Nitrogen nutrition of Douglas-fir (Pseudotsuga menziesii) on strongly acid sandy soil

Plant and Soil 126, 53-61 (1990). © Kluwer Academic Publishers. Printed in the Netherlands. PLSO 8528

Nitrogen nutrition of Douglas-fir (Pseudotsuga menziesii) on strongly acid sandy soil I. G r o w t h , nu tr ien t up take a n d ionic balance

ARJAN J. GIJSMAN Institute for Soil Fertility Research, P.O. Box 30003, 9750 RA Haren, The Netherlands

Received 12 December 1989. Revised April 1990

Key words: NH X deposition, ammonium toxicity, carboxylates, cell pH regulation

Abstract

Through ionic balance calculations, the effect of different sources and levels of nitrogen on nutrient uptake by Douglas-fir was studied. With ammonium as the sole source of N, growth of the plants was very poor. Increasing the levels of ammonium supply strongly decreased the surplus of total inorganic cations (C) over total inorganic anions (A). This decrease in C-A value, corresponding to the level of carboxylates in the plant, implies that in the long term the plant will run out of carboxylates and will then no longer be able to eliminate protons in the cytoplasm, produced during assimilation of ammonium. This can lead to internal acidification of the plant, toxic concentrations of free ammonium and an unbalanced amino acid composition. Values for the ratio of net carboxylate production and organic nitrogen production were in the same range as commonly found for other species. This did not support the theory of a conifer-specific ionic balance regulation as posed by others.

Introduction

The vitality of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) in The Netherlands has decreased considerably during the last decade (Staatsbosbeheer, 1987). Factors related to air pollution, covered by the term acid rain, probably play an important role in forest decline. Especially deposition of NH X (i.e. ammonia plus ammonium), originating from intensive livestock farming, is regarded as one of the major causes of decreased vitality. In some regions the mean annual deposition of NH× has reached levels of up to 3000mol h a -1 (Erisman et al., 1987; Schneider and Bresser, 1987), but locally even much higher levels are found. Apart from the possible acidifying effect of NH x during nitrification (Van Breemen et al., 1982; 1984) the deposition of NH x also leads to a strongly increased

nitrogen supply to trees which, mostly growing on poor sandy soils, were conditioned to a rather low nitrogen availability. Depending on the de- gree of nitrification, nitrogen will be available to the plant as ammonium, nitrate or a mixture of the two sources. The form in which nitrogen is absorbed largely determines the acidifying or alkalizing effect of plant nutrient uptake (Smiley, 1974; Troelstra et al., 1985). From ionic balance studies it can be calculated that ammonium nutrition always leads to H + excretion, while nitrate nutrit ion-depending on the level of supply- can give rise to H + or O H - (HCO3-) excretion.

From the nutrients absorbed, nitrogen and sulphate will be (partly) assimilated into organic form. The remaining content of inorganic cations (C) and anions (A) -including free NH~, NO 3, SO] -usually will not be in stoichiometric balance. The difference C-A reflects the surplus in

54 Gijsman

positive charge of inorganic ions in the plant, which is compensated for by organic anions or carboxylates (Breteler, 1973; Keltjens, 1981). If the phosphorus content used in calculating the total sum of anions is obtained from both inorganic (H2PO4) and organic (RHPO4) phosphorus, then C-A refers to salts of carboxylic acids only, rather than organic anions in general. Therefore the term carboxylates is preferred to organic anions (Van Tuil, 1965).

Rygiewicz et al. (1984a; b) and Bledsoe and Rygiewicz (1986) determined nutrient uptake and H+/OH--excretion of Douglas-fir and several other coniferous species. They found that the amount of H+/OH--ions excreted per unit nitrogen taken up was very much higher than commonly reported for other plant species; they also found a very high production of carboxylates. The authors concluded that the ionic balance in conifers might be regulated in a different way than in other species.

The present study was carried out to investi- gate the ionic balance and rhizosphere pH of Douglas-fir, fertilized with different sources and levels of nitrogen. This paper (part I) considers plant nutritional and physiological aspects of changes in the ionic balance. In part II H+/OH - excretion will be calculated and compared with actual measurements of the rhizosphere pH.

