Author for correspondence: Luis Gabriel Wall Tel: +54 11 4365 7136 Fax: +54 11 4365 7132 Email: firstname.lastname@example.org
• After nitrogen (N), phosphorus (P) is the nutrient that most limits plant productivity. The role of P on growth and root nodulation was studied in the actinorhizal symbiosis between Discaria trinervis and Frankia, an intercellular infected N2–fixing association.
• Growth, nodulation and nutrient content (N and P) were analysed in symbiotic plants receiving different supplies of P in nutrient solutions. The relative requirement of P for nodulation was analysed in P-deficient plants.
• Nodule initiation was less impaired than general plant growth by low P. However, low P impaired nodule growth to a greater extent than plant growth. The proportion of nodule biomass, although not the number of nodules per plant, was stimulated by P supply. Autoregulation of nodulation was not affected by P. Use of N was limited by availability of P. Reserves of P in seeds were enough for the seedling to establish nodules. However nodule (and plant) growth was limited in the absence of exogenous P.
• It is possible that P interacts with the feedback control of nodule growth that is associated with the plant demand for N. Leaf N : P ratio is negatively correlated with the proportion of nodule tissue.
After nitrogen (N), phosphorus (P) is the nutrient that most commonly limits plant productivity (Marschner, 1995). It is well established that N2-fixing plants, either legumes or actinorhizal plants, require phosphate for adequate growth and root nodulation (Robson, 1983; Marschner, 1995; Huss-Danell, 1997). However, it is not clear whether P is needed more for plant growth than for nodule growth or for N2 fixation itself. In addition, no reference has been made in the literature to a role in nodulation for P reserves in seeds. It has been argued that P indirectly stimulates nodulation by a positive effect on Casuarina plant growth (Yang, 1995; Reddell et al., 1997). On the other hand, a direct and positive stimulation of P on nodulation has been reported for pea (Jakobsen, 1985), soybean (Israel, 1987, 1993), red clover (Hellsten & Huss-Danell, 2001) and also for Casuarina (Sanginga et al., 1989). Moreover, the effect of P seems to be related to N effects, suggesting that the N : P ratio modulates symbiotic development (Wall et al., 2000; Hellsten & Huss-Danell, 2001). Thus, different experimental approaches where mineral N was added to nutrient solutions, and the lack of time-course observations, could have hampered interpretation of the effects of P on the ability of host plants to nodulate, to fix N2 and to regulate these processes.
Seeds of Discaria trinervis (Hooker et Arnot) Reiche were separated from dry fruits collected in 1998 from Pampa de Huenuleo (41°10′ S, 71°12′ W, Bariloche, Rio Negro, Argentina). Surface sterilization was performed by scarification (Valverde & Wall, 1999a). Seeds were transferred to sterile perlite moistened with sterile distilled water and kept at 4°C for 5 d. Germination and further plant growth were carried out in a glasshouse, with a mean maximum temperature of 24°C, a mean minimum temperature of 20°C and rh ranging from 45 to 90%. Incandescent lamps (400 W, Osram, Brazil) supplemented natural light such that the photoperiod was of 16 h. Light intensity on the plants, under the lamps, was 13000 lx.
Experiment 1: growth with different P supply
Seedlings at the cotyledonary stage (12–14 d after the start of germination), were aseptically transferred to growth pouches (typically four seedlings per pouch) containing 13 ml of modified Evans 1/10 full strength solution (E1/10; Huss-Danell, 1978). Nutrient solution was initially supplemented with 0.071 mM N (as NH4NO3). The concentration of P in treatment solutions was adjusted to 1, 5, 10, 50, 100, 500 or 1000 µM, with different amounts of potassium phosphate. The concentration of K was adjusted in all solutions to 2.2 mM with KCl and pH was corrected to 6.8 before autoclaving. Each group consisted of six growth pouches.
