41

Plant and Soil 190: 4146, 1997.

c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

The effects of aluminium on nodulation and symbiotic nitrogen fixation in

Casuarina cunninghamiana Miq.
J.M. Igual1 , C. Rodrguez-Barrueco and E. Cervantes
Instituto de Recursos Naturales y Agrobiologia, C.S.I.C. Apdo 257, 37071 Salamanca, Spain. Present address:
1
Department of Natural Resources and Environmental Sciences, 1025 Plant Sciences Lab., 1201 South Dorner
Drive, University of Illinois, Urbana, IL 61801-47783, USA
Received 4 September 1996. Accepted in revised form 21 January 1997

Key words: actinorhizal plants, aluminium toxicity, Casuarina cunninghamiana, Frankia, nitrogen fixation, nodulation
Abstract
In order to investigate the effects of Al on nodule formation and function in the Casuarina-Frankia symbiosis,
inoculated plants were grown in sand culture at five nominal Al concentrations (0-880 M Al) at pH 4.0. There
was an Al-free control at pH 6.0 to assess the effects of pH 4.0 treatments. Mean N concentration of nodules was
significantly less at pH 4.0 (1.83%) than at pH 6.0 (2.01%). There were nodulated plants at all Al levels, though
there were fewer nodulated plants at 440 and 880 M Al. Dry weights of nodules, shoots and roots were not
reduced by Al concentrations at or below 220 M Al, but were decreased by Al concentrations at or above 440 M
Al. Nodule weight expressed as a percentage of total weight did not differ significantly with respect to an Al-free
control at pH 4. N concentrations of shoots and whole plants were significantly reduced at 440 M Al. Nodular
specific acetylene reduction activity (ARA) did not differ significantly among Al treatments. However, N2 -fixation
efficiency was decreased from 0.20 to 0.10 mg N fixed mg nodule dry weight 1 at 880 M Al.
Introduction
Acid soils occupy approximately 30% of the worlds
ice free land area and occur mainly in two global belts:
one in the humid northern temperate zone that is covered predominantly by coniferous forests; another in
the humid tropics occupied by savanna and tropical
rainforest (Von Uexkull and Mutert, 1995). Furthermore, human activity is increasing soil acidification
throughout the world, especially in the developing
countries. The poor fertility of acid soils is due in part
to high H+ concentrations and, especially below pH 5,
to Al, Mn and Fe toxicity, and limited availability of
Ca, Mg, K and P (Von Uexkull and Mutert, 1995).
Aluminium is the third most abundant element in
the earths crust after oxygen and silicon. It is found
in soils predominantly as insoluble alumino-silicates
or oxides (Martin, 1988). In acid soils, Al (primarily
in the form of Al3+ ) is mobilized into soil solution

impairing the growth of most plant species (Foy et al.,

1978; Kochian, 1995). Soluble Al reduces plant growth
because its targeted action at the root apex (Ryan et al.,
1993) inhibits root growth. Although not completely
understood, several mechanisms have been proposed
to explain Al toxicity (for reviews, see Delhaize and
Ryan, 1995; Kochian, 1995). Aluminium can lower P
availability and block the normal uptake of Ca2+ and
Mg2+ causing an imbalance in plant mineral nutrition;
Al produces rigidity in the actin cytoskeleton (Grabski and Schindler, 1995), and its binding to nucleic
acids inhibits cell division (Matsumoto et al., 1977;
Morimura et al., 1978). Certain plant species or genotypes show resistance to Al achieved in two different
ways: Al exclusion or Al tolerance. In the process of
exclusion, Al is immobilized outside the plant by complexation with organic acids, such as malic and citric
acid, released from roots (Delhaize et al., 1993; Pellet
et al., 1995; Ryan et al., 1995). In tolerant plants the