Materials and methods

The experiment was carried out with three-year old Douglas-fir trees (Pseudotsuga menziesii [Mirb.] Franco, provenance Arlington 202), planted in 10-liter pots with 12.75 kg sandy soil (on an oven-dry soil weight basis). On each pot one tree was planted with clean roots (without adhering soil); there were ten pots per treatment. The soil was covered with a 1-cm layer of polyethylene beads to reduce evaporation. The soil was taken from the forest research site for the Dutch acidification research program near Kootwijk, The Netherlands. It was collected from the upper 20-cm after the litter layer was removed, and sieved to remove stones and twigs. The particle-size distribution was as follows: <2/~m, 3.7%; 2-50/xm, 5.6%; 50-210/xm,

58.9% and >210/~m, 31.8%. The organic matter content as determined by loss on ignition was 3.2g per 100g air-dry soil. The CEC was determined by successively leaching the soil with a 1:1 (v:v) mixture of 1 M NH4AC and 96% ethanol, 1 M NaAc, 96% ethanol and finally 1 M NH4Ac; all, solutions adjusted to pH 7.0. Its value was 58 meq kg -1 on an air-dry soil weight basis. The pH-HaO and pH-KCI determined in a 1:5 (w:v) suspension of air-dry soil shaken with water or 1 M KC1, respectively, were 3.87 and 3.28. Further physical and chemical characteris- tics of the soil are given by Tiktak et al. (1988).

Three different forms of nitrogen were applied at N-levels of 10, 50 and 100mg kg -1 (on an oven-dry soil weight basis): NO 3 as Ca(NO3)2, NH 4 as (NH4)2SO4, and NHaNO 3. Other nutrients and trace elements were added as a basal fertilization. A control series without nitrogen but with the basal fertilization was included. All nutrients were added as solutions, and were mixed with the soil. The basal fertilization con- sisted of the following nutrients (per kg oven-dry soil): 30mg P as Ca(H2PO4)2, 50mg K as K2804, 70mg Ca as CaSO 4 (not on the pots receiving nitrate as the sole N-source), 25 mg Mg as MgSO4, 5 mg Fe as FeEDTA, 0.8 mg of each of the following trace elements: B as Na2B40 7 10H20, Mo as Na2MoO 4 2H20 , Cu as CuSO 4 7H20, Mn as MnSO 4 5H20 , Zn as ZnSO 4 7H20.

The nitrification inhibitor N-Serve 24E (2- chloro-6-trichloro-methylpyridine; Dow Chemi- cal) was mixed with the soil in all treatments at a rate of 10 mg per kg field-moist soil, in order to prevent conversion of NH 4 to NO 3. During the following growth period application of N-Serve was repeated every 10 weeks by injecting a 100 mL N-Serve solution into the soil at different depths with a 20-cm long needle. Regular checks on nitrification in fertilized pots without plants showed that indeed no NO 3 had formed. After the trees were planted in November 1987 the pots were placed in an unheated greenhouse (kept frost-free) until March. They were then transferred to a greenhouse with removable roof and walls. The pots were maintained at a fixed weight corresponding to a volumetric moisture content of 0.18cm 3 cm -3. Water lost due to evapotranspiration was regularly replenished by

adding demineralized water. In August 1988 the trees were harvested.

No special measures were taken to ensure the presence of mycorrhiza fungi in the soil. Inspec- tion of the roots at the start of the experiment and at harvest time revealed that mycorrhizae were virtually absent in all treatments.

Harvest

Shoot and roots were separated just above the first branch root. The shoot was divided into needles and woody parts; the latter fraction also included the young branches that had not yet lignified. Both fractions were dried at 70°C. The roots, collected from the soil by hand and by passing the soil through a l-ram sieve, were lightly rinsed with water and deep-frozen (-20°C) prior to thorough cleaning later and further examination. After thawing, sand and organic material were removed by washing. Liv- ing roots were separated from dead roots by manually checking their firmness and elasticity; only the living root material was used for calculations. The roots were dried at 70°C and weighed. During sampling, cleaning and storage of roots a considerable loss of dry matter may occur (Grzebisz et al., 1989; Van Noordwijk and Floris, 1979). Root dry weights were therefore correc- ted, assuming a 20% loss. Thus, the final root dry weights were obtained by multiplying the measured weights by 1.25. There also may have been leaching of e.g. K + ions and organic solutes with low molecular weight during the cleaning of the roots. Grzebisz et al. (1989) showed that the washing itself only slightly affected the N- concentration of sugar beet roots; the effect of freezing the roots before washing was not determined. Since nutrient losses from the roots could not be quantified, no correction factors on the concentrations were used.

