# Nitrogen nutrition of ectomycorrhizal symbiosis

Claude Plassard, Pascale Scheromm, Pascal Tillard, Louis Salsac

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Claude Plassard, Pascale Scheromm, Pascal Tillard, Louis Salsac. Nitrogen nutrition of ectomycorrhizal symbiosis. Agriculture, Ecosystems & Environment, 1989, 10.1016/0167-8809(90)90068-O. hal-04275850

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## NITROGEN NUTRITION OF ECTOMYCORRHIZAL SYMBIONTS

PLASSARD C., SCHEROMM P., TILLARD P. and SALSAC L.

Laboratoire de Recherches sur les Symbiotes des Racines. INRA. 1, Place P. VIALA,

34060 MONTPELLIER - FRANCE

### ABSTRACT

Inorganic nitrogen nutrition was studied on isolated partners of ectomycorrhizal associations. The growth of maritime pines (*Pinus pinaster*) was measured with $NO_3^-$ or $NH_4^+$ as N source. Seedling weights were greater on $NH_4^+$ than on $NO_3^-$ . The ionic balances were established in both types of nutrition suggesting that nitrate reduction was mainly carried out in the roots.

The uptake rates of $NO_3^-$ or $NH_4^+$ by fungal species were measured by using assimilation of 15N labelled nitrogen : with suitable carbon source, the uptake rates of $NH_4^+$ were higher than the uptake rates of $NO_3^-$ . The ionic balances were established in both types of nutrition and suggested that nitrate reduction does not result in a high level of carboxylate production

### INTRODUCTION

Mineral nitrogen nutrition was studied mainly in herbaceous angiosperms and less often on woody plants or in ectomycorrhizal fungi. However, in forest soils, nitrogen is often a limiting element of plant productivity, and mycorrhization may have a beneficial effect on the nitrogen nutrition of the host plant. Therefore, in order to further understand the positive effect of the mycorrhizal association, we decided firstly to study nitrate and ammonium nutrition on the isolated symbionts.
# MATERIAL AND METHODS

## 1 - MATERIAL

Maritime pine seedlings (*Pinus pinaster* Soland. inAlt.) were grown from seeds. The plants were grown in axenic conditions (SCHEROMM et PLASSARD, 1988) for growth measurements and mineral studies. The ammonium solution used was: 0.2 mM NaCl, 0.5 mM CaCl₂, 1.0 mM KH₂PO₄, 0.6 mM MgSO₄, 2.0 mM NH₄Cl, 1.5 mM (NH₄)₂SO₄, 3.0 mM KCl, 1.0 mM K₂SO₄, thiamin hydrochloride: 50.0 µg l⁻¹, Fe-citrate 1%: 0.5 ml l⁻¹; and micronutrients: 0.2 ml l⁻¹ of Morizet et Mingeau (1976) solution. The nitrate solution was: 0.2 mM NaCl, 0.5 mM CaCl₂, 1.0 mM KH₂PO₄, 0.6 mM MgSO₄, 5.0 mM KNO₃, thiamin hydrochloride: 50.0 µg l⁻¹, Fe-citrate 1%: 0.5 ml l⁻¹; and micronutrients: 0.2 ml l⁻¹ of Morizet et Mingeau (1976) solution.

The two ectomycorrhizal fungi used were *Hebeloma cylindrosporum* Romagn. and *Pisolithus tinctorius* Cooker and Couch which were grown *in vitro* on liquid medium, with ammoniacal medium containing: 1.0 mM NaCl, 0.5 mM CaCl₂, 3.0 mM (NH₄)₂HPO₄, 1.0 mM MgSO₄, 10.0 mM KCl, thiamin hydrochloride: 50.0 µg l⁻¹, Fe-citrate 1%: 1.0 ml l⁻¹; and micronutrients: 0.2 ml l⁻¹ of Morizet et Mingeau (1976) solution. When mentionned, pyruvic acid(10 mM) is added to the ammoniacal medium. Nitrate medium was: 1.0 mM NaCl, 0.5 mM CaCl₂, 6.0 mM KNO₃, 3.0 mM KH₂PO₄, 1.0 mM MgSO₄, 4.0 mM KCl, thiamin hydrochloride: 50.0 µg l⁻¹, Fe-citrate 1%: 1.0 ml l⁻¹; and micronutrients: 0.2 ml l⁻¹ of Morizet et Mingeau (1976) solution. pH of all media were adjusted to 6.0.

## 2 - METHODS

Mineral analysis of pine seedlings were carried out as described in SCHEROMM and PLASSARD (1988); ionic balances were established for the fungi using the same methods as for with the pine seedlings.

