Influence of Glomus etunicatum/Zea mays mycorrhiza on atrazine
degradation, soil phosphatase and dehydrogenase activities, and soil
microbial community structure
Huang, H. L., Zhang, S. Z., Wu, N. Y., Luo, L., & Christie, P. (2009). Influence of Glomus etunicatum/Zea mays
mycorrhiza on atrazine degradation, soil phosphatase and dehydrogenase activities, and soil microbial
community structure.
Soil Biology and Biochemistry
,
41
(4), 726-734. https://doi.org/10.1016/j.soilbio.2009.01.009
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Download date:09. Aug. 2022
Influence of Glomus etunicatum/Zea mays mycorrhiza on atrazine
degradation, soil phosphatase and dehydrogenase activities,
and soil microbial community structure
Honglin Huang
a
, Shuzhen Zhang
a
,
*
, Naiying Wu
a
, Lei Luo
a
, Peter Christie
b
a
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, PO Box 2871,
Beijing 100085, China
b
Agri-Environment Branch, Agri-Food and Biosciences Institute, Newforge Lane, Belfast, BT9 5PX, UK
article info
Article history:
Received 18 June 2008
Received in revised form
24 December 2008
Accepted 11 January 2009
Available online 5 February 2009
Keywords:
Arbuscular mycorrhiza
Atrazine dissipation
Maize
PLFA profiles
Soil enzymes
abstract
The effects of an arbuscular mycorrhizal (AM) fungus (Glomu s etunicatum) on atrazine dissipation, soil
phosphatase and dehydrogenase activities and soil microbial community structure were investigated. A
compartmented side-arm (‘cross-pot’) system was used for plant cultivation. Maize was cultivated in the
main root compartment and atrazine-contaminated soil was added to the side-arms and between them
650 or 37
m
m nylon mesh was inserted which allowed mycorrhizal roots or extraradical mycelium to
access atrazine in soil in the side-arms. Mycorrhizal roots and extraradical mycelium increased the
degradation of atrazine in soil and modified the soil enzyme activities and total soil phospholipid fatty
acids (PLFAs). Atrazine declined more and there was greater stimulation of phosphatase and dehydro-
genase activities and total PLFAs in soil in the extraradical mycelium compartment than in the mycor-
rhizal root compartment when the atrazine addition rate to soil was 5.0 mg kg
1
. Mycelium had a more
important influence than mycorrhizal roots on atrazine degradation. However, when the atrazine
addition rate was 50.0 mg kg
1
, atrazine declined more in the mycorrhizal root compartment than in the
extraradical mycelium compartment, perhaps due to inhibition of bacterial activity and higher toxicity to
AM mycelium by atrazine at higher concentration. Soil PLFA profiles indicated that the AM fungus
exerted a pronounced effect on soil microbial community structure.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Arbuscular mycorrhizal (AM) fungi are among the most ubiq-
uitous soil microorganisms and they form mutualistic associations
with 80–90% of vascular plant species in ecosystems throughout
the world (Harrison, 1997; Smith and Read, 1997). Most previous
studies have found that AM fungi have positive effects on the
dissipation of organic contaminants such as atrazine (Huang et al.,
20 07), PAHs (Joner et al., 2001; Joner and Leyval, 20 03; Xu et al.,
20 06; Wu et al., 2008a), DDT (Wu et al., 2008b) and weathered p,p-
DDE in soils (White et al., 2006), although no impact of AM fungi on
PAH dissipation was observed by Binet et al. (2000) and a depres-
sion in PAH dissipation in the presence of ectomycorrhizas (Joner
et al., 2006; Genney et al., 2004) has also been reported. AM fungi
may therefore play a critical role in the degradation of organic
contaminants in soils.
