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Keywords:

  • actinorhizal;
  • Hippophaë rhamnoides;
  • Frankia;
  • nitrogen;
  • nodulation;
  • phosphorus;
  • split-root;
  • nutrient interactions

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  •  The effects of N (ammonium nitrate), P (phosphate) and their interactions on nodulation were studied in the intercellularly infected actinorhizal plant Hippophaë rhamnoides.
  •  A split-root design, with pots receiving different concentrations of N and P, was used to determine whether the effects of N and P are local or systemic, if they are specific to nodulation or general, and whether P could counteract N inhibition. H. rhamnoides plants were grown for 6–10 wk after inoculation with Frankia.
  •  Inhibition of nodulation by N was systemic for both nodule number and nodule biomass in H. rhamnoides.
  •  By contrast, high P had a systemic stimulation on nodule number and biomass and P prevented systemic, but not local, N inhibition. Stimulation by P was specific to nodulation and not simply mediated via plant growth. Whether N and P alter not only nodulation but also N2-fixation in the nodules requires further investigation.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Hippophaë is one of the 25 genera (eight families) of actinorhizal plants forming N2-fixing root nodules when infected with the actinomycete Frankia (Huss-Danell, 1997). Depending on plant genus, actinorhizal plants can be infected by Frankia in one of two ways: either root hair infection or intercellular penetration (Berry & Sunell, 1990). Hippophaë is infected intercellularly (Miller & Baker, 1985a). In the plant, Frankia is always surrounded by the host cell membrane and by the capsule, a modified plant cell wall (Huss-Danell, 1990).

As in legumes (Streeter, 1988), N (nitrogen) inhibits nodulation and N2-fixation in actinorhizal plants (Huss-Danell, 1997). Nitrate strongly inhibited nodule biomass and nitrogenase activity in Hippophaë rhamnoides and Coriaria arborea (Bond & Mackintosh, 1975). Split-root cultures have been used to distinguish between local and systemic effects of N in actinorhizal plants as well as in legumes and have shown that nodulation, measured as nodule number per plant was inhibited locally by nitrate (Pizelle, 1965; Carroll & Gresshoff, 1983; Arnone et al., 1994), while nodule biomass was inhibited systemically by nitrate (Carroll & Gresshoff, 1983; Arnone et al., 1994).

Other nutrients also affect nodulation. Phosphorus (P) has been shown to increase plant growth and stimulate nodulation in actinorhizal plants (Quispel, 1958; Sanginga et al., 1989; Ekblad & Huss-Danell, 1995; Yang, 1995; Reddell et al., 1997) as well as in legumes (Gates, 1974; Gates & Wilson, 1974; Robson et al., 1981; Jakobsen, 1985; Israel, 1987, 1993; Hellsten & Huss-Danell, 2000). However, the effects of P on nodulation and nitrogenase activity were often ascribed to a general stimulation via plant growth (Robson et al., 1981; Jakobsen, 1985; Yang, 1995;Reddell et al., 1997). On the other hand, a specific stimulation of nodulation by P was found in soybean (Israel, 1987) and in Trifolium pratense (Hellsten & Huss-Danell, 2000). When all six macronutrients (N, P, K, S, Mg, Ca) were varied in a multivariate study of N2 fixation in Alnus incana, P had an especially strong effect on nodule biomass and N2 fixation (Ekblad & Huss-Danell, 1995). The ratio between N and P in the nutrient solution was important for nodulation in Alnus incana and in Trifolium pratense (Wall et al., 2000). These plants are representatives of those infected through root-hairs. Much less is known about nutrient effects on symbioses with an intercellular infection pathway. The aims of this work were therefore: to study the effects of N and P and the interactions between N and P on nodulation; to distinguish between local and systemic effects of the two macronutrients on nodulation; and to distinguish between specific effects on nodulation and general effects (exerted via plant growth) in Hippophaë rhamnoides.

