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Transfer of N fixed by a legume tree to the associated grass in a tropical silvopastoral system
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Soil Biology & Biochemistry 38 (2006) 1893–1903 www.elsevier.com/locate/soilbio
Transfer of N fixed by a legume tree to the associated grass in a tropical silvopastoral system Jorge Sierraa,, Pekka Nygrenb,1 a
Unite´ Agrope´doclimatique de la Zone Caraı¨be, INRA Antilles-Guyane, Domaine Duclos (Prise d’eau), 97170 Petit-Bourg, Guadeloupe, France b Center for Agroforestry, University of Missouri, Columbia, MO 65211, USA Received 13 October 2005; received in revised form 1 December 2005; accepted 5 December 2005 Available online 28 February 2006
Abstract Below-ground transfer of nitrogen (N) fixed by legume trees to associated non-N2-fixing crops has received little attention in agroforestry, although the importance of below-ground interactions is shown in other ecosystems. We used 15N natural abundance to estimate N transfer from the legume tree Gliricidia sepium (Jacq.) Kunth ex Walp. to C4 grass Dichanthium aristatum (Poir.) C.E. Hubb. in a silvopastoral system, where N was recycled exclusively by below-ground processes and N2 fixation by G. sepium was the sole N input to the system. Finding a suitable reference plant, a grass without contact with tree roots or litter, was problematic because tree roots invaded adjacent grass monocrop plots and soil isotopic signature in soil below distant grass monocrops differed significantly from the agroforestry plots. Thus, we used grass cultivated under greenhouse conditions in pots filled with agroforestry soil as the reference. A model of soil 15N fractionation during N mineralization was developed for testing the reliability of that estimate. Experimental and theoretical results indicated that 9 months after greenhouse transplanting, the percentage of fixed N in the grass decreased from 35% to o1%, due to N export in cut grass and dilution of fixed N with N taken up from the soil. The effect of soil 15N fractionation on the estimate of the reference value was negligible. This indicates that potted grass is a suitable reference N transfer studies using 15N natural abundance. About one third of N in field-grown grass was of atmospheric origin in agroforestry plots and in adjacent D. aristatum grassland invaded by G. sepium roots. The concentration of fixed N was correlated with fine root density of G. sepium but not with soil isotopic signature. This suggests a direct N transfer from trees to grass, e.g. via root exudates or common mycorrhizal networks. r 2006 Elsevier Ltd. All rights reserved. Keywords: Dichanthium aristatum; Gliricidia sepium;
15
N natural abundance; Soil
1. Introduction Serious nitrogen (N) deficits develop in many tropical agroecosystems because of heavy N export in crop harvest. Agroforestry involving dinitrogen-fixing legume trees has been proposed as a promising agricultural practice for coping with the N export problems (Lal, 2004). Legume trees are used in agroforestry as shade for plantation crops; living hedgerow fences and fence posts; support for shadetolerant climber crops; or they are harvested for fodder, green manure, and timber. The beneficial effects of legume Corresponding author. Tel.: +590 590 255949; fax: +590 590 941663.
E-mail address: [email protected] (J. Sierra). Current address: Department of Forest Ecology, University of Helsinki, PO Box 27, 00014, Finland. 1
0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.12.012
15
N fractionation; Fine root density
trees on tropical soils include increased soil and microbial C and N content in comparison to annual cropping (Sierra et al., 2002), and long-term accumulation of soil organic matter and N (Dulormne et al., 2003). It is assumed that companion crops benefit from the N2 fixation of trees by reabsorbing N mineralised from decomposing N-litter and prunings (Catchpoole and Blair, 1990; Xu et al., 1993; Beer et al., 1998). In other ecosystems, common mycorrhizal networks (He et al., 2003), and root exudates (Paynel et al., 2001) have been proposed as alternative, direct pathways for plant–plant N transfer. Studies using 15N labelling have indicated net N transfer from legume tree saplings to grass in shared containers (Rao and Giller, 1993), and from herbaceous legumes to grass under field conditions (Farnham and George, 1994). Isotopic studies also revealed N transfer from alders (Alnus
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J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903
spp.) to pines (Pinus spp.) via ectomycorrhizae (Arnebrandt et al., 1993; Ekbland and Huss-Danell, 1995) and via mycorrhizae or root exudates from Alnus subcordata C.A. Mey and Eleagnus agnustifolia L. to associated Prunus avium L. (Roggy et al., 2004). An alternative method to estimate N transfer from the N2-fixing plant is the analysis of 15N natural abundance (Mariotti et al., 1981). The apparent merit of this method is that no 15N needs to be added to the soil or the plant and N transfer may be estimated in situ without disturbing the plant-soil system. The method was first used for estimating N transfer from alders to conifers (Binkley et al., 1985). The estimate was based on the 15N natural abundance measured in plants and in the soil inorganic N pool, and it was concluded that isotopic fractionation in soil biased the estimates. van Kessel et al. (1994) showed that the deviation of the sample 15N/14N ratio from that of atmosphere (d15N) decreased in understorey vegetation after introducing N2-fixing Leucaena leucocephala (Lam.) de Wit trees, which is an indication of increasing presence of N fixed from atmosphere in the non-N2-fixers. They could not quantify the N transfer from trees to understorey because of the lack of a reference plant; an understorey plant species growing in the same soil without the N2-fixing species. Snoeck et al. (2000) were able to estimate N transfer in several coffee (Coffea arabica L.) plantations with legume shade trees by using coffee plants distant enough from the legume trees to have no contact with the legume roots or litter as reference plants. They estimated that N transfer contributed 13–42% of total N in coffee plants, depending on the legume species. Changes in 15N natural abundance in the non-N2-fixing plant may be related to transfer from the N2-fixing species or soil processes like nitrification, NH3 volatilization, and denitrification, which discriminate against 15N causing decrease (nitrification) or increase (volatilization, denitrification) in 15N enrichment of the plant-available soil N (Ho¨gberg, 1997). Interpretation of the variation in the isotopic signature of the non-N2-fixing and the N2-fixing plant requires knowledge on the isotope fractionation within the studied plant–soil system (Mariotti et al., 1981). Another constraint of the 15N natural abundance method is the availability of suitable reference plants. Because a part of the fixed N may be transferred to the soil (Dulormne et al., 2003), its 15N natural abundance may vary over time. In this way, the soil of the site selected for the reference non-N2-fixing plant (without fixed-N input) and for the intercropping (with fixed-N input) may differ greatly in a few years after introduction of the N2-fixer (cf. van Kessel et al., 1994). The objective of this study was to test the 15N natural abundance method for quantifying the presence of N fixed from atmosphere in the fodder grass Dichanthium aristatum (Poir.) C.E. Hubb., over a natural gradient of fine root density of associated legume tree Gliricidia sepium (Jacq.) Kunth ex Walp. An experimental procedure for estimating the d15N of the reference plant and a model of soil 15N
fractionation for calculating the error estimate for N transfer are presented. Grass and tree production (Nygren and Cruz, 1998), symbiotic N2 fixation by the trees (Nygren et al., 2000), and soil N and C dynamics (Sierra et al., 2002; Sierra and Nygren, 2005) have been earlier studied in the same agroforestry system. The applicability of the 15N natural abundance method for studying N transfer is discussed in the light of the knowledge gained on this system during 12 years of experimentation. 2. Materials and methods 2.1. Experimental site The study was conducted at the Godet Experimental Station of the French National Institute for Agronomic Research (INRA) in Guadeloupe, French Antilles (161250 N, 611300 W, 10 m a.s.l.). The climate is warm and subhumid with annual mean air temperature of 26 1C and the annual mean rainfall of 1300 mm. The soil is a Vertisol with 80% clay rich in smectite, developed over coral reef limestone. Three adjacent 20 m 13 m plots from a 0.5-ha experiment of silvopastoral and grass monocrop plots were selected for this study, which had similar and relatively homogeneous soil depth of ca. 0.5 m. Some chemical and physical characteristics of the soils are presented in Table 1. Agroforestry plots were established with cuttings of G. sepium in natural grassland of D. aristatum in 1989. They were separated by an open grass plot (OG) of D. aristatum (Fig. 1). The plots were not trenched. The trees were planted in rows aligned N–S, at 0.3 m 2 m spacing. Because of mortality, actual tree density was about 12,000 trees ha1 in 2001. Trees were partially pruned every 2 months and the grass was cut every 40–50 d in all plots from 1989 to 1996. From April 1996, trees were partially pruned every 6 months and the grass was cut every 80 d in the P6 agroforestry plot, and the old management regime was maintained in the P2 plot and in OG (Fig. 1). All three plots were managed from the beginning of the experiment as cut-and-carry systems. Fertilizer (100 kg [P] ha1, 150 kg [K] ha1) was applied four times between 1989 and 1998. No N fertilizer was applied. 2.2. Field experiment The soil was sampled with a 0.06-m diameter auger in January 2001 on three transect lines across the plots (Fig. 1). Only the 0–0.2 m layer was analyzed because no differences in C and N content between plots below that soil depth were detected in an earlier study and about 80% of the mineral N observed in the soil profile (0–0.5 m) was concentrated in the first 0.2-m layer (Sierra et al., 2002). Soil samples were dried at 70 1C for 72 h and ground too0.2 mm. Total N and 15N content of soil samples were determined by an element analyzer (Carlo Erba EA 1110, Carlo Erba Strumentazione, Milan, Italy) connected to a
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Table 1 Chemical and physical properties of the 0–0.2 m soil layer in the open grassland (OG) and in the agroforestry plots of Guadeloupe (French Antilles) Plot
P2 OG P6
Organic C (g kg1)
Total N (g kg1)
28.3c 29.7b 33.1a
2.6b 2.7b 3.1a
pH
7.4a 7.8a 7.7a
CEC (cmol(+) kg1)
52.2a 51.5a 50.8a
Particle size distribution o2 mm (%)
2–50 mm (%)
450 mm (%)
78 79 80
15 13 12
7 8 8
P2 and P6 are two agroforestry plots with tree pruning every 2 or 6 months, respectively. Within columns, values followed by different letters are significantly different at Po0:05.