The mechanisms involved in interactions between AM fungi and
organic contaminants in soil remain unclear. It is reasonable to
expect that soil microbial activity enhanced and soil microbial
communities modified by AM fungi play a key role in the degra-
dation of organic contaminants. Once arbuscular mycorrhizal
association has developed, AM hyphae influence the surrounding
soil which has been termed the mycorrhizosphere (Linderman,
1988), resulting in the development of distinct microbial commu-
nities in the rhizosphere and bulk soil (Andrade et al., 1997;
So
¨
derberg et al., 2002; Cheng and Baumgartner, 2006; Purin and
Rillig, 2008). Phospholipid fatty acid (PLFA) analysis has revealed an
important qualitative difference in microbial community structure
in mycorrhizosphere soil as affected by AM fungi in PAH-spiked soil
(Joner et al., 2001). Furthermore, the AM fungal hyphosphere, the
zone of soil affected by the extraradical hyphae (Marschner, 1995),
may support a distinct microbial community within the mycor-
rhizosphere and exert effects on degradation of organic compounds
*
Corresponding author. Tel.: þ86 10 62849683; fax: þ86 10 62923563.
E-mail address: szzhang@rcees.ac.cn (S. Zhang).
Contents lists available at ScienceDirect
Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
0038-0717/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2009.01.009
Soil Biology & Biochemistry 41 (2009) 726–734
in soil. There have been no published reports on attempts to
distinguish the specific role of extraradical hyphae as well as to
separate the roles of arbuscular mycorrhizal roots, extraradical
mycelium and non-mycorrhizal roots on the dissipation of organic
contaminants in soil.
Studies have indicated that AM fungi can increase the activities
of soil enzymes such as phosphatase and dehydrogenase (Dodd
et al., 1987; Kothari et al., 1990; Va
´
zquez et al., 2000). Dehydroge-
nase, a soil oxidoreductase, is an intracellular enzyme catalyzing
oxidoreduction reactions of organic compounds. Several studies
have demonstrated that the dehydrogenase enzyme activity of
microorganisms is one of the most sensitive parameters available
for toxicity evaluation and alkaline phosphatase is involved in the
process of phosphate acquisition in mycorrhizal plants (Gianinazzi
et al., 1992). Both of these enzymes are considered to play key
metabolic roles in mycorrhizal function (Vivas et al., 2003; Lo
´
pez-
Gutie
´
rrez et al., 2004). However, there have been few reports that
deal with the effects of AM inoculation on soil enzymes and
microbial community structure in soils containing organic
contaminants.
Atrazine is one of the agricultural herbicides most frequently
detected in soils and waters (Kazemi et al., 2008). It has been
reported that in rhizosphere soil atrazine degradation is associated
with higher dehydrogenase (Seibert et al., 1991; Singh et al., 2004)
and phosphatase activities (Perucei et al., 1988; Bielinska and Pra-
nagal, 2007), and atrazine can influence the populations of certain
microbial groups (Ros et al., 2006). We observed in our previous
studies that atrazine dissipation in soil was enhanced by AM
inoculation (Huang et al., 2006). We therefore hypothesized that
increased dissipation of atrazine in soil might contribute to: (1)
specific effects of mycorrhizal roots and extraradical mycelium on
atrazine dissipation; and/or (2) modification of the effects on soil
enzyme activities and microbial community structure resulting
from mycorrhizal inoculation. The present study was therefore
carried out to identify the specific effects of mycorrhizal roots and
extraradical mycelium on dissipation of atrazine in soil using
a compartmented cultivation system. Phosphatase and dehydro-
genase activities and microbial community structure in soils under
the influence of mycorrhizal roots and extraradical mycelium were
examined in order to elucidate the interaction between atrazine
degradation and soil enzymes and microbial communities.
2. Materials and methods
2.1. Host plants and AM fungus
Seeds of maize (Zea mays L. cv. Nongda 108) were surface ster-
ilized in a 10% (v/v) solution of hydrogen peroxide for 10 min,
rinsed with sterile distilled water, and soaked in a 3 mM solution of
Ca(NO
3
)
2
for 4 h in the dark. Then they were germinated on moist
filter paper for about 48 h. Seedlings of uniform size were selected
and were ready for sowing.
Original inoculum of the AM fungus Glomus etunicatum (BGC
USA01), which was kindly provided by the Institute of Plant
Nutrition and Fertilizers, Beijing Academy of Agronomy and
Forestry, was propagated in pot culture on sorghum for 10 weeks in
a zeolite–sand mixture in a greenhouse. The inoculum, a mixture of
spores, mycelium, sand and root fragments, was air-dried and
sieved (<2 mm). For the extraction of spores and sporocarps, 20 mL
of inoculum were treated by the wet sieving and decanting method
(Gerdemann and Nicolson, 1963). The resulting material was
centrifuged with 80% saccharose (Walker et al., 1982). Quantifica-
tion was carried out in 9-cm-diameter Petri dishes with gridlines of
1 cm per side under a stereoscopic microscope at 50 magnifica-
tion. The inoculum contained about 2200 spores per 20 mL.
