Cuellar-Sánchez et. al.

/Revista Latinoamericana el Ambiente y las Ciencias, 2 (1):1-11, 2011

PRODUCTION OF EXTRARADICAL MYCELIUM AND ITS METAL

IMMOBILIZATION CAPACITY IN THE ASSOCIATION GLOMUS
MOSSEAE BEG25-SORGHUM

Cuellar-Sánchez Alma, González-Chávez Ma. del Carmen A*, Carrillo-González

Rogelio
Colegio de Postgraduados en Ciencias Agrícolas, Instituto de Recursos Naturales,
Campus Montecillo. Carretera México-Texcoco, km 36.5. Texcoco, México, 56230.
*E-mail: carmeng@colpos.mx

Abstract: The production of the extraradical mycelium (ERM) by Glomus mosseae BEG25
and its capacity to sequester Cd, Pb and Zn under symbiotic growth in sorghum plants
was studied. Inoculated plants were watered with nutrient solution containing four different
concentrations of Cd (0, 0.5, 1 and 2 mg L-1), Pb (0, 5, 10 and 20 mg L-1) and Zn (0, 10, 20
and 40 mg L-1). Results showed that metals decreased ERM production, especially in high
metal concentrations. Cd was the most toxic metal to the ERM production (88% decrement
at 2 mg L-1 of Cd). For Zn, reduction in 53% was observed at the highest doses (40 mg L-
1
), while for Pb concentrations of 20 mg L-1, it was reduced up to 38%. The sequestration
capacity of the ERM varied according to the metal involved. Sequestration was between
12 to 18 μg Cd g-1 mycelium; 11 to 59 μg of Pb g-1 mycelium and 9 to 22 μg Zn g-1
mycelium. The metals structurally bound to the ERM represent an immobilization in the
rhizosphere of mycorrhizal plants, which may have relevant implications in metal
phytostabilization.

Keywords: arbuscular mycorrhizal fungi, metal sequestration, metal stabilization

Introduction rhizosphere and beyond it, and they

Microorganisms are abundant represent the single group of
in the rhizosphere and may play a microorganisms that are able to
relevant role in modifying the behavior transport mineral elements from the
of heavy metals (HM) in the soil soil solution to plants (Joner et al.,
(Lasat, 2002; Bollag and Bollag, 2000). These fungi produce a well
1994). Arbuscular mycorrhizal fungi elaborated hyphal mycelium network
(AMF) are found both in the in soil, the extraradical mycelium

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(ERM). Audet and Charest (2007) time (Joner et al., 2000; González-
resumed two roles of AMF in polluted Chávez et al., 2002). In vitro
soils: 1) an enhanced metal uptake at experiments have also showed that
low soil-HM concentrations, and 2) a the ERM is directly involved in the
reduced metal bioavailability via uptake and sequestration of metals
hyphae fungal participation (metal- (Giasson et al., 2005). However, the
binding) at high levels of metals in the mechanisms underlying this process
soil. They emphasized the importance are largely unknown. The amount of
of AMF in buffering the soil metals sequestered in the ERM when
environment for plants, which the fungus is symbiotically connected
provides an advantage to a number of to its host plant has not quantified,
organisms in the food chain because which may give more information of
it reduces metal exposure and fungal capacity to deal with HM.
toxicity. In a field study, González- The aim of this research was to
Chávez et al. (2008) showed that the study the production of the ERM of an
ERM and mycorrhizal plants, growing arbuscular mycorrhizal fungus
in a fertility island, are strongly related (Glomus mosseae BEG25) and its
to natural remediation in a slag heap capacity to sequester Cd, Pb and Zn
polluted with Cd. when symbiotically associated to
Joner and Leyval (1997) sorghum plants.
showed that the ERM of Glomus
mosseae transport Cd from soil to 1 Materials and methods
plants, but the transference of this 1.1 Biological material
metal from fungus to plant was The capacity of the extraradical
restricted due to fungal mycelium (ERM) of Glomus mosseae
immobilization. Some authors have BEG25 to sequester Cd, Pb and Zn
determined sequestration of metals in was studied. This fungus was isolated
excised ERM, which represents metal from a non polluted soil and was
sorption in the first minutes of metal propagated, using onion as a host
exposition and some metal plant, for three months. 15 g of this
accumulation in a longer period of inoculum, containing colonized roots