N
Agroforestry P2
Open grassland (OG) of Agroforestry P6 D. aristatum tree row line 1
13 m line 2
line 3
time on the second soil sampling transect (Fig. 1) by manually cutting a 0.2 m 0.2 m quadrant at 1 cm aboveground. These grass shoots samples were used to estimate in situ N transfer from the legume tree. The plant samples were dried for 48 h at 70 1C, and ground to o0.2 mm for isotopic analyses. The components analyzed were mixed fine roots, individual grass and tree fine roots and grass shoots. Total N, 15N, and 13C to 12C ratio were analyzed by element analyzer-mass spectrometer as described above. Only dry mass was determined for grass fine roots separated at sampling.
29 m
2m
2.3. Greenhouse experiment Soil sampling Grass shoot sampling Root mixture sampling Tree root sampling Harvest at 12 wk
21 wk
34 wk
Whole grass sampling
Greenhouse transplanting
Fig. 1. Design of the field and the greenhouse experiments carried out in Guadeloupe on the Gliricidia sepium—Dichanthium aristatum intercrop. P6 and P2 ¼ G. sepium pruned every six and two months, respectively. Grass transplanted from the field site to greenhouse was harvested at 12 weeks, 21 weeks, and 34 weeks after transplanting (n ¼ 5).
mass spectrometer (Thermo-Finnigan Deltaplus, TermoQuest, Bremen, Germany) through a ConFlo II interface (Thermo-Finnigan) in the Stable Isotope Laboratory of the Kansas State University (USA). Fine roots (diameter p2 mm) of G. sepium and D. aristatum were sampled in February 2001 at 0.5 m from the second transect line selected for soil samples (Fig. 1). Soil monoliths 0.5 m long 0.2 m wide 0.2 m deep were taken and watered for 24 h. Grass roots directly connected to the respective shoot were separated and washed, dried and weighed separately in order to simplify the analyses. The remaining mixed roots were carefully extracted by washing over a 0.2-mm sieve, and separated into fine and coarse roots. Two samples of tree and grass fine roots directly connected to the respective tree or grass shoots were collected for determining reference values for estimating the tree and grass fine root proportions in the mixed root samples (Eq. (2)). Grass shoots were sampled at the same
This experiment was carried out in order to estimate the N natural abundance in grass without contact with tree roots or litter for using it as the reference non-N2-fixing plant (Eq. (4)). The prescribed method to estimate the d15N value of the reference plant is to sample plants growing in the same soil and growing season as the plants intercropped in contact with the N2-fixer. However, this procedure is difficult to apply in agroforestry systems where fine roots of legume trees are everywhere. Soil was taken from the P6 plot by removing the 0–0.2 m layer on a quadrant of 0.5 m2 (Fig. 1). Shoots and large roots were removed in the field. The soil was manually ground for breaking soil aggregates to less than 1 cm. Fine roots were removed, and soil was divided into five 15-l pots in a greenhouse. Twenty-five 15-cm-high D. aristatum plants were gently transplanted from the P6 plot to the pots on 15 January 2001; e.g. five plants per pot. Soil moisture was maintained between 0.3 and 0.4 kg kg1 (60–80% of water holding capacity). Temperature in the greenhouse was ca. 2 1C higher than outside. Shoots were cut to 1 cm on 11 April, 12 June and 10 September 2001; the same pots were followed throughout the experiment. Shoots from each individual pot were mixed, dried for 48 h at 70 1C and ground to o0.2 mm. Total N and 15N content were determined as described above. Nitrate-N and NH+ 4 –N in potting soil was extracted by shaking a sample 20 g of moist soil for 2 h in 100 ml of 0.5 M KCl. Mineral N was analyzed colorimetrically with an autoanalyzer (Technicon Industrial Systems, USA) using hydrazine reduction for NO 3 –N (Kampshake 15
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et al., 1967) and the nitroprusside method for NH+ 4 –N (Kaplan, 1965). 2.4. Calculations The percentage of G. sepium fine roots out of total fine root mass in mixed root samples was calculated using the different C isotope ratios in G. sepium (a C3 plant) and D. aristatum (a C4 plant). The deviation of the 13C to 12C ratio in the sample from that in the PDB (a limestone) standard (d13C) was calculated (Balesdent, 1996): 13
d C ¼ ½ðRx =Rr Þ 1 1000; 13
(1) 12
where Rx and Rr are the C– C ratios in the sample and PDB standard, respectively. The percentage of G. sepium fine roots (%Mgro) was calculated (Sierra and Nygren, 2005): %M gro ¼ ½ðd13 Ci d13 Cd Þ=ðd13 Cg d13 Cd Þ 100; 13
(2)
13
where d Ci is the d C in the fine root sample from intercropping (roots of G. sepium and D. aristatum mixed), d13Cd in pure D. aristatum root sample and d13Cg in pure G. sepium root sample. The mass of G. sepium fine roots in the sample was obtained by multiplying the total mass of the mixed root sample by %Mgro. The fine root mass of D. aristatum was obtained by deducting the mass of G. sepium roots from total mixed root sample mass, and adding the mass of D. aristatum fine roots that were separated at sampling because of direct connection to a D. aristatum shoot. The fine root density was calculated by dividing the root mass by the sample volume (20 dm3). The 15N natural abundance method was used for detecting fixed N in the grass because 15N natural abundance was also used for quantifying N2 fixation by G. sepium in the site (Nygren et al., 2000), we did not want to disturb the N cycle with labeled fertilizer, and 12 years under the same production system was assumed to have stabilized the 15N signature of the soil. The deviation of the sample 15N proportion from that of atmosphere (d15Nx) was calculated (Mariotti et al., 1981): d15 Nx ¼ ½ð%15 Nx %15 Na Þ=%15 Na 1000;
(3)
where %15Nx and %15Na are the percentages of 15N in the sample and in the air (0.3663%; Mariotti, 1984), respectively. The percentage of N fixed from atmosphere out of total N in D. aristatum (%Nf) was calculated (Snoeck et al., 2000): %Nf ¼ ½ðd15 Nr d15 Ni Þ=ðd15 Nr d15 Nfr Þ 100;
(4)
where subscript r (reference) refers to D. aristatum growing without contact with G. sepium, i to D. aristatum intercropped with G. sepium, and fr to G. sepium growing in N-free medium. The d15Nfr value for G. sepium of same genetic origin as the trees in the study site is 2.07 (Nygren et al., 2000). The d15Nr value for D. aristatum was determined in the
greenhouse experiment. Eq. (4) gives the percentage of fixed N in D. aristatum associated with G. sepium, not the percentage of total N transfer from the tree, because a part of the legume N is taken up from the soil. Concentration of fixed N in D. aristatum shoots (g [Nf] kg1 [DM]) was calculated multiplying the total N content of the sample (g [Nt] kg1 [DM]) by %Nf. 2.5. Statistical analyses Because tree roots were present in all plots, including the OG, the plots may not be considered strictly as treatments, and plant variables were analyzed as a function of a root density gradient of G. sepium. Selected grass features were related to the gradient by Pearson correlation and linear regression. Differences in soil variables along the tree fine root density gradient were tested by analysis of variance followed by separation of means by Duncan’s Multiple Range Test using the 19 east–west transect positions as treatments and the three soil sampling transects as replications (Fig. 1). Statistical analyses were conducted with SAS v. 8.02 software (SAS Institute, 1999). 2.6. Model of soil 15N fractionation and N partitioning in D. aristatum A model was developed for simulating 15N fractionation in N mineralization and N distribution inside D. aristatum following transplanting from the agroforestry plot to a pot in the greenhouse (Fig. 2). The simulated effect of 15N fractionation in soil on the d15Nr estimate was used to assess the reliability of the experimental procedure. Nitrogen mineralization was described by a first-order reaction: 14
Nm ¼14 N0 ½1 expðk14 tÞ;
(5)
15
Nm ¼15 N0 ½1 expðk15 tÞ;
(6)
where 14N0 and 15N0 are the mineralizable pools of the two N isotopes at time 0 (mg kg1), 14Nm and 15Nm are the cumulative mineralization of 14N and 15N (mg kg1) at time t (week), and k14 and k15 (week1) are the mineralization rate constants of each isotope pool. The fractionation coefficient for mineralization (b) is calculated as (Mariotti et al., 1981): b ¼ k14 =k15 .
