9 Agus Rohyadi_405.pmd

Volume 3, Number1, April 2009

p 42-46
ISSN 1978-3477

Neighboring Plants Alleviate Aluminum Toxicity

on The External Hyphae of Gigaspora margarita

AGUS ROHYADI

Faculty of Agriculture, Universitas Mataram, Jalan Majapahit 62, Mataram 83125, Indonesia

Phone: +62-370-621435, Fax: +62-370-640189, Email: arohyadi01@telkom.net

Excessive soluble aluminum (Al3+) in acidic soils is toxic to the external hyphae of arbuscular mycorrhizal fungi but it can be

alleviated by other soil factors. A glasshouse experiment was conducted to study the effect of increased Al3+ concentration on the

growth of the external hyphae of Gigaspora margarita in the presence of other plants near the host plants. The experiment used

compartmentalized pots to facilitate the growth of mycorrhizal-inoculated host plants, external hyphae of the fungus and not

mycorhizal-inoculated neighboring plants in different compartments; and measuring the effects of Al3+ and of the neighboring

plants on the growth of the fungal hyphae independently. Increased concentration of Al3+ in soil affected the growth of external

hyphae of G. margarita negatively. However, the hyphal length density of the fungus was much higher in the pots with

neighboring plants than that in the other ones, despite the Al toxicity. This indicates that the hyphae could be taken away from

the toxic effect of Al3+ by the stimulating growth from roots of the neighboring plants.

Key words: aluminum, cowpea, external hyphae, compartmentalized pot system, Gigaspora margarita

Crops often suffer from adverse conditions of acid soils.

Aluminum (Al) is considered the major stress factor because

of its toxicity under acidic conditions. Excessive soluble-Al

(Al3+) in soil might create a poor plant root system by

inhibiting lateral root and root hair formation, and limit the

solubility and, therefore, the availability of some essential

mineral nutrients, especially P (Baligar et al. 1995). Also, high

Al3+ concentration can be detrimental to soil microbes that

are involved in nutrient cycling and/or forming symbiotic

associations with the plant roots (Wood 1995; Rohyadi 2006).

As a result, the plants may have limitation in taking up

nutrients and water in sufficient quantity, and consequently

they grow stunted.

Arbuscular mycorrhizas (AM) are mutualistic symbioses

mostly benefiting plants that have a small and/or coarse root

with a lack of root hairs, and those are grown on infertile soils

with particularly low available-P. Hence the symbioses can

be of great importance in acid soils, where poorly developed

root systems and P deficiency are the main constraints for

plant growth (Miyasaka and Habte 2001). However, the AM

fungi themselves are subjected to detrimental effects of

mainly high Al3+ concentration in acidic soils. Rohyadi (2006)

indicated that decreased effectiveness of an AM fungus,

Gigaspora margarita on cowpea plant grown on acidic soils

was related to the poor growth of the external fungal hyphae

because of Al toxicity. It is well known that the external hyphae

of AM fungi growing out from colonized plant roots play a

crucial role. They develop to form a hyphal net-work in soils

and function as an extension of root system to explore soil

particularly beyond the depletion of the root zone, take up

water and element nutrients there and then transport them to

the roots of the host plants (Smith et al. 2000). Therefore, the

inhibited growth of the hyphae may directly reduce the

symbiotic effectiveness.

Only a few studies were done on the effect of Al3+ on the

external hyphae of the AM fungi. It was reported that the

growth responses of the AM fungi to Al toxicity are complex

and much dependent on the fungal species (Siqueira et al.

1985). Recently, several studies in vitro showed that factors

such as CO
2
level, some chemical compounds and volatiles

released by plant roots, and bacterial products, influence the

growth of the fungal hyphae (Tawaraya et al. 1996; Nagahashi

and Douds 2000, 2003; Xavier and Germida 2003; Scervino et

al. 2005). Therefore, it is believed that the presence of other

plants near their hosts will alter the response of the external

fungal hyphae to toxic Al3+ in situ.

The objective of this study was to examine if the presence

of neighboring plants could modify the growth response of

the external hyphae of G. margarita to the detrimental effects

of Al3+.