Nitrogen nutrition of Douglas-fir. I. 55

ried out according to Vierveijzer et al. (1979). Total nitrogen and phosphorus were determined after digestion of the plant material with a mixture of salicylic acid and sulfuric acid plus thiosulfate, with a Technicon autoanalyzer. For determination of the cations the plant material was ashed at 400°C (K and Na) or 600°C (Mg and Ca), whereafter the ash was taken up in an HC! solution and analyzed by flame emission (Eppendorf) and AAS (Perkin Elmer), respectively. For chloride, the plant material was ashed at 550°C, the ash was taken up in HNO 3 and analyzed with a Marius Chlor-o-counter. For NO 3 the plant material was extracted with an Ag2SO4/CuSO 4 solution and the nitrate was bound by adding o-xylenol. The resulting nitrox- ylenol was measured spectrophotometricaIIy. Free NH] was determined in a water extract with a gas diffusion ammonia electrode (Orion model 95-10), as described by Novozamsky and Houba (1977). Free SO] was measured gravimetrically after digestion of the plant material with HNO.~ (slightlly modified after Novozamsky et al., 1986).

If no plants (or no more than 2) had died, 8 of the original 10 plants were randomly chosen and used for measurements and analyses; if more than 2 plants had died, all of the remaining ones were so used. Since not enough plant material

+

was left for the NH 4 analyses in the shoot, replicates were grouped before analysis. There- fore, NH2 concentrations are based on only 1 sample per treatment.

For determination of initial dry weight and chemical composition, 10 trees were randomly selected from the planting material at the start of the experiment. All root cleaning procedures described above were also performed on these trees. The dry weight of each was determined, while for chemical analysis the trees were com- bined into two groups of five.

Analyses

Shoots (needles+woody parts together) and roots were analyzed for N, P, K, Ca, Mg, Na, C1, NO3-N, NH4-N, SO4-S, which are the elements needed to calculate the ionic uptake balance. Unless stated otherwise, all analyses were car-

Statistics

The data were analyzed with ANOVA and, if significant treatment effects were found, with Duncan's Multiple Range Test, as adapted to unequal numbers of replications by Kramer (1956). Statistical operations were performed on

56 Gijsman

log-transformed data, since these fitted better to the condition of normal distribution and homogeneous variance.

Results

After planting, the roots had about 4 months for establishment before the weather became warm and growth started. All plants survived this period without any visible problem. Early in the spring differences between the treatments soon became visible. All plants fertilized with ammonium alone had a poor appearance and grew considerably more slowly than plants fertilized with one of the other two nitrogen sources. The needle color was dull green or even yellowish and young shoots did not elongate. Seven out of 10 plants grown at the highest NH4-N level soon died. There was little difference in plant appearance between the nitrate and ammonium nitrate treatments.

Dry matter production

Dry matter production of the plants is given in Table 1. In the nitrate treatments, 10 and 50 mg kg -1 N stimulated growth compared with the control; at 100 mg kg -~ the production declined 'again to the same level as in the control treatment. Growth of the ammonium fertilized plants was poor. With increasing ammonium concentration, both shoot and root growth decreased.

In the 100 mg kg-1 NH4_ N treatment hardly any net gain in root dry weight occurred, but, when the plants were harvested, a large amount of dead roots was found, indicating a high rate of root turnover. Growth of the plants fertilized with ammonium nitrate did not differ significantly from that in the control or nitrate treatments, but was usually much better than that in the ammonium treatments.

The relative distribution of the dry matter production over wood, needles and roots was clearly different for the ammonium treatment compared with the rest. The ratio of shoot growth to root growth strongly increased with N-level in the case of ammonium (3.2, 4.3 and 10.8, respectively), while with both other N- sources only slight changes were observed (in the range of 2.6-3.8). In the control treatment this ratio was 1.9. In the shoot, an increasing fraction of the dry matter production was found in the needles in the case of the ammonium treatment, while with the other treatments the distribution of dry matter between needles and wood re- mained more or less constant: the ratio between the production of needles and wood increased from 1.4 to 3.3 in the ammonium treatments, and varied from 1.5 to 1.8 in the other treatments (control: 1.9).