Hippophaë rhamnoides is a multipurpose plant that has been exploited in Asia and East Europe for many years (Li & Schroeder, 1996) and is now receiving increased interest in the western world. Its berries are very rich in vitamin C and carotenes. The seed oil is rich in unsaturated fatty acids and is used as an ingredient in cosmetics, phytopharmaceuticals or UV skin protectants (Beveridge et al., 1999). Like many other actinorhizal plants it is used as an ornamental plant and in soil restoration.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material, split-root design and growth conditions

Seeds of Hippophaë rhamnoides (L.) were of mixed Swedish geographical origin. They were mixed with gravel (1 : 1 v/v), immersed in ethanol (70% v/v) and shaken for 15 min, then rinsed in water and shaken for a further 15 min in hydrogen peroxide (30% v/v) followed by several rinses in water. Seeds were then sown in Petri dishes containing perlite and moistened with modified Evans solution (Huss-Danell, 1978) diluted to 1 : 10 of full strength and with 0.71 mM of N added as ammonium nitrate. All material and solutions were sterile. Seedlings grew for c. 3 wk before transfer to 6 × 6 cm pots carefully cleaned with perlite. During the next 5 wk, the concentration of N in the nutrient solution was increased from 0.71 to 2.1 mM (3.5 mM in experiment 1) to support growth of the seedlings.

When the seedlings were 8-wk-old they were transplanted into split-root pots taking care to divide the root system into two parts as similar as possible. Two 8 × 8 cm square pots were taped together in order to make split-pots. A slot of c. 1 cm was made in the middle wall to support the plant at the point where the roots were divided. The surface of the perlite was a few mm below this slot. Approx. 1 wk later the N concentration of the nutrient solution was returned to 0.71 mM in experiments 1 and 2.

During the whole growth period (mid-December to mid-May in experiment 1, late March to mid-July in experiments 2 and 3) the plants were maintained in a glasshouse in Umeå, Sweden (63°45′ N). They received 17 h of supplemental light (Philips HPI/T 400 W lamps) at c. 25°C and 7 h of darkness at c. 15°C. Relative humidity was approx. 40%.

Frankia and inoculation

Seedlings were inoculated when 10-wk-old-with Frankia strain E15b grown in K-medium (Lumini & Bosco, 1996) for 2.5 wk. Cells harvested by centrifugation were suspended in modified Evans solution (Huss-Danell, 1978) diluted to 1 : 10 of full strength and with 0.71 mM of N added as ammonium nitrate. Approx. 9 mg (wet weight) of Frankia cells were added to the base of each root system.

N and P treatments

Treatments with different N and P concentrations began at the time of inoculation in experiments 1 and 2 but 2 wk before inoculation in experiment 3 (pretreatment). N was added as ammonium nitrate while P (as K-phosphate) was used at two different concentrations in the modified Evans solution (Table 1). The N : P ratio (Table 1) varied from 0.71 to 71. Plants were watered 3–4 times a week with such a large volume that a surplus of nutrient solution drained through all pots. Pots were suspended on a large mesh-size net so that no transfer of solutions could occur from one side to the other in a split-pot. At the start of each experiment, plants had similar shoot height in all treatments. Experiment 1 lasted for 10 wk after inoculation and each of the six treatments involved 7–10 plants (Table 1). Experiments 2 and 3 lasted for almost 6 wk after inoculation, and each of the seven treatments (Table 1) involved 10 plants. The pH of the solutions was adjusted to 6.8 ± 0.2. Electrical conductivity of the solutions showed no sign of salt accumulation in the solution draining through the pots.

Table 1.  Concentrations of N and P in the nutrient solutions used
SolutionN (mM)P (mM)N : P
  1. The prefix ‘h’ refers to presence of a particular nutrient at its higher concentration. The solutions were given to one or both sides of a split-root system to make up seven different treatments: NP NP (control); NP hNP; hNP hNP; NP NhP; NhP NhP; NP hNhP; hNhP hNhP. In experiment 1 the treatment hNhP hNhP was not present.

NP0.710.1 7.1
hNP6.450.09171
NhP0.711 0.71
hNhP6.450.91 7.1

Harvest and chemical analyses

At harvest, shoot height, nodule number, and d. wt (60°C for 24 h) of leaves, stem, roots and nodules were determined. Each half of the root system was measured separately. Nodules were excised from roots very carefully and any multilobed nodules were counted as one nodule.

