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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambient. 22 (1) 39-47, 2006
INFLUENCE OF TWO POLYCYCLIC AROMATIC HYDROCARBONS ON SPORE
GERMINATION, AND PHYTOREMEDIATION POTENTIAL OF
Gigaspora margarita
-
Echynochloa polystachya
SYMBIOSIS IN BENZO[
a
]PYRENE-POLLUTED SUBSTRATE
Alejandro ALARCÓN
1,2
, Julián DELGADILLO-MARTÍNEZ
1
, Alicia FRANCO-RAMÍREZ
1
,
Frederick T. DAVIES Jr.
2
and Ronald FERRERA-CERRATO
1
1
Microbiología de Suelos. Colegio de Postgraduados. Montecillo. Carretera México-Texcoco km 36.5, Montecillo
56230, Estado de México. México
2
Department of Horticultural Sciences. Texas A&M University. College Station 77843-2133, Texas. EUA
(Recibido enero 2006, aceptado abril 2006)
Key words: arbuscular mycorrhizal fungi, PAH, rhizosphere
ABSTRACT
Arbuscular mycorrhizal fungi (AMF) are ubiquitous microorganisms that occur in
contaminated soils. However, little is known about the responses of AMF with or-
ganic contaminants such as polycyclic aromatic hydrocarbons (PAH). The first ob-
jective of this study was to evaluate the influence of two PAH on spore germination
of
Gigaspora margarita
Becker & Hall. Water-agar plates were contaminated with
phenanthrene (PHE) and benzo[
a
]pyrene (BaP) at several concentrations: 0, 25 (0.1
mM BaP & 0.15 PHE), 50 (0.2 mM BaP & 0.3 mM PHE), 75 (0.3 mM BaP & 0.45
mM PHE), and 100 μg mL
-1
(0.4 mM BaP & 0.6 mM PHE), respectively. The second
objective consisted on the evaluation of the responses of the symbiosis between
G.
margarita
and
Echinochloa polystachya
(H.B.K.) Hitch. to increased concentra-
tions of BaP (0, 25 (0.1 mM), 50 (0.2 mM), 75 (0.3 mM), and 100 mg kg
-1
(0.4 mM)
under plant growth chamber conditions. Spore germination and hyphal length were
drastically reduced by PHE. Reduction of spore germination was higher than 90% in
presence of PHE. In presence of BaP, spore germination reduction was 42.8% when
exposed at 100 μg mL
-1
(0.4 mM).
Spores that germinated in presence of 75 (0.3
mM) and 100 (0.4 mM) μg BaP mL
-1
had greater hyphal elongation. BaP did not affect
shoot dry mass of non-mycorrhizal or mycorrhizal
E. polystachya
. Mycorrhizal plants
showed higher dehydrogenase activity in the rhizosphere soil at 0, 0.2 and 0.3 mM
BaP, but reduced root polyphenol oxidase activity at 0 and 0.1 and higher at 0.3 mM
BaP than non-mycorhizal plants. Dissipation of BaP was higher in non-mycorrhizal
plants than mycorrhizal plants.
Echinochloa polystachya
showed an intrinsic capa-
bility on dissipating PAH from its rhizosphere.
Palabras clave: hongos micorrízicos arbusculares, HAP, rizósfera
RESUMEN
Los hongos micorrízicos arbusculares (HMA) son microorganismos cosmopolitas
que se pueden encontrar en suelos contaminados. No obstante, pocos estudios se han
enfocado a la evaluación de las respuestas de HMA ante contaminantes orgánicos
A. Alarcón
et al.