Mineral composition

Table 2 shows the concentrations of cations and anions in shoots and roots. An increase in N-

Table 1. Average dry mat ter weights (g plant -~) of wood, needles and roots at harvest, and total dry mat ter production during the experimental period (g plant -a) by Douglas-fir, as affected by different sources and levels of N-supply (mg kg -1 oven-dry soil) a.

Trea tment Wood Needles Shoot Roots Total growth n

Initial 4.73 3.56 8.29 4.20 * 10 Control 7.93 c 9.57 bc 17.50 bc 8.97 ab 13.97 bc 8 NO3 10 10.05 ab 13.24 a 23.29 a 10.04 a 20.84 a 8 NO 3 50 11.12 a 13.02 a 24.14 a 9.35 ab 21.00 a 8 NO 3 100 8.01 c 9.52 be 17.53 bc 6.91 c 11.95 c 8 NH 4 10 8.36 bc 8.54 c 16.90 bc 6.87 c 11.27 c 8 NH4 50 6.26 d 8.15 c 14.41 c 5.63 cd 7.55 d 8 NH 4 100 5.87 d 7.37 c 13.24 c 4.66 d 5.42 d 3 NH4NO3 10 9.45 abc 11.01 ab 20.46 ab 7.42 bc 15.39 abe 7 NH4NO 3 50 10.13 ab 11.91 a 22.04 a 9.29 ab 18.84 ab 8 NH4NO 3 100 8.78 abc 10.77 ab 19.54 ab 7.33 bc 14.39 abc 8

" Any two means in one column having a common letter are not significantly different at the 5% level of significance, according to Duncan ' s Multiple Range Test.


Table 2. Total nitrogen, cation (C), anion (A) and carboxylate (C-A) concentrat ion (meq.kg t dry matter) of shoot and roots of Douglas-fir, as affected by different sources and levels of N-supply (meq.kg ~ oven-dry soil). ~(C-A)/ANo,~ = the ratio of carboxylate production and organic ni trogen production with respect to the nutrient contents at the start of the experiment. SED = standard error of differences of means

N NH 4 K Ca Mg Na ~,C NO3 SO 4 P CI .ZA C-A A(C-A)/AN,,r~

Shoots Initial 700 31 178 146 97 6 459 Initial 0 95 51 7 153 30~ * Control 787 32 296 127 121 21 597 Control 7 180 37 19 244 353 (1.48 NO 3 10 808 31 270 138 119 17 575 NO~ 10 6 139 30 14 190 385 0.52 NO~ 50 1106 49 243 172 131 25 618 NO 3 50 8 111 23 t8 161 457 (t.43 NO~ 100 1250 34 259 258 150 32 733 NO, 100 37 77 25 21 159 574 0.50 NH 4 10 668 33 219 138 116 26 531 NH~ 10 7 225 32 20 283 248 (I.33 NH 4 50 876 57 2(19 132 114 24 536 NH4 5(1 7 206 36 26 275 26l (1.12 NH 4 100 938 58 209 141 106 24 538 NH~ 100 7 264 36 19 326 213 (t.(14 NH~NO, 10 752 34 257 132 120 20 563 NH~NO 3 10 6 197 28 18 249 314 (I.42 NH4NO 3 50 1103 51 255 141 129 20 596 NH4NO 3 50 8 176 25 17 226 37l 0.32 NH~NO~ i00 1159 40 229 159 127 24 579 NH4NO 3 100 12 185 31 17 245 334 0.24

SED 50.8 * 16.4 13.5 8.5 1.8 29.2 SED 4.6 23.0 3.6 2.2 26.0 29.8

Roots Initial 535 26 99 289 62 23 498 Initial 1 51 41 17 110 388 * Control 746 25 23 341 45 14 446 Control (I 64 36 7 106 340 0.33 NO 3 10 771 24 22 296 49 12 403 NO 3 10 0 62 35 8 104 298 0.25 NO 3 50 801 22 22 307 44 14 409 NO 3 50 1 76 31 8 116 294 0.19 NO~ 100 805 29 17 285 43 12 387 NO 3 100 2 71 3l 7 110 277 ().04 NH 4 10 709 24 17 323 44 15 422 NH 4 10 0 61 35 7 103 319 0.15 NH 4 50 751 37 11 339 38 13 439 NH~ 5(J 0 100 38 5 142 297 0.0l NH 4 100 900 33 13 360 44 10 460 NH 4 100 0 114 41 3 158 302 - 0 . 1 2 NH4NO 3 10 775 31 18 237 33 12 330 NH4NO 3 10 0 71 34 6 111 219 - 0 . 1 2 NH4NO ~ 50 851 27 34 320 47 15 442 NH~NO 3 50 0 85 35 11 131 311 0.21 NH4NO~ 100 862 30 15 267 38 11 361 NH4NO 3 100 0 76 35 6 116 245 0.00