N and P concentrations were analysed in leaves of five randomly chosen plants from each treatment in experiment 2 and 3. In experiment 1, leaves and roots of three randomly chosen plants were analysed for N and P. Nodules from all plants belonging to the same treatment in experiment 1 were pooled for N and P analysis. The N concentration was analysed in dried, ball milled (Retsch, MM2000, Haan, Germany) samples using a Europa Scientific CN-analyser (Europe Scientific, Chichester, UK) at Department of Forest Ecology, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden. Samples were digested in HNO3 and HClO4 at 130°C before measurement of P concentration using a Tecator autoanalyser (The Environmental Research Laboratory, SLU, Umeå, Sweden).

Statistical analyses

The paired t-test was used to identify statistical differences between the two sides of root systems receiving different nutrient solutions in the two root sides. The t-test and ANOVA were used to identify statistical differences between control plants and all other treatments. Statistical calculations were performed with Minitab software (Minitab Inc., State College Pennsylvania, PA, USA, 2000).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The three experiments showed many similarities and, for simplicity, only data from experiments 2 and 3 are presented in the graphs.

Nodulation

Compared to control plants (NP NP), high N to one side (NP hNP) inhibited nodulation completely (experiment 3) or decreased nodule biomass (d. wt) to approx. 20% (experiments 1, 2) at the hN side (Fig. 1a,b). Nodule biomass in the NP side of NP hNP plants was strongly decreased in experiment 3 and partly decreased in experiment 2. High N at both sides (hNP hNP) drastically decreased nodule biomass in experiment 2 or completely inhibited nodule biomass in experiments 1 and 3 (Fig. 1a,b).

image

Figure 1. Nodule biomass and nodule number on each root side in Hippophaë rhamnoides grown with split-root systems receiving different concentrations of N and P. The prefix ‘h’ refers to the presence of a particular nutrient at its higher concentration. Mean ± SE for 10 plants. Plants receiving same N and P concentrations in both sides of the root system are represented by the mean value of the two sides (single column). Plants were grown for: (a–c) 6 wk after inoculation (experiment 2); and (b–d) 6 wk after inoculation but pretreated with the different solutions 2 wk before inoculation (experiment 3). a on the top of a column stands for no difference, b for statistically significant inhibition and c for statistically significant stimulation when compared to the control (NP NP), while s shows statistically significant difference between the two root sides receiving different nutrient solutions. P < 0.05 was used as significance level.

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High P at one side (NP NhP) increased nodule biomass (Fig. 1) in both sides (strongly, up to 100% increase, in experiment 1 and weakly in experiment 2) or at the NP side (experiment 3). Plants receiving high P at both sides (NhP NhP) never showed a significant change in nodule biomass as compared to controls. When both N and P were high at one side (NP hNhP) nodule biomass strongly decreased at the hNhP side. By contrast, nodule biomass at the NP side was strongly stimulated and reached approx. 160–200% of values in control plants (Fig. 1a,b). Thus hP could prevent a systemic inhibition by N at the side kept under control conditions (cf. NP hNP and NP hNhP, Fig. 1). High N and high P at both sides of the root system produced a drastic decrease in nodule biomass in experiment 2 (Fig. 1a) or a total inhibition of nodulation in experiment 3 (Fig. 1b), similar to the effect of hN at both root sides (cf. hNP hNP and hNhP hNhP). Thus hP did not counteract the inhibition of nodulation by N when high N was given to both sides.

Effects of high N on nodule number (Fig. 1c,d) showed the same general pattern as effects on nodule biomass. In plants receiving high N at one side (NP hNP), the inhibition of nodule number by N was comparably stronger than the inhibition of nodule biomass. High P gave the same general pattern as for nodule biomass but effects on nodule biomass were comparably stronger, especially in experiment 1. When both N and P were high at one side, the nodule number was drastically decreased at that side while the other side, receiving N and P at control concentrations, was barely affected. Thus, within these plants there was a significant local inhibition by N but a stimulation by P at the opposite side (compare NP hNP and NP hNhP; Fig. 1c,d). When compared to control plants (NP NP), the stimulation of P at the opposite side (NP side in NP hNhP plants) was stronger for nodule biomass (Fig. 1a,b) than for nodule number (Fig. 1c,d). When both N and P were high at both sides, nodule number was very drastically decreased or was zero (Fig. 1c,d).