2.2. Compartmented cultivation system
The compartmented cultivation system (‘cross-pots’) (Chen
et al., 2007; Joner and Leyval, 1997; Joner et al., 2000 ) comprised
a vertical root compartment (PVC tube, 6.0 cm diameter 25 cm
long) for plant growth and two symmetrical horizontal side-arm
compartments (PVC tube, 4.5 cm diameter 9.0 cm long). The
side-arm compartments were separated from the main root
compartment by 37- or 650-
m
m nylon mesh, the fine mesh to
prevent the entry of roots and only allow free passage of mycelium
into the side compartment and the coarse mesh to allow the
passage of mycorrhiza together with any extraradical mycelium.
2.3. Experimental design
The experiment was a 2 2 3 factorial design with mycor-
rhizal colonization (þM/M), coarse mesh/fine mesh separating
the main root compartment from the side-arm compartment, and
atrazine-free and two addition levels of atrazine (5.0 and 50.0 mg
kg
1
) in the side-arm compartment soil. The two atrazine addition
levels were selected according to the range of contamination levels
in soil (0.017 mg kg
1
– 482.7 mg kg
1
) reported by Grigg et al.
(1997) as well as its limited phyto-availability because only the
roots penetrating through the mesh would be exposed to atrazine
in the side-arm compartment. Four replicate pots of each treatment
were set up.
2.4. Growth medium
A brown soil (Alfisol containing 1.35% organic matter) was
collected from the top 15 cm of the soil profile in an experimental
field at Beijing Academy of Agriculture and Forestry. The soil
was air-dried, ground, and passed through a 2-mm nylon sieve.
A 1:1 (v/v) mixture of sand (1–2 mm) and soil was used as the
growth medium in order to enhance the permeability of the soil
(Biermann and Linderman, 1983) and the mixture was sterilized by
g
-radiation (10 kGy, 10 MeV
g
-rays) to inactivate the native AM
fungi. The soil mixture (henceforth referred to as the soil) had a pH
of 7.89 (1:2.5 soil/water) and a 0.5 M NaHCO
3
-extractable P content
of 9.98 mg kg
1
. P was added to the soil at a rate of 50.0 mg kg
1
and portions of the soil were then artificially dosed with atrazine.
HPLC grade atrazine (Sigma Chemical Co.) was dissolved in reagent
grade acetone and added to portions of the soil at concentrations of
0, 5.0 and 50.0 mg kg
1
(dry matter basis). The soil mixtures were
then allowed to dry in a fume hood until the acetone had volatilized
completely and were shaken, homogenized, and incubated for
4 weeks to allow the contaminant to equilibrate.
2.5. Pot experiment
Each side-arm of the cross-pot was filled with 135 g sterile
atrazine-contaminated soil and then with 45 g atrazine-free soil to
establish a buffer layer to minimize possible movement of atrazine
from the side-arm compartment to the main root compartment.
Deionized water was added to the side-arm compartment soil to
adjust the moisture content to 60% of water holding capacity
(WHC). The side-arm tubes were then sealed with nylon mesh and
allowed to stand overnight. Soil amended with basal nutrients was
placed in the main root compartment to support plant growth. For
mycorrhizal treatments, 60 g inoculum was thoroughly mixed with
850 g soil and each pot contained about 6600 spores. Above the
side-arm compartment a further 10 g inoculum was placed on the
soil. The side-arm compartments were fixed tightly to the main
root compartment which was then sealed at the bottom with
a plastic bag. Non-mycorrhizal treatments received the same
H. Huang et al. / Soil Biology & Biochemistry 41 (20 09) 726–734 727
amount of sterilized inoculum (70 g) together with a 2-mL aliquot
of a filtrate (<20
m
m) of the AM inoculum to provide a general
microbial population free of AM propagules. Four pre-geminated
maize seeds were sown in each pot and five days after emergence
the seedlings were thinned to two of uniform size. All pots were
lined with polyethylene bags to avoid cross-contamination and
water loss and the surface of each pot was covered with a black
plastic bag to minimize algal growth. The experiment was con-
ducted in a controlled-environment growth chamber that main-
tained a daily 14-h light periodat a light intensityof 250
m
mol m
2
s
1
provided by supplementary illumination. The day/night tempera-
ture regime was 25/20
C. The relative humidity was maintained at
70%. The plants grew for 8 weeks. Deionized water was added as
required to maintain soil moisture content at 60–70% WHC.