FAX No:+12172443469. E-mail:igual@staff.uiuc.edu

*134280*

JS: PIPS No. 134280 BIO2KAP

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42
Al cation is tolerated within the symplasm (Kochian,
1995).
With respect to the symbioses between RhizobiumBradyrhizobium and legumes, Al has been shown to
adversely affect the process of nodulation through inhibition of root hair formation and nodule initiation (Flis
et al., 1993). The susceptibility of the symbiotic relationships of actinorhizal plants to damage by Al has not
been studied, though some research has been focused
on the role of pH in nodulation (Dixon and Wheeler,
1983).
Among the N2 -fixing trees, members of the Casuarinaceae family are recognized as versatile species capable of tolerating extreme environmental conditions
such as waterlogging, variation in soil pH, salinity
and drought. Some Casuarina species, such as C.
deplancheana, thrive in soils so rich in Al and Fe that
they are toxic to most plants (NRC, 1984). They possess root nodules caused by the nitrogen-fixing actinomycete Frankia and hence are self-sufficient with
regard to N nutrition. For these reasons, they are successfully used in the afforestation of unproductive soils
(Subbarao and Rodrguez-Barrueco, 1995). Casuarina
cunninghamiana is one of the largest of the casuarinas.
Native to eastern and northern Australia, this species is
adapted to climates varying from temperate to tropical,
and it is able to tolerate up to 50 light frosts per year
(NRC, 1984). Casuarina cunninghamiana is extensively planted in Argentina and neighboring countries
for windbreaks and to protect stream bands. In Hawaii,
it grows well on histosols (pH 5.0) developed over
acidic lava (NRC, 1984).
The present study was designed to determine the
effects of Al on the symbiosis between Frankia and C.
cunninghamiana, and thereby to test its potential for
the afforestation of acid soils.

Materials and methods

Plant material and stress treatments
Seeds of Casuarina cunninghamiana obtained from
native stands of trees in Queensland (Australia) were
surface-sterilized for 10 minutes using 5% sodium
hypochlorite, and then repeatedly washed with sterile, distilled water. After sterilization, the seeds were
sown in a plastic tray containing autoclaved perlite.
Seeds were germinated in a growth chamber at 28  C,
80% relative humidity, and with a day/night cycle of 16
h/8 h. One week after germination, the seedlings were

transferred to 50 mL sterile plastic tubes (one seedling

per tube) filled with acid washed sand. Each seedling
was watered every two days with 5 mL of a solution
containing (M ): Ca 1000, Cl 2025, Mg 370, S 372,
Na 455, P 330, EDTA 250, Fe 250, K 2520, N 2520,
Mn 1.25, Cu 0.25, Zn 0.25, B 12.5, Mo 0.125.
Three weeks after transfer, each tube was watered
with 10 mL of sterile distilled water, in order to eliminate nitrate, and the treatments were started with the
application of 5 mL of the appropriate solution without
combined nitrogen. The nutrient solution consisted of
(M ): Ca 400, Cl 1820, Mg 148, S 149, Na 182, P 132,
EDTA 100, Fe 100, K 1010, Mn 0.5, Cu 0.1, Zn 0.1,
B 5, Mo 0.05. Aluminium was added as AlCl3
6H2 O
and the solutions were adjusted to pH 4.0 or 6.0 with
HCl and NaOH.
Inoculation and culture of plants
Eighteen hours after the first Al application, the
seedlings were inoculated with crushed nodule suspension. The suspension was prepared with fresh and
healthy nodules less than 7 mm in diameter obtained
directly from plants of C. cunninghamiana grown in
a greenhouse for 3 years. Nodule material (6.5 g) was
surface-sterilized in 5% sodium hypochlorite for 20
minutes, rinsed repeatedly in sterile distilled water and
then ground to a paste in a mortar and pestle with 5 mL
sterile distilled water. The resultant suspension was
made up to 300 mL with sterile distilled water, and 5
mL of this suspension was added to each tube. For the
uninoculated tubes, 5 mL of sterile distilled water was
applied instead of the nodule suspension. The plants
were kept unwatered for 3 days following inoculation
to avoid loss of inoculum by watering, after which the
treatments were renewed with a volume of solution to
field capacity of the tubes. Twelve weeks after inoculation, individual plants were transferred to 2 L plastic
pots containing acid-washed sand and kept in a growth
room with a day/night cycle of 16 h/8 h (200 mE m 2
s 1 ) at 23  C and 17  C, respectively. Nutrient solutions containing the different Al concentrations were
applied in adequate volume to permit leaching of pots
every day until harvest at 32 weeks after inoculation.
At harvest, the plants were withdrawn from the pots
and the roots carefully washed with distilled water.
Roots were excised and used for the acetylene reduction assay, after which, roots, nodules and shoots were
dried at 80  C for 24 h and weighted. Samples of 50
mg of ground and dried plant material were digested using the Kjeldahl method, and total N determined