Plant growth

Effects of N and P on the biomass production of leaves (data not shown), roots and total plants showed a similar pattern (Fig. 2). When compared to the control (NP NP), high N strongly stimulated root growth and plant growth, in particular when high N was given to one side of the root system. High P stimulated root growth and plant growth when it was given to one side of the root system at time of inoculation (experiments 1 and 2). When high P was given to one side or both root sides before inoculation, root biomass and plant biomass was decreased (Fig. 2b,d). High N and high P at one or both sides produced strong stimulation of root biomass (Fig. 2a,b) and total plant biomass (Fig. 2c,d).

image

Figure 2. Root biomass on each root side and plant biomass in Hippophaë rhamnoides grown with split-root systems receiving different concentrations of N and P. The prefix ‘h’ refers to the presence of a particular nutrient at its higher concentration. Mean ± SE for 10 plants. Plants receiving same N and P concentrations in both sides of the root system are represented by the mean value of the two sides (single column). Plants were grown for: (a–c) 6 wk after inoculation (experiment 2); and (b–d) 6 wk after inoculation but pretreated with the different solutions 2 wk before inoculation (experiment 3). a on the top of a column stands for no difference, b for statistically significant inhibition and c for statistically significant stimulation when compared to the control (NP NP), while s shows statistically significant difference between the two root sides receiving different nutrient solutions. P < 0.05 was used as significance level.

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Nodulation in relation to plant growth

Both nodulation (Fig. 1) and plant growth (Fig. 2) responded to N and P. To separate specific effects on nodulation from general effects via plant growth, nodule biomass and nodule number were related to root biomass (Fig. 3) and to plant biomass (Fig. 4). Effects of N and P followed the same general trend as previously seen for nodule biomass and nodule number per root side (Fig. 1). Inhibition by N was more pronounced when nodule biomass or nodule number was related to root biomass (Fig. 3) or plant biomass (Fig. 4) than when expressed per root side (Fig. 1). The fact that high P or high N at one side produced a great response at the other side (NP) clearly demonstrated the systemic nature of stimulation by P and the inhibition by N (Fig. 3). When the concentrations of both N and P were increased at one (NP hNhP) or both sides (hNhP hNhP) their effects (Fig. 3) followed the same trend as previously seen for nodule d. wt and nodule number per root side (Fig. 1). The same effects were noticed when nodulation was related to total plant biomass (Fig. 4). Thus, stimulation by high P was still evident when nodulation was related to root biomass or plant biomass. Furthermore, P stimulation was comparably stronger on nodule biomass than on nodule number (Figs 3 and 4; experiment 1).

image

Figure 3. Nodule biomass and nodule number per root biomass on each root side in Hippophaë rhamnoides grown with split-root systems receiving different concentrations of N and P. The prefix ‘h’ refers to the presence of a particular nutrient at its higher concentration. Mean ± SE for 10 plants. Plants receiving same N and P concentrations in both sides of the root system are represented by the mean value of the two sides (single column). Plants were grown for: (a–c) 6 wk after inoculation (experiment 2); and (b–d) 6 wk after inoculation but pretreated with the different solutions 2 wk before inoculation (experiment 3). a on the top of a column stands for no difference, b for statistically significant inhibition and c for statistically significant stimulation when compared to the control (NP NP), while s shows statistically significant difference between the two root sides receiving different nutrient solutions. P < 0.05 was used as significance level.

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image

Figure 4. Nodule biomass and nodule number per plant biomass on each root side in Hippophaë rhamnoides grown with split-root systems receiving different concentrations of N and P. The prefix ‘h’ refers to the presence of a particular nutrient at its higher concentration. Mean ± SE for 10 plants. Plants receiving same N and P concentrations in both sides of the root system are represented by the mean value of the two sides (single column). Plants were grown for: (a–c) 6 wk after inoculation (experiment 2); and (b–d) 6 wk after inoculation but pretreated with the different solutions 2 wk before inoculation (experiment 3). a on the top of a column stands for no difference, b for statistically significant inhibition and c for statistically significant stimulation when compared to the control (NP NP), while s shows statistically significant difference between the two root sides receiving different nutrient solutions. P < 0.05 was used as significance level.