# RESULTS AND DISCUSSION

## 1 - THE HOST PLANT

### 1.1 - Growth in controlled conditions

Fresh weight of roots and shoots of maritime pine seedlings was always greater with ammonium than with nitrate (Fig. 1), which is consistent with most observations for
<figure><img src="image_2.png" /><figcaption>FIGURE 1- Fresh matter production by the maritime pine seedlings grown aseptically on ammoniacal (■, □) or nitric (▲, △) medium - Roots (■, ▲), shoots (□, △).</figcaption></figure>

Gymnosperms (VAN DEN DRIESSCHE, 1971; KRAJINA et al., 1973; VAN DEN DRIESSCHE and DANGERFIELD, 1975; BIGG and DANIEL, 1978). When seedlings are less than two months old, relative growth with ammonium was approximately twice that with nitrate. Thereafter it remains slightly higher with ammonium. The ionic balances were established in roots and shoots for both types of nutrition, to determine whether they varied according to nitrogen nutrition, as reported for numerous species (COIC et al., 1961; KIRKBY and MENGEL, 1967; WOOLHOUSE and HARDWICK, 1966; CHAILLOU et al., 1986; ARNOZIS and FINDENEGG, 1986; SALSAC et al., 1987).

## 1.2 - Ionic balances

The sum of cations (Fig. 2A) is greater in roots of pines grown on nitrate, resulting from considerable accumulation of $K^+$, $Mg^{2+}$, $Ca^{2+}$. This accumulation is accompanied by a smaller accumulation of mineral anions (Fig. 2B). Therefore, the difference
<figure><img src="image_3.png" /><figcaption>FIGURE 2- Accumulation of cations, organic and inorganic anions in roots and shoots of maritime pine seedlings grown on ammoniacal or nitric medium. (A,B,C): roots -(D,E,F): shoots - (A,D) : cation content - (B,E) : inorganic anion content - (C,F) : organic anion content (O) ammoniacal medium - (●) nitric medium.</figcaption></figure>
<table><caption>TABLE I - MEASUREMENTS OF MINERAL NITROGEN UPTAKE RATES
BY *H. cylindrosporum* THALLI BY USING 15N ASSIMILATION OF
($^{15}$NH4)2SO4 (6mM) + pyruvic acid (10 mM) OR K15NO3(6mM)</caption><thead><tr><th rowspan="2">INCUBATION TIME<br>(hours)</th><th colspan="2">UPTAKE RATES<br>(µmol/h/gFW)</th></tr><tr><th>NH<sub>4</sub></th><th>NO<sub>3</sub></th></tr></thead><tbody><tr><td>12</td><td>3,58</td><td>1,53</td></tr><tr><td>24</td><td>3,54</td><td>1,78</td></tr><tr><td>48</td><td>2,17</td><td>1,17</td></tr><tr><td>72</td><td>1,69</td><td>1,16</td></tr></tbody></table>

between the sum of cations and anions, which represents the sum of organic anions, is very high under nitrate nutrition (Fig. 2C). The synthesis of organic anions and their vacuolar accumulation are normally attributed to the necessity of eliminating hydroxyl anions produced from nitrate reduction (RAVEN and SMITH, 1976): so these results strongly suggest that nitrate reduction is mainly carried out in the roots.

In shoots, the difference between the sum of cations (Fig. 2D) of pines grown on nitrate and on ammonium is significantly less high than in roots. However, the excess of organic anions under nitrate nutrition (Fig. 2F) indicates a significant nitrate reduction in shoots (10 to 20% of assimilated nitrate). On the other hand, the sum of mineral anions in shoots is higher with ammonium nutrition than with nitrate nutrition (Fig. 2E), which expresses the low intensity of the transport of nitrate in the xylem.

## 2 - THE FUNGUS

### 2.1 - Mineral nitrogen uptake

Uptake rates of NH4 and NO3 by *H. cylindrosporum* thalli were estimated by 15N labelling. 15 days old thalli were transferred on fresh media containing K15NO3 (6 mM) or ($^{15}$NH4)2SO4 (6 mM) with pyruvic acid (10 mM) and the results obtained (Tab. 1) show that in all cases, if suitable carbon source is provided in the culture medium, the uptake rates of NH4 are always higher (two times more) than uptake rates of NO3.
<table><caption>TABLE 2 - TOTAL NITROGEN, SOLUBLE CATION AND SOLUBLE ANION CONTENTS IN TWO ECTOMYCORRHIZAL FUNGI</caption><caption>Pisolithus tinctorius</caption><thead><tr><th>CULTURE MEDIUM</th><th>TOTAL N CONTENT (µmol/g DW)</th><th colspan="3">SOLUBLE CATION CONTENT (µeq/g DW)</th><th colspan="3">SOLUBLE ANION CONTENT (µeq/g DW)</th></tr><tr><th></th><th></th><th>POTASSIUM</th><th>CALCIUM</th><th>MAGNESIUM</th><th>TOTAL</th><th>CHLOR PHOSPORUS</th><th>TOTAL ORGANIC ACIDS</th></tr></thead><tbody><tr><td>NITRATE</td><td>1928</td><td>405</td><td>40</td><td>58</td><td>503</td><td>44</td><td>236 280 223</td></tr><tr><td>AMMONIUM + PYRUVATE</td><td>2500</td><td>395</td><td>34</td><td>50</td><td>479</td><td>74</td><td>319 393 86</td></tr><tr><td>AMMONIUM</td><td>3357</td><td>412</td><td>16</td><td>40</td><td>468</td><td>114</td><td>321 435 33</td></tr></tbody></table>