2.6. Harvest and analysis
2.6.1. Sample preparation
At harvest the side-arm compartments were separated from the
main root compartment and the buffer soil layer was discarded. Soil
samples were collected from the main root and the side-arm
compartments, freeze-dried and stored at 4
C. Soil PLFA, dehy-
drogenase activity and phosphatase activity were determined
within 1 month. Plant shoots and roots were harvested separately.
Root samples were first carefully washed with tap water to remove
any adhering soil particles. Then the shoot and root samples were
thoroughly rinsed with distilled water, wiped with tissue paper,
and immediately weighed. A subsample of fresh roots was taken
from each treatment for the determination of the proportion of root
length colonized by the AM fungus. The remaining plant samples
were freeze-dried, weighed, and stored at 4
C.
2.6.2. Determination of root colonization
The proportion of root length colonized by the fungus was
estimated by randomly taking a subsample of 1.0 g of fresh roots
and cutting them into 0.5–1.0-cm pieces. Root segments were
cleared in 10% KOH for 10 min at 90
C in a water bath, rinsed in
water, and then stained with 0.1% Trypan Blue for 3–5 min at 90
C
in a water bath. Percentage root length colonization and total root
length were determined by the gridline intersect method (Gio-
vannetti and Mosse, 1980).
2.6.3. Dehydrogenase activity
Twenty grams of freeze-dried soil were thoroughly mixed with
0.2 g CaCO
3
and three replicate samples of 5.0 g soil were placed in
three test tubes. Three milliliters of 1% 2,3,5-triphenyltetrazolium
chloride (TTC) and 2.0 mL of distilled water were added to each
tube. Samples were then incubated at 37
C for 24 h with constant
shaking at medium speed. Afterward, 10 mL of methanol were
added to each tube and the samples were vortexed. The soil
suspensions were filtered through glass funnels plugged with
absorbent cotton. The filtrates were diluted with methanol to
100 mL volume and the intensity of the reddish color was
measured at 485 nm using a spectrophotometer. Dehydrogenase
activities in the samples were calculated by using calibration
graphs prepared from 500, 1000, 1500 and 2000 mg triphenyl for-
mazan (TPF) per 100 mL standards. Results are presented as mg TPF
kg
1
soil.
2.6.4. Phosphatase activity
The assay of phosphatase activity was determined by measuring
the p-nitrophenol released by phosphatase activity when soil was
incubated with buffered (pH 9.4) sodium p-nitrophenyl phosphate
solution and toluene at 37
C for 24 h using the modified method
proposed by Tabatabai and Bremner (1969). The p-nitrophenol
formed was determined using a spectrophotometer at 660 nm.
Controls were prepared in the same way.
2.6.5. PLFA analysis
Fine root fragments were teased out and then the soil samples
were sieved (2.0 mm mesh). Five grams of freeze-dried fresh soil
were transferred to a test tube for lipid extraction according to the
method of Frostegård et al. (1993). The samples were extracted with
a one-phase mixture of chloroform, methanol and citrate buffer
(0.15 M, pH 4) (1:2:0.8 v/v/v). The phases were separated after
adding 3.0 mL of chloroform and 3.0 mL of the buffer. The lower
phase was collected, dried and used in lipid fractionation. The dried
lipid extract was dissolved in 100 mL chloroform and fractionated
on pre-packed columns with 100 mg silicic acid (Bond Elut
Extraction Cartridges, Varian, USA). Phospholipid fractions were
collected and methyl nonadecanoate was added as the internal
standard and then transesterified by a mild alkaline methanolysis
(Dowling et al., 1986). Blanks without soil were subjected to the
same lipid extraction protocol in order to detect possible contami-
nation errors. The fatty acid methyl esters (FAMEs) 16:1cis11 are
often used as a biomarker for AM fungi in roots and soils (Olsson
and Johansen, 2000; Olsson, 1999), i-15:0, a-15:0, i-16:0, i-17:0, and
a-17:0 for Gram-positive bacteria (O’Leary and Wilkinson, 1988),
18:1
u
7, cy-17:0, and cy-19:0 for Gram-negative bacteria (Wilkin-
son, 1988), and 18:2
u
6,9 for fungi (Federle, 1986).