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N and P concentration

In experiment 1, leaf N concentration and root N concentration were highest in plants given high N at both root sides (Table 2). The concentration of N in nodules was always higher than in roots and leaves. At control concentrations of P in the nutrient solution, the P concentration in plants was fairly similar, approx. 0.1% of d. wt in all N treatments (Table 2). When a high concentration of P was given to one side of the root system, the P concentration in leaves and roots increased, especially at the root side receiving high P. High P at both root sides gave the highest concentration of P in all plant parts. When P in the solution was kept at its control concentration, the concentration of P was higher in nodules than in all other plant parts. Also, within each treatment, nodules were often the plant part having the highest P concentration. In experiments 2 and 3 (Table 3), the concentration of N in leaves increased when high N was given at one side and to an even greater extent when high N was given at both sides. The concentration of P in leaves was approx. 0.1% of d. wt at control concentrations of P but increased up to almost 10-fold when high P was given to plants. In plants receiving high P at one or both sides (NP NhP, NhP NhP), the leaf N concentration was highest in experiment 1 (10 wk treatment) and lowest in experiment 2 (6 wk treatment), with experiment 3 (6 + 2 wk treatment) being intermediate. By contrast, the concentration of P was stable in all three experiments irrespective of pretreatment or duration of experiment. The N : P ratio in solutions varied 100-fold, from 0.71 to 71 (Table 1). Leaf N : P ratio varied within a smaller range, 1.8–29.8 at harvest (Tables 2 and 3).

Table 2.  Concentration of N and P in leaves, roots and nodules on each root side of Hippophaë rhamnoides grown with split-root systems receiving different concentrations of N and P (data for experiment 1). The prefix ‘h’ refers to the presence of a particular nutrient at its higher concentration
TreatmentSide of split-root systemLeaf N (%)Root N (%)Nodule N (%)Leaf P (%)Root P (%)Nodule P (%)Leaf N : PRoot N : P
  1. Values for leaves and roots are mean ± SE of three plants. Values for nodules are from a pooled sample of all 7–10 plants per treatment. Treatments began at inoculation and plants were grown for 10 wk after inoculation. Concentrations are percentage of d. wt. NA indicates too few nodules to be analysed.

NP NPNP2.22 ± 0.171.64 ± 0.124.520.10 ± 0.0010.09 ± 0.0020.2821.417.9
NP hNP 2.21 ± 0.13  0.10 ± 0.01  21.9 
 NP 1.79 ± 0.125.84 0.13 ± 0.01NA 13.6
 hNP 2.32 ± 0.16  0.13 ± 0.01  18.1
hNP hNPhNP3.06 ± 0.062.67 ± 0.10 0.10 ± 0.010.10 ± 0.01 29.727.1
NP NhP 2.62 ± 0.09  0.51 ± 0.01   5.1 
 NP 1.90 ± 0.064.75 0.27 ± 0.040.53  7.4
 NhP 1.75 ± 0.154.43 0.77 ± 0.050.60  2.4
NhP NhPNhP2.30 ± 0.171.77 ± 0.054.570.84 ± 0.080.79 ± 0.110.89 2.8 2.3
NP hNhP 2.77 ± 0.04  0.38 ± 0.09   8.1 
 NP 2.07 ± 0.165.28 0.28 ± 0.040.60  7.5
 hNhP 2.24 ± 0.10NA 0.59 ± 0.10NA  4.0
Table 3.  Concentration of N and P in leaves of Hippophaë rhamnoides grown with split-root systems receiving different concentrations of N and P (data for experiments 2 and 3). The prefix ‘h’ refers to the presence of a particular nutrient at its higher concentration
Treatment (experiment 2)Leaf N (%)Leaf P (%)N : PPre-treated (experiment 3)Leaf N (%)Leaf P (%)N : P
  1. Values are means ± SE of leaf samples from five plants. Treatments began at inoculation (experiment 2) or 2 wk before inoculation (experiment 3). Plants were grown for almost 6 wk after inoculation.