The bottom edge of each growth pouch was cut off and all pouches belonging to the same treatment were placed together into a separate bigger plastic bag to give seedlings access to a larger volume of nutrient solution. The nutrient solution (10 ml of fresh solution per pouch) was replaced weekly. The pH of nutrient solution (measured before renewal) started to drop from the original value of 6.8, at 5 wk after inoculation. The final pH was lower with increasing concentration of P in nutrient solutions, reaching a value of 4 at 8 wk after inoculation, for P concentrations of 50 µM or higher (data not shown).
Experiment 2: growth without external supply of P
Four seedlings (12–14 d-old) were aseptically transferred to each growth pouch containing 13 ml of E1/10 solution without potassium phosphate and supplemented with 0.71 mM N (as NH4NO3). Control plants were grown in E1/10 containing 100 µM P and 0.71 mM N. The bottom edge of each growth pouch was cut off and all pouches belonging to the same treatment were placed together into a separate bigger plastic bag to facilitate renewal of nutrient solution (10 ml per pouch were added weekly). Each experimental group consisted of four pouches.
Frankia growth and plant inoculation
Frankia isolate BCU110501 was used for inoculation (Chaia, 1998; Valverde & Wall, 1999a). Inoculum was prepared from bacteria grown at 28°C for 28 d in static BAP minimal medium containing 55 mM glucose as C source (Valverde & Wall, 1999a). Cells were harvested by centrifugation (2000 g, 10 min), washed twice, and resuspended in 2–3 ml of N-free nutrient solution, containing the lowest concentration of P (1 µM). The cell suspension was homogenized by repeated passage through needles, first with 0.8-mm gauge and then with 0.5-mm gauge (three times each). Frankia biomass in the homogenate was estimated by determination of packed cell volume (Nittayajarn & Baker, 1989). Seedlings were inoculated 21 d after growing in pouches by dripping 200 µl of homogenate (containing the equivalent of 1.5 µl of Frankia packed cells) onto the roots from the apex to the base. At the moment of inoculation the position of each root tip (RT) was marked on the plastic pouch (RT mark). For every concentration of P tested in experiment 1, groups of seedlings were left uninoculated controls for N2 fixation. In the same experiment, N was removed from nutrient solutions after inoculation.
Growth and nodulation
Shoot height was measured weekly to the nearest 1 mm. Plants were harvested at 10 wk after inoculation. Nodules on all roots were counted. Nodule position on taproots was marked on the pouch, and their distance from RT was measured. Position of nodules relative to RT on lateral roots was not considered. Shoots, roots and nodules were dried separately (72 h, 60°C) and weighed to estimate biomass production.
N and P content
Nitrogen content of dried leaves, and seeds, was estimated by colourimetric detection of ammonia after Kjeldahl mineralization (Jackson, 1958). To estimate P content, ashes of this material, incinerated at 500°C, were dissolved in 1 M HCl. Phosphate was determined by molybdate colourimetry in the presence of ascorbate (Israel, 1987).
Light microscopy of nodules
Nodules were fixed in 2.5% (w/v) glutaraldehyde in 45 mM potassium phosphate, pH 7.2, for 30 min at reduced pressure and then for at least 3–4 h at atmospheric pressure. Fixed nodules were prestained with 2% (w/v) osmium tetroxide and dehydrated with ethanol (50, 70, 80, 95 and 100% v/v). Dehydrated samples were embedded in Epon-Araldite and polymerization was carried out for 3 d at 70°C. Longitudinal sections (1–1.5 µm) were mounted in glass slides and stained with methylene blue-Azur II, and examined in a Nikon Alphaphot-2 YS2-H (Japan) light microscope.
The experimental unit was a growth pouch. However, all variables were expressed on a single plant basis. ANOVA was performed to compare group means using Number Cruncher Statistical System v5.7 (NCSS, 1988, Kaysville, UT, USA). Regression analysis was performed with SigmaPlot v4.01 (Jandel Scientific, Erkrath, Germany).
