The Effects of Al on Nodulation and Nitrogen Fixation in Casuarina

8/10/2019 The Effects of Al on Nodulation and Nitrogen Fixation in Casuarina

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Plant and Soil 190:4146, 1997. 41c

1997Kluwer Academic Publishers. Printed in the Netherlands.

The effects of aluminium on nodulation and symbiotic nitrogen fixation inCasuarina cunninghamianaMiq.

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:1Department 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, nodu-

lation

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 cov-

ered predominantly by coniferous forests; another in

the humid tropics occupied by savanna and tropical

rainforest (Von Uexkull and Mutert, 1995). Further-

more, 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

FAX No:+12172443469. E-mail:[email protected]

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

1978; Kochian, 1995). Soluble Al reduces plant growthbecause 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 (Grab-

ski and Schindler, 1995), and its binding to nucleic

acids inhibits cell division (Matsumoto et al., 1977;

Morimura et al., 1978). Certain plant species or geno-

types show resistance to Al achieved in two differentways: Al exclusion or Al tolerance. In the process of

exclusion, Al is immobilized outside the plant by com-

plexation 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


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Al cation is tolerated within the symplasm (Kochian,

1995).

With respect to the symbioses betweenRhizobium-

Bradyrhizobiumand legumes, Al has been shown to

adverselyaffect the processof nodulation throughinhi-

bition of root hair formation and nodule initiation (Flis

et al., 1993). The susceptibility of the symbiotic rela-tionshipsof actinorhizal plants to damageby 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 Casuar-

inaceae family arerecognized as versatile species capa-

ble 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 pos-

sess root nodules caused by the nitrogen-fixing acti-

nomycete Frankia and hence are self-sufficient with

regard to N nutrition. For these reasons, they are suc-

cessfully 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 fromtemperate to tropical,

and it is able to tolerate up to 50 light frosts per year

(NRC, 1984). Casuarina cunninghamiana is exten-

sively 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 betweenFrankiaandC.

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) weresurface-sterilized for 10 minutes using 5% sodium

hypochlorite, and then repeatedly washed with ster-

ile, 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/nightcycle 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 wateredwith 10 mL of sterile distilled water, in order to elim-

inate 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, Mg148,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 6H2O

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 sus-

pension. The suspension was prepared with fresh and

healthy nodules less than 7 mm in diameter obtained

directly from plants of C. cunninghamianagrown 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 wasapplied 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 inocu-

lation, 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 solu-

tions 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 reduc-

tion 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 digest-

ed using the Kjeldahl method, and total N determined


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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 20min at 25 C in hermetically sealed flasks (310 mL)

containing 10% v/v acetylene. A flask without acety-

lene 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 tem-

perature 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 growth and nitrogen concentration

Nitrogen fixation by the inoculated plants was reflect-

ed in the dry weight of the plants (Table 1). Inoculat-

ed control plants had about 43 times greater total dry

weight than uninoculated plants.

Only at Al concentrations at or above 440 M

wasthere 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 concentra-

tions relative to the Al-free treatment. Shoot and total

plant N concentrations were 78% and 84% of the con-

trol, respectively (Table 2). The treatment of 880 M

Al yielded mean N concentrations of shoot and whole

plant less than those at the 440 M

Al level (Table

2). However, the reduction in N concentration relative

to the control, according to Studentst

-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

C2H4 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 nod-

ule 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 were80% and 50% of the control, respectively.

Discussion

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

nodule formation and function in actinorhizal sym-

bioses. There are numerous papers on this subject

for thesymbiosesbetweenRhizobium-Bradyrhizobium

and legumes.

The Al concentrations mentioned in this article are

nominal values. There could have been considerable

precipitationof aluminium phosphate, as a result of the

high P concentration in the nutrient solution. At 880

M

Al, the summed concentration of Al monomers

Al3+ , Al(OH)+2 and Al(OH)2+ was estimated to be

279 M

, according to the chemical speciationprogram

GEOCHEM-PC (Parker et al., 1996).


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44

Table 1. Effect of Al concentration on shoot, root, nodule and total dry weight of Casuarina cunninghamiana32 weeks after the

commencement of Al treatment (values are means of 8 replicates S.E.)

Nodulated/

Treatment nonnodulated Dry weight (g plant 1 ) Nodule weight as

pH M Al Inoculationa Ratio Shoot Root Total % of total weightb

4 0 - 0 /8 0.05

0.01** 0.04

0.01** 0.09

0.02**

6 0 + 8 / 8 3.68 0.72 1.34 0.35 5.36 1.03 6.26 0.80

4 0 + 8 / 8 2.39

0.57 1.15

0.28 3.83

0.90 7.67

0.58

4 110 + 8 / 8 3.10 0.44 1.25 0.16 4.72 0.60 8.07 0.89

4 220 + 8 / 8 2.71

0.47 1.31

0.25 4.28

0.73 6.46

0.64

4 440 + 7 / 8 0.44 0.13* 0.21 0.07* 0.71 0.22* 7.18 0.53

4 880 + 4 / 8 0.13

0.05** 0.07

0.01** 0.21

0.07** 8.54

2.19

a +inoculated, -uninoculated.b Nodulated plants only.

