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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.
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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.