Plant and Soil 152: 207-214, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands. PLSO 9933

Studies of in the foliage reveal interactions between nutrients and water in forest fertilization experiments

PETER HOGBERG 1, CHRISTIAN JOHANNISSON 1 and JAN-ERIK H.~LLGREN 2 1Department of Forest Ecology, Section of Forest Soils and e Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S-901 83 Umed, Sweden

Received 25 January 1993. Accepted in revised form 24 April 1993

Key words: 13C, forests, N deposition, N fertilization, water stress

Abstract

Addition of N to an initially N-limited forest increases foliage biomass, demand for water and the probability of water stress. Effects of water and N on tree growth are thus compounded. The 13C abundance of plant tissues is directly correlated with water use efficiency (WUE), and could be used to disentangle the effect of water alone on carbon fixation. However, the 13C abundance may also be directly influenced by changes in rates of photosynthesis related to variations in N status, and by variations in N metabolism via non-RuBisCo carboxylations, and indirectly by effects of N source on WUE. We studied the 13C abundance of current needles from top whorls in two long-term fertilization experiments, one in Norway spruce (Picea abies Karst.) and one in Scots pine (Pinus sylvestris L.). As predicted, N fertilization increased foliage biomass and 6 13C (%@ In the experiment with spruce this effect on 13C abundance was correlated with volume production and foliage biomass in a dry year, but was not seen in a wet year after 19 years of continuous annual N fertilization, which rules out the possible influences of N metabolism and changes in rates of photosynthesis. In the experiment with pine, which was at a drier site, needles from N-fertilized plots had a higher 13C abundance in three dry years, but not significantly so in a wet year. We suggest that effects of N source (NH~ or NO3) on 13C abundance are unlikely to be important under these experimental conditions. The balance between demand and supply of water should thus be the major determinant of the ~3C abundance of current needles on top whorls. This opens possibilities to conduct retrospective studies of the role of water supply in fertilization experiments.

Introduction

Numerous fertilization trials on mineral soils in the temperate zone have shown that forest production is limited by a low supply of nitrogen (e.g. Tamm, 1964, 1991). Under natural condi- tions, a high supply of available N is often associated with groundwater discharge (flushed) areas (Tamm 1991). Effects of N on tree growth are thus confounded by possible effects of water. A positive effect of water in irrigation experi- ments can, on the other hand, be due to in-

creased N mineralization and transport of N to roots, rather than a physiological effect of water on the trees. For example, a strong interaction between irrigation and fertilization was shown by Brix (1972). Hence, it would be of considerable interest to disentangle the effect of water alone from that of its effects on transformations and flow of N in soils.

A consequence of increasing deposition of N on initially N-limited forests will be an increase in foliar biomass (Abet et al., 1989, Agren, 1983), and hence an increased risk of tree water

208 H6gberg et al.

deficit resulting in shedding of foliage and re- tarded stem growth (Linder et al., 1987).

In C3-plants like most trees, the enzyme RuBisCo discriminates strongly against 13C dur- ing photosynthesis, but this effect becomes less expressed as stomata close because of water stress (e.g. Farquhar et al., 1982; Farquhar and Richards, 1984; Farquhar, 1991). This effect on the ~3C abundance of fixed C is directly and positively correlated with the ratio between the concentrations of CO 2 intracellularly and in ambient air (Ci/Ca). Measurements of the 13C abundance of plant tissue thus provide an inte- grated retrospective measure of the effect of water stress on carbon fixation. In contrast to growth analysis, e.g. measurements of tree diam- eter etc., this gives direct evidence of the effect of water alone on carbon fixation.