40
como los hidrocarburos poliaromáticos (HAP). El primer objetivo de este trabajo
consideró la evaluación del efecto de dos HAP sobre la germinación de esporas de
Gigaspora margarita
Becker & Hall. Placas con agar-agua fueron contaminadas con
fenantreno (PHE) o benzo[
a
]pireno (BaP) en diferentes concentraciones:
0, 25 (0.1
mM BaP y 0.15 PHE), 50 (0.2 mM BaP y 0.3 mM PHE), 75 (0.3 mM BaP y 0.45 mM
PHE), y 100 μg mL
-1
(0.4 mM BaP y 0.6 mM PHE), respectivamente. El segundo
objetivo consistió en la evaluación de las respuestas de la simbiosis entre
G. marga-
rita
y
Echinochloa polystachya
(H.B.K.) Hitch. ante la presencia de diferentes con-
centraciones de BaP (0, 25 (0.1 mM), 50 (0.2 mM), 75 (0.3 mM), y 100 mg kg
-1
(0.4
mM), bajo condiciones de cámara de crecimiento. La germinación de esporas y lon-
gitud hifal fueron significativamente inhibidas por PHE, el cual produjo una disminu-
ción del 92%. En el caso de BaP, la germinación de esporas disminuyo en 42.8% ante
100 μg mL
-1
(0.4 mM). Las esporas que germinaron en presencia de 75 (0.3 mM) y
100 (0.4 mM) μg BaP mL
-1
, tuvieron mayor elongación hifal. La presencia del BaP no
produjo efectos negativos en el peso seco de la parte aérea de
E. polystachya
con la
inoculación o no del HMA. Las rizosferas de plantas micorrizadas presentaron ma-
yor actividad deshidrogenasa ante 0, 0.2 y 0.3 mM BaP, pero reducida actividad
polifenoloxidasa en la raíz ante 0 y 0.1, y mayor ante 0.3 mM BaP,
en comparación
con plantas no inoculadas. La disipación del BaP de la rizosfera fue mayor en plantas
no inoculadas en comparación con plantas micorrizadas.
Echinochloa polystachya
al parecer tiene una capacidad intrínseca para disipar BaP de su rizosfera.
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAH) are ubiq-
uitous environmental pollutants that have toxic, mu-
tagenic, and carcinogenic properties (Sutherland
et al
.
1995). To reduce human health risks for these com-
pounds some innovative bioremediation techniques
have been developed using phytoremediation
(Reynolds and Skipper 2005). However, the extent
of the dissipation/degradation of PAH depends on their
physical and chemical properties. Benzo[
a
]pyrene is
one of the most recalcitrant PAH due to its complex
molecular structure, however its biodegradation has
been enhanced by free aerobic microorganisms
(Juhasz and Naidu 2000) and microorganisms inhab-
iting the rhizosphere of selected plant species
(Newman and Reynolds 2004). High concentrations
of PAH in petroleum-contaminated soils limit plant
growth and survival by affecting water and nutrient
uptake to the roots (De Jong 1980, Merkl
et al
. 2005).
Studies have demonstrated that these stressful condi-
tions may be alleviated by the association of plants
with certain beneficial rhizosphere microorganisms that
contribute to nutrient availability as well as to im-
prove phytoremediation of soil pollutants (Sarand
et
al
. 1998, Siciliano and Germida 1998). Microbial as-
sociations in the rhizosphere provide higher plant tol-
erance to inorganic and organic contaminants and con-
tribute to detoxify and degrade soil contaminants
(Johansson
et al
. 2004, Barea
et al
. 2005). Arbuscular
mycorrhizal fungi (AMF) are ubiquitous microorgan-
isms in most ecosystems and form a mutually benefi-
cial symbiosis with the roots of approximately 80%
of all extant plant species (Smith and Read 1997).
Benefits of AMF to plants are often related with in-
creased nutrition, water uptake, and enhanced toler-
ance and survivability to cultural and environmental
stresses (Jeffries
et al
. 2003).
Phytoremediation of heavy metals can be en-
hanced by AMF (Meharg and Cairney 2000, Davies
et al
. 2001). However, some studies have demon-
strated that the presence of heavy metals and petro-
leum hydrocarbons in soils are detrimental to the for-
mation/expression of AMF associations with the roots
of higher plants (Davies
et al
. 2001, Cabello 1997,
Leyval and Binet 1998). Some species of AMF such
as
Gigaspora margarita
Becker & Hall and
Acaulospora delicata
Walker, Pfeiffer & Bloss have
been detected in soils chronically contaminated by
petroleum in Veracruz, México (Varela
et al
. 2000).