MATERIALS AND METHODS

The fungus used in this study was G. margarita Becker

& Hall (BEG collection), which is tolerant to and working

effectively under acidic soil conditions (Rohyadi et al. 2004).

The fungus was propagated in pot cultures of Trifolium

subterranean L. in a sand and soil (90:10, w/w) mixture for 4

months. Another pot without the fungus to provide

mycorrhiza-free inoculums was included. The cultivar of

cowpea used was Red Caloona supplied by CSIRO Tropical

Agriculture, Brisbane, Australia. The growth medium was a

mixture of sand and acidic podsolic soil of grey sandy loam

(90:10, w/w). The mixture was firstly fertilized with (in mg kg-

1 medium) 59.4 NH
4
-N, 178.2 NO

3
-N, 36 P, 54 S, 214.2 K, 18.9

Mg, 114.3 Ca, 13.5 Na, 8.1 Cl, 2.7 Fe, 0.45 B, 0.45 Mn, 0.225 Zn,

0.036 Cu and 0.009 Mo. Then it was treated with 0, 75, 150 or

300 Al
2
(SO

4
)

3
. The concentrations of Al3+ (measured based

on the method of Close and Powell 1989) in the final

established media (A
0
, A

1
, A

2
or A

3
) were 0.4, 1.1, 4.1 or 7.3 mg

kg-1 soil respectively.

This experiment was conducted under glasshouse

conditions. It was a 2 x 3 x 2 factorial experiment consisting of

two levels of mycorrhiza with (M
1
) and without (Mo)

inoculation. Three concentrations of Al3+ established in soil

were 1.1, 4.1 and 7.3 mg kg-1 soil (denoted as A
1
, A

2
and A

3
),

and the presence and absence of neighboring plants were

denoted as N
1
and N

0
respectively. There were six replicates

per treatment combination.

Microbiol Indones 43Volume 3, 2009

The pot system used was made of a PVC pipe (Fig 1),

divided into three compartments by two vertical 30 µm screen

meshes that allow hyphae to pass but exclude plant roots.

The first compartment, the host plant compartment (HPC),

was for plants inoculated or not inoculated with mycorrhiza

fungal structures, designated as the host plants. They

supplied the external fungal hyphae being tested. The second

compartment in the middle of the pot, the external hyphal

compartment (EHC), was a root free compartment provided

for the external hyphae extending from mycorrhizal-colonized

roots of the host plants in the HPC. The third compartment,

the neighboring plant compartment (NPC), was for non-

inoculated plants, denoted as neighboring plants, which

acted as a growth promoter for the fungal hyphae to traverse

the EHC. In order to provide favorable conditions for plant

roots, the HPC and NPC were filled with growth medium not

treated with Al, A
0
(pH 5.3, containing 0.4 mg Al3+ kg-1 soil

and 26 ppm Bray-1 P), whereas the medium in EHC was treated

with Al at different concentrations. Therefore, the basic

purpose of using the compartmentalized pot system is to

allow the external fungal hyphae from mycorrhizal-colonized

roots of the host plants in the HPC to extend into the EHC,

interact with Al and possibly traverse to the NPC, and finally

colonize roots of neighboring plants there. The effects of the

Al3+ concentrations in the EHC and the presence of

neighboring plants in the NPC on the growth of the external

hyphae was determined by the extending of the external

hyphae in the EHC and the colonization of neighboring plants

in the NPC by the AM fungus.

At the beginning of the experiment, HPC and NPC were

filled with 320 g of A
0
soil, but only the HPC was inoculated

with 10% (w/w) of pot culture inoculums of G. margarita or

was left not-inoculated to provide mycorrhizal and non-

mycorrhizal colonized host plants. Two pre-germinated seeds

of cowpea (cv. Red Caloona) were transplanted into all of the

HPC and a half of the NPC, and then left grown for two weeks.

The EHC was then filled with 260 g of A
1
, A

2
or A

3
soils in the

same way. Reverse Osmotic (RO) water, adjusted to pH 4.6

with H
2
SO

4
, was used on a weight loss basis to maintain the

moisture content of the growth media at the field capacity

(about 0.1 g g-1 soil) and the pH of soil in the EHC, at about

4.7 throughout the experiment.