There are, however, a few problems to con- sider in this context. First of all, the 13C abun- dance changes vertically in the canopy as a result of self-shading (Ehleringer et al., 1986; Francey et al., 1985), and in branches close to the ground as a result of refixation of C respired from the soil (Medina and Minchin, 1980; Vogel, 1978). Secondly, theoretically there is a variation in a3C abundance related to the use of different sources of N and N assimilation pathways because of non-RuBisCo carboxylations (Raven and Far- quhar, 1990), and possibly also via effects of N source on water use efficiency (WUE; Raven et al., 1992). Thirdly, rates of photosynthesis may increase with increased availability of N and internal N concentration (Field and Mooney, 1986), and thereby influence carbon isotope discrimination irrespective of variations in stomatal conductance. Genotypic differences in WUE may be another source of variation (Far- quhar and Richards, 1984).

We attempted to see if N fertilizer treatments induced changes in the ~3C abundance of trees, and if these could be ascribed to water stress or to change in N metabolism and rates of photo- synthesis. We used needle samples from top whorls, because these should not be shaded or refix significant amounts of C respired from the soil. Virtually all C in current needles is derived from their own photosynthesis or from the same years photosynthesis in C + 1 (last years) needles (Ericsson, 1978). The stands we studied can be

assumed to be composed of a range of geno- types, which together with heterogeneity of the soils should superimpose variations on the treat- ment effects.

Materials and methods

Sites

We studies two of the Swedish Forest Optimum Nutrition Experiments (Tamm, 1985), one in Norway spruce (Picea abies Karst.) at Strfisan (60°55'N, 16°01'E, 360-410m a.s.l.), and the other in Scots pine (Pinus sylvestris L.) at Lis- selbo (60°28'N, 16°57'E, 80m a.s.1.). The Str~san site is on a rather steep (up to c. 20%) slope with a glacial till soil dominated by medium and fine sand. The Lisselbo site is on gentle slopes on and around a glacifluvial esker, and has sandy soils, which range from gravelly to fine sandy. Mean annual temperature and rain- fall (1931-60) at nearby weather stations were 3.1°C and 745mm at StrSsan, as compared to 4.8°C and 593mm at Lisselbo. StrSsan was planted with Norway spruce in 1958 one year after a prescribed burning. The Scots pine forest at Lisselbo was established by natural regenera- tion, but with some additional sowing, around 1955. Further details about the sites are given by Tamm et al. (1974a,b).

Treatments'

Experimental treatments started in 1967 at Str~san and in 1969 at Lisselbo. At both sites individual plots were 30 x 30 m, but all sampling have been conducted on the central 20 x 20 m. At Str~san, we studied the experiment E26A, which is a factorial experiment on 55 plots comprising additions of N a s NH4NO 3 at four levels (N0-N3), of P at three levels (P0-P2), of K plus Mg and micronutrients at two levels (K0- K1), and some plots with additional lime (Ca). The total additions of N during the period concerned here, 1967-1985, amounted to 730, 1460 and 2190kg N ha -1, to N1, N2 and N3 plots, respectively. Further details about treat- ments at Str~san are given by Tamm et al.

(1974a), Tamm (1991) and H6gberg et al. (1992). At Lisselbo, we studied two experi- ments, E40 and E41, comprising together 52 plots. In E40, four levels of N as NH4NO 3 were tested (N0-N3), with and without PK (P2K2). In E41, P, K, Mg and S were added to N- fertilized (N2) plots, of which a few also received micronutrients. The total additions of N to N1, N2 and N3 plots in E40 at Lisselbo 1969-1988 were 580, 1160 and 1740 kg N ha -l, respectively. In E41, the N2 treatment resulted in a total addition of 1040 kg N ha-l ; this experiment was terminated in 1985. Further details about the treatments are given in Tamm et al. (1974b).