Vegetative growth of nodulated plants was stimulated by increasing P supply between 10 and 100 µM (Fig. 1a). P had a positive effect on whole plant biomass production (Fig. 1a). Biomass allocation was preferentially diverted to shoot over root with increases in [P] (Fig. 1b). The effect of P concentration on shoot growth was not evident until 5 wk after inoculation (Fig. 2a). Shoot height at harvest was positively and linearly correlated with log [P] (r2 = 0.90, [P] = 1–500 µM; Fig. 2b). Final shoot height for [P] = 1000 µM was lower than for [P] = 500 µM indicating that supraoptimal P supply affected shoot development (Fig. 2a).
All inoculated plants formed nodules for every tested concentration of P (Fig. 3a). Noninoculated plants did not form nodules. The rate of nodule initiation and the final number of nodules per plant were not significantly affected by P concentration (P > 0.05, Fig. 3a). The individual d. wt of nodules increased with P concentrations above 10 µM (Fig. 3b). Thus, the total nodule biomass per unit of plant d. wt also increased (Fig. 3c). This positive effect of P was statistically significant for [P] ≥ 100 µM (P < 0.05).
Less P was needed to achieve optimal plant biomass production (100 µM; Fig. 1a) than to obtain maximal nodule growth (500 µM; Fig. 3b). At any concentration of P tested, > 90% of nodules developed on the tap roots. Moreover, the nodule distribution profiles along taproots were similar for all groups (Fig. 4).
Nitrogen and phosphorus content of leaves
Leaf N content increased almost linearly with log [P] for the entire tested range of P-concentrations (r2 = 0.78; Fig. 5a), which suggests that use of symbiotically fixed N was limited by P availability. The N content of leaves showed a high hyperbolic correlation (r2 = 0.95) with P content (Fig. 5b). The P content of D. trinervis leaves also increased with P concentrations in nutrient solution, but the increment was steepest for concentrations > 100 µM (Fig. 5a). Consequently, the N : P ratio in leaves of symbiotic plants dropped with log [P].
Growth and nodulation of P-deficient seedlings
The fact that seedlings supplied with low concentrations of P were still able to form a high number of nodules (Fig. 3a), and moreover, to autoregulate their number and distribution (Fig. 4), suggested that D. trinervis possesses a very efficient system for acquisition of phosphate. Alternatively, D. trinervis might contain enough P at the moment of inoculation to ensure the formation of nodules. In order to test these hypotheses, axenic seedlings were grown in pouches with no supply of P in mineral solution and supplemented with 0.71 mM N (−P group). Control seedlings grew in pouches with 100 µM P and 0.71 mM N (+P group). The shoot height of both groups of seedlings increased similarly during the first 8 wk (Fig. 6). Thereafter, −P seedlings grew no further, while +P seedlings grew at a sustained rate (Fig. 6). No structural changes (proteoid root formation) were detected in roots of P-deficient seedlings. Since phosphate was omitted in nutrient solutions that were used to irrigate −P plants, the buffering capacity of this solution was greatly decreased. However, −P plants did not show any differential growth until P became limiting (Fig. 6; up to week 8). In typical nodulation experiments (Valverde & Wall, 1999b; Valverde et al., 2000; Figs 1–5), seedlings were inoculated 3 wk after transfer to pouches, that is, 5 wk before P deficit was evident in this experiment. By 6 wk after the onset of P deficit (or 14 wk after the start of the experiment), −P and +P seedlings were inoculated with cells of Frankia BCU110501. Following inoculation, N was omitted from nutrient solution to avoid inhibitory effects on nodulation (Valverde et al., 2000). As expected, +P seedlings rapidly formed nodules (Fig. 6). These seedlings developed a high number of nodules, most of them on lateral roots that were present at the moment of inoculation. As previously observed (Valverde et al., 2000), +P plants ceased to nodulate at approx. 6 wk after inoculation. It was noteworthy that, even though their growth was impaired, −P plants could still nodulate, although at a much lower rate and number of nodules per plant (Fig. 6). These nodules remained small, but light microscopy showed typical hypertrophied infected cells with Frankia filaments and vesicles (Fig. 7a,b; Valverde & Wall, 1999a). Noninfected cells in the symbiotic zone of the nodules contained numerous starch-like bright refractive corpuscles (Fig. 7c). These starch-like corpuscles were much less abundant in P-sufficient nodules (Valverde & Wall, 1999a).