Arbuscular mycorrhizal fungal species isolated from
petroleum-contaminated soils have been shown to be
more efficient in promoting plant growth and nutrient
uptake than introduced fungal species (Cabello 1999).
In addition, plant survival and growth has been en-
hanced by the presence of AMF in PAH-contami-
nated soil (Leyval and Binet 1998). The establish-
ment of AMF in the root system of some plants may
represent an important biological process for the dis-
sipation/degradation or alleviation of PAH toxicity
(Binet
et al
. 2000, Joner
et al
. 2003). However, the
role of AMF on the performance of plants utilized in
RESPONSE OF
Gigaspora
margarita
SPORE AND SYMBIOSIS TO BENZO[
a
]PYRENE
41
phytoremediation of petroleum hydrocarbons has not
been well understood.
The objectives of this study were to evaluate the
response of
Gigaspora margarita
spores to two PAH
compounds (phenanthrene and benzo[
a
]pyrene) and
to determine the response of
Gigaspora margarita
-
Echinochloa polystachya
symbiosis to the presence
of benzo[
a
]pyrene in the rhizosphere.
MATERIAL AND METHODS
Spore germination response to phenanthrene and
benzo[
a
]pyrene
Spores of
Gigaspora margarita
were collected from
the rhizosphere of
Sorghum vulgare
L. trap cultures,
and surface sterilized as described by Becard and Fortin
(1988). Spores were maintained in microtubes at 4 °C
during five days.
Four solutions of phenanthrene (PHE, 96% purity;
Sigma-Aldrich
®
P-2558) using acetone as solvent, were
prepared in order to obtain the following concentrations
(μg mL
-1
): 25 (0.15 mM), 50 (0.30 mM), 75 (0.45 mM),
and 100 (0.60 mM). Four solutions of benzo[
a
]pyrene
(BaP, 97% purity; Sigma-Aldrich
®
B-1760) were also
prepared to obtain the following concentrations (μg mL
-
1
): 25 (0.1 mM), 50 (0.2 mM), 75 (0.3 mM), and 100
(0.4 mM). Each contaminant solution was sprayed on
to individual petri dishes containing water-agar (1%).
Non-contaminated petri dishes were used as a control
treatment. After 48 hours, spores were placed on the
water-agar surface. Each plate contained 20 spores, and
four plates (n=4) were utilized for each treatment of
contamination with either PHE or BaP.
Spores were incubated at 24 °C in the dark for 25
days. The number of germinated spores was evalu-
ated under dissecting microscope. Germinated spores
were gently extracted from the water-agar and set
them on glass slides to evaluate the hyphal total length
in each spore by using a light microscope Reichert
Microstar IV, Model 410, and American Optical 10X
micrometer.
Data were analyzed as a two-ways factorial experi-
ment through the analysis of variance procedure, and
Tukey´s test (a=0.05%) for means separation and mul-
tiple regression model were performed (SAS Institute,
2000).
Phytoremediation potential of
Gigaspora
margarita
-
Echinochloa polystachya
symbiosis in a
benzo[
a
]pyrene-polluted substrate
River sand was washed and sterilized by three hours
of exposition to steam pressure (124.1 kPa, 121 °C).
Sand was contaminated with benzo[
a
]pyrene (Sigma-
Aldrich
®
, 97% purity) at doses from 0, 0.1, 0.2, 0.3 to
0.4 mM (0, 25, 50, 75, and 100 mg kg
-1
, respectively).
Contaminant solutions were prepared by the dilution
of BaP in 50 mL of acetone. Then, the solution was
applied directly to the sand and manually mixed until
acetone evaporation. Contaminated sand with its re-
spective concentration of BaP was kept under labora-
tory conditions during 24 h to allow the solvent evapo-
ration. Glass-dark containers were filled up with 700 g
of sand and watered with 100 mL of sterilized (124.1
kPa, 121 °C, 20 minutes) Long Asthon Nutrient Solu-
tion (LANS) modified to supply 11 μg mL
-1
of phos-
phorus to avoid interference on the symbiosis estab-
lishment.