Sampling and chemical analysis

Starting from one year before the experimental treatments, samples of current needles were taken from top whorls of 10 trees from each plot in the autumn every year, mixed into one composite sample per plot, dried (70°C, 24h) and ground in a ball mill. We selected at random needle samples from the years 1966, 1975 and 1985 at Str~san, and from 1968, 1970, 1973, 1979 and 1988 at Lisselbo. Analysis of ~3C abundance was carried out by ANCA-MS (Barrie 1991). Results are reported in 613C (%o):

6 ~3C = 1 0 0 0 x (R~.,op,~ - R ~ t a n a a . a ) / R . . . . . . . d ( % o ) .

where R = 13C/12C, and the working standard was sawdust calibrated against Pee Dee Belem- nite. A high 13C abundance means a less nega- tive 613C (%@ Plant physiologists commonly use the inverse derived unit /',, which is convenient in discussions about plant carbon isotope dis- crimination. We aim at linking our observations on the foliage with future studies of litter de- composition and soil carbon dynamics and, therefore, use the 6 unit directly refering to isotopic composition.

Analysis of data

First of all, we studied the distribution of 13C data the year before treatments at both sites. Secondly, we studied the variation between years, and compared our results with data on the

Studies of 13C in foliage 209

climate. Thirdly, we tested if there was any variation due to the treatments. Fourthly, we tested if the position of plots along the slope at Strhsan was of any significance, i.e. if plots on the upper slope were driest.

We used ANOVA and t-tests throughout in the case of Strfisan, because pretreatment data as well as data from 1975 and 1985 were normally distributed (Fig. 1). The experimental design allowed a three-way ANOVA on the effects of N, P and K. In the case of Lisselbo, pretreat- ment data showed a considerable skewness, if not a bimodal distribution, but other years data were normally distributed (Fig. 1). As shown below, we found a strong spatial component in the pretreatment data from Lisselbo, and de- cided to remove nine exceptional plots from the further analyses (the framed plots in Fig. 2), although this did not affect the analyses sig- nificantly other years. We thereafter used parametric statistics as above when analyzing data from 1970, 1973, 1979 and 1988. For the test of effects of N we used E40 only, because there were no NO plots in E41, while for tests of effects of K and P in N-fertilized forest we used E41, which comprises N2 plots with and without additions of K and P.

Results

Norway spruce at Strdtsan

Data on ~3C in current needles were normally distributed each of the studied years (Fig. 1). There were no difference between 1966, the year before treatments, and 1985, i.e. after 19 years of treatment, but 1975 differed from the other two years (Table 1). 1975 was one of the driest years during the experiment, i.e. the rainfall was low while the temperature was high (Fig. 3). However, needles from treatments not receiving any N had almost the same 6 13C (%o) all three years (Table 1). The deviation in 1975 was due to a higher 613C (%o) on plots fertilized with N, and was positively correlated with annual stem volume production (Fig. 4). A correlation (r 2= 0.23, p = 0.022) was also found if only plots fertilized with N were used in this regression. At

210 HOgberg et al.

t -

O

8

0

co

Str&san

- 2 5

- 2 7 01 Ill

kisselbo

- 2 0 II 1968

-22"

-24"

-26.

~ 985

1970 1973 1979 1988

Fig. 1. Distribution of data on 13C abundance of current needles of Norway spruce at Str~san (top; n = 55) and of current needles of Scots pine at Lisselbo (bottom; n = 52) before (1966 and 1968, respectively) and during treatments.

Str~san, volume production is strongly correlated with needle biomass (Axelsson, 1985; Tamm, 1985). A weak (p = 0.06) positive correlation was found between the ~ 13C (%0) of needles in 1975 and the needle biomass of ten plots in 1975, as estimated by interpolation of data from sam- plings in 1973 (Tamm 1985) and 1978 (Axelsson, 1985; Fig. 4). However, removal of an outlier improved the correlation significantly (r 2= 0.80, p = 0.000). Additions of P and K plus other elements had no effect on the 13C abundance of needles (Table 2). Neither was there any indica- tion that the position along the slope was of any importance for the ~3C abundance of needles (Table 1).

needles from plots not receiving any N (Table 3). As said above, we decided to remove these plots from the further analyses. After the treat- ments had commenced, the 13C abundance of needles from plots treated with N was always 0.6-1.4 ~ 3 C (%o) higher than those from plots receiving no N (Table 3). Analyses of data from 1970 and 1973 revealed that the N additions had the major influence on 13C abundance; effects of K or P were never significant. The needle biomasses on N1-N3 and N2PK plots varied between 5.0 and 5.8 tonnes ha -1 in 1975, as compared to 2.8 tonnes ha -1 on NO (control) plots (A. Albrektsson and A. Aronsson, pers. comm.).