P-deficient seedlings that could not further increase their shoot height, but could develop some nodules (Fig. 8), were challenged to recover from P deficit. Plants were divided in two groups, one of which was supplied with 100 µM P in nutrient solution (−P+P group) while the other one remained deprived of P (−P−P group). After 3 wk, −P+P seedlings resumed growth (Fig. 8). Simultaneously, more nodules appeared in the proximity of the pre-existing small nodules.
Phosphorus content of P-deficient seedlings and seeds of Discaria trinervis
Before restoring the P supply to −P seedlings (Figs 6, 8), their P content was estimated to be 0.03% of d. wt, which corresponded to approx. 8.4 µg of total P per seedling. The amount of P stored in D. trinervis seeds used for this study was 4.0 µg mg−1 (Table 1) and 3.8 µg mg−1 without seed-coats. Given that the average weight of coated D. trinervis seeds was 2.4 mg, this represents approx. 9 µg of P available for germination. This balance directly confirmed that −P plants used the stored P in the seed to grow until it became exhausted.
Table 1. Nitrogen (N) and phosphorus (P) content of actinorhizal seeds
Host plant (family)
N [‰ d. wt]
P [‰ d. wt]
N : P ratio [‰ d. wt (‰ d. wt)−1]
Alnus incana (Betulaceae)
K. Huss-Danell (Umeå, Sweden)
Alnus acuminata (Betulaceae)
R. Enrico (Tucumán, Argentina)
Casuarina cunninghamiana (Casuarinaceae)
L. G. Wall (La Plata, Argentina)
Discaria trinervis (Rhamnaceae)
E. Chaia (Bariloche, Argentina)
In the absence of any source of N other than atmospheric N2, the rate of N2 fixation could be adjusted in response to environmental changes either through a variation in the amount of nodule tissue (number of nodules and/or their size) or through a variation in nitrogenase activity (Hartwig, 1998). In D. trinervis, the number and distribution of root nodules is tightly regulated by an autoregulatory feedback mechanism that is induced before onset of N2 fixation (Valverde & Wall, 1999b; Wall, 2000). The rate of nodule growth (and the proportion of nodule biomass) is more likely to be regulated by another feedback mechanism, related to the N content of the plant (Valverde et al., 2000; Wall, 2000). In this study, we show that the general plant growth (shoot height, plant d. wt and shoot biomass allocation) of symbiotic D. trinervis was limited to a greater extent than nodule formation by P (Figs 1, 2, 4). Whereas accumulation of plant biomass was impaired at concentrations of P < 50 µM (Fig. 1), nodule initiation was not affected by such low P supply (Fig. 3). Moreover the ability of seedlings to regulate the number and location of nodules was not altered (Figs 3, 4), suggesting that autoregulation of nodulation (Valverde & Wall, 1999b) was not affected by the concentration of P in mineral solution. However the size of nodules and the proportion of nodule tissue was stimulated by P (Fig. 3). These data suggest that the feedback regulation of nodulation in D. trinervis, which might be mediated by a systemic mechanism linked to the N status of the plant (Parsons et al., 1993; Wall, 2000), is modulated by the availability of P. In order to support this hypothesis we found that when the proportion of nodule biomass in the plant was plotted as a function of the N : P ratio in leaves, a strong correlation was found (Fig. 9), suggesting that this ratio affects the feedback regulation of nodulation and nodule growth. Similar results were observed in soybean (Israel, 1987; Drevon & Hartwig, 1997), clover (Almeida et al., 2000; Hellsten & Huss-Danell, 2001) and alfalfa (Drevon & Hartwig, 1997). A negative correlation between leaf or shoot N : P ratio and the proportion of nodule tissue was also evident in studies where other N sources where present, or where mycorrhizal fungi colonized nodulated roots (Jha et al., 1993; Russo et al., 1993). Thus, although other fluxes of N and P are present in a symbiotic plant, the leaf N : P ratio would seem to be a better indicator of nutrient status for the purpose of feedback regulation of nodulation (Wall et al., 2000) through a systemic response as proposed by Parsons et al. (1993). A possible mechanism for this response would be that a phloem-delivered signal of N-status would interact with a metabolic pathway whose rate is limited by P (Wall, 2000). In this way, the positive effect of P on the growth of symbiotic plants could be explained as a specific effect of P on the regulation of nodule growth, rather than as a general positive effect on host plant growth (Robson, 1983). The fact that the proportion of nodule tissue was dependent on P content (Figs 3c, 9) gives support to such an explanation. If nodule growth were not affected by P, there is no reason why the proportion of nodule biomass in the plant should have changed in response to P supply, that is, nodule growth would have been accommodated to plant growth rate. A time course analysis of changes in plant biomass and nodule biomass following a change in P supply (Israel, 1993) would give additional support to such hypothesis. Variation of P supply would modify the rate of nodule growth and, subsequently the rate of plant growth would reach a new stationary growth rate value. The underlying biochemical mechanism for the participation of P in this proposed regulation of nodulation remains unknown and deserves further studies.