Plants were harvested 6 and 10 weeks (H
1
and H

2
) after

transplanting. The dry biomass and root length of the host

plants, the dry biomass of the neighboring plants, the

mycorrhizal colonization on the roots of these plants, and

the length density of the external fungal hyphae in the EHC

were measured. Plant biomass was weighed after it was dried

at 70 oC for 48 hours. To measure root length and percentage

of mycorrhizal colonization, root samples were cleared with

10% KOH, and stained with trypan blue in lacto-glycerol and

then assessed under a dissecting microscope using a gridline

intersect method (Giovannetti and Mosse 1980). To measure

length density of the external fungal hyphae in the HPC,

representative samples of the growth media were taken from

the compartment. The hyphae in the soil samples were then

extracted following the aqueous membrane-filtration method

of Jakobsen et al. (1992), and assessed under microscopic

observation (see detail in Rohyadi 2006).

The data were statistically analyzed using ANOVA after

grouping them into plant compartments and harvest times.

The LSD tests at p < 0.05 were then applied to determine the

significant differences among ways of treatment.

RESULTS

Growth of Host Plant. Dry biomass of the host plants,

measured at any harvest was not affected by the Al3+

concentration in the EHC or by the presence (N
1
) and the

absence (N
0
) of neighboring plants in the NPC, but was

significantly affected by mycorrhizal inoculation. The dry

biomass of mycorrhizal (M
1
)-plants was significantly higher

than that of non-mycorrhiza (M
0
) (Table 1). Similarly,

mycorrhizal inoculation significantly increased the root length

of the host plants in the HPC regardless of the presence or

absence of neighboring plants. Aluminum concentration and

the presence of neighboring plants had no significant effect

on the growth of roots irrespective of the mycorrhizal status

of plants.

Mycorrhizal Colonization on Host Plants. Plants not

inoculated with AM fungus exhibited no evidence of AM

fungal colonization, while the extent of roots colonized by

AM fungus and the root length of host plants were stimulated

by inoculation (Table 1). In general, mycorrhizal colonization

increased from the first to the second harvest. However, the

increases were not related to the Al3+ concentration in the

EHC. The presence of neighboring plants reduced mycorrhizal

colonization observed in the HPC at the second but not at

the first harvest.

The effect of Al3+ concentration and neighboring plants

on the length of the mycorrhizal colonized roots in the HPC

was nil at the first harvest. At the second harvest, the adverse

effect of Al3+ on the mycorrhizal-colonized root length was

dependent on the presence or absence of neighboring plants.

It was found that the presence of neighboring plants was

more detrimental than that the adverse effect of Al3+ on the

root colonization by the fungus.

H
o

s
t

p
la

n
t

c
o

m
p

a
rt

m
e
n

w
it

h
t

h
e
f

u
n

g
a
l

in
o

c
u

lu
m

E
x

te
rn

a
l

h
y

p
h

a
l

c
o

m
p

a
rt

m
e
n

w
it

h
d

if
fe

re
n

t
A

l-
tr

e
a
te

d
s

o
il

N
e
ig

h
b

o
ri

n
g

p
la

n
t

c
o

m
p

a
rt

m
e
n

1
2

c
m

3.5 cm 2.0 cm 3.5 cm

Fig 1 Schematic representation of the compartmentalized pot

system. The compartments are separated by two vertical 30 µm

nylon screen meshes ( ) that can be crossed by fungal hyphae

but not by plant roots.

Microbiol Indones44 ROHYADI

Growth of External Hyphae. The growth of external fungal

hyphae in the EHC increased with harvest times; the extent

was dependent on the Al3+ concentration and the presence

of neighboring plants in the NPC. However, there was no a

significant interaction between the two factors. The hyphal

length density (HLD) decreased with increased Al3+

concentration, but the adverse effect of Al3+ was nullified by

the presence of neighboring plants (Fig 2).