*Significantly different from the inoculated control (pH 4; 0 M

Al) atp

005 according to Studentst

-test.

**Significantly different from the inoculated control (pH 4; 0 M

Al) atp

001 according to Studentst

-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.)

Nodulated/

Treatment Nonnodulated N concentration (%)

pH M

Al Inoculationa Ration

Shoot Root Nodules Total

4 0 - 0 / 8 8 0.81

0.06** 0.82

0.07 0.81

0.03**

6 0 + 8 / 8 8 1.63

0.11 1.01

0.09 2.01

0 .05* 1.49

0.11

4 0 + 8 / 8 8 1.69

0.07 0.94

0.07 1. 83

0.06 1.47

0.05

4 110 + 8 / 8 8 1.74

0.10 0.87

0.04 1. 98

0.09 1.53

0.09

4 220 + 8 / 8 8 1.60 0.12 0.97 0.05 1. 88 0.16 1.43 0.08

4 440 + 7 / 8 7 1.31

0.06** 0.85

0.05 2. 05

0.09 1.23

0.05**

4 880 + 4 / 8 4 1.16 0.25 1.04 0.17 1. 66 0.15 1.15 0.22

a +inoculated; -uninoculated.

*Significantly different from the inoculated control (pH 4; 0 M Al) at p 005 according to Students t -test.

**Significantly different from the inoculated control (pH 4; 0 M Al) at p 001 according to Students t -test.

Table 3. Effect of Al concentration on N2fixation ofCasuarina cunninghamianaas estimated by three different methods (mean

S.E.)

Nodulated/

Treatment nonnodulated Estimated N fixedb ARA nodules ARA plant

pH M

Al Inoculationa Ration

(mg N mg nodule d.wt 1) (

mol C2H4 g d.wt 1 h 1) (

mol C2H4 plant 1 h 1)

4 0 - 0 / 8

6 0 + 8 / 8 8 0.26

0.02 40.38

5.22 14.32

3.40

4 0 + 8 / 8 8 0.20 0.02 39.20 4.15 11.23 2.72

4 110 + 8 / 8 8 0.21

0.03 37.11

3.63 13.05

1.71

4 220 + 8 / 8 8 0.23

0.02 60.41

9.16 17.99

4.84

4 440 + 7 / 8 7 0.16

0.02 38.23

6.31 2.23

0.83*

4 880 + 4 / 8 4 0.10

0.02** 22.48

11.71 0.72

0.46**

a +inoculated; -uninoculatedb 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; 0 M Al) at p 005 according to Students t -test.

**Significantly different from the inoculated control (pH4; 0 M Al) at p 001 according to Students t -test.


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There was a significant difference (p

0:

05) in N

concentrationof nodules between Al-free treatments at

pH6 andpH 4 (Table2). The rateof 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 sug-

gest that the nodule efficiency was higher at pH 6. Nofurther 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. cun-

ninghamiana. In the rhizosphere, it is known that the

pH of the soil solution can be greatly altered to val-

ues 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 ofFrankia 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 tol-

erate some Al in the soil solution. When assayed in

vitro, the optimum growth ofFrankia 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 apparent-

ly exists between tolerance to acid pH and tolerance

to free Al3+

in the culture medium (Faure-Raynaud etal., 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; Hecht-

Buchholz et al., 1990). As do most actinorhizal plants,

Frankia gains entry into Casuarina via root hairs (Sub-

barao and Rodrguez-Barrueco, 1995). Therefore, apossible detrimental effect of Al on root hair devel-

opment might explain how Al impairs nodulation in

Casuarina.

Two estimates of nodular nitrogenase activity

(Table 3) were done in order to avoid the uncertain-

ty 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 estima-

tion. 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 moder-

ate Al concentrations,the high noduledry weight (nod-

ule weight as % of total dry weight, Table 1) and the

efficiency of N2-fixation (Table 3) indicate that a highly

effectiveC. cunninghamiana-Frankia association was

achieved. It is therefore important for the successful

introduction ofC. cunninghamiana to acid soils high in

soluble Al that tolerant genotypes are inoculated with

the most effective nitrogen-fixing Frankia. Casuari-

na cunninghamianaappears 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 sampleanalyses, 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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Section editor: F R Minchin

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The Effects of Al on Nodulation and Nitrogen Fixation in Casuarina