(7)
We assumed that b is constant over time and independent of the amount of 14N0 and 15N0 remaining in soil. About 95–98% of mineral N was present as NH+ 4 in the field site (Sierra et al., 2002). Further, some tropical grasses release compounds from roots which reduce population or activity of nitrifying bacteria (Ishikawa et al., 2003), and this was also observed in D. aristatum (J. Sierra, unpublished data). Thus, neither nitrification nor denitrification was included in the model.
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U s ¼ U 0s Ptr U 0s ;
Shoot
(10) 1
15
N
14
N
where Uro and Us (mg [N] week ) are the rates of current N uptake to root and shoot, respectively. Nitrogen uptake rate between two grass cuts was assumed to be constant over time. The d15N of the N retranslocated from root to shoot at time t was assumed to be equal to the d15N of roots at time t1. The d15N at time t in each plant compartment was calculated as a dilution process by taking into account the d15N of each compartment at time t1 and the d15N corresponding to N taken from the soil (d15Nm in Fig. 2) and that of the N metabolized in roots and retranslocated to shoot. The part of the N in shoot and root biomass remaining after grass cut (Psa and Proa in Fig. 2) was calculated as a proportion of the respective N content before cut. The d15N of the remaining shoot and root biomass was set equal to the d15N of each compartment before cut. Calculations were made at a time step of a week.
Ns, δ15Ns
Ps-a
Shoot after cut
Ptr
Roots 15
N 14 N
Nro,δ15Nro Roots after cut
Pro-a Us
Uro
N uptake
2.7. Model parameters Organic matter 15 14
N N
δ15No
Mineral N
Mineralization (fractionation)
15
N
14
N
δ15Nm
Fig. 2. Model of soil 15N fractionation and N partitioning in the grass for simulating the temporal change of 15N content in soil and in a grass without contact with legume tree. The values of the parameters used in simulations are presented in Table 2.
Nitrogen uptake was calculated in two steps. First, the increase rate of root N content (U0 ro in mg [N] week1) was assumed to be proportional to the increase rate of shoot N content (U0 s in mg [N] week1) and to be constant over time (Cruz, 1997). Thus, U0 ro was calculated from U0 s: U 0ro ¼ U 0s Pu ;
(8)
where Pu is the ratio of the increase rate of root N content to the increase rate of shoot N content. Nitrogen taken up by grass was divided into three components (Cruz, 1997): direct N transfer to shoot in xylem flow, N metabolized and retained in roots, and N metabolized in roots and recycled to shoot. Direct N transfer in xylem flow does not affect the isotopic ratios (Handley et al., 1999) and this component was assumed to retain the d15N of soil N. Nitrogen recycled from roots to shoot was assumed to have the d15N of roots, because N metabolism within a plant may strongly alter the isotopic signature (Handley et al., 1999; Ho¨gberg, 1997). Under the scheme of frequent cutting, the ratio (Ptr) of the N root–shoot retranslocation rate to the increase rate of shoot N content was assumed to be constant between two cuttings (Cruz, 1997). Thus, the distribution of current N uptake into root and shoot was calculated from the increase rates of root and shoot N content: U ro ¼ U 0ro þ Ptr U 0s ;
(9)
Grass growth was simulated during a 54-weeks-period including grass cut every 12 weeks applying parameter values shown in Table 2. The initial d15N of the mineralizable soil organic N pool (d15N0) was assumed to be equal to d15N determined in this study for the soil organic matter. The initial d15Nm was set to 3.50, which is close to the d15Nr determined at the end of the greenhouse experiment. This is based on the assumption that the d15N of the grass without tree contact was representative of the 15 N natural abundance of the soil available N at 34 weeks after transplanting. With these values of d15N0 and d15Nm, the coefficient b was equal to 1.0025, which is within the range of fractionation values for mineralization reported by other authors (Ho¨gberg, 1997). The value of k14 was estimated from the results reported by Sierra et al. (2002) for the same soil used in this study; N0 was set so that the amount of mineralized N was equal to the N absorbed by the grass during the simulated 54-weeks-period. The increase rate of shoot N content was set equal to 200 mg N for each 12-weeks regrowth period. This value and those selected for the initial d15Ns and d15Nro (Table 2) were close to those observed in the greenhouse experiment. The values of Pu, Ptr, Psa, and Proa were estimated from the results reported by Cruz (1997) on N partitioning in frequently defoliated D. aristatum. 3. Results 3.1. Greenhouse experiment The d15N value of greenhouse grown D. aristatum shoots was 1.6470.05 (mean7standard error of mean, SEM) 12 weeks after transplanting, which was close to the value in grass in the P6 plot where the greenhouse plants originated (1.5070.13; ‘‘initial value’’ in Fig. 3). The d15N value
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Table 2 Values of the model parameters used to simulate the temporal change of the isotopic signature of soil mineral N and grass in the greenhouse experiment Parameter
Explanation
Unit
Value
Source
Soil d15No N0 14 N0 15 N0 k14 k15 b
d15N for soil organic N Mineralizable soil N at time ¼ 0 Mineralizable soil 14N at time ¼ 0 Mineralizable soil 15N at time ¼ 0 Mineralization rate constant of 14N Mineralization rate constant of 15N Fractionation coefficient for mineralization
— mg [N] kg1 mg [N] kg1 mg [N] kg1 week1 week1 —
6.0 500 498.19 1.81 2.37 103 2.3641 103 1.0025
This study This study This study Calculated from N0 and 14N0 Sierra et al. (2002) Calculated from k14 and b This study
Plant d15Ns, d15Nro U0 s Pu Ptr Ps–a Pro–a
d15N in grass shoot and root at time ¼ 0 — Increase rate of shoot N content mg [N] week1 Ratio of increase rate of root N content to increase rate of shoot N content — Root-to-shoot N retranslocation coefficient — Proportion of shoot N retained after cut — Proportion of root N retained after cut —
of the greenhouse experiment. In both cases, 95% of the mineral N was present as NH+ 4 –N.
4.0
3.2. Root biomass
3.0
δ Shoot 15N
1.5 (at time 0) This study 16.67 This study 0.30 Cruz (1997) 0.15 Cruz (1997) 0.10 Cruz (1997) 0.50 Cruz (1997)
2.0
1.0
0.0 0
9
18
27
36
Weeks after transplanting
Fig. 3. Greenhouse experiment: d15N of grass shoots as a function of the time after transplanting. Swards of Dichanthium aristatum were transplanted from P6 agroforestry plot to monoculture pots filled with soil from the same plot. Vertical bars indicate the standard error (n ¼ 5).
increased from 12 to 34 weeks in greenhouse, with a strong variation between pots at 21 weeks after transplanting (Fig. 3). The d15N values were quite uniform 34 weeks after transplanting, and appeared to reach the highest value. The mean7SEM of d15N value was 3.4470.24, which was higher than any value observed in the field (maximum 2.70). The mean of d15N in D. aristatum shoots in the last greenhouse harvest was used as the reference value without presence of a N2-fixing plant, term d15Nr in Eq. (4). Total N content in shoots was 14.370.5 mg [N] g1 [DM] at transplanting, and 9.070.2, 5.670.3, and 7.370.2 mg [N] g1 [DM] at 12, 21 and 34 weeks after transplanting, respectively. Soil mineral N content was 18.271.2 mg [N] kg1 at transplanting and 31.072.6 mg [N] kg1 at the end
The d13C values in the pure roots of D. aristatum and G. sepium were 12.1 and 26.1, respectively. This formed a sufficient contrast for application of Eq. (2) in estimation of %Mgro in the mixed root samples. Fine root density of G. sepium formed a decreasing gradient from P6 plot through OG plot to a slight increase in the P2 plot (Fig. 4a). The fine root density gradient of D. aristatum was about the reverse. Fine root biomass varied from 1.3 to 6.3 Mg ha1 in D. aristatum, and from 0.1 to 3.0 Mg ha1 in G. sepium. Fine roots densities of G. sepium and D. aristatum were negatively correlated (R2 ¼ 0:37, Po0:05). Nitrogen content in the pure G. sepium fine root sample was 21.7 mg [N] g1, and the d15N value was 0.8. 3.3. Total N and
15
N content in soil and plant in the field
Soil total N content was significantly higher in the sampling points in the P6 plot than in those of the P2 or OG plots (Duncan’s MRT at Po0:05; Fig. 4b). Differences in soil d15N between sampling points were not significant. The mean7SEM of soil d15N value was 6.0170.05 (Fig. 4b). Soil total N was positively correlated with tree fine root density (R2 ¼ 0:78, Po0:05), but soil d15N was not correlated with either G. sepium or D. aristatum fine root density in the adjacent transect line. Total N concentration in shoots of D. aristatum averaged 1.43 mg [N] g1 in P6, 1.14 mg [N] g1 in the open grassland, and 1.56 mg [N] g1 in P2 (Fig. 4c). It had a weak but significant positive correlation with tree fine root density (R2 ¼ 0:30, Po0:05) and soil total N content (R2 ¼ 0:30, Po0:05). The d15N of grass shoot did not present any clear spatial pattern across the transect line,
ARTICLE IN PRESS J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903
3.4. Estimation of transfer of fixed N from legume trees to grass
D. aristatum
3.5
1899
G. sepium
Fine root density (g dm-3)
3.0 2.5 2.0 1.5 1.0 0.5 0.0
(a)
0
10
30
Total N
4.0
7
δ15 N
3.5
6
3.0
5
Soil 15N
Soil total N (g kg-1)
20
2.5
(b)
4 0
10
20
30
3.5. Simulation results
Total N 20
3.0
15
δ N
2.5 15 2.0 1.5
10
Shoot 15N
Shoot total N (mg g -1)
The percentage of N fixed from atmosphere out of total N in shoots of D. aristatum, calculated according to Eq. (4), averaged 36% in P6 and in OG, and 27% in P2. Percentage of fixed N in grass shoot was not correlated with either tree fine root density or soil N content. Because total N concentration was high in P6, the concentration of fixed N was the highest in the shoots of D. aristatum growing in this plot. The averages were 0.52, 0.38 and 0.39 mg [Nf] g1 in P6, OG, and P2, respectively. Considering the average annual grass N yield of 195 kg ha1 year1 (Dulormne et al., 2003), the percentages of N fixed from atmosphere in D. aristatum correspond to 53 and 70 kg ha1 year1 of grass N of atmospheric origin in P2 and P6 plots, respectively. A significant correlation was observed between the fine root density of G. sepium and concentration of fixed N in shoots of adjacent D. aristatum samples (Fig. 5). The three points with highest fine root densities of G. sepium correspond to P6 plot, while data for P2 and OG plots is mixed within each others in the points with low fine root densities. The concentration of fixed N in grass shoot was not correlated with soil d15N.