plso6553.tex; 4/06/1997; 16:18; v.7; p.2

43
using an Orion Research Ioanalyzer 901 equipped with
an ammonia electrode.
Nitrogenase activity
Nitrogenase activity was estimated by the acetylene
reduction assay. Excised roots were incubated for 20
min at 25  C in hermetically sealed flasks (310 mL)
containing 10% v/v acetylene. A flask without acetylene was also incubated for the assay of endogenous
ethylene. Ethylene was quantified using a Varian Gas
Chromatograph 2700 equipped with a hydrogen flame
ionization detector and one alumina column. High
purity N2 served as the carrier gas. The column temperature was 150  C. The endogenous production of
ethylene was negligible.
Statistical analysis
There were eight replicates per treatment and the
experiment was arranged in a completely randomized
design. The results of each treatment were compared
to the control (pH = 4,0 M Al, inoculated) using
a Students t-test according to Snedecor and Cochran
(1989).

Results
Although all plant mass and N concentration values
tended to be higher at pH 6 than at pH 4, only the
N concentration of the nodules differed significantly
(p  0:5) between these two pH levels.

plant less than those at the 440 M Al level (Table

2). However, the reduction in N concentration relative
to the control, according to Students t-test, was not
significant, probably because the degrees of freedom
were lower than at 440 M Al.
Nodulation and N2 -fixation
There were nodulated plants at all Al levels, though
the number of nodulated plants was reduced at 440
and 880 M Al. Seven of eight plants nodulated at
440 M Al, and only four at 880 M Al (Table 3).
The mean nodule dry weight as a percentage of total
dry weight was not significantly less (p  0:05) than
that of the Al-free, pH 4, control treatment (Table 1).
Specific nitrogenase activity (Table 3) did not differ
significantly (p  0:05) between any treatment and the
control, although at 220 M Al it was 54% greater
than that of the control (p = 0:062). At 880 M Al,
with only four nodulated plants, it decreased to 57%
of the control (p = 0:122). The acetylene reduction
activity (ARA), considered on a per plant basis (mol
C2 H4 plant 1 h 1 ), was significantly reduced at the
two highest Al concentrations (Table 3). At 440 M
Al ARA was only 20% of the control, and was only
6% that of the control at 880 M Al.
An additional estimation of N2-fixation was derived
by calculating the mg N in the plants per mg of nodule dry weight (Table 3). N2 -fixation estimated in this
manner remained fairly constant with increasing Al
concentrations up to 220 M Al. At 440 M Al and
880 M Al, the estimated N2 -fixation values were
80% and 50% of the control, respectively.

Plant growth and nitrogen concentration

Discussion
Nitrogen fixation by the inoculated plants was reflected in the dry weight of the plants (Table 1). Inoculated control plants had about 43 times greater total dry
weight than uninoculated plants.
Only at Al concentrations at or above 440 M
was there a significant reduction in C. cunninghamiana
dry weight (Table 1). The total dry weight at 440 and
880 M Al decreased to 19% and 5% of the control,
respectively.
At 440 M there was a significant reduction
(p  0:01) in shoot and whole plant N concentrations relative to the Al-free treatment. Shoot and total
plant N concentrations were 78% and 84% of the control, respectively (Table 2). The treatment of 880 M
Al yielded mean N concentrations of shoot and whole

This is the first published report on the effect of Al on

nodule formation and function in actinorhizal symbioses. There are numerous papers on this subject
for the symbioses between Rhizobium-Bradyrhizobium
and legumes.
The Al concentrations mentioned in this article are
nominal values. There could have been considerable
precipitation of aluminium phosphate, as a result of the
high P concentration in the nutrient solution. At 880
M Al, the summed concentration of Al monomers
2+
Al3+ , Al(OH)+
was estimated to be
2 and Al(OH)
279 M , according to the chemical speciation program
GEOCHEM-PC (Parker et al., 1996).

plso6553.tex; 4/06/1997; 16:18; v.7; p.3

44
Table 1. Effect of Al concentration on shoot, root, nodule and total dry weight of Casuarina cunninghamiana 32 weeks after the
commencement of Al treatment (values are means of 8 replicates S.E.)