NP NP1.65 ± 0.240.12 ± 0.0113.1NP NP1.59 ± 0.130.15 ± 0.0210.6
NP hNP1.78 ± 0.100.10 ± 0.00217.4NP hNP1.62 ± 0.130.09 ± 0.00517.4
hNP hNP2.49 ± 0.080.09 ± 0.00527.6hNP hNP2.84 ± 0.140.09 ± 0.00329.8
NP NhP1.31 ± 0.060.53 ± 0.05 2.6NP NhP2.03 ± 0.130.54 ± 0.05 3.9
NhP NhP1.77 ± 0.220.97 ± 0.07 1.8NhP NhP2.14 ± 0.200.88 ± 0.08 2.5
NP hNhP1.87 ± 0.030.33 ± 0.03 5.9NP hNhP1.95 ± 0.030.35 ± 0.03 5.6
hNhP hNhP2.77 ± 0.040.62 ± 0.055 4.6hNhP hNhP2.55 ± 0.080.59 ± 0.04 4.4

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The present work on Hippophaë rhamnoides has confirmed the well-known inhibition of nodulation by high N previously shown in other actinorhizal plants (Bond & Mackintosh, 1975; Kohls & Baker, 1989; Thomas & Berry, 1989; Arnone et al., 1994; Wall et al., 2000). Inhibition by N was systemic for both nodule biomass and nodule number (Fig. 1). In part, this contrasts with what has previously been found in root-hair infected Casuarina, where a high concentration of nitrate inhibited nodule number locally but nodule biomass systemically (Arnone et al., 1994). However, even if the two studies covered a similar period of growth, they dealt with different plants and with different growing techniques; pots with perlite in the present experiments but water culture in the work by Arnone et al. (1994).

The slightly weaker response in experiment 2 than in experiment 3 (pretreatment) may be explained by the shorter exposure time to high N and P in experiment 2. Indeed the exposure time was almost 6 wk in experiment 2, and Frankia began to fix N2 3–4 wk after inoculation in A. incana (Huss-Danell, 1978) and in Discaria trinervis (Valverde et al., 2000). In experiment 3, plants treated with high N (NP hNP, hNP hNP, NP hNhP and hNhP hNhP) could increase their N content by using N in nutrient solution for a longer time than could plants in experiment 2. The highest nodule biomass and nodule number was nearly twice as high in experiment 3 as in experiment 2. This refers to nodulation per root side as well as to nodulation per root d. wt or per plant d. wt. The internal N concentration of plants governed the infection process, as has earlier been observed in the other actinorhizal plants, Ceanothus griseus (Thomas & Berry, 1989) and Discaria trinervis (Valverde et al., 2000). That internal N triggered some metabolic pathways that prevent or limit the infection process appeared to be a systemic event and was particularly obvious for nodule number (Figs 1c,d, 3c,d, 4c,d). How this might work is not clear but it could resemble the auto-regulation of nodulation in actinorhizal plants and legumes (Caetano-Anollès & Gresshoff, 1991; Wall & Huss-Danell, 1997; Valverde & Wall, 1999) where a systemic messenger was proposed to prevent nodulation.

Only local, but not a systemic, inhibition by N was evident when hN was combined with hP (NP hNhP). Such a counteracting effect of high P on systemic inhibition was not however, found with hN and hP at both root sides (hNhP hNhP). Plants receiving hN at both root sides had the highest leaf N concentrations (Tables 2 and 3) so, perhaps, the internal concentration of N was so great that the concentrations of added P used in this experiment could no longer counteract the effects of N. Our results are in agreement with a recent study on beans where increase in P could not improve nodulation at high nitrate concentration (Leidi & Rodriguez-Navarro, 2000). Thus, not only the concentrations of N and P but also the N : P ratio is important for nodulation (Wall et al., 2000).