Mycorrhizal Colonization and Growth of Neighboring

Plant. Table 2 clearly shows the fungal hyphae, which traverse

the EHC, were able to definitely colonize roots of plants in

the NPC. At H
1
, the colonization was less than 20%, but

increased to more than 40% at H
2
. It also shows that different

Al3+ concentrations in the EHC affect the colonization rates

differently. At H
1,

compared to the root colonization in pots

with A
1
, a significant reduction in the percentage of colonized

roots only occurred in pots with A
3
; meanwhile, at H

2
such

reduction was already evident in pot with A
2
. In general, the

growth of the neighboring plants increased, but was not

related to soluble-Al concentration in the EHC. Differences

in plant growth occurred between plants in pots inoculated

with (M
1
) and without mycorrhiza (M

0
). The dry biomass of

the neighboring M
1
-plants was higher than that of the

neighboring M
0
-plants regardless of the Al3+ concentration

in the EHC, even though it was only significant at H
2

(Table 2).

DISCUSSION

The results of this study showed that the toxicity of high

Al3+ concentrations in soil solution to the external hyphae of

G. margarita was evident. The growth of the hyphae was

depressed particularly when neighboring plants were absent.

This supports and extends previous results of work in vitro

indicating the inhibitory effects of Al3+ on spore germination,

germ tube elongation (Siqueira et al. 1985), and root

colonization of the fungus (Rohyadi 2005). On the other hand,

in pots with neighboring plants the growth of the external

fungal hyphae considerably increased (Fig 2), which

corresponds with the decrease in percentage of the

mycorrhizal colonization on roots of their host plants (Table

1). This suggests the importance of the presence of

neighboring plants for the growth of external hyphae of the

fungus. Previously, Mummey et al. (2005) showed the

Table 1 Growth and AM fungal colonization on roots of host plants in the HPC in response to mycorrhizal inoculation, Al3+ concentration in

the EHC and the presence of neighboring plants in the NPC

N o t

inoculated

Inoculated

1 . 1

4 . 1

7 . 3

1 . 1

4 . 1

7 . 3

N
0

N
1

N
0

N
1

N
0

N
1

N
0

N
1

N
0

N
1

N
0

N
1

Treatments

245 a 441b

272 a 460b

260 a 411b

248 a 419b

249 a 361b

235 a 392b

311 a 829 a

327 a 815 a

341 a 792 a

345 a 814 a

361 a 797 a

325 a 745 a

Plant dry

biomass (mg)

627 b 1202 b

706ab 1107b

682 b 1072 b

647 b 1049 b

657 b 1026 b

639 b 1035 b

876a 1420a

849 a 1488 a

872 a 1535 a

829 a 1483 a

882 a 1496 a

791 a 1458 a

Root length(cm)

0 0

42a 77a

46a 57b

50a 76a

42a 56b

48a 73a

42a 51b

Root

colonization

(%)

Mycorrhizal

root length (cm)

0 0

368a b 1093 a

391a b 848 b

436 a 1167 a

349 b 830 b

423 a 1092 a

332 b 744 c

Neighboring plants

in the NPC

mg Al3+ kg-1 soil

in the EHC

Mycorrhizal

inoculation

in the HPC

aMeans within a selected column followed by different superscripts are significantly different based on the LSD-test at p ≤ 0.05. HPC: host plant

compartment; EHC: external hyphal compartment; NPC: neighboring plant compartment.H
1
and H

2
: harvest at 6 and 10 weeks after

transplanting.

H
1

H
2 H

1
H

2
H

1
H

2
H

1
H

Table 2 Dry biomass and root colonization of the neighboring

plants relating to mycorrhizal inoculation in the HPC and Al 3+

concentrations in the EHC observed at two harvest times

Treatments

Mycorrhizal

inoculation

in the HPC

Not inoculated

mg Al3+ kg-1 soil

in the EHC

210 a 382 b

210 a 399 b

195 a 397 b

255 a 509 a

247 a 532 a

242 a 589 a

0 0

18a 59a

17a 48b

8b 39bc

Root

Colonization

(%)

Plant dry

biomass (mg)

1 . 1

4 . 1

7 . 3

1 . 1

4 . 1

7 . 3

Inoculated

H
1

H
2

H
1

H
2

aMeans within a selected column followed by different superscripts

are significantly different based on the LSD-test at p < 0.05; HPC:

host plant compartment; EHC: external hyphal compartment; H
1

and H
2
: harvest at 6 and 10 weeks after transplanting.