1.0
Simulated d15Nm increased from 3.50 to 3.81 (Fig. 6a) and simulated d15N0 increased from 6.00 to 6.32 (data not shown) in 54 weeks. This was related to 15N fractionation during N mineralization. The temporal change in d15N differed slightly between the shoot and root biomass (Fig. 6a). The increase of d15Ns was faster than that of d15Nro because more soil mineral N was allocated to the shoot. However, at the end of the third regrowth period (36
5
8
0.5 0
0.0 0
10
P2
20
OG
30
P6
Transect position (m)
Fig. 4. Field experiment: (a) Fine root density of Dichanthium aristatum and Gliricidia sepium on the studied transect; (b) total N content and d15N in soil; (c) total N content and d15N in shoots of D. aristatum. Values in (b) and (c) correspond to the second transect line (see Fig. 1). Vertical dashed lines indicate tree rows.
6 Fixed N (mg [N] g -1)
(c)
4
y = 1.98 x + 3.47
2
R2 = 0.55, P < 0.05
0
except in P2 where d15N was higher in the alleys (Fig. 4c). The d15N values varied from about 2.6 in the alleys of P2 to 0.85 in the open grassland next to P6 plot. Shoot d15N was not correlated with either tree fine root density or soil d15N.
0.0
0.4
0.8
1.2
1.6
-3
Fine root density of G. sepium ( g dm )
Fig. 5. Concentration of N fixed from atmosphere in the shoots of the grass Dichanthium aristatum as a function of fine root density of the associated legume tree Gliricidia sepium.
ARTICLE IN PRESS J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903 4.0
60
3.5
50
2.5
δ15Nm
2.0
δ15Nro
δ15Ns
Estimate with δ15Nm at t = 0
10 0
0
12
(a)
24
36
48
0.5
4.0
3.5
3.5 δ15Nm 15
δ Ns (initial simulation)
δ15N
4.0
2.5
δ15Ns (Ps-a = 0.2) δ15Ns (Ptr = 0.3)
2.0
1
(b)
Time (weeks)
3.0 δ15N
30 20
1.5
1.5 2 δ15Ns
2.5
3
3.0 2.5 δ15Nm
2.0
1.5
δ15Ns
1.5 0
(c)
Estimate with δ15Ns at t = 36 weeks
40
3.0
%Nf
δ15N
1900
12
24
36
48
Time (weeks)
0
12
(d)
24
36
48
Time (weeks)
Fig. 6. Simulated temporal change in soil and plant d15N in the greenhouse experiment: (a) grass and soil mineral d15N as a function of time. Parameter values in Table 2 were used. (b) Percentage of fixed N in the grass (%Nf) as a function of grass d15N. The %Nf was estimated with two different values of d15N for the reference grass plant: d15Nm at time 0 or d15Ns at time 36 weeks. Differences between the estimates were o3%. (c) Effect of a change in the values of the model parameters Psa and Ptr (Table 2) on the d15N of the grass. Value of d15Ns (initial simulation) corresponds to d15Ns in (a). (d) Effect of a four-time reduction in plant N uptake in the first regrowth period (0–12 weeks) on the d15N of the grass. Subscripts m, s, and ro refer to soil mineral N, grass shoot, and grass root, respectively. Vertical bars in (a) and (d) indicate the time of grass harvest (every 12 weeks).
weeks) both d15N values were similar and close to the d15Nm value; 3.60 for d15Ns, 3.59 for d15Nro, and 3.66 for d15Nm. At this time, the fixed N represented only 0.1% of the grass shoot N content. To calculate the error involved for using d15Nr such as it was determined in this study, the estimate of %Nf (Eq. (4)) was performed with two values of d15Nr: (i) d15Nr was set equal to d15Nm at time 0, and (ii) d15Nr was set equal to d15Ns at time 36 weeks; e.g. the time corresponding to the end of the greenhouse experiment. The first estimate corresponds to the theoretical value of %Nf because no 15 N fractionation was assumed for N uptake; that is, d15Nm at time 0 is equal to the d15Ns of a plant without contact with legume roots and without any effect of soil 15N fractionation. The second estimate corresponds to the experimental procedure proposed in this study. The theoretical results indicated that even if 15N fractionation during N mineralization induces an overestimate of the fixed N transferred to the grass, the error is small for a large range of d15Ns (Fig. 6b). For example, the overestimate was 1.0 and 1.5% for N transfer of 50 and 10%, respectively. The error of the estimate was not affected by the plant parameter values used in the model. Fig. 6c shows that changes in Psa and Ptr caused the initial increase in d15Ns, but it was very similar for all the simulations at 36 weeks.
4. Discussion 4.1. Soil N and
15
N content
Soil N content has increased 2.5 Mg [N] ha1 in P6, and about 1.8 Mg [N] ha1 in P2 and OG in 12 years after planting G. sepium in the current study plots (Sierra and Nygren, 2005). Because tree pruning residues were removed from the site and litterfall was small because of frequent pruning, it may be concluded that N released from tree roots was the most important N input in the three plots. In the present study, no correlation was observed between soil d15N and tree root density. Considering the typically rapid fine root dynamics of periodically pruned trees (Mun˜oz and Beer, 2001), there is no reason to assume that soil d15N would be correlated with the current tree fine root density in a point in the field. The isotopic signature of the soil rather reflects the cumulative effect of presence and absence of tree roots during the 12-years experimentation. Soil d15N may be expected to stabilize in 4–6 years after introducing a dense legume tree plantation (van Kessel et al., 1994). The change in soil d15N is a result of long-term accumulation of tree litter depleted in 15N, and counteracting soil microbial processes that tend to enrich the soil in 15N (Ho¨gberg, 1997).