pH

Treatment
M Al Inoculationa

4
6
4
4
4
4
4

0
0
0
110
220
440
880

Nodulated/
nonnodulated
Ratio

+
+
+
+
+
+

Dry weight (g plant

Root

Shoot

0 /8
8/8
8/8
8/8
8/8
7/8
4/8

0.05
3.68
2.39
3.10
2.71
0.44
0.13

 0.01**
 0.72
 0.57
 0.44
 0.47
 0.13*
 0.05**

0.04
1.34
1.15
1.25
1.31
0.21
0.07

1)

 0.01**
 0.35
 0.28
 0.16
 0.25
 0.07*
 0.01**

Nodule weight as
% of total weightb

Total
0.09
5.36
3.83
4.72
4.28
0.71
0.21

 0.02**
 1.03
 0.90
 0.60
 0.73
 0.22*
 0.07**

6.26
7.67
8.07
6.46
7.18
8.54

 0.80
 0.58
 0.89
 0.64
 0.53
 2.19

a +inoculated, -uninoculated.
b Nodulated plants only.
*Significantly different from the inoculated control (pH 4; 0 M Al) at p 0:05 according to Students t-test.
**Significantly different from the inoculated control (pH 4; 0 M Al) at p 0:01 according to Students t-test.




Table 2. Effect of Al concentration on N concentration in shoot, root, nodules and total N content of Casuarina cunninghamiana
32 weeks after the commencement of Al treatment (mean S.E.)

Treatment
M Al Inoculationa

pH
4
6
4
4
4
4
4

0
0
0
110
220
440
880

Nodulated/
Nonnodulated
Ratio

0/8
8/8
8/8
8/8
8/8
7/8
4/8

8
8
8
8
8
7
4

+
+
+
+
+
+

N concentration (%)
Root
Nodules

Shoot
0.81
1.63
1.69
1.74
1.60
1.31
1.16

 0.06**
 0.11
 0.07
 0.10
 0.12
 0.06**
 0.25

0.82
1.01
0.94
0.87
0.97
0.85
1.04

 0.07
 0.09
 0.07
 0.04
 0.05
 0.05
 0.17

2.01
1.83
1.98
1.88
2.05
1.66

Total
0.81
1.49
1.47
1.53
1.43
1.23
1.15

 0.05*
 0.06
 0.09
 0.16
 0.09
 0.15

 0.03**
 0.11
 0.05
 0.09
 0.08
 0.05**
 0.22

a +inoculated; -uninoculated.
*Significantly different from the inoculated control (pH 4; 0 M Al) at p 0:05 according to Students t-test.
**Significantly different from the inoculated control (pH 4; 0 M Al) at p 0:01 according to Students t-test.




Table 3. Effect of Al concentration on N2 fixation of Casuarina cunninghamiana as estimated by three different methods (mean

 S.E.)

Nodulated/
Treatment
nonnodulated
pH M Al Inoculationa
Ratio

4
6
4
4
4
4
4

8
8
8
8
7
4

0
0
0
110
220
440
880

+
+
+
+
+
+

0/8
8/8
8/8
8/8
8/8
7/8
4/8

Estimated N fixedb
(mg N mg nodule d.wt

0.26
0.20
0.21
0.23
0.16
0.10

1)

ARA nodules
(mol C2 H4 g d.wt 1 h

 0.02
 0.02
 0.03
 0.02
 0.02
 0.02**

40.38
39.20
37.11
60.41
38.23
22.48

 5.22
 4.15
 3.63
 9.16
 6.31
 11.71

1)

ARA plant
(mol C2 H4 plant

1)

 3.40
 2.72
 1.71
 4.84
 0.83*
 0.46**

14.32
11.23
13.05
17.99
2.23
0.72

a +inoculated; -uninoculated
b Estimated mg N fixed per mg nodule dry weight calculated as :
total N content of inoculated plant - N content of uninoculated plant
total nodule dry weight per plant




*Significantly different from the inoculated control (pH4; 0M Al) at p 0:05 according to Students t-test.
**Significantly different from the inoculated control (pH4; 0M Al) at p 0:01 according to Students t-test.