SED 17.6 2.0 4.1 31.3 2.3 0.9 34.4 SED 0.3 5.4 1.0 0.9 5.9 35.0 *

* All values are the results of analysis of individual plants and were averaged afterwards. However, A(C-A)/AN ~ was calculated from the t reatment means, and therefore cannot be analyzed statistically.

supply, regardless of source, raised the N- concentrations in the plant. In the shoot, these increases were considerable in the case of the nitrate and ammonium nitrate treatments, and a little smaller for the ammonium treatment. In the roots the increases in N-concentration were small for all forms of nitrogen supplied. Free nitrate concentrations in the plant were usually low; only at the highest level of nitrate and - to a lesser ex ten t -ammonium nitrate, did shoot concentrations exceed very low levels. Free- ammonium concentrations exceed very low levels. Free-ammonium concentrations in the plant were rather high (compared with, for in- stance, Na, P and C1), also in the nitrate treatments.

In the shoot, the concentrations of Mg, Na and especially Ca increased with increasing nitrate supply, while the K concentration slightly decreased. In the ammonium and the ammonium nitrate treatments cation concentrations in the shoot varied little with N-level. The total cation concentration in the shoot increased with increasing nitrate supply and was unaffected by the level of supply in the other two N-sources, ammonium giving the lowest value.

The treatments had little effect on the anion concentrations in the shoots, with the exception of sulfate. The much higher sulfate concentrations in the case of the ammonium treatment was probably due to the fact that ammonium was supplied as ammonium sulfate. Compared with

58 Gijsman

the control, total anion concentration decreased with nitrate supply and increased with ammonium supply.

In the roots variations in cation as well as anion concentrations were very small, except for Ca and SO 4. With nitrate, the Ca concentrations in the roots were somewhat lower than with ammonium, resulting in a lower total cation concentration. The sulfate concentration was higher with ammonium, and the same was true for the total anion concentration.

For some nutrients there were differences in distribution between shoot and roots. Nutrient concentrations in the shoot were usually higher than in the roots, which was most pronounced for K; its concentration in the shoot was some- times more than 15 times higher than that in the roots. In contrast, Ca concentrations in the roots were much higher than those in the shoot.

Carboxylate production

Table 3 shows that the total cation contents of the whole plants receiving nitrate were higher, and total anion contents equal to or slightly lower than those of the plants given ammonium. This resulted in a considerably higher carboxylate content (C-A) in the nitrate-fertilized plants. The ratio between the production of carboxy-

lates and the production of organic nitrogen, A(C-A)/ANorg , was fairly constant in the nitrate- fertilized plants. With ammonium fertilization, A(C-A)/ANorg rapidly decreased with increasing ammonium supply, finally reaching a value of zero. Thus, at the 100mg kg -1 NH4-N level there was no net production of carboxylates. With the mixed nitrogen source A(C-A)/ANorg values were intermediate between those of the nitrate- and the ammonium-fed plants.

Discussion

As hardly any mineral nitrogen was present in the soil at the start of the experiment, almost all nitrogen taken up by the plant in the control pots must originate from mineralization. Table 3 shows that this amount of nitrogen was considerable. Assuming that the N-Serve completely blocked nitrification, all mineralized nitrogen was available in the ammonium form. Leaving possible interactions between mineral nitrogen content of the soil and rate of mineralization out of consideration, it may be assumed that in all other treatments a comparable amount of ammonium from mineralization was added to the fertilizer nitrogen pool. So in all except the ammonium treatments, a mixture of both nitro-

Table 3. Total inorganic cation (C) and anion (A) contents (meq plant 1), total organic ni trogen content (Norg; mmol plant ~) and A(C-A)/ANorg ( - ) with respect to the nutr ient contents at the start of the experiment , as affected by different sources and levels of N-supply (mg kg -1 oven-dry soil) a