Rooted cuttings of
Echinochloa polystachya
(H.B.K.) Hitchcock (Poaceae) were transplanted to the
containers and inoculated with 500 spores of
Gigaspora
margarita
(AMF-plant). In addition, non-mycorrhizal
(Non-AMF) plants were utilized as a control treatment.
Plants were grown in a growth chamber (28 °C, 75
% relative humidity; 12 h photoperiod; Biotronette
850H, Lab-Line Instruments Inc.), and watered ap-
proximately every seven days with a sterilized LANS
for the duration of the study. Plants were harvested
after 70 days. At this time, shoot dry mass, dehydroge-
nase (DEH) activity was measured at the rhizosphere
(Casida
et al
. 1964), and root polyphenoloxidase (PPO)
activity was assessed (Racusen and Foote, 1965). Tol-
erance of AM symbiosis to BaP was estimated by per-
forming a vital stain technique to determine the fungal
alkaline phosphatase activity in roots (Tisserant
et al
.
1993). Fractional infection of
G. margarita
was esti-
mated microscopically as the intensity of colonization
of the root cortex (Trouvelot
et al
. 1986).
The extraction of BaP from the rhizosphere sand
(100 g) was performed with 200 mL of acetone (Sigma-
Aldrich
®
, 99.5% purity) by the mechanical-shaking ex-
traction procedure (Schwab
et al
. 1999). Extracts were
purified by filtering through in a silica gel column, and
concentrated to a volume about 3 mL at 60 °C. UV-
Absorbance readings were taken at 297 nm (Soroka
and Soroka, 2002) with a UV-VIS Hewlett Packard
Spectrophotometer (ChemStation 8453). Calibration
solutions with BaP were prepared at six concentrations
ranging from 0 to 50 μg mL
-1
. BaP-dissipation was
expressed as percentage which was calculated by sub-
tracting the final concentration of BaP (at time 70-days)
to the initial recovered concentration of BaP (time zero)
of each treatment.
A factorial (2
5
) experiment with 10 treatments was
set in a completely randomized design. There was
one plant per container which was a single replication
A. Alarcón
et al.
42
(n=5). Statistical analysis was performed with the sta-
tistical analysis system (SAS Institute, 2000). Data
were analyzed by variance analysis (ANOVA), and
Tukey´s test (
α
=0.05) for mean comparison and mul-
tiple regression model were performed (SAS Insti-
tute, 2000).
RESULTS
Spore germination response to phenanthrene and
benzo[
a
]pyrene
Spore germination was drastically reduced at all
concentrations of PHE (0.15 to 0.6 mM). Concen-
trations
25 μg PHE mL
-1
were detrimental to the
germination of the
G. margarita
spores (
Fig. 1a
). Spore
germination reduction by PHE was >92 % in com-
parison with the spore germination observed at non-
contaminated medium (
Fig. 1a
). Hyphal growth and
elongation were also significantly (
P
0.01) reduced
by PHE (
Fig. 1b
).
Benzo[
a
]pyrene reduced spore germination per-
centage (
Fig. 2a
) at all concentrations but not to the
same extent as PHE. For instance, spore germination
showed a reduction > 40 % in the presence of 100
(0.4 mM) μg BaP mL
-1
in comparison with the ger-
mination percentage obtained from non-contaminated
spores (
Fig. 2a
). In contrast to the PHE effects, ger-
minated spores showed non-significant enhanced hy-
phal elongation in response to this contaminant. The
average longest hyphal lengths (69.2 and 102.0 mm)
were observed at the two highest concentrations of
BaP, 75 (0.3 mM) and 100 (0.4 mM) μg mL
-1
, re-
spectively (
Fig. 2b
).
Phytoremediation potential of
Gigaspora
margarita
-
Echinochloa polystachya
symbiosis in a
benzo[
a
]pyrene-polluted substrate
Echinochloa polystachya
roots were colonized by
G. margarita
when growing at BaP-contaminated sub-
strate. Shoot dry mass was not significantly enhanced
by the mycorrhiza nor reduced by BaP-contamina-
tion (
Fig. 3
).