When nodule biomass or number was related to root biomass (Fig. 3) the systemic inhibition by N was even more pronounced in all experiments. When related to plant biomass it appeared clear that N inhibition was specific for nodulation and not simply mediated via plant growth (Fig. 4). P stimulation was particularly evident for nodule biomass. A possible explanation of systemic P stimulation, or prevention of N inhibition, in the opposite root side (NP side of NP hNhP plants) could be that high availability of P made N become limiting. To get more N, and establish a good N : P ratio, plants could stimulate nodulation and N2-fixation only at the root side receiving low concentrations of both nutrients.

In experiment 3, high P was inhibitory to root growth and total plant growth when both root sides received hP (Fig. 2b,d). P is involved in the regulation of many enzymatic activities. One of these is starch synthesis, and it has been found that an excess of P can inhibit this process by two separate mechanisms located in the chloroplast (Marschner, 1995). Consequently this inhibition results in decreased growth. In experiment 3, plants probably accumulated P (Table 3) without being able to balance the N : P ratio until N2 fixation commenced (Wall et al., 2000). The ratio between P and other nutrients, including micronutrients, was not studied but may have been important as well (Jones, 1998).

P stimulation in this work was systemic and specific to nodulation. A specific P stimulation of nodulation was also found for Casuarina, where the P requirement for certain nodulation phases was greater than for plant growth (Sanginga et al., 1989) and for Trifolium pratense where P had a specific effect on nodulation (Hellsten & Huss-Danell, 2000). By contrast, the stimulatory effect of P on nodulation was considered a general effect, exerted via plant growth, in Casuarina (Yang, 1995; Reddell et al., 1997), Trifolium (Robson et al., 1981) and Pisum (Jakobsen, 1985). A greater need for P in nodules as compared to other plant parts, can be expected. For example, in nodules, Frankia is always surrounded by a membrane continuous with the plasmalemma. At nodule primordium formation (cell division), and when Frankia invades primordium cells, the plant produces secretory vesicles whose membranes will fuse into the plasmalemma (Miller & Baker, 1985b). This demands a great amount of P. In addition Frankia is a Gram-positive bacterium, and Gram-positive bacteria have P, in teichoic acids, in the cell wall (Alexander, 1998). The concentration of P in Frankia E15b is much higher than that in the plant, approx. 7.4% of d. wt when grown in liquid medium (F. Gentili, unpublished).

In all three experiments, the P concentration in leaves was almost the same, independent of the time of nutrient treatment (Tables 2 and 3). By contrast, N concentration in general increased over time in all treatments. The greatest increase in leaf N concentration occurred in plants receiving hP at one side of the root system (NP NhP). These doubled their leaf N concentration over a period of 4 wk (compare experiment 1 (10 wk treatment) and experiment 2 (6 wk treatment)). During the same period of time plants had more than doubled their nodule biomass and most likely increased their rate of N2 fixation. For a more complete understanding of N and P effects on nodulation it seems important to study the time effect in more detail.

In conclusion, the present work has shown that N had a systemic inhibitory effect on nodule number and biomass in H. rhamnoides. Mechanisms for N inhibition are poorly understood, but Parsons et al. (1993) and Baker et al. (1997) proposed that the concentration of reduced N compounds, probably amino acids, is involved in an internal feedback mechanism from phloem to nodule. For actinorhizal plants, Wall (2000) proposed a model where N inhibition could occur at different steps of the infection process. In the present work, high P could counteract N inhibition of nodulation at the opposite side of the split-root system. P stimulated both nodule biomass and number, but the biomass was more strongly affected. Furthermore stimulation by P was specific for nodulation and not simply mediated via plant growth. It will be of interest to investigate whether N and P alter not only nodulation but also N2-fixation in the nodules.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Ann-Sofi Hahlin for valuable technical help, Dr Erica Lumini and Dr Marco Bosco for providing the Frankia strain E15b, Dr Bo Nilsson for providing seeds, Dr Ari Jumpponen for valuable statistical advice, Georg Carlsson and Anna Wiklund for valuable discussions and Tina Johansson for secretarial help. This work was financially supported by the Swedish Natural Science Research Council, the Swedish Council for Forestry and Agricultural Research and EC FAIR-BM-972009. F.G. would like to dedicate this paper to the memory of Felice Pedarzini.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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