£C .ZA C-A No~ * A(C-A)/ANob,~

Initial 5.89 1.73 4 .17 7 .68 *

Control 14.34 bcde 5.13 ab 9.21 bc 19.39 c 0.43 NO 3 10 17.38 ab 5.51 ab 11.87 a 25.32 b 0.44 NO 3 50 18.66 a 4.89 ab 13.77 a 32.76 a 0.38 NO 3 100 15.45 abcd 3.45 c 12.00 ab 26.23 ab 0.42 NH 4 10 11.7l ef 5.38 ab 6.33 de 15.19 d 0.29 NH 4 50 10.18 f 4.67 b 5.51 ef 15.74 d 0.17 NH 4 100 9.26 f 5.08 ab 4.19 f 15.59 d 0.00 NH4NO 3 10 14.10 cde 5.90 ab 8.20 cd 20.03 c 0.33 NH4NO 3 50 17.09 abc 6.13 a 10.96 ab 30.49 ab 0.30 NH4NO 3 100 13.93 de 5.59 ab 8.34 cd 27.66 ab 0.21

a Any two means in one column having a common letter are not significantly different at the 5% level of significance, according to Duncan ' s Multiple Range Test. b All values are the results of analysis of individual plants and were averaged afterwards. However, A(C-A)/ANorg was calculated from the t rea tment means , and therefore cannot be analyzed statistically.

gen sources at different ratios will have been available to the plant. Elsewhere, calculations on the ammonium/nitrate uptake ratio of the plant will be presented, which are based on the amounts of mineral nitrogen taken up from the soil and left in the soil at harvest (Gijsman, 1990).

Growth and nitrogen uptake

In the present experiment ammonium alone was clearly an inferior source of nitrogen. Plants in the ammonium treatments absorbed not more than 35--45% of the amount of nitrogen absorbed by plants in the corresponding nitrate treatment. Since nitrogen absorption and dry matter production by the ammonium-fertilized plants was even lower than that of the unfertil- ized control plants, it is likely that the additional ammonium supply led to disturbance of certain physiological functions. These results are in agreement with those of Smit et al. (1987), who also found a very high mortality and a low growth rate of Douglas-fir in ammonium treatments at low pH.

At the lowest N-level plants fed with ammonium nitrate did not grow as well as those fed with nitrate alone. An input/output balance of the pots (Gijsman, 1990) showed that in the 10mg kg 1 NH4-N treatment nitrate uptake could only meet one third of the total amount of nitrogen taken up by the plant. So, most nitrogen must have been taken up as ammonium, which means that the same adverse effects of pure ammonium nutrition will also hold for the 10 mg kg-~ NH4NO3-N treatment. With increasing supply of ammonium nitrate, there was suf- ficient nitrate in the soil to cover most of the plants N demand.

Carboxylate production

During the cleaning of the roots, leakage of nutrients may have occurred, so actual (C-A) concentrations in the root can be slightly different from the values measured. However, the effect of rather large corrections of the measured C-A concentrations (say 20%) on A(C-A)/AN,,rg


of the total plant is small; this ratio only changes with a few hundredths. The interpretation of the data therefore is not seriously affected.

When assimilation of nitrate occurs in the shoot, OH ions produced during nitrate reduction are converted into carboxylates, thus pre- venting large changes in cytoplasmic pH (Davies, 1973a; b). After transportation of the carboxylates to the roots and subsequent decarboxylation, OH ions can be excreted to the medium (Ben Zioni et al., 1971; Dijkshoorn, 1969; 1970; Dijkshoorn et al., 1968). When reduction occurs in the roots, OH ions can be directly excreted and carboxylate production is usually low. In woody species the main site of nitrate reduction is considered to be in the roots (Lee and Steward, 1978; Pate, 1973). Most of the carboxylate production will then be the result of the surplus of cation over anion uptake (other than nitrate).