Dehydrogenase activity was significantly (
P
0.05
)
higher in the rhizosphere of AMF-plants at 0, 0.2, and
0.3 mM BaP when compared to non-AMF plants (
Fig.
4a
). BaP-contamination did not significantly affect
DEH activity of AMF-plants, which showed similar
DEH activity than AMF-plants at non-contaminated
substrate. No significant differences on rhizosphere
0123456
Phenanthrene concentration
100
75
50
25
0
µ
g mL
-1
0
0.15
0.3
0.45
0.6 mM
80
70
60
50
40
30
20
10
0
-10
-20
Total hyphal length (mm)
50
45
40
35
30
25
20
15
10
5
0
Spore germination (%)
a)
b)
y = 38.287 - 0.9973x + 0.0069x
2
r
2
= 0.8029
y = 61.253 - 2.0509x + 0.0151x
2
r
2
= 0.8601
Fig. 1.
Effect of phenanthrene concentrations on spore germi-
nation (a), and total hyphal length (b) of
Gigaspora
margarita
, after 25 days of incubation. n=4. Means
±
Standard Error
0123456
b)
a)
100
75
50
25
0
0
0.1
0.2
0.3
0.4
µ
g mL
-1
mM
Benzo[
a
]pyrene concentration
Total hyphal length (mm)
Spore germination (%)
160
140
120
100
80
60
40
20
0
50
45
40
35
30
25
20
15
10
5
0
a)
b)
y = 68.974 - 1.5855x + 0.0195x
2
r
2
= 0.9309
y = 53.766 - 13.051x + 1.2171x
2
r
2
= 0.9706
Fig. 2.
Effect of benzo[
a
]pyrene concentrations on spore ger-
mination (a), and total hyphal length (b) of
Gigaspora
margarita
, after 25 days of incubation. n=4. Means
±
Standard Error
RESPONSE OF
Gigaspora
margarita
SPORE AND SYMBIOSIS TO BENZO[
a
]PYRENE
43
soil DEH activity between AMF- and non-AMF plants
were observed at 0.1 and 0.4 mM BaP (
Fig. 4a
). Root
PPO activity was significantly (
P
0.05
) diminished in
AMF-plants in the presence of 0 and 0.1 mM BaP in
comparison to non-AMF plants. However, AMF-plants
show significant greater PPO (
P
0.05
) than non-AMF
plant exposed to 0.3 mM BaP (
Fig. 4b
). No significant
differences on root PPO activity between AMF- and
non-AMF plants were observed at 0.2 and 0.4 mM
BaP (
Fig. 4b
).
Arbuscular mycorrhizal symbiosis was not affected
by BaP-contamination (
Fig. 5
). However, the appli-
cation of BaP at 0.3 mM produced significant
(
P
0.05
) increases in AMF-colonization when com-
pared to the colonization in AMF-plants at 0 mM BaP.
Colonization was greater than 35%, as indicated by
the fungal metabolic activity measured by alkaline
phosphatase vital stain. No mycorrhizal colonization
was observed in non-AMF plants.
Benzo[
a
]pyrene dissipation from the rhizosphere
was significantly higher in non-AMF plants at 0.1,
0.2, and 0.3 mM BaP than AMF-plants (
Fig. 6
). No
significant differences were observed in the dissipa-
tion of BaP between Non-AMF and AMF-plants ex-
posed at 0.4 mM BaP (
Fig. 6
). In general, the extent
of BaP-dissipation from the rhizosphere of either non-
AMF or AMF-plants decreased as BaP-contamina-
tion increased (
Fig. 6
).
*
BaP application (mM)
0.4
0.3
0.2
0.1
0
50
40
30
20
10
0
Fractional colonization
(ALP %)
Fig. 5.
Fractional infection of
Gigaspora margarita
in roots of
Echinochloa polystachya
exposed at several doses of
benzo[
a
]pyrene (BaP) revealed as alkaline phosphatase
vital stain, after 70 days. n=5. Means
±
Standard Error.
