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Plant and Soil 190: 41–46, 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. Rodr´ıguez-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 world’s 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 Uexk¨ull 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 Uexk¨ull and Mutert, 1995). Aluminium is the third most abundant element in the earth’s 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
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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 Rodr´ıguez-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
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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 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 Student’s 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 Student’s 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).
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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 Student’s t-test. **Significantly different from the inoculated control (pH 4; 0 M Al) at p 0:01 according to Student’s 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
n
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 Student’s t-test. **Significantly different from the inoculated control (pH 4; 0 M Al) at p 0:01 according to Student’s 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
n
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
h
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 Student’s t-test. **Significantly different from the inoculated control (pH4; 0M Al) at p 0:01 according to Student’s t-test.
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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 Rodr´ıguez-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 Gonz´alez 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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