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and an average of 400 spores, was
used for the metal sequestration
experiments.
1.2 Substrate and experimental
units
Coarse sand (80-110 mesh
sieves) was used as substrate. It was
acid-washed with a 3 N HCl solution,
and then rinsed with deionized water
up to pH 6.1. Sand was sterilized
three times for 1 h before use. Two-
compartment pots were prepared.
The root compartment was made with
a 9 x 4 cm (high x diameter) plastic
bottle with 37-μm nylon mesh on the
bottom. This bottle was inserted in a
12 cm internal diameter
containing the sand (total volume
1000 cm3) to form a root-free area for
the ERM or hyphal compartment (Fig.
1). Sorghum vulgaris was used as a
host plant. Seeds were
disinfested with a 5% commercial
sodium hypochlorite (NaHClO4)
solution for 2 min. Inoculum and
seeds were placed together in the
center of the root compartment.
Plants were maintained in a plant
growth chamber (25 °C, 12/12 h
day/night photoperiod, with
horizontally mounted 20
fluorescent lamp 250 fc). Plants from
experiment 2 were arranged in a
random design in two blocks: one to
Zn sequestration (upper part of the
chamber) and the second one to Pb
sequestration (lower side of the
chamber).
1.3 Experiments establishment
Two experiments were set up. In
the first experiment Cd sequestration
was studied and in the second one Zn
and Pb. In the first experiment plants
were watered manually once a day
with 50 mL standard nutrient solution
(Millner and Kitt, 1992), while in the
second experiment, plants were
pot watered by hand twice a day (25 mL
each one). 50 mL of nutrient solution
was calculated as the volume
required for keeping pots at 90% of
water capacity. In all cases 0.5 mM
surface MES (2-[N-Morpholino]ethanesulfonic
acid) was added in the nutrient
solution to maintain the pH at 6.1±0.2.
High P (40 mM) nutrient solution was
used for the first week for all plants
and then low P (20 mM) nutrient
solution (Millner and Kitt, 1992) was
used for the next eight weeks of the
one experiments.
watt
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On the third week
planting, the nutrient solution was
amended with three levels of Cd (0.5,
1 and 2 mg L-1); of Pb (5, 10 and 20
-1
mg L ) or Zn (10, 20 and 40 mg L ),
using nitrate salts as metals sources
[Cd(NO3)2; Pb(NO3)2 ; Zn(NO3)2,
respectively]. In all the treatments 0.5
mM MES was used to maintain the
pH at 6.1±0.2. Controls received the
basic nutrient solution that contained
1 μM of Zn, 0 μM of Pb and 0 μM of
Cd, respectively.
1.4 Harvest and analysis
Plants were grown for a total of
9 weeks (6 weeks of exposure to
different metal levels). At harvest
hyphal compartment (the
contained in the sand)
submerged in water and stirred by
hand to release hyphae. These were
collected by decanting the water over
a sieve (43 μm) as soon as the sand
sedimented. The sand was washed
and decanted three times. Hyphae
were trapped on the 53-μm sieves.
Glomalin was extracted from the ERM
following the method described by
González-Chávez et al., (2004). After
glomalin extraction, the ERM was
dried for two days at 60 °C and
after weighed in order to calculate dry
biomass and metal content (mycelium
metal structural content).
Metal content in the ERM and
-1
sequestered by glomalin was
determined by AAS (Perkin Elmer)
after digestion in concentrated 3 mL
of nitric acid (65%) during 3 h at 85
°C. Reagent blanks and internal
standards controls were carried out
when appropriate to ensure accuracy
and precision in the analysis of
metals. The experiments had four
metal levels with six replicates in a
randomized design. Differences
among dry biomass of the ERM and
ERM-metal bound were tested by
ERM ANOVA analysis. When differences
was were found, Tukey’s test (P=0.05)
was performed in SAS (SAS Institute,
2000).
2 Results and discussion
Cadmium at levels of 1 and 2
mg significantly decreased the
amount of the ERM (Fig. 2). In
contrast, significant reduction of the
ERM was just observed in the highest
concentration of Pb (20 mg L-1) and
Zn (40 mg L-1) (Fig. 3). Cadmium was
the most toxic metal to the ERM
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production, followed by Zn and Pb. twice a day (25 mL each one). During
Mycelium biomass decreased 35%, the harvesting of the first experiment
71% and 88% when 0.5, 1 and 2 mg we realized that a small amount of
L-1 of Cd was added to the pots, mycelium was produced. Water
respectively. While, mycelium availability and dryness during the
production decreased 3%, 7% and day are strong factors that affect
38% at 5, 10 and 20 mg of Pb L -1 of mycelium production in propagation of
nutrient solution added, respectively AMF (Sara Wright personal
(Fig. 3a). For Zn, the addition of 10 communication). Hence, it was
and 20 mg L-1 decreased biomass decided to keep the pots wet longer
mycelium by 1% and 12%, but the with two watering stages: one in the
highest doses (40 mg L-1) decreased morning and another in the evening.
it 53% (Fig. 3b). González-Chávez et This increased the production of
al. (2004) observed a reduced mycelium in the second experiment in
production of mycelium of 45% as a comparison with the first one.
result of the highest addition of Cu However, in the second experiment,
-1
(1.2 mg L ); however no negative half of the plants growing in the plant
effect was observed at 0.6 mg L-1. growth chamber occupied the upper
This suggests that Cd at small side of the chamber, while the other
concentrations causes stronger half were in the lower compartment of
reduction on the ERM production than the chamber. Although all plants
Pb and Zn do. share a lamp in each side of the
Differences in amount of ERM chamber, it appears that plants
produced between experiments were occupying the upper side, tended to
found (Fig. 2, 3). This is possible due produce higher amounts of mycelium
to watering and plants light incidence. (plants set to Zn sequestration
In the first experiment (Cd experiment; Fig. 3b). This reflects that
sequestration), pots were only mycelium production may be strongly
watered once a day (50 mL), while in influenced by environmental factors.
the second experiment (Pb and Zn In the present research
sequestration), pots were watered glomalin, extracted from the ERM,