*
Indicate significant differences among treatments at
5% level
*
*
*
BaP application (mM)
0.4
0.3
0.2
0.1
100
80
60
40
20
0
BaP dissipation (%)
Control
Mycorrhizal
Fig. 6.
Benzo[
a
]pyrene (BaP) dissipation from the rhizosphere
of
Echinochloa polystachya
with or without the
inoculation of
Gigaspora margarita
, after 70 days. n=5.
Means
±
Standard error.
*
Indicate significant differences
between Non-AMF and AMF plants at 5% level
AMF
BaP-application (mM)
0.4
0.2
0.1
0
Shoot dry mass (g)
2
1.5
1
0.5
0
Non-AMF
AMF
Fig. 3.
Effect of benzo[
a
]pyrene (BaP) application on shoot dry
matter of
Echinochloa polystachya
without (Non-AMF)
or with the inoculation of
Gigaspora margarita
(AMF),
after 70 days. n=5. No significant differences were
observed among treatments at 5% level
DEH (
µ
g formazan g
-1
day
-1
)
0
0.1
0.2
0.3
0.4
Non-AMF
AMF
a)
b)
*
*
*
*
*
*
BaP-application (mM)
0.3
0.4
0.2
0.1
0
PPO (units g
-1
min
-1
)
20
16
12
8
4
0
20
16
12
8
4
0
a)
b)
Non-AMF
AMF
Fig. 4.
Rhizosphere dehydrogenase (DEH) (a) activity and root
polyphenoloxidase (PPO) activity (b) of
Echinochloa
polystachya
with benzo[
a
]pyrene (BaP) contamination
(Non-AMF) or with the inoculation of
Gigaspora
margarita
(AMF), after 70 days. n=5. Means
±
Standard
Error.
*
Indicate significant differences between Non-
AMF and AMF plants at 5% level
A. Alarcón
et al.
44
DISCUSSION
Spore germination response to phenanthrene and
benzo[
a
]pyrene
This is one of the first reports detailing the re-
sponse of
Gigaspora margarita
spore germination to
the presence of phenanthrene and benzo[
a
]pyrene.
Spore germination was significantly reduced by all
tested concentrations of PHE and BaP. However, al-
though no significant differences were observed
among treatments, hyphal length was stimulated by
the presence of BaP at 75 (0.3 mM) and 100 (0.4
mM) μg mL
-1
.
Although not available information was found in
the literature about the effects of petroleum hydro-
carbons on spore germination, some studies have dem-
onstrated that factors such as temperature, pH, soil
moisture, root exudates, and presence of inorganic
and organic chemicals may affect the spore germina-
tion, hyphal growth and branching from several AMF
species (Tommerup and Briggs 1981, Tommerup
1984, Clark 1997, Chiocchio
et al
. 2000, Akiyama
et
al
. 2005). It was observed that crystals of PHE had
larger size when compared to crystals produced by
BaP when applied to the water-agar (data not pre-
sented). The toxic effect of PHE to the spores may
potentially be explained by the particle size of this
contaminant allowing higher spore surface area cov-
ered by the crystals. Such exposure could have cre-
ated unfavorable conditions related with dehydration
of the surrounding zones of the spores due to the
hydrophobic properties of PHE and other PAH
(Cerniglia 1992). Thus, limiting spore hydration and
consequently inhibiting its germination under this
stressful condition. In this respect, arbuscular mycor-
rhizal spores have four developmental phases during
germination, which appear to have different hydra-
tion requirements that may affect spore germination
and hyphal growth/branching (Tommerup 1984).
Based on the achieved spore germination from non-
contaminated medium, we suggest that the presence
of PAH on the medium may prevent the hydration of
the spores, thus inhibiting or delaying germination.
However, further studies are suggested to clarify the
effect of PAH on both germination and viability of
AMF spores.
Phytoremediation potential of
Gigaspora
margarita
-
Echinochloa polystachya
symbiosis in a
benzo[
a
]pyrene-polluted substrate
Petroleum hydrocarbons, including PAH, may re-
tard plant growth by affecting water and nutrient up-
take (De Jong 1980, Merkl
et al
. 2005), which may
alter plant physiology and therefore reduce plant tol-
erance and survival to organic contaminants. Under
these conditions, AMF may enhance plant water and
nutrient uptake and subsequent growth and adapta-
tion in contaminated substrates (Smith and Read,
1997). However, under our experimental conditions,
BaP-contamination and AMF-inoculation did not sig-
nificantly reduce or enhance plant growth.
