed varieties : triangles, Exceed replicates;
squares, Innovator replicates; diamonds, Invigor replicates; circles,
Quest replicates. Error bars represent the standard error of the mean.
Principal components marked with a P value indicate a signi¢cant variety e¡ect as determined by ANOVA.
Fig. 8. PCA of CLPP obtained from the rhizosphere soil-associated microbial communities of canola (A) and from the root interior-associated
microbial communities of canola (B), grown at Denholm and Watrous,
Saskatchewan, in 1999. Each symbol is the average of four replicates
from the two combined ¢eld sites (n = 8). Closed symbols represent conventional varieties: triangles, Excel replicates ; squares, Fairview replicates. Open symbols represent genetically modi¢ed varieties : circles,
Quest replicates. Error bars represent the standard error of the mean.
Principal components marked with a P value indicate a signi¢cant variety e¡ect as determined by ANOVA.
Table 4
Substrate utilization rates of GN functional groups for the root interior-associated microbial communities from canola varieties grown at Melfort in
1998
Variety
Excel
Fairview
Hyola
Quest
Innovator
Exceed
P value
a
b
Substrate utilization rate ((no. wells)1=2 day31 )
Amine/polymers
Carbohydrates
Carboxylic acids
Amino acids
Miscellaneous
0.78bca
0.73b
0.78bc
0.50a
0.52a
0.87c
0.04
1.50b
1.62bc
1.54b
1.27a
1.20a
1.72c
0.02
1.34b
1.37b
1.16a
1.08a
1.10a
1.46b
0.09
1.08
1.08
0.95
0.77
1.04
1.11
NSb
0.96bc
1.08d
0.92b
0.82a
1.01cd
1.08d
0.13
Means in each column followed by the same letter are not signi¢cantly di¡erent at the P value indicated.
No signi¢cant di¡erences between varieties.
FEMSEC 1283 14-12-01
K.E. Dun¢eld, J.J. Germida / FEMS Microbiology Ecology 38 (2001) 1^9
the Watrous ¢eld site these communities were separated by
principal components analysis (Fig. 6). Furthermore, at
Watrous, principal components analysis separated communities associated with Quest from Excel and Fairview
(data not shown).
3.3. CLPP of microbial communities
Substrate utilization rates of several functional groups
were di¡erent for communities associated with di¡erent
plant varieties. In the rhizosphere of plants grown at Eyebrow and Melfort in 1998 the carbohydrates, amino acids
and miscellaneous functional guilds were used at a greater
rate by the microbial community associated with Quest
than the one associated with Excel. However, the communities associated with the transgenic varieties Invigor
and Innovator had lower utilization rates of these functional guilds (Table 3). In contrast, the communities associated with the root interior of Quest plants grown at
Melfort in 1998 used the polymer, carbohydrate and carboxylic acid functional guilds at lower rates than the community associated with Excel (Table 4).
There were no di¡erences in substrate utilization rates
of the rhizosphere or root interior communities of Quest
as compared to other plant varieties at Denholm and Watrous in 1999, data not shown. In addition, principal components analysis of the CLPP at these sites indicated that
there were no signi¢cant di¡erences in the ability of the
communities to use carbon substrates in either the rhizosphere or the root interior communities (Figs. 7A and 8A).
However, communities associated with the transgenic variety Quest separated out non-signi¢cantly from the other
varieties in 1998 and 1999. In addition, there were di¡erences (P 6 0.17 and P 6 0.14) in the CLPP of both the
rhizosphere and root interior communities of Quest, Excel
and Fairview (Figs. 7B and 8B).
4. Discussion
Recent studies by Siciliano et al. [10] and Siciliano and
Germida [11] assessed the root interior and rhizosphere
bacterial communities associated with a ¢eld-grown genetically modi¢ed canola variety (Quest) and two conventional varieties (Excel and Parkland). Those studies used
FAME and CLPP analyses, together with isolation and
characterization of representative bacteria, to show that
the root interior and rhizosphere bacterial communities
of the genetically modi¢ed B. napus variety Quest were
di¡erent from a closely related B. napus variety Excel.
Excel supported microbial communities more similar to
the microbial communities supported by a B. rapa variety
Parkland. Our results con¢rm that the root interior and
rhizosphere bacterial community associated with Quest
was di¡erent from Excel and another B. rapa variety Fairview. Moreover, this di¡erence was noted at di¡erent ¢eld
7
sites and during two di¡erent growing seasons. However,
this ¢nding was not generalized for the other genetically
modi¢ed canola varieties we assessed. In fact, in most
cases, Quest seemed to be unique and supported microbial
communities quite di¡erent from the three other genetically modi¢ed and four conventional canola varieties we
assessed. The di¡erences noted for these other varieties
appeared to be signi¢cantly in£uenced by ¢eld sites, i.e.
soil type.
A variety of studies have shown that soil type can in£uence rhizosphere microbial community diversity [23^27].
Our results from this study demonstrated that ¢eld site
in£uenced microbial community composition and interacted with plant varieties in their in£uence on the microbial community. The e¡ect of plant variety on the microbial community at one ¢eld site was sometimes entirely
di¡erent in another ¢eld site. For example, both fatty
acid composition of the root-associated microbial community and substrate utilization rates of the rhizosphere
microbial community were a¡ected by ¢eld site and year.
Further work to assess the interactive e¡ects of ¢eld site
and genetically modi¢ed plants on the rhizosphere and
root-associated microbial community is currently underway.