ARTICLE IN PRESS J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903
In spite of the gradient of soil N content (Fig. 4b), soil d15N was quite homogeneous throughout the experimental site, and no differences were detected between sampling sites. This was an essential condition for using the 15N natural abundance method for estimating in situ N transfer across the tree fine root density gradient. Further, the d15N value of the P6 soil used in greenhouse was representative for the homogeneous experimental site. The difference between the d15N values of the reference plant and N2-fixer in N-free environment should be at least 5%-units in order to obtain a reliable estimate of N2 fixation by the 15N natural abundance method (Ho¨gberg, 1997). If the same condition is applied for N transfer studies using 15N natural abundance, the d15N values of the potted D. aristatum reference and G. sepium in N-free medium, +3.44 and 2.07 (Nygren et al., 2000), respectively, provided sufficient contrast in this study. 4.2. Estimate of d15N of the reference plant Using of a field-grown reference plant was not suitable in this study because tree roots were present everywhere. Wide horizontal extension of tree root systems has been observed in several agroforestry systems (Hauser, 1993; Schroth and Zech, 1995). In plots cultivated with D. aristatum monocrop for at least 20 years in the Godet Experimental Station, the soil d15N was 9.570.6 (mean7SEM, n ¼ 5) (J. Sierra, unpublished data). Because the reference plant should grow in a soil with similar 15N signature as the plant intercropped with N2-fixers, D. aristatum from a monocrop plot was not a suitable reference. Therefore, we proposed a method based on the determination of d15N of the grass in a pot filled with the soil of the agroforestry site. In such pot system with no external N input, the d15N of the grass is expected to increase over time for two reasons: (i) frequent grass cut causes a dilution of the fixed N transferred from the tree, which has a d15N much lower than that of the soil N, and (ii) 15N fractionation during mineralization causes increase in the d15N of the soil mineral N. A model was developed for separating the effects of both processes on the d15Nr value estimate. Simulations indicated that 3% of the calculated increase in grass d15N was associated to 15N fractionation during mineralization, and 97% was due to the dilution of the initial fixed N present in the grass. Thus, it may be concluded that the increase of d15Ns observed in the greenhouse experiment (Fig. 3) was mainly due to the decrease in the proportion of N derived from G. sepium because of N export in grass harvest. Simulations also indicated that the amount of fixed N in the grass was negligible at 36 weeks after transplanting. The increase of d15Ns in pots also indicates that rhizospheric diazotrophs were not the source of fixed N in grass, because diazotrophs would have been transferred to the pots in the field soil and mostly intact grass root systems. Therefore, both experimental and theoretical
1901
results suggest that the procedure proposed in this study provided a suitable estimate of d15Nr for estimating the transfer of fixed N from the tree to grass (Eq. (4)). Differences between the greenhouse-observed and simulated trend of d15Ns during the first regrowth period (Figs. 3 and 6a) were probably due to grass transplanting stress. Poor grass growth during the first regrowth period probably reduced N uptake and dilution of the fixed N content causing the small increase in the d15Ns value (Fig. 3). The simulation, in which N uptake rate for this period was set four times lower than the initial value (U0 s in Table 2), reproduced well the small increase in d15Ns during that period (Fig. 6d). The estimate error was similar to that calculated in the basic simulation (Fig. 6b) because the differences in d15Ns at 36 weeks were negligible between simulations. Denitrification and volatilization discriminate against 15 N causing soil mineral 15N enrichment, and NH+ 4 becomes relatively enriched by 15N in comparison to 15 NO N fractionation during nitrification 3 because of (Ho¨gberg, 1997). These potential changes in the isotopic signature of available soil N may affect plant d15N and must be considered when interpreting the observed d15N increase in shoots of potted grasses. About 95–98% of mineral N was present as NH+ 4 in our soil (Sierra et al., 2002; this study). Some tropical grasses release compounds from roots, which reduce population or activity of nitrifying bacteria (Ishikawa et al., 2003), and this was also observed in D. aristatum (J. Sierra, unpublished data). For this reason, neither nitrification nor denitrification was included in the model. Volatilization of NH3 has been observed to be negligible in a non-N-fertilized Vertisol similar to that used in this study (Courtaillac et al., 1998). Thus, soil mineral 15N enrichment by these processes may be discarded as a source of 15N enrichment in potted grass. 4.3. Transfer of fixed N in the field The greenhouse experiment provided indirect evidence of the transfer of fixed N from the tree to grass. In the field, all the shoot samples of D. aristatum appeared to be depleted in 15N in relation to the reference value obtained in greenhouse. This implies that N transfer occurred along the entire grass transect. The percentage of N derived from atmosphere in D. aristatum, 26–35% along the gradient of G. sepium fine root density, was within the range of values obtained by 15N-labeling of soil or plant (e.g. Rao and Giller, 1993; Farnham and George, 1994), and 15N natural abundance (Snoeck et al., 2000). Higher percentage, ca. 68%, has been observed in grass associated with alfalfa (Medicago sativa L.; Brophy et al., 1987). These data indicate that N2-fixing legumes may make important contributions to N nutrition of associated non-legumes. Nitrogen content in the grass shoot biomass was higher under the trees than in the open grassland (Fig. 4c). In an earlier long-term study this phenomenon was attributed to (i) higher shoot N/root N ratio in the grass under the trees
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because of morphogenetic effects induced by shade, and (ii) higher N mineralization and better soil N availability in P6 because of a higher N content in soil (Dulormne et al., 2003; cf. Fig. 4b). The significant correlation between the concentration of fixed N in grass shoots and tree fine root density (Fig. 5), together with the lack of correlation between these variables and soil d15N, indicate that transfer of fixed N may have involved a more direct mechanism than a transfer via the complete N mineralization cycle in soil. This hypotheses was supported by direct measurements of soil mineral d15N performed in a later work carried out in the same OG plot. Soil mineral d15N averaged 2.7170.08 in this plot, and no correlation was observed between soil mineral d15N and the distance from the nearest tree line (J. Sierra, unpublished). The value was higher than that observed in the present study for the grass shoots in the OG plot (1.3570.16; Fig. 4c), which also suggests that a part of N transfer occurred directly. Soil mineral d15N was also lower than the d15N of the reference plant, which may be associated to tree root exudates released to the soil solution of the OG plot. About 95% of N in root exudates of white clover (Trifolium repens L.) is in the form NH+ 4 (Paynel et al., 2001). If this was the case of G. sepium, NH+ 4 could be easily absorbed by the grass because it is the current N source in our soil. Nitrogen may also have been transferred from trees to grass via common mycorrhizal networks (He et al., 2003). G. sepium forms symbioses with arbuscular mycorrhizae (Okon et al., 1996). We could not find any information on mycorrhizae in D. aristatum, a little studied yet important fodder grass in the Caribbean basin, but most grasses have arbuscular mycorrhizae. In most studies, the N transfer from a N2-fixer is expressed as percentage of fixed N out of the total N in the associated plant (e.g. Brophy et al., 1987; Snoeck et al., 2000). Our results indicated that fixed N by unit of dry matter is a more suitable indicator for analyzing N transfer. For example, the amount of fixed N and N taken up from the soil were highest in P6, but grass d15N and the percentage of fixed N were similar to those observed in the OG plot. The change in soil available N as a function of the distance from the tree (Sierra et al., 2002) may explain the lack of correlation between tree fine root density and the percentage of fixed N in the grass. However, soil d15N as measured in this study is an indicator of the 15N natural abundance in the bulk soil and not in the tree rhizosphere. Further work is necessary to discriminate between direct and indirect pathways of N transfer, including the study of N recycling in the rhizospheric soil of the legume tree, and the role of mycorrhizal networks. Acknowledgements We thank Saint-Ange Sophie for skilful technical assistance and for the management of the experimental plots since 1989, and Roxane Fagan (Stable Isotope
Laboratory of the Kansas State University, USA) for the mass spectrometry of 15N and 13C samples. This work was funded by the University of Missouri Life Sciences Mission Enhancement Programme (PN) and the De´partement Environnement et Agronomie of the Institut National de la Recherche Agronomique, France (JS). References Arnebrandt, K., Ek, H., Finlay, R.D., So¨derstro¨m, B., 1993. Nitrogen translocation between Alnus glutinosa (L.) Gaertn. seedlings inoculated with Frankia sp. and Pinus contorta Doug. ex Loud seedlings connected by a common ectomycorrhizal mycelium. New Phytologist 124, 231–242. Balesdent, J., 1996. The significance of organic separates to carbon dynamics and its modelling in some cultivated soils. European Journal of Soil Science 47, 485–493. Beer, J., Muschler, R., Kass, D., Somarriba, E., 1998. Shade management in coffee and cacao plantations. Agroforestry Systems 38, 139–164. Binkley, D., Sollins, P., McGill, W.B., 1985. Natural abundance of nitrogen-15 as a tool for tracing alder-fixed nitrogen. Soil Science Society of America Journal 49, 444–447. Brophy, L.S., Heichel, G.H., Russelle, M.P., 1987. Nitrogen transfer from forage legumes to grass in a systematic planting design. Crop Science 27, 753–758. Catchpoole, D.W., Blair, G., 1990. Forage tree legumes I. Productivity and N economy of leucaena, gliricidia, calliandra and sesbania and tree/green panic mixtures. Australian Journal of Agricultural Research 41, 521–530. Courtaillac, N., Baran, R., Oliver, R., Casabianca, H., Ganry, F., 1998. Efficiency of nitrogen fertilizer in the sugarcane-vertisol system in Guadeloupe according to growth and ratoon age of the cane. Nutrient Cycling in Agroecosystems 52, 9–17. Cruz, P., 1997. Effect of shade on the carbon and nitrogen allocation in a perennial tropical grass, Dichanthium aristatum. Journal of Experimental Botany 48, 15–24. Dulormne, M., Sierra, J., Nygren, P., Cruz, P., 2003. Nitrogen-fixation dynamics in a cut-and-carry silvopastoral system in the subhumid conditions of Guadeloupe, French Antilles. Agroforestry Systems 59, 121–129. Ekbland, A., Huss-Danell, K., 1995. Nitrogen fixation by Alnus incana and nitrogen transfer from A. incana to Pinus sylvestris influenced by macronutrients and ectomycorrhiza. New Phytologist 131, 453–459. Farnham, D.E., George, J.R., 1994. Dinitrogen fixation and nitrogen transfer in birdsfoot trefoil-orchardgrass communities. Agronomy Journal 86, 690–694. Handley, L.L., Austin, A.T., Robinson, D., Scrimgeour, C.M., Raven, J.A., Heaton, T.H.E., Schmidt, S., Stewart, G.R., 1999. The 15N natural abundance (d15N) of ecosystem samples reflects measures of water availability. Australian Journal of Plant Physiology 26, 185–199. Hauser, S., 1993. Root distribution of Dactyladenia (Acioa) barteri and Senna (Cassia) siamea in alley cropping on Ultisol. I. Implications for field experimentation. Agroforestry Systems 24, 111–121. He, X.H., Critchley, C., Bledsoe, C., 2003. Nitrogen transfer within and between plants through common mycorrhizal networks (CMNs). Critical Reviews in Plant Science 22, 531–567. Ho¨gberg, P., 1997. 15N natural abundance in soil–plant systems. New Phytologist 137, 179–203. Ishikawa, T., Subbarao, G.V., Ito, O., Okada, K., 2003. Suppression of nitrification and nitrous oxide emission by the tropical grass Brachiaria humidicola. Plant and Soil 255, 413–419. Kampshake, L.J., Hannah, S.J., Cohen, J.M., 1967. Automated analysis for nitrate by hydrazine reduction. Water Resources Research 1, 205–216. Kaplan, A., 1965. Standard Methods of Clinical Chemistry. Academic Press, New York, USA 249p.