Soil rhizosphere enzymatic activities have been pro-
posed as indicators of contamination and detoxifica-
tion of organic pollutant in soils (Gianfreda
et al
.
2005). In general, BaP-contamination and
Gigaspora
margarita
inoculation resulted in modifications on the
rhizosphere DEH and root PPO activities. Inocula-
tion of
G. margarita
resulted in a 275 % increase in
DEH activity when compared with non-AMF control
plants (0 mM BaP). At the contaminated rhizosphere,
the benefit of AMF inoculation on DEH was evident at
0.2 and 0.3 mM BaP. Inoculated plants had an 84 %
and 66 % increase in DEH, respectively, when com-
pared with their respective non-AMF plants. In addi-
tion, AMF-plants had a 68% increase in PPO activity
at 0.3 mM BaP when compared to respective non-
AMF plants. These results may be an indication of
potential benefits of
G. margarita
on plants growing
at BaP-contaminated substrate. It has been suggested
that soil enzymatic activities including DEH and PPO,
are affected by both concentration and type of PAH
in the soil, and soil properties including organic mat-
ter content and pH (Ma
et al
. 2003, Baran
et al
.
2004). The DEH activity has been demonstrated to
have more consistent response to organic contami-
nants, suggesting that it would be a sensitive indicator
to soil contamination (Trasar-Cepeda
et al
. 2000).
Dehydrogenase activity is recommended to assess
overall microbial activities (Günther
et al
. 1996), and
it may be an indicator of the activity of AMF in the
rhizosphere contaminated with PAH.
This is one of the first reports detailing the mycor-
rhizal condition of
Echinochloa polystachya
. This
plant has been experimentally utilized in phytoreme-
diation of petroleum- and BAP-contaminated soil
(Rivera-Cruz 2001). Benzo[
a
]pyrene did not adversely
affect root colonization of
Gigaspora margarita
and
its efficiency on phosphate transfer (Tisserant
et al
.
1993) from the contaminated soil to the roots, as in-
dicated by the alkaline phosphatase activity of this
fungus. However, it has been shown that AMF colo-
nization is negatively affected by the presence of pe-
troleum hydrocarbons as well as by mixtures or single
PAH in soils (Cabello 1997, Leyval and Binet 1998,
Gaspar
et al
. 2002, Liu
et al
. 2004).
The efficiency of BaP dissipation from the rhizo-
RESPONSE OF
Gigaspora
margarita
SPORE AND SYMBIOSIS TO BENZO[
a
]PYRENE
45
sphere of
E. polystachya
was dependent on the con-
centration of this PAH in the substrate as well as on
the mycorrhizal condition. In the first case,
E.
polystachya
contributed on 56 % and 45 % of BaP-
dissipation at 0.1 mM and 0.2 mM BaP concentration
in the substrate, respectively. This efficiency was de-
creased when plants were exposed to 0.3 and 0.4 mM
whose BaP-dissipation extent was 33% and 25 %, re-
spectively. In contrast, mycorrhizal condition of
E.
polystachya
had lower BaP dissipation at 0.1 mM
(47 %). At 0.2, 0.3 and 0.4 mM BaP,
Gigaspora
margarita
inoculation significantly reduced the BaP-
dissipation in the rhizosphere presenting in average
23.9 % of efficiency. These results are contrary to
those reported for the inoculation of alfalfa with
Glo-
mus caledomiun
in BaP-contaminated substrate (Liu
et al
. 2005). Although it has been demonstrated that
AMF species from contaminated areas are more ef-
fective on stimulating plant growth and phytoreme-
diation or petroleum hydrocarbons than introduced
AMF species (Cabello, 1999),
Gigaspora margarita
(isolated from a chronically contaminated soil in
Mexico, Quiñones
et al
. 2004) did not enhance either
plant growth or BaP-dissipation from the rhizosphere
of
E. polystachya
. It seems that differences on eco-
logical adaptations of AMF and their host plant com-
bination may result in weak or strong indirect ben-
efits on phytoremediation of petroleum-contaminated
soils as suggested by Cabello (2001) and Joner and
Leyval (2003b).