It is assumed that P is required for nodule initiation (Marschner, 1995; Huss-Danell, 1997). However, our results suggest that this would not be the case for D. trinervis. The formation of nodules was not altered when the supply of P was limiting plant growth (Figs 1, 3). Seedlings that formed a normal number of nodules with low P supply could also regulate their distribution (Fig. 4), whilst, seedlings that did not receive any external supply of P and that showed a clear P deficiency for growth, could develop some nodules (Fig. 6). Differentiation of Frankia into vesicles was observed within these nodules, although carbohydrate metabolism was apparently impaired in noninfected cells of the symbiotic tissue (Fig. 7). These results collectively indicate that D. trinervis has a higher P requirement for vegetative and nodule growth than for nodule initiation and early development. This was not the case for other actinorhizal plants like Alnus and Casuarina, which are more dependent on an adequate P supply to establish the symbiosis with Frankia (Sanginga et al., 1989; Yang, 1995; Wall et al., 2000). In these actinorhizal hosts, a good P supply stimulates the formation of nodules, as was noted for Alnus glutinosa by Quispel (1958). The response of early nodulation to P supply might be related to the content of P in the seeds. A high P content in the seeds could facilitate the establishment of the symbiosis though further growth would be more dependent on external P supply. In account with this possibility, we observed that nodule development was re-established in P deficient seedlings once P nutrition was resumed (Fig. 8), suggesting that some infection events had occurred at the moment of inoculation but that their development was arrested by lack of P (Valverde & Wall, 1999b).
Seeds of D. trinervis had 6–16 times more P than those of Alnus incana, Alnus acuminata and Casuarina cunninghamiana (Table 1). In these genera, and contrary to what we present here for D. trinervis, positive effects of P supply on early nodulation have been observed (Yang, 1995; Wall et al., 2000).
The average content of extractable P in the site where seeds were collected was 4.4 µg per gram of soil (Chaia, 1997). Thus, seeds of D. trinervis (with a P content of approx. 4 mg of P per g) concentrate P approx. 1000-fold. This accumulation of P in seeds could be interpreted as an adaptive strategy to ensure initial growth of the seedling after germination and establishment of the symbiosis with Frankia. The requirement for external P would arise later on and may be acquired directly by root exploration of soil or be complemented by the establishment of an additional symbiosis with mycorrhizal fungi. Nodulated roots of Discaria trinervis were always found to be mycorrhizal in nature (S. Fontenla pers. comm.).
Financial support was obtained through grants from Universidad Nacional de Quilmes (Argentina), Agencia Nacional de Promoción Científica y Tecnonlógica (Argentina) and Fundación Antorchas (Argentina). We thank Gabriela Massa for her technical help. C.V. holds a fellowship from CONICET (Argentina) and L.G.W. is member of the Scientific Research Career of CONICET (Argentina).