2.6.6. Atrazine analysis
Five grams of the soil samples (dry matter basis) were extracted
twice with 50.0 mL of 80% aqueous methanol by shaking the
suspension on a reciprocating shaker for 48 h. The extracts were
filtered and combined and then extracted successively with 50 mL
of petroleum ether/dichloromethane (65:35, v/v) three times.
Supernatants were passed through anhydrous Na
2
SO
4
columns and
collected. The volumes of the eluates were reduced to 1–2 mL. Then
they were solvated with 30 mL of petroleum ether and re-extracted
three times with 20 mL of acetonitrile. The acetonitrile fractions
were combined, concentrated, and evaporated off. The residues
were solvated with petroleum ether and cleaned with Florisil
columns. The concentrations of atrazine in extracts were analyzed
with an Agilent 6890 gas chromatograph equipped with a detector
of NPD using a HP-5 capillary column (0.32 mm 30 m, 0.25
m
m
film thickness). The column oven was programmed from an initial
temperature of 70
C for 2 min to 220
C at a rate of 20
C min
1
,
held for 1 min, and then ramped at a rate of 4
C min
1
to 240
C
with a final hold time of 10 min. The detector and injector were
maintained at 300 and 250
C, respectively. The injector was in the
splitless mode for nitrogen–phosphorus detection. To determine
analytical recovery, aliquots of soil were spiked with pesticides.
Recoveries ranged from 85 to 90% (RSD ¼ 6.8%, n ¼ 5).
2.7. Statistical analysis
The data were analyzed by three-way analysis of variance using
the SPSS version 11.5 software package. Means and standard errors
of four replicates were calculated. The data were examined for the
significance of AM treatment, atrazine application and coarse
mesh/fine mesh separation between the main root compartment
and the side-arm compartment. A 95% confidence limit (P < 0.05)
was chosen to indicate differences between samples and least
significant differences (LSD) were calculated when samples were
significantly different. The PLFA data (24 distinct PLFAs identified)
were subjected to principal component analysis (PCA) to examine
patterns of compartment and inoculation combinations. PCA scores
were subjected to three-way analysis of variance (ANOVA) to test
the significance of the effects of AM treatment, atrazine application
H. Huang et al. / Soil Biology & Biochemistry 41 (20 09) 726–734728
and mesh separation and their interactions on microbial commu-
nity structure.
3. Results
3.1. Colonization of roots by Glomus etunicatum and plant
biomass
Roots of inoculated plants were extensively colonized by Glomus
etunicatum but non-inoculated controls remained non-mycorrhizal
(Table 1). The percentage of root length colonized in the main root
compartment ranged from 45 to 64% across all the inoculated
treatments. Rate of root colonization was higher when the side-arm
compartment was separated from the main root compartment by
the 37-
m
m nylon mesh than by the 650-
m
m nylon mesh (P < 0.05)
and decreased with increasing atrazine application rate in the side-
arm compartment. Atrazine application did not significantly affect
the dry weights of shoots and roots (P > 0.05). Colonization
significantly increased the root dry weight ( P < 0.001) but did not
affect the shoot dry weight.
3.2. Residual atrazine in soil and accumulation in maize
After plant harvest the atrazine concentration in the side-arm
compartment soils decreased by 26.4 to 77.3% compared with the
initial concentrations (Table 1). Inoculation significantly decreased
the amount of residual atrazine in the side-arm soil (P < 0.05). The
residual atrazine concentration decreased by 22.7 and 36.1% in the
mycorrhizal root compartment soil and in the mycelium compart-
ment soil respectively when the application rate of atrazine to soil
was 5.0 mg kg
1
, with corresponding values of 53.4 and 46.2%
when the atrazine was applied at 50.0 mg kg
1
. No atrazine was
found in the main root compartment soil when the application rate
was 5.0 mg kg
1
and only a very small amount was detected at the
application rate of 50.0 mg kg
1
(data not shown). No atrazine was
detected in either the roots or shoots of maize.