Echinochloa polystachya
seems to have an inher-
ent root mechanism for the BaP removal, but it has
not been clarified. Phytoremediation performance of
PAH in soils is dependent on the synthesis and activ-
ity of oxidoreductases at the contaminated rhizosphere,
which can be selectively enhanced by the presence of
AMF (Criquet
et al
. 2000). Nakajima
et al
. (1996)
suggested that plant cells may accumulate glycosil con-
jugates presumably derived from pyrene uptake by
leaves or roots. However, Ryan
et al
. (1988) sug-
gested that the extent of absorption of lipophilic com-
pounds by roots is a complex but not significant pro-
cess. However, more recently, Binet
et al
. (2000)
demonstrated that some PAH can be either adsorbed
to the root surface or slightly accumulated in both
roots and shoots of
Lolium perenne
L. cv. Barclay.
In summary, the fate of PAH-transformation prod-
ucts in plant tissue is still unclear and we suggested
that plant species and ecotypes may be determinant
factors on phytoremediation of PAH in soils.
In our experimental conditions, the inoculation of
Gigaspora margarita
did not induce more dissipa-
tion of BaP in the rhizosphere even though mycor-
rhizal plants showed more stable DEH and root PPO
activities. There was no apparent correlation between
enzymatic activities and BaP-dissipation in the rhizo-
sphere of
E. polystachya
. Binet
et al
. (2000) reported
that
Glomus mosseae
did not affect either plant growth
or dissipation/degradation of selected PAH in the rhizo-
sphere of
Lolium perenne
. Nevertheless, the ability
of AMF-plants to dissipate/degrade PAH in the rhizo-
sphere may be dependent on the mycorrhizal depen-
dency of the host (Leyval and Binet, 1998). For in-
stance, AMF-inoculation to alfalfa has been demon-
strated to enhance the degradation of BaP (Liu
et al
.
2004), but it did not stimulate more PAH dissipation/
degradation in the
Lolium perenne
rhizosphere (Bi-
net
et al
. 2000).
Although AMF have not been demonstrated to
posses a specific physiological mechanism to contrib-
ute directly on PAH-degradation, it is clear that
Gigaspora margarita
may influence plant tolerance
under contaminated substrates with PAH. It has been
proposed that AMF might reduce the root adsorption
and thus, the toxicity of PAH (Binet
et al
. 2000) by
unknown physiological fungal mechanisms. Due to
the limited dissipation of BaP in the mycorrhizosphere
of
E. polystachya
, it is suggested that AMF may con-
tribute on the stabilization/sequestration of PAH in
the soil. We concur with Gaspar
et al
. (2002) in sug-
gesting that one probable mechanism for PAH stabili-
zation is the accumulation of PHE in spores of AMF;
furthermore we suggest that the external mycelium
may also play a significant role in sequestration and
accumulation of PAH. In addition, AM-fungal biom-
ass may indirectly create favorable microhabitat for
microbial activity (Rilling and Steinberg 2002), which
may also stimulate hydrocarbon degradation. Further-
more, the induction of higher synthesis of oxidative
enzymes responsible for the degradation or transfor-
mation of PAH by plants may represent another pos-
sible indirect benefit of AMF in contaminated rhizo-
sphere (Joner and Leyval 2003a). However, further
studies are required in order to clarify the physiologi-
cal role of these symbiotic fungi on phytoremediation
of organic contaminants, and to identify the physi-
ological mechanisms of either AMF or AMF-plants,
that contribute to improve their tolerance and adapta-
tion under petroleum hydrocarbon-contaminated con-
ditions.
ACKNOWLEDGEMENTS
Authors thank financial support from International
Foundation for Science (IFS) Grant C/3193-1 (2001).
A. Alarcón
et al.
46
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