Scots pine at Lisselbo

In 1968, the pretreatment year, very high 613C (%o), which caused the skewed distribution of da~a (Fig. 1), were found within a fairly confined area comprising nine plots (Fig. 2). These nine plots were less exceptional other years, which appeared to be wetter, as indicated by data on climate (Fig. 3), and the 13C abundances of

Discussion

We assume that the variations in 13C abundance before treatments largely reflected spatial differ- ences in soil water supply and to a minor extent variations in WUE between genotypes. Both experiments are located in groundwater recharge areas. It is, therefore, no surprise that there were no differences along the slope at Str~san

-24.2 -24.3

-25.0 -24.9

-24.7 -24.4

-24.4 -24.6

I -24.7 -24.4 -24.6 1-24. -24

-24.6 -24.6 -24.3 -24.5 -24.£

-22.1 -20.5 -20.2 [-25.1 -25.(

-20.6 -20.4 -20.4 -25.0 -24.!

-21.2 -21.8 -22.7 -25.0 -24.~

-23.3 -24.8 -24.7 -24.~

-24.3 n.d. -24.41 -24.9

-24.~

-24.~

-24.~

-24., ~ -24.6

25.0

-24.5

Fig. 2. Spatial distribution of data on 13C abundance (613C (%0)) of current needles (composite samples from 10 trees per plot -~) of Scots pine from each of the 52 plots at Lisselbo in 1968, which was the year before treatments started. The area with needles of very high ~3C abundance is framed by bold lines. The arrow points to the North. Each plot is 30 x 30 m.

(Table 1). In a study where the hydrology was more varied, Garten and Taylor (1992) demon- strated that trees on xeric ridge sites had a relatively high 13C abundance.

With the exception of Strfisan in 1985, N fertilization increased the 613C (%~) of needles (Tables 1-3). At Strfisan this was thus seen in a dry year, but not in a wet year, and the possible influence of changes in non-RuBisCo carboxyla-

Studies of 13C in foliage 211

tions via a change in N metabolism (Raven and Farquhar, 1990), as well as influences of changes in rates of photosynthesis can be ruled out. The increase in rates of photosynthesis with increas- ing leaf N concentration is remarkably smaller for evergreens than for deciduous and annual species (Field and Mooney, 1986). Observations on N-fertilized and non-fertilized Norway spruce in Sweden confirm that the response in rates of photosynthesis is small (M. Strand, T. Lundmark and J.-E. H/illgren, pers. comm.). At Lisselbo, which has much more coarse-textured soils, we could not ascribe the effect of N fertilization on 613C %o to a lower water supply only, because the difference between needles from N-fertilized plots and plots not fertilized with N was found irrespective of climatic variations although it was not fully significant in the comparatively wet year 1988 (Fig. 3 and Table 3).

An appreciable increment in ~ 13C (%~) due to a change in N metabolism would only be seen if there was a shift from assimilation of NH 4 or NO~- in roots to a predominance of shoot assimi- lation of NO3 (Raven and Farquhar, 1990). In a similar experiment in Scots pine in northern Sweden, there was only a minor induction of shoot nitrate reductase activity on plots heavily fertilized with N (T. Nfisholm, pers. comm.). There are no major differences in N metabolism between Norway spruce and Scots pine. Accord- ingly, the difference between needles from N- fertilized and non-N-fertilized plots at Lisselbo should also reflect variations in water stress rather than variations in N metabolism.

According to Raven and Farquhar (1990), effects of N metabolism on ~3C abundance should be small in trees considering their high C/N ratios. They referred to C/N ratios of wood, which should be irrelevant since carboxy- lations take place in active assimilating tissues. C/N ratios of conifer needles are, nevertheless, clearly higher than that of the model herbaceous plant discussed by Raven and Farquhar (1990).