3.3. Soil phosphatase activity
Phosphatase activities in soils in both the side-arm and the main
root compartments were enhanced by AM inoculation (P < 0.05)
(Fig. 1). Compared with the non-mycorrhizal control, the soil
phosphatase activity in the inoculation treatment increased by
27.1–71.1% and 8–111% in the main root compartment with mesh
separation of 650 and 37
m
m(P < 0.05), respectively. Soil phos-
phatase activity increased by 35.8–70.1% and 90.2–179% in the
mycorrhizal and mycelium side-arm compartments (P < 0.05),
respectively. Application of atrazine activated the phosphatase
activities in the side-arm compartment soil. No consistent effect of
atrazine application on phosphatase activity in the main root
compartment soil was observed.
3.4. Soil dehydrogenase activity
When no atrazine was applied, AM inoculation had no signifi-
cant effect on dehydrogenase activity in the control main root
compartment soil (P > 0.05; Fig. 2). When atrazine was applied at
5.0 mg kg
1
the dehydrogenase activity in the main root
compartment soil decreased by 17.0 and 19.4% by inoculation in the
coarse and fine mesh treatments (P < 0.05), respectively. When
atrazine was applied at 50.0 mg kg
1
the dehydrogenase activities
in the main root compartment soil increased by 20.0 and 12.9% by
inoculation in the coarse and fine mesh treatments (P < 0.05).
Dehydrogenase activity was higher in soil in the mycelium
compartment than in the mycorrhizal root compartment when
atrazine was applied at 5.0 mg kg
1
(P < 0.05), the opposite trend
to that when the atrazine application rate was 50.0 mg kg
1
(P < 0.05). Dehydrogenase activity in the side-arm compartment
soil was decreased by 6.0–22.9% comparing the treatment with
atrazine application to the atrazine-free control treatment
including both mesh treatments and atrazine application levels.
Table 1
Biomass of maize (dry matter basis), mycorrhizal colonization rates of roots and residual atrazine in the side-arm compartment soil in association with Glomus etunicatum as
influenced by different sizes of mesh and various concentrations of atrazine added to the side-arm compartment soil.
Mycorrhiza inoculation treatment Type of nylon mesh Added ATR
a
(mg kg
1
) Dry matter (g) Root colonization (%) Residual ATR (mg kg
1
)
Roots Shoots
Non- inoculated Coarse mesh 0 1.26 (0.24)cde 7.50 (0.59)a 0 0
5 1.22 (0.17)de 7.61 (0.86)a 0 3.15 (0.14)f
50 1.18 (0.26)e 7.37 (0.68)a 0 21.18 (1.70)b
Fine mesh 0 1.38 (0.07)bcde 5.67 (1.18)b 0 0
5 1.46 (0.32)abcde 6.86 (1.02)a 0 3.68 (0.15)e
50 1.23 (0.16)de 7.41 (0.21)a 0 26.59 (2.12)a
Inoculated Coarse mesh 0 1.65 (0.14)abc 7.31 (0.30)a 64 (3)ab 0
5 1.72 (0.25)ab 7.02 (0.02) 56 (2)cd 2.67 (0.35)g
50 1.58 (0.16)abcde 7.52 (0.14)a 45 (5)e 11.34 (0.91)d
Fine mesh 0 1.81 (0.34)a 7.24 (0.43)a 67 (5)a 0
5 1.62 (0.14)abcd 6.78 (0.44)a 58 (3)bc 2.31 (0.10)h
50 1.49 (0.10)abcde 6.90 (0.25)a 48 (3)de 18.06 (1.54)c
Significance of
b
Inoculation (I) *** NS *** ***
Mesh type (M) **** *
ATR addition (A) NS NS *** *
I M *** ** ** **
I A ** NS *** *
M A NS * * *
I M A ** NS *** **
Values denote standard error of the mean (SEM) in parentheses; n ¼ 4. Means within each column with the same letter are not significantly different at the 5% level. The root
colonization data of non-inoculated controls were excluded from the statistical analysis.
a
ATR, atrazine.
b
By analysis of variance, ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant.
H. Huang et al. / Soil Biology & Biochemistry 41 (20 09) 726–734 729