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was purified achieving very low at 10 and 20 mg L-1 increased the

concentrations; in consequence metal sequestration between 129% and
sequestration by this bio fungal 414% in comparison to the lowest Pb
product could not be accurately concentration used (5 mg L-1). While
analyzed (data no presented). In this the ERM sequestered between 9 and
extraction process, sodium citrate and 22 μg Zn g-1 mycelium, which
hydrochloric acid were used. These represented between 11% and 136%
two chemical compounds are strong more Zn sequestered than the 10 mg
treatments that desorb an important L-1 of Zn treatment (Fig. 3b).
amount of metals bound to glomalin Metal saturation in the ERM
and hypha surface (González-Chávez was not observed in any of the
et al. 2004). Then, in this research the treatments. Hence, the metal binding
amount of metal structurally bound to capacity of the ERM may increase
the EM is reported (Fig. 2, 3). The with more concentrated metal
results showed that the ERM has solutions; however based on the
capacity to sequester metals (Zn, Cd results, the biomass of the ERM is
and Pb); however it depended on the strongly decreased at higher metal
metal tested (Fig. 2, 3). This capacity concentrations. The magnitude of
improved as the level of metal sequestration by the ERM was
increased in the nutrient solution Pb>Zn=Cd. Similar sequestration
added to the pots. At the 1 and 2 mg trends were showed by González-
-1
L of Cd treatment, mycelium bound Chávez et al. (2004). They observed
between 12 to 18 μg Cd g-1 mycelium. higher affinity to sequester Pb (600 –
The ERM grown with 1 mg L-1 of Cd 1120 μg g-1 glomalin) than Cd (80 -
sequestered 165% more Cd than 200 μg g-1 glomalin) by the protein
when it was grown at 0.5 mg L-1, while produced in the hyphae of AMF
at 2 mg L-1 Cd sequestration presented in polluted soils. Vodnik et
increased in 263%. al. (2008) also found a similar
Pb sequestration ranged preferential trend to sequester more
between 11 and 59 μg Cd g-1 Pb than Zn by glomalin extracted from
mycelium (Fig. 3a). The addition of Pb heavy metal polluted soil. Chern et al.