H
y

p
h

a
l

le
n

g
th

(
c
m

g
-1

s
o

il
) 1 2 0 0

9 0 0

6 0 0

3 0 0

0
N 0 N 1

Harvest-1 Harvest-2
N 0 N 1

Fig 2 The influence of Al3+ concentrations and neighboring plants

on hyphal length density of Gigaspora margarita in the external

hyphal compartment (Bars represent means ± sem, n=3); o, A
1
; n, A

2
,

and , A
3
are growth media with 1.1, 4.1 and 7.3 mg Al3+ kg-1 soil; N

and N
0
are pots with and with no neighboring plants.

Microbiol Indones 45Volume 3, 2009

significance of neighboring plants on the diversity and

community composition of the AM fungi in the field.

Most studies on the interaction of AM fungi and plant

roots have been carried out using in vitro systems. The

significant effects of the presence of plant roots on hyphal

growth have been demonstrated. Sbrana and Giovannetti

(2005) showed a chemotropism in elongation of germ tube of

Glomus mossea in response to the presence of plant roots. It

seems that the plant roots produced influential stimuli for the

growth of the AM fungal hyphae. Root exudates, in this case,

play a key role during the pre-symbiotic phase. For instance,

it was reported that hyphal growth of G. margarita was

stimulated by root exudates of onion (Tawaraya et al. 1996)

and tomato (Scervino et al. 2005). Also, a component of root

exudates of Lotus japonicum, identified as strigolactone,

induced an extensive branching of the fungal hyphae at very

low concentration (Akiyama et al. 2005). In general, some

phenolic and flavonoid compounds, and/or volatile

metabolite exuded by roots of mycotrophic plants have been

recognized as chemical stimuli for hyphal elongation and

branching (Nagahashi and Douds 2003; Akiyama et al 2005),

or as a chemical signal leading the hyphae to grow forward

(Vierheilig et al. 1998; Steinkellner et al. 2007). Therefore, in

the present study in situ it was possible that some eliciting

factors produced by roots of the neighboring plants have

triggered most of the external hyphae of the fungus to grow

out of the HPC, and so it left a small number of the fungal

inoculums to initiate new root colonization in the plant

compartment.

Moreover, in this study it was found that colonization of

the roots of neighboring plants by the fungal hyphae

traversing the EHC reached about 40% (Table 2), suggesting

that exposure to increasing Al3+ concentrations did not really

affect the function of the external hyphae of G. margarita as

inoculums. The growing hyphae remained viable to initiate

new colonization. This is very important for field practice

since spores begin most colonization by this fungus. Other

results on the increased growth of neighboring plants (Table

2) also indicate that the fungal hyphae did not suffer from the

loss of their ability to improve the growth of cowpea plant

after being exposed to high concentrations of Al3+, even

though there was a delay in seeing a significant contribution

to the plants.

To summarize, the results of this study have broadened

our understanding on eco-physiological aspects of AM

symbiosis in acid soils particularly with problems of Al

toxicity. It is clear that increasing concentrations of soluble-

Al might directly reduce the growth rate of the external hyphae

of G. margarita in soil, but functions of the hyphae as

inoculums and as an extension of the plant root system were

not really affected. Meanwhile, the presence of the roots of

neighboring plants stimulated the external hyphae to grow

further, irrespective of the toxic Al3+ in soil. These results, in

general, suggest that plants need more AM symbioses to

grow better particularly under adverse soil conditions, which

is comparable to the requirement of the AM fungi themselves

to get carbon from the plants. Therefore, from this point of

view, developing a suitable cropping system is essential for

empowering AM fungi to be persistent and be able to

continually develop their external hyphae. Further studies

however are needed to investigate deep mechanisms and

other soil factors involved in triggering the growth of the

fungal hyphae under regimes of different mycotrophic crop

species, and of adverse soil conditions.

ACKNOWLEDGEMENTS

I wish thank Sally S Smith, F Andrew Smith, and Rob S

Murray (The University of Adelaide, Waite Campus, The

South Australia) for their help in many ways thoughout this

work. I also thank AusAID Scholarship, Australia for the

financial support.

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