plso6553.tex; 4/06/1997; 16:18; v.7; p.4

45
There was a significant difference (p  0:05) in N
concentration of nodules between Al-free treatments at
pH 6 and pH 4 (Table 2). The rate of N2 -fixation (Table
3) was higher and the nodule weight/total weight ratio
(Table 1) was lower at pH 6 than pH 4. Although these
differences were not statistically significant, they suggest that the nodule efficiency was higher at pH 6. No
further significant differences (p  0:05) were found
between Al-free treatments at pH 6 and pH 4; thus,
solutions as acid as pH 4 did not significantly impair
growth, nodulation and nitrogen fixation in C. cunninghamiana. In the rhizosphere, it is known that the
pH of the soil solution can be greatly altered to values more suitable for plant growth (Marschner, 1995).
The utilization of unbuffered solutions and sand as
substrate could reflect more accurately what happens
in the plant-soil system, and it can explain why there
were not more appreciable differences due to pH.
There were nodulated plants at all Al levels. This
indicates that the growth of Frankia in the rhizosphere,
infection and nodule development occurred at low pH
with Al concentrations up to 880 M Al. The presence
of nodulated actinorhizal plants on acid soils (Dixon
and Wheeler, 1983) indicates that Frankia can grow
saprophytically on these soils; therefore, they may tolerate some Al in the soil solution. When assayed in
vitro, the optimum growth of Frankia is achieved at
pH values near to neutrality (Burggraaf and Shipton,
1982; Murry et al., 1984). However, some Frankia
strains can grow at pH 4.6, and a correlation apparently exists between tolerance to acid pH and tolerance
to free Al3+ in the culture medium (Faure-Raynaud et
al., 1986).
Nodulation was noticeably reduced at 880 M Al,
where only 50% of the plants were nodulated (Table
3). In legumes nodulation has often been shown to be
affected by Al. In addition to its effects on molecular
interactions between rhizobia and plants (Richardson
et al., 1988a, 1988b) it has been suggested that the
reduction caused by Al in root hair formation might
lessen nodulation (Brady et al., 1993, 1994; HechtBuchholz et al., 1990). As do most actinorhizal plants,
Frankia gains entry into Casuarina via root hairs (Subbarao and Rodrguez-Barrueco, 1995). Therefore, a
possible detrimental effect of Al on root hair development might explain how Al impairs nodulation in
Casuarina.
Two estimates of nodular nitrogenase activity
(Table 3) were done in order to avoid the uncertainty associated with the closed acetylene reduction assay
(Minchin et al., 1994; Vessey, 1994; Winship and Tjep-

kema, 1990). Both methods showed similar trends, but

ARA values were higher with respect to the control
than was the N2 -fixed per unit nodule mass estimation. A relatively high ARA value at 220 M Al (54%
over the control) is at odds with plant dry weight and
total nitrogen content. The estimation of N2 -fixed had
higher correlation coefficients with total dry weight (r
= 0.62) and total nitrogen content (r = 0.60) than with
nodular ARA (r = 0.45 and r = 0.41, respectively).
Therefore, the estimation of N2 -fixed per unit nodule
mass is a better estimator of nitrogen fixed.
An effective Casuarina-Frankia association is
characterized by high nodule weight and high nitrogen
fixation activity (Reddell and Bowen, 1985). At moderate Al concentrations, the high nodule dry weight (nodule weight as % of total dry weight, Table 1) and the
efficiency of N2-fixation (Table 3) indicate that a highly
effective C. cunninghamiana-Frankia association was
achieved. It is therefore important for the successful
introduction of C. cunninghamiana to acid soils high in
soluble Al that tolerant genotypes are inoculated with
the most effective nitrogen-fixing Frankia. Casuarina cunninghamiana appears to be a good species for
the selection of Al resistant host and endosymbiont
genotypes in order to obtain suitable combinations for
reclamation of acid soils.

Acknowledgements
The authors wish to thank M V Sevillano Gonzalez for
technical assistance in pot experimentation and sample
analyses, and Professor J O Dawson, University of
Illinois at Urbana-Champaign, for manuscript reading
and valuable discussions. We thank Dr D R Parker,
University of California, Riverside for supplying the
GEOCHEM-PC program. This research was supported
by European Union (Program STD-II)

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