ARTICLE IN PRESS J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903 Lal, R., 2004. Soil carbon sequestration impacts on global climate change and food security. Science 304, 1623–1627. Mariotti, A., 1984. Natural 15N abundance measurements and atmospheric nitrogen standard calibration. Nature 311, 251–252. Mariotti, A., Germon, J.C., Hubert, P., Kaiser, P., Letolle, R., Tardieux, A., Tardieux, P., 1981. Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes. Plant and Soil 62, 413–430. Mun˜oz, F., Beer, J., 2001. Fine root dynamics of shaded cocoa plantations in Costa Rica. Agroforestry Systems 51, 119–130. Nygren, P., Cruz, P., 1998. Biomass allocation and nodulation of Gliricidia sepium under two cut-and-carry forage production regimes. Agroforestry Systems 41, 277–292. Nygren, P., Cruz, P., Domenach, A.M., Vaillant, V., Sierra, J., 2000. Influence of forage harvesting regimes on dynamics of biological dinitrogen fixation of a tropical woody legume. Tree Physiology 20, 41–48. Okon, I.E., Osonubi, O., Sanginga, N., 1996. Vesicular–arbsucular mycorrhiza effects on Gliricidia sepium and Senna siamea in a fallowed alley cropping system. Agroforestry Systems 33, 165–175. Paynel, F., Murray, P.J., Cliquet, J.B., 2001. Root exudates: a pathway for short-term N transfer from clover and ryegrass. Plant and Soil 229, 235–243. Rao, A.V., Giller, K.E., 1993. Nitrogen fixation and its transfer from Leucaena to grass using 15N. Forest Ecology and Management 61, 221–227.
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Roggy, J.C., Moiroud, A., Lensi, R., Domenach, A.M., 2004. Estimating N transfer between N2-fixing actinorhizal species and the non-N2fixing Prunus avium under partially controlled conditions. Biology and Fertility of Soils 39, 312–319. SAS Institute, 1999. SAS User’s Guide. Statistics. SAS Inst., Cary, NC, USA. Schroth, G., Zech, W., 1995. Root length dynamics in agroforestry with Gliricidia sepium as compared to sole cropping in the semi-deciduous rainforest zone of West Africa. Plant and Soil 170, 297–306. Sierra, J., Nygren, P., 2005. Role of root inputs from a dinitrogen-fixing tree in soil carbon and nitrogen sequestration in a tropical agroforestry system. Australian Journal of Soil Research 43, 667–675. Sierra, J., Dulormne, M., Desfontaines, L., 2002. Soil nitrogen as affected by Gliricidia sepium in a silvopastoral system in Guadeloupe, French Antilles. Agroforestry Systems 54, 87–97. Snoeck, D., Zapata, F., Domenach, A.M., 2000. Isotopic evidence of the transfer of nitrogen fixed by legumes to coffee trees. Biotechnology, Agronomy, Society and Environment 4, 95–100. van Kessel, C., Farrell, R.E., Roskoski, J.P., Keane, K.M., 1994. Recycling of the naturally occurring 15N in an established stand of Leucaena leucocephala. Soil Biology and Biochemistry 26, 757–762. Xu, Z.H., Saffigna, P.G., Myers, R.J.K., Chapman, A.L., 1993. Nitrogen cycling in leucaena (Leucaena lecuocephala) alley cropping in semiarid tropics. 1. Mineralization of nitrogen from leucaena residues. Plant and Soil 148, 63–72.
Soil Biology & Biochemistry 38 (2006) 1893–1903 www.elsevier.com/locate/soilbio
Transfer of N fixed by a legume tree to the associated grass in a tropical silvopastoral system Jorge Sierraa,, Pekka Nygrenb,1 a
Unite´ Agrope´doclimatique de la Zone Caraı¨be, INRA Antilles-Guyane, Domaine Duclos (Prise d’eau), 97170 Petit-Bourg, Guadeloupe, France b Center for Agroforestry, University of Missouri, Columbia, MO 65211, USA Received 13 October 2005; received in revised form 1 December 2005; accepted 5 December 2005 Available online 28 February 2006
Abstract Below-ground transfer of nitrogen (N) fixed by legume trees to associated non-N2-fixing crops has received little attention in agroforestry, although the importance of below-ground interactions is shown in other ecosystems. We used 15N natural abundance to estimate N transfer from the legume tree Gliricidia sepium (Jacq.) Kunth ex Walp. to C4 grass Dichanthium aristatum (Poir.) C.E. Hubb. in a silvopastoral system, where N was recycled exclusively by below-ground processes and N2 fixation by G. sepium was the sole N input to the system. Finding a suitable reference plant, a grass without contact with tree roots or litter, was problematic because tree roots invaded adjacent grass monocrop plots and soil isotopic signature in soil below distant grass monocrops differed significantly from the agroforestry plots. Thus, we used grass cultivated under greenhouse conditions in pots filled with agroforestry soil as the reference. A model of soil 15N fractionation during N mineralization was developed for testing the reliability of that estimate. Experimental and theoretical results indicated that 9 months after greenhouse transplanting, the percentage of fixed N in the grass decreased from 35% to o1%, due to N export in cut grass and dilution of fixed N with N taken up from the soil. The effect of soil 15N fractionation on the estimate of the reference value was negligible. This indicates that potted grass is a suitable reference N transfer studies using 15N natural abundance. About one third of N in field-grown grass was of atmospheric origin in agroforestry plots and in adjacent D. aristatum grassland invaded by G. sepium roots. The concentration of fixed N was correlated with fine root density of G. sepium but not with soil isotopic signature. This suggests a direct N transfer from trees to grass, e.g. via root exudates or common mycorrhizal networks. r 2006 Elsevier Ltd. All rights reserved. Keywords: Dichanthium aristatum; Gliricidia sepium;
15
N natural abundance; Soil
1. Introduction Serious nitrogen (N) deficits develop in many tropical agroecosystems because of heavy N export in crop harvest. Agroforestry involving dinitrogen-fixing legume trees has been proposed as a promising agricultural practice for coping with the N export problems (Lal, 2004). Legume trees are used in agroforestry as shade for plantation crops; living hedgerow fences and fence posts; support for shadetolerant climber crops; or they are harvested for fodder, green manure, and timber. The beneficial effects of legume Corresponding author. Tel.: +590 590 255949; fax: +590 590 941663.