However, a shift from NH 4 to NO~ could also lead to a higher WUE (Raven and Farquhar, 1990), and thereby increase the 13C abundance, as indicated by empirical evidence, but not considerations of biochemical pathways (Raven et al., 1992). This issue can be made even more complex by taking into account interactions

212 HOgberg et al.

Table 1. Abundance of ~3C in current needles of Norway spruce in the forest fertilization experiment E26A at Str&san. Plots are stratified according to (A) their position on the slope, or (B) whether they were fertilized with N or not in various combinations with other elements. Upper slope, >385 m a.s.l.; mid-slope, 375 > x > 385 m a.s.1.; lower slope, <375 m a.s.l. Data are mean values ± I S .E . 1966 was the year before treatments started

Year

1966 1975 1985 (f~ 13C (~oo))

( A ) Position on slope Upper slope (n = 13) -26.1 -+ 0.1 -25 .0 ± 0.2 -25 .9 ± 0.1 Mid-slope (n = 18) -26.1 ± 0.1 -25 .1 ± 0.2 -26 .0 ± 0.1 Lower slope (n = 24) -25 .8 ± 0.2 -25 .0 ± 0.2 -25 .9 +-- 0.2

(B) Treatment Treatments without N (n = 15) Treatments with N (n = 40)

- 2 6 . 2 ~ 0 . 2 - 2 5 . 9 ± 0 . 1 - 2 6 . 1 ± 0 . 2 - 2 6 . 0 ± 0 . 1 - 2 4 . 7 ± 0 . 1 ~ - 2 5 . 9 ± 0 . 1

Total average (n = 55) -26 .0 --- 0.1 -25 .0 ± 0.12 -25 .9 ± 0.1

Treatments with N are different from treatments without N in 1975 (ANOVA: p < 0.001). 2 Pooled data from 1975 are different from data from 1966 and 1985 (ANOVA: p < 0.001).

800-

t,~, f~% '""......../ A t -4- ~"" ~ - ~ ~ T ' - ---: ~

A 8 -2z

0

600- ~ -2q E E ~"

E o 400- = 0 ~ , , , , 0

6 "5 8

800- 0 - 2 4 e - < co

600-

6

o

8" 3 -2 "0

e-

-8 I

o

O"

v

-4 6

-2 -2{

B

,

400-

1965 1475 1985 0

Year

Fig. 3. Variations in annual rainfall (solid line, the horizontal one shows the mean during the period) and mean tempera- ture from April to October (broken line, the horizontal one shows the mean during the period) at the weather station Falun, 60 km from Strgtsan A, and at the station G~ivle, 30 km from Lisselbo and 70 km from Str&san B.

o3 7

~-o o

o

A • • • c o . ~ __

• o -~

J

r 2= 0.64 p = 0.000

12 18 Annual volume production, m 3 ha- lyr -t

i i i

B

0 0

• •

r 2 = 0.30 6) p = 0.06

' 6 Needle biomass, t ha -1

18

Fig. 4. 13C abundance of current needles at Strgtsan in a dry year, 1975, plotted versus A mean annual stem volume production 1973-1975 (n = 2-5 plots), and B needle biomass in 1975 as estimated by interpolating between data collected in 1973 and 1978 (n = 1 plot). Open symbols denote plots not given any N; those given N are denoted by closed symbols.

Table 2. Results of a three-way ANOVA on effects of N, P and K on the 13C abundance of current needles of Norway spruce in the forest fertilization experiments at Strhsan in

1975. The treatment N2P2K1Ca was removed to obtain a more balanced factorial design

Source df F p

N 3 5.00 0.006 P 2 0.71 0.498 K 1 0.03 0.859 N x P 6 0.43 0.850 N x K 3 1.31 0.290 P x K 2 0.58 0.568 N x P x K 6 1.00 0.444 Error 24 Total 47

between water supply, plant demand and pref- erence for NH4, and the fluxes of NH4 and NO 3 in a buffered medium (e.g. Marschner et al., 1991), e.g. shifts from diffusion to mass flow as major N transport mechanisms in the soil-root system. The effects of N source on 613C (%Q via effects on WUE may be superimposed on this pattern, but the relevant data set is as yet too restricted to allow generalizations (Raven et al., 1992). It does, for example, not include any tree species.