<table><caption>Hebeloma cylindrosporum</caption><thead><tr><th>CULTURE MEDIUM</th><th>TOTAL N CONTENT (µmol/g DW)</th><th colspan="3">SOLUBLE CATION CONTENT (µeq/g DW)</th><th colspan="3">SOLUBLE ANION CONTENT (µeq/g DW)</th></tr><tr><th></th><th></th><th>POTASSIUM</th><th>CALCIUM</th><th>MAGNESIUM</th><th>TOTAL</th><th>CHLOR PHOSPORUS</th><th>TOTAL ORGANIC ACIDS</th></tr></thead><tbody><tr><td>NITRATE</td><td>1728</td><td>382</td><td>14</td><td>32</td><td>428</td><td>25</td><td>210 235 193</td></tr><tr><td>AMMONIUM + PYRUVATE</td><td>2228</td><td>366</td><td>16</td><td>38</td><td>420</td><td>57</td><td>271 328 92</td></tr><tr><td>AMMONIUM</td><td>2928</td><td>285</td><td>10</td><td>24</td><td>319</td><td>74</td><td>238 312 7</td></tr></tbody></table>
<table><caption>TABLE 3 - PHYSIOLOGICAL CAPACITIES OF THE ISOLATED PARTNERS OF ECTOMYCORRHIZAL SYMBIOSIS GROWN ON NITRATE MEDIUM</caption><thead><tr><th>PHYSIOLOGICAL CAPACITIES</th><th>HOST - PLANT</th><th>ECTOMYCORRHIZAL FUNGUS</th></tr></thead><tbody><tr><td>NO3 ACCUMULATION (µmol/g FW)</td><td>1 to 2</td><td>0,5 to 2</td></tr><tr><td>UPTAKE RATE (µmol/h/g FW)</td><td>0,4 to 0,6</td><td>1 to 2</td></tr><tr><td>NITRATE REDUCTASE ACTIVITY (µmol/h/g FW)</td><td>0,5 to 1,0 (in vivo NRA)</td><td>3 to 5 (in situ NRA)</td></tr><tr><td>ORGANIC ANION ACCUMULATION (A)</td><td>0,8 to 2,0</td><td>0,1</td></tr></tbody></table>

A: RATIO BETWEEN ORGANIC ANION ACCUMULATION AND TOTAL NITROGEN ACCUMULATION
## 2.2 - Ionic balances

The ionic balances were established for two ectomycorrhizal fungi, *Pisolithus tinctorius* and *Hebeloma cylindrosporum* which have different growth responses depending on culture medium. In both cases, ammoniacal medium supplemented with pyruvate (10 mM) gives the best growth. But *P. tinctorius* assimilates NH₄⁺ better than NO₃⁻. Conversely, *H. cylindrosporum* does not assimilate NH₄⁺ without organic acid in the culture medium, presenting a growth ten times lower than on nitrate medium. However, the ionic balances were similar for both species which mainly accumulate K⁺ and P(Tab.2). The both species produce very little organic acid quantities as compared with the nitrogen (nitrate form) assimilated, assuming that one OH⁻ can be produced when one NO₃⁻ is assimilated (RAVEN and SMITH, 1976). This is quite different from what is observed for Angiosperms (COIC et al., 1961; KIKBY and MENGEL, 1967; WOOLHOUSE and HARDWICK, 1966; CHAILLOU et al., 1986; ARNOZIS and FINDENEGG, 1986; SALSAC et al., 1988) and for maritime pine seedlings. This means that the fungus may eliminate a very high quantity of OH⁻ in the medium.

## 3 - CONCLUSION

Some works showed the positive effect of ectomycorrhizal association on dry matter production of the host plant grown on nitrate medium (CLEMENT et al. 1977; BLEDSOE and ZAZOSKI, 1983; PLASSARD, 1983). By comparing the physiological capacities of the isolated partners of ectomycorrhizal association (Tab.3), we can see that the fungus can act in different ways to improve the nitrate nutrition of the higher plant. Although the nitrate accumulation capacities were very low in both types of organisms(Tab.3), the fungus can improve the nitrate uptake rate directly or the nitrate reductase activity of the ectomycorrhizal plant. On the other hand the fungus can act indirectly by economizing carbohydrates production by the host plant, preventing organic acid synthesis and accumulation in the roots by means of very high OH⁻ excretion in the medium, as suggested by BOWEN and SMITH (1981).

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