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(2007) reported that glomalin, fungal structures and their bioproduct:

extracted from embankment soils at the ERM, spores and glomalin
seven locations along an (González-Chávez et al., 2006;
urban/coastal watershed, Sánchez-Viveros et al., 2004). This
sequestered Cd, Fe, Pb and Mn in represents an immobilization of
concentration ranges of 0-0.34, 0.5- metals in the rhizosphere of
228, 0.11-189 and 2.23-784 µg mg-1 mycorrhizal plants with relevant
protein, respectively. All these results implications to the development of
showed higher sequestration affinity phytostabilization technology.
for Pb, than for Zn and Cd; however Recently, Gonzalez-Chavez et
these authors did not quantify the al. (2008) showed that the ERM of
metal sequestration capacity directly AMF was very abundant (range from
in the ERM, which makes it difficult to 1.03 – 26.3 mg/g with average of
compare the results. 10.29 ± 9.7 mg/g) in a slag heap
anou kov et al. (2006) contaminated with Cd, where natural
performed an experiment where the remediation has occurred. Results
ERM of Glomus intraradices was from the present research are
exposed in vivo to Cd. They reported important because contribute to the
that the ERM accumulated 10-20 knowledge of the role of the ERM in
times more Cd per unit of biomass the sequestration of metals, which
than tobacco roots exposed to can infer its participation in metal
different Cd concentrations in the polluted sites. Nowadays is
growth substrate. However, these recognizable that the ERM is a key
authors, in fact, measured Cd fungal structure with importance not
immobilization in the ERM and only in soil microbial ecology, plant
glomalin produced by the hyphae of community and agroecosystem
G. intraradices, rather than just Cd functioning, but also in biogechemical
accumulation of the ERM. Hence, cycles. This acquires paramount
one of the roles of AMF in polluted importance as AMF represent the
soils may be their involvement in the dominant beneficial symbiotic fungi
sequestration of metals through and are widely distributed among

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higher plant species in many metal sequestered higher amounts of Pb

polluted soils. than Zn and Cd. This fungal structure
represents another way (in addition to
3. Conclusions spores and glomalin) to reduce metal
This study analyzed the availability in the rhizosphere. Hence,
capacity of the ERM to sequester AMF participate in metal
metals such as Zn, Pb and Cd under immobilization. More studies are
symbiotic association with sorghum necessary including different AMF
plants. The ERM of G. mosseae and other metals in order to obtain
BEG25 was strongly affected by Cd in more evidence of their role in metal
the three doses studied (0.5, 1 and 2 polluted soils.
mg L-1). In contrast, low levels of Pb
(5-10 mg L-1) and Zn (10-20 mg L-1) Acknowledgements: This research
did not affect the amount of the ERM was part of the project research
produced for this fungus. While, SEMARNAT-CONACyT CO-01-2002-
higher doses of Pb (20 mg L-1) and Zn 739. The authors thank Eng. Daisy
-1
(40 mg L ) significantly decreased Daiana Díaz Sánchez for her
production of ERM. The ERM technical help.

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Figure 1. Experimental compartment pots to study production and metal-immobilization of the

extraradical mycelium of Glomus mosseae BEG25 symbiotically growing with sorghum in sand
irrigated with nutrient solution containing Cd, Pb or Zn.

Figure 2. Addition of different levels of Cd(NO 3)2 on production and Cd immobilization of the
extraradical mycelium of Glomus mosseae BEG25 symbiotically bound to sorghum plants. Bars
indicate sd, n=6.

Figure 3. Addition of different levels of Pb(NO3)2 (a) and Zn(NO3)2 (b) on production and Cd
immobilization of the extraradical mycelium of Glomus mosseae BEG25 symbiotically bound to
sorghum plants. Bars indicate sd, n=6.

Root compartment
Fungal inoculum

Nylon mesh 37
μm

Hyphal compartment

Figure 1.

180 Mycelium 70
Metal in mycelium
160
60
140
Mycelium biomass (mg) DW

50
Cd in mycelium (μg g -1)

120

100 40

80 30

60
20
40
10
20

0 0
0 0.5 1 2
Cd added (mg L-1)

Figure 2.

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a) 180 Mycelium 70
Metal in mycelium
a) 160
60
140
Mycelium biomass (mg) DW

50

Pb in mycelium (μg g -1)

120

100 40

80 30

60
20
40
10
20

0 0
0 5 10 20
Pb added (mg L-1)

b)

a) 180 70

160
Mycelium biomass (mg) DW

60
140
50

Zn in mycelium ( μg g -1)
120

100 40

80 30

60
20
40
10
20

0 0
1 10 20 40
Zn added (mg L-1)

Figure 3.

11