We find it interesting that pretreatment data on ~3C abundance at Lisselbo showed a consider- able variation (Fig. 1). In the case of very oligotrophic temperate forest environments as these in areas with low deposition of N, NO 3 should be a rare N species (H6gberg et al., 1990; Read, 1983). We repeat our suggestion that the data from Strhsan in 1985 rule out an influence of N source on 13C abundance via non-RuBisCo carboxylations (Table 1). This is further corrobo-

Studies of 13C in foliage 213

rated by the low shoot nitrate reduction in these conifers. As regards effects on WUE of NH~ vs NO3 based nutrition, the situation is much more complex as discussed above. It is unlikely that there could be a total shift to a NO 3 based nutrition on N-fertilized plots, in particular under dry conditions. Nor does our data indicate any influence of possible changes in rates of photosynthesis caused by N fertilization on 613C %~, i.e. there was no effect on N fertilization in a wet year (Table 1). We thus suggest that varia- tions in 13C abundance in top whorls in forests fertilized with N are primarily related to effects of balances between demand and supply of water on carbon isotope discrimination during fixation.

At Str~san, these variations were directly related to needle biomass in the dry year of 1975 (Fig. 4), which supports the hypothesis that additions of N to a N-limited forest will increase the risk of water stress (Aber et al., 1989). Based on a transpiration model, it has been calculated that a 50% increase in needle area would double the probability of drought stress in Sweden (Lindroth, 1987).

In conclusion, additions of N increased the needle biomass and thereby also the probability of water stress. Additions of other elements to correct for nutrient imbalances, so called revitali- zation fertilization, may increase the needle biomass further, and increase the risk of water stress. Studies of variations in 13C abundance of needles and tree-rings of material from experi- ments like these can obviously disentangle the role of water as such, and thereby help us to analyse the effects of N deposition and measures against acid rain on the water balance of forests.

Table 3. Effects of N-fertilization on 13C abundance of current needles of Scots pine in the experiment E40 at Lisselbo (means -+S.D., n = 6 - 8 ) . Treatments started in 1969

Year Treatment

NO NI N2 N3 ((~ 13C (%~))

Level of significance (p)

1968 -24 .5 -+ .2 -24 .4 -+ .7 1970 -24 .3 - .5 -23 .3 -+ .6 1973 -25 .4 -+ .3 -24 .6 -+ .4 1979 -25 .9 + .4 -25 .4 - .5 1988 -26 .2 -+ .5 -25.5 -+ .7

-24 .9 -4-. 1 -24 .5 -+ .3 0.077 -23 .7 -+ .4 -22 .9 -+ .7 0.000 -24.5 + .3 -24 .3 -+ .4 0.000 -25 .3 -+ .3 -24 .9 -+ .5 0.002 -25 .7 -+ .7 -25 .4 -+ .3 0.053

" One ANOVA for each year.

214 Studies o f J3C in fol iage

Acknowledgements

W e w o u l d l ike to t h a n k A A l b r e k t s s o n , A

A r o n s s o n , L H 6 g b o m a n d C - O T a m m fo r va lu -

a b l e i n f o r m a t i o n , a n d A H a y s t e a d and A R a -

j e n d r a a r fo r e x c e l l e n t ana ly t i c a l se rv ices . T h i s

s t u d y was f u n d e d by t h e S w e d i s h C o u n c i l fo r

F o r e s t r y a n d A g r i c u l t u r a l R e s e a r c h , and is a lso a

c o n t r i b u t i o n to t h e E C p r o j e c t N i P h y s .

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Section editor: H Lambers