It is important to note that Quest was the only glyphosate-tolerant variety we studied, as the other genetically
modi¢ed varieties were glufosinate ammonium-tolerant.
These varieties have di¡erent genes that confer resistance
to the plants, these genes products are minor components
of the total protein produced by the genetically modi¢ed
plants; however, it is possible that they may be released as
root exudates. Several authors have speculated that the
reason for the di¡erences in the communities of genetically
modi¢ed plants is due to di¡erences in the root exudate
patterns of these plants [9,10,12]. A recent study examining soybean varieties showed enhanced colonization by
Fusarium spp., a soil-borne pathogen, on glyphosate-tolerant soybean varieties compared to conventional varieties
[28]. It is possible that the exudation of the gene product,
found in both the glyphosate-tolerant soybean variety
tested and Quest, may be one mechanism for the alterations in the community diversity caused by these two plant
varieties. However, characterization of root exudates and
additional studies with a more diverse sample of glyphosate-tolerant plant varieties are required to con¢rm this
idea.
A number of fatty acids were found in signi¢cantly
higher quantities in the root-associated communities of
genetically modi¢ed canola plants. Three of these were
hydroxyl fatty acids, derived primarily from Gram-negative bacteria, especially Pseudomonas sp. [14,29]. Also included was the cyclopropane fatty acid, cy17:0, another
indicator of certain groups of Gram-negative bacteria such
as Chromatium, Legionella, Rhodospirillum and Campylobacter [22,31]. Transgenic root-associated communities
also had higher amounts of the branched fatty acid,
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a15:0, commonly thought to be a marker for Gram-positive bacteria such as Clostridium and Bacillus [22,30], and
18:3g6,9,12c, a fatty acid primarily found in lower fungi
[31]. Siciliano and Germida [11] also found a greater abundance and diversity of Pseudomonas and Flavobacter species in the root interior of the genetically modi¢ed variety,
Quest, compared to conventional varieties, Excel and
Parkland. These results suggest that the genetically modi¢ed varieties have di¡erent organisms within their roots.
The changes in the fatty acid composition found in our
study may be an indicator of increased presence of Gramnegative, Gram-positive and fungal isolates in the root
interior of the transgenic canola varieties. However,
Haack et al. [32] caution that conclusions about community structure derived from analysis of signature fatty acids
may be oversimpli¢ed. In addition, Buyer and Drinkwater
[33] suggest that soil fatty acid composition may change
without accompanying changes in the microbial species
composition, due in part to extraction of fatty acids associated with soil organic matter and plant roots. In order to
minimize the number of fatty acids derived from canola
roots in our pro¢les we have eliminated fatty acids that
were found in a sample of sterile canola roots.
The changes in fatty acid composition coincided with
changes in the ability of microbial communities to utilize
a variety of carbon substrates. A di¡erence in the substrate utilization rate of functional groups is a predictor
of functional diversity [10]. In 1998, we found di¡erences
in the substrate utilization rates of the carbohydrates, amino acids and miscellaneous functional guilds in the microbial communities associated with Quest. CLPP has been
previously used to describe soil microbial communities
[13,33,34]. However, the utility of CLPP has been questioned due to its reliance on studying culturable microorganisms, the ecological relevance of the substrates tested
and metabolic redundancy caused by changes in species
genetic diversity that are not accompanied by changes in
functional diversity as determined by substrate utilization
patterns [35]. The advantage to both FAME and CLPP is
that they are fast and reliable, and useful for describing
the overall soil microbial community structure. Further
studies using methods such as phospholipid fatty acid
analysis or 16S rDNA analysis would be useful in order
to determine if the changes to the FAME and CLPP pro¢les due to soil type and plant variety are present in the
non-culturable microbial community.
The complex interaction between soil microorganisms
and plants is well known, and can be bene¢cial, harmful,
or neutral for the plant [36]. Microorganisms play major
roles in nutrient transformations and element cycling, thus
a¡ecting the availability of these nutrients for plant uptake. In addition, a number of di¡erent bacteria have been
identi¢ed as plant growth promoting rhizobacteria
(PGPRs) for canola [37]. These organisms enhance plant
growth by preventing deleterious e¡ects of phytopathogenic organisms, or they may provide nutrients to plants
or facilitate their uptake [36]. In contrast, many soil organisms cause economically important canola diseases including blackleg (Leptosphaeria maculans (Desmaz.) Ces.
and De Not), seedling blight and root rot (Rhizoctonia
solani Ku«hn), stem rot (Sclerotinia sclerotiorum (Lib.) de
Bary), alternaria blight or blackspot (Alternaria brassicae
(Berk.) Sacc., and A. raphani Groves and Skolko), and
white rust and staghead (Albugo candida (Pers.) Kuntze)
[38]. Any impact that plant variety may have on the dynamics of the rhizosphere and root interior microbial community could have either positive or negative e¡ects on
plant growth and health and in turn ecosystem sustainability. Ongoing studies in our lab are attempting to address the question by assessing whether speci¢c bacterial
populations associated with genetically modi¢ed canola
roots are potential deleterious rhizobacteria, PGPR or
biocontrol agents.
Acknowledgements
This research was supported by the Natural Sciences
and Engineering Research Council of Canada and the
Saskatchewan Wheat Pool. K.E.D. was supported by a
Canadian Wheat Board Fellowship. The technical help
of Sonia Cyrenne, Bobbi Helgason, Amy Misko, Julie
Roy and Arlette Seib is gratefully appreciated. Contribution no. R869, Saskatchewan Center for Soil Research.
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