Wollenweber and Kinzel (1988) determined carboxylate and organic nitrogen contents for a variety of plant species from 5 different habitats. They found lowest (C-A)/Norg ratios (down to 0.15) in plants from soils with NH~ as the main N source and higher values (up to 1.1) in plants from nitrate-dominated sites. Data on carboxylate levels in Picea abies were reported by Evers (1964), working with trees grown on nutrient solutions at different pH with ammonium, nitrate or the mixed form as the N-source. With ammonium-fed plants, the surplus concentration of cations over anions dropped very rapidly when the pH was lowered from 8 to 6, becoming negative below pH 6. In nitrate-fed plants vari- ation in C-A value was much smaller and values were in the same range as reported here. When the pH was about neutral, the pattern in C-A concentration with ammonium nitrate nutrition resembled that with nitrate nutrition. In the acid range the C-A concentration in ammonium nitrate-fed plants dropped. These results agree very well with those reported here, showing a deple- tion of carboxylates with ammonium nutrition in acid medium, as can be seen from the A(C-A)/ ANorg ratio of the whole plant (Table 3). At the 100 mg kg -j NH4-N level there was no net carboxylate production at all (A(C-A)/ANorg =0) and a net decrease in carboxylate content was observed in the roots of these plants, In this

60 Gijsman

treatment most plants died and growth of those remaining was very poor.

Cell pH control

Assimilation of ammonium gives rise to a production of protons in the plant which have to be excreted to the medium, or neutralized by reac- tion with OH- ions from decarboxylation of previously formed carboxylates. From Nernst's Law it follows that at a pH of the rooting medium lower than about 9, a passive H ÷ influx into the root takes place. In very acid soils the capacity of the proton extrusion pump will be largely used for neutralization of the passive H ÷ influx. The ability to exrete additional protons can then be small, so they have to be neutralized within the plant. However, with ammonium nutrition net carboxylate production is small, and so a situation may arise in which a large demand for carboxylates exists, but only a very small supply. Further elimination of protons- and thus pH control- is then no longer possible, so internal acidification of the plant will occur.

A way to avoid this situation might be not to assimilate ammonium ions. This would lead to accumulation of (toxic) free NH 4 ions in the plant and interference with the normal amino acid composition. Analysis of amino acids in needles from vital and less vital Pinus sylvestris, growing in an area with a high level of NH x deposition, showed a large increase in the concentration of the N-rich amino acid arginine in the less vital trees (Boxman and Van Dijk, 1988). This increased arginine production may have a function in getting rid of ammonium.

Results from Troelstra et al. (1990) support the above theory. They showed that Plantago plants under ammonium nutrition reduced the pH of the nutrient solution to a value as low as 3.4; at this pH nutrient uptake ceased. Appar- ently, the combination of ammonium nutrition with the very low pH seriously impaired nutrient uptake by the roots. Plants pretreated with nitrate were able to delay the harmful effects of ammonium for some time, indicating that they could benefit from carboxylates, which were produced during reduction of nitrate from an internal nitrate pool.

Ionic balance

Bledsoe and Rygiewicz (1986) measured the rate of influx and efflux of several nutrients for roots of Douglas-fir, exposed to an ammonium- containing solution in a 4-hour experiment. From the ionic balance of the plant they calculated the carboxylate production; it was several times higher (i.e. A(C-A)/ANorg = 4 to 5) than that commonly found in other (non-coniferous) plant species. Earlier they stated that also the rate of H÷/OH -excretion per unit nitrogen absorbed was very high in several coniferous species (Rygiewicz et al., 1984a; b). The authors suggested that the ionic balance in conifers may be regulated differently from that of agricultural species (Bledsoe and Rygiewicz, 1986).

The present results do not support this hypoth- esis, since carboxylate levels and H+/OH - excretion per unit nitrogen absorbed (see also Gijs- man, 1990) were in the same range as those found for other plants. Indeed, it is difficult to accept that the situation described by Rygiewicz and colleagues can exist for more than a few hours, since the measured anion uptake (Bledsoe and Rygiewicz, 1986) was so low- 1 anion for every 650 cations in the case of mycorrhizal roots - or even negative (non-mycorrhizal roots), that a shortage of essential anions seems un- avoidable.

Acknowledgements

I thank Dr S R Troelstra, Institute for Ecological Research, Prof Dr Ir P J C Kuiper and Dr G Stulen, University of Groningen, for their criti- cisms and comments on an earlier version of the text, and Dr Ir A de Jager, who initiated this research.

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Nitrogen nutrition of Douglas-fir (Pseudotsuga menziesii) on strongly acid sandy soil