E-mail address: [email protected] (J. Sierra). Current address: Department of Forest Ecology, University of Helsinki, PO Box 27, 00014, Finland. 1
0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.12.012
15
N fractionation; Fine root density
trees on tropical soils include increased soil and microbial C and N content in comparison to annual cropping (Sierra et al., 2002), and long-term accumulation of soil organic matter and N (Dulormne et al., 2003). It is assumed that companion crops benefit from the N2 fixation of trees by reabsorbing N mineralised from decomposing N-litter and prunings (Catchpoole and Blair, 1990; Xu et al., 1993; Beer et al., 1998). In other ecosystems, common mycorrhizal networks (He et al., 2003), and root exudates (Paynel et al., 2001) have been proposed as alternative, direct pathways for plant–plant N transfer. Studies using 15N labelling have indicated net N transfer from legume tree saplings to grass in shared containers (Rao and Giller, 1993), and from herbaceous legumes to grass under field conditions (Farnham and George, 1994). Isotopic studies also revealed N transfer from alders (Alnus
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J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903
spp.) to pines (Pinus spp.) via ectomycorrhizae (Arnebrandt et al., 1993; Ekbland and Huss-Danell, 1995) and via mycorrhizae or root exudates from Alnus subcordata C.A. Mey and Eleagnus agnustifolia L. to associated Prunus avium L. (Roggy et al., 2004). An alternative method to estimate N transfer from the N2-fixing plant is the analysis of 15N natural abundance (Mariotti et al., 1981). The apparent merit of this method is that no 15N needs to be added to the soil or the plant and N transfer may be estimated in situ without disturbing the plant-soil system. The method was first used for estimating N transfer from alders to conifers (Binkley et al., 1985). The estimate was based on the 15N natural abundance measured in plants and in the soil inorganic N pool, and it was concluded that isotopic fractionation in soil biased the estimates. van Kessel et al. (1994) showed that the deviation of the sample 15N/14N ratio from that of atmosphere (d15N) decreased in understorey vegetation after introducing N2-fixing Leucaena leucocephala (Lam.) de Wit trees, which is an indication of increasing presence of N fixed from atmosphere in the non-N2-fixers. They could not quantify the N transfer from trees to understorey because of the lack of a reference plant; an understorey plant species growing in the same soil without the N2-fixing species. Snoeck et al. (2000) were able to estimate N transfer in several coffee (Coffea arabica L.) plantations with legume shade trees by using coffee plants distant enough from the legume trees to have no contact with the legume roots or litter as reference plants. They estimated that N transfer contributed 13–42% of total N in coffee plants, depending on the legume species. Changes in 15N natural abundance in the non-N2-fixing plant may be related to transfer from the N2-fixing species or soil processes like nitrification, NH3 volatilization, and denitrification, which discriminate against 15N causing decrease (nitrification) or increase (volatilization, denitrification) in 15N enrichment of the plant-available soil N (Ho¨gberg, 1997). Interpretation of the variation in the isotopic signature of the non-N2-fixing and the N2-fixing plant requires knowledge on the isotope fractionation within the studied plant–soil system (Mariotti et al., 1981). Another constraint of the 15N natural abundance method is the availability of suitable reference plants. Because a part of the fixed N may be transferred to the soil (Dulormne et al., 2003), its 15N natural abundance may vary over time. In this way, the soil of the site selected for the reference non-N2-fixing plant (without fixed-N input) and for the intercropping (with fixed-N input) may differ greatly in a few years after introduction of the N2-fixer (cf. van Kessel et al., 1994). The objective of this study was to test the 15N natural abundance method for quantifying the presence of N fixed from atmosphere in the fodder grass Dichanthium aristatum (Poir.) C.E. Hubb., over a natural gradient of fine root density of associated legume tree Gliricidia sepium (Jacq.) Kunth ex Walp. An experimental procedure for estimating the d15N of the reference plant and a model of soil 15N
fractionation for calculating the error estimate for N transfer are presented. Grass and tree production (Nygren and Cruz, 1998), symbiotic N2 fixation by the trees (Nygren et al., 2000), and soil N and C dynamics (Sierra et al., 2002; Sierra and Nygren, 2005) have been earlier studied in the same agroforestry system. The applicability of the 15N natural abundance method for studying N transfer is discussed in the light of the knowledge gained on this system during 12 years of experimentation. 2. Materials and methods 2.1. Experimental site The study was conducted at the Godet Experimental Station of the French National Institute for Agronomic Research (INRA) in Guadeloupe, French Antilles (161250 N, 611300 W, 10 m a.s.l.). The climate is warm and subhumid with annual mean air temperature of 26 1C and the annual mean rainfall of 1300 mm. The soil is a Vertisol with 80% clay rich in smectite, developed over coral reef limestone. Three adjacent 20 m 13 m plots from a 0.5-ha experiment of silvopastoral and grass monocrop plots were selected for this study, which had similar and relatively homogeneous soil depth of ca. 0.5 m. Some chemical and physical characteristics of the soils are presented in Table 1. Agroforestry plots were established with cuttings of G. sepium in natural grassland of D. aristatum in 1989. They were separated by an open grass plot (OG) of D. aristatum (Fig. 1). The plots were not trenched. The trees were planted in rows aligned N–S, at 0.3 m 2 m spacing. Because of mortality, actual tree density was about 12,000 trees ha1 in 2001. Trees were partially pruned every 2 months and the grass was cut every 40–50 d in all plots from 1989 to 1996. From April 1996, trees were partially pruned every 6 months and the grass was cut every 80 d in the P6 agroforestry plot, and the old management regime was maintained in the P2 plot and in OG (Fig. 1). All three plots were managed from the beginning of the experiment as cut-and-carry systems. Fertilizer (100 kg [P] ha1, 150 kg [K] ha1) was applied four times between 1989 and 1998. No N fertilizer was applied. 2.2. Field experiment The soil was sampled with a 0.06-m diameter auger in January 2001 on three transect lines across the plots (Fig. 1). Only the 0–0.2 m layer was analyzed because no differences in C and N content between plots below that soil depth were detected in an earlier study and about 80% of the mineral N observed in the soil profile (0–0.5 m) was concentrated in the first 0.2-m layer (Sierra et al., 2002). Soil samples were dried at 70 1C for 72 h and ground too0.2 mm. Total N and 15N content of soil samples were determined by an element analyzer (Carlo Erba EA 1110, Carlo Erba Strumentazione, Milan, Italy) connected to a
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Table 1 Chemical and physical properties of the 0–0.2 m soil layer in the open grassland (OG) and in the agroforestry plots of Guadeloupe (French Antilles) Plot
P2 OG P6
Organic C (g kg1)
Total N (g kg1)
28.3c 29.7b 33.1a
2.6b 2.7b 3.1a
pH
7.4a 7.8a 7.7a
CEC (cmol(+) kg1)
52.2a 51.5a 50.8a
Particle size distribution o2 mm (%)
2–50 mm (%)
450 mm (%)
78 79 80
15 13 12
7 8 8
P2 and P6 are two agroforestry plots with tree pruning every 2 or 6 months, respectively. Within columns, values followed by different letters are significantly different at Po0:05.
N
Agroforestry P2
Open grassland (OG) of Agroforestry P6 D. aristatum tree row line 1
13 m line 2
line 3
time on the second soil sampling transect (Fig. 1) by manually cutting a 0.2 m 0.2 m quadrant at 1 cm aboveground. These grass shoots samples were used to estimate in situ N transfer from the legume tree. The plant samples were dried for 48 h at 70 1C, and ground to o0.2 mm for isotopic analyses. The components analyzed were mixed fine roots, individual grass and tree fine roots and grass shoots. Total N, 15N, and 13C to 12C ratio were analyzed by element analyzer-mass spectrometer as described above. Only dry mass was determined for grass fine roots separated at sampling.
29 m
2m
2.3. Greenhouse experiment Soil sampling Grass shoot sampling Root mixture sampling Tree root sampling Harvest at 12 wk
21 wk
34 wk
Whole grass sampling
Greenhouse transplanting
Fig. 1. Design of the field and the greenhouse experiments carried out in Guadeloupe on the Gliricidia sepium—Dichanthium aristatum intercrop. P6 and P2 ¼ G. sepium pruned every six and two months, respectively. Grass transplanted from the field site to greenhouse was harvested at 12 weeks, 21 weeks, and 34 weeks after transplanting (n ¼ 5).
mass spectrometer (Thermo-Finnigan Deltaplus, TermoQuest, Bremen, Germany) through a ConFlo II interface (Thermo-Finnigan) in the Stable Isotope Laboratory of the Kansas State University (USA). Fine roots (diameter p2 mm) of G. sepium and D. aristatum were sampled in February 2001 at 0.5 m from the second transect line selected for soil samples (Fig. 1). Soil monoliths 0.5 m long 0.2 m wide 0.2 m deep were taken and watered for 24 h. Grass roots directly connected to the respective shoot were separated and washed, dried and weighed separately in order to simplify the analyses. The remaining mixed roots were carefully extracted by washing over a 0.2-mm sieve, and separated into fine and coarse roots. Two samples of tree and grass fine roots directly connected to the respective tree or grass shoots were collected for determining reference values for estimating the tree and grass fine root proportions in the mixed root samples (Eq. (2)). Grass shoots were sampled at the same
This experiment was carried out in order to estimate the N natural abundance in grass without contact with tree roots or litter for using it as the reference non-N2-fixing plant (Eq. (4)). The prescribed method to estimate the d15N value of the reference plant is to sample plants growing in the same soil and growing season as the plants intercropped in contact with the N2-fixer. However, this procedure is difficult to apply in agroforestry systems where fine roots of legume trees are everywhere. Soil was taken from the P6 plot by removing the 0–0.2 m layer on a quadrant of 0.5 m2 (Fig. 1). Shoots and large roots were removed in the field. The soil was manually ground for breaking soil aggregates to less than 1 cm. Fine roots were removed, and soil was divided into five 15-l pots in a greenhouse. Twenty-five 15-cm-high D. aristatum plants were gently transplanted from the P6 plot to the pots on 15 January 2001; e.g. five plants per pot. Soil moisture was maintained between 0.3 and 0.4 kg kg1 (60–80% of water holding capacity). Temperature in the greenhouse was ca. 2 1C higher than outside. Shoots were cut to 1 cm on 11 April, 12 June and 10 September 2001; the same pots were followed throughout the experiment. Shoots from each individual pot were mixed, dried for 48 h at 70 1C and ground to o0.2 mm. Total N and 15N content were determined as described above. Nitrate-N and NH+ 4 –N in potting soil was extracted by shaking a sample 20 g of moist soil for 2 h in 100 ml of 0.5 M KCl. Mineral N was analyzed colorimetrically with an autoanalyzer (Technicon Industrial Systems, USA) using hydrazine reduction for NO 3 –N (Kampshake 15
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et al., 1967) and the nitroprusside method for NH+ 4 –N (Kaplan, 1965). 2.4. Calculations The percentage of G. sepium fine roots out of total fine root mass in mixed root samples was calculated using the different C isotope ratios in G. sepium (a C3 plant) and D. aristatum (a C4 plant). The deviation of the 13C to 12C ratio in the sample from that in the PDB (a limestone) standard (d13C) was calculated (Balesdent, 1996): 13
d C ¼ ½ðRx =Rr Þ 1 1000; 13
(1) 12
where Rx and Rr are the C– C ratios in the sample and PDB standard, respectively. The percentage of G. sepium fine roots (%Mgro) was calculated (Sierra and Nygren, 2005): %M gro ¼ ½ðd13 Ci d13 Cd Þ=ðd13 Cg d13 Cd Þ 100; 13
(2)
13
where d Ci is the d C in the fine root sample from intercropping (roots of G. sepium and D. aristatum mixed), d13Cd in pure D. aristatum root sample and d13Cg in pure G. sepium root sample. The mass of G. sepium fine roots in the sample was obtained by multiplying the total mass of the mixed root sample by %Mgro. The fine root mass of D. aristatum was obtained by deducting the mass of G. sepium roots from total mixed root sample mass, and adding the mass of D. aristatum fine roots that were separated at sampling because of direct connection to a D. aristatum shoot. The fine root density was calculated by dividing the root mass by the sample volume (20 dm3). The 15N natural abundance method was used for detecting fixed N in the grass because 15N natural abundance was also used for quantifying N2 fixation by G. sepium in the site (Nygren et al., 2000), we did not want to disturb the N cycle with labeled fertilizer, and 12 years under the same production system was assumed to have stabilized the 15N signature of the soil. The deviation of the sample 15N proportion from that of atmosphere (d15Nx) was calculated (Mariotti et al., 1981): d15 Nx ¼ ½ð%15 Nx %15 Na Þ=%15 Na 1000;
(3)
where %15Nx and %15Na are the percentages of 15N in the sample and in the air (0.3663%; Mariotti, 1984), respectively. The percentage of N fixed from atmosphere out of total N in D. aristatum (%Nf) was calculated (Snoeck et al., 2000): %Nf ¼ ½ðd15 Nr d15 Ni Þ=ðd15 Nr d15 Nfr Þ 100;
(4)
where subscript r (reference) refers to D. aristatum growing without contact with G. sepium, i to D. aristatum intercropped with G. sepium, and fr to G. sepium growing in N-free medium. The d15Nfr value for G. sepium of same genetic origin as the trees in the study site is 2.07 (Nygren et al., 2000). The d15Nr value for D. aristatum was determined in the
greenhouse experiment. Eq. (4) gives the percentage of fixed N in D. aristatum associated with G. sepium, not the percentage of total N transfer from the tree, because a part of the legume N is taken up from the soil. Concentration of fixed N in D. aristatum shoots (g [Nf] kg1 [DM]) was calculated multiplying the total N content of the sample (g [Nt] kg1 [DM]) by %Nf. 2.5. Statistical analyses Because tree roots were present in all plots, including the OG, the plots may not be considered strictly as treatments, and plant variables were analyzed as a function of a root density gradient of G. sepium. Selected grass features were related to the gradient by Pearson correlation and linear regression. Differences in soil variables along the tree fine root density gradient were tested by analysis of variance followed by separation of means by Duncan’s Multiple Range Test using the 19 east–west transect positions as treatments and the three soil sampling transects as replications (Fig. 1). Statistical analyses were conducted with SAS v. 8.02 software (SAS Institute, 1999). 2.6. Model of soil 15N fractionation and N partitioning in D. aristatum A model was developed for simulating 15N fractionation in N mineralization and N distribution inside D. aristatum following transplanting from the agroforestry plot to a pot in the greenhouse (Fig. 2). The simulated effect of 15N fractionation in soil on the d15Nr estimate was used to assess the reliability of the experimental procedure. Nitrogen mineralization was described by a first-order reaction: 14
Nm ¼14 N0 ½1 expðk14 tÞ;
(5)
15
Nm ¼15 N0 ½1 expðk15 tÞ;
(6)
where 14N0 and 15N0 are the mineralizable pools of the two N isotopes at time 0 (mg kg1), 14Nm and 15Nm are the cumulative mineralization of 14N and 15N (mg kg1) at time t (week), and k14 and k15 (week1) are the mineralization rate constants of each isotope pool. The fractionation coefficient for mineralization (b) is calculated as (Mariotti et al., 1981): b ¼ k14 =k15 .
(7)
We assumed that b is constant over time and independent of the amount of 14N0 and 15N0 remaining in soil. About 95–98% of mineral N was present as NH+ 4 in the field site (Sierra et al., 2002). Further, some tropical grasses release compounds from roots which reduce population or activity of nitrifying bacteria (Ishikawa et al., 2003), and this was also observed in D. aristatum (J. Sierra, unpublished data). Thus, neither nitrification nor denitrification was included in the model.
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U s ¼ U 0s Ptr U 0s ;
Shoot
(10) 1
15
N
14
N
where Uro and Us (mg [N] week ) are the rates of current N uptake to root and shoot, respectively. Nitrogen uptake rate between two grass cuts was assumed to be constant over time. The d15N of the N retranslocated from root to shoot at time t was assumed to be equal to the d15N of roots at time t1. The d15N at time t in each plant compartment was calculated as a dilution process by taking into account the d15N of each compartment at time t1 and the d15N corresponding to N taken from the soil (d15Nm in Fig. 2) and that of the N metabolized in roots and retranslocated to shoot. The part of the N in shoot and root biomass remaining after grass cut (Psa and Proa in Fig. 2) was calculated as a proportion of the respective N content before cut. The d15N of the remaining shoot and root biomass was set equal to the d15N of each compartment before cut. Calculations were made at a time step of a week.
Ns, δ15Ns
Ps-a
Shoot after cut
Ptr
Roots 15
N 14 N
Nro,δ15Nro Roots after cut
Pro-a Us
Uro
N uptake
2.7. Model parameters Organic matter 15 14
N N
δ15No
Mineral N
Mineralization (fractionation)
15
N
14
N
δ15Nm
Fig. 2. Model of soil 15N fractionation and N partitioning in the grass for simulating the temporal change of 15N content in soil and in a grass without contact with legume tree. The values of the parameters used in simulations are presented in Table 2.
Nitrogen uptake was calculated in two steps. First, the increase rate of root N content (U0 ro in mg [N] week1) was assumed to be proportional to the increase rate of shoot N content (U0 s in mg [N] week1) and to be constant over time (Cruz, 1997). Thus, U0 ro was calculated from U0 s: U 0ro ¼ U 0s Pu ;
(8)
where Pu is the ratio of the increase rate of root N content to the increase rate of shoot N content. Nitrogen taken up by grass was divided into three components (Cruz, 1997): direct N transfer to shoot in xylem flow, N metabolized and retained in roots, and N metabolized in roots and recycled to shoot. Direct N transfer in xylem flow does not affect the isotopic ratios (Handley et al., 1999) and this component was assumed to retain the d15N of soil N. Nitrogen recycled from roots to shoot was assumed to have the d15N of roots, because N metabolism within a plant may strongly alter the isotopic signature (Handley et al., 1999; Ho¨gberg, 1997). Under the scheme of frequent cutting, the ratio (Ptr) of the N root–shoot retranslocation rate to the increase rate of shoot N content was assumed to be constant between two cuttings (Cruz, 1997). Thus, the distribution of current N uptake into root and shoot was calculated from the increase rates of root and shoot N content: U ro ¼ U 0ro þ Ptr U 0s ;
(9)
Grass growth was simulated during a 54-weeks-period including grass cut every 12 weeks applying parameter values shown in Table 2. The initial d15N of the mineralizable soil organic N pool (d15N0) was assumed to be equal to d15N determined in this study for the soil organic matter. The initial d15Nm was set to 3.50, which is close to the d15Nr determined at the end of the greenhouse experiment. This is based on the assumption that the d15N of the grass without tree contact was representative of the 15 N natural abundance of the soil available N at 34 weeks after transplanting. With these values of d15N0 and d15Nm, the coefficient b was equal to 1.0025, which is within the range of fractionation values for mineralization reported by other authors (Ho¨gberg, 1997). The value of k14 was estimated from the results reported by Sierra et al. (2002) for the same soil used in this study; N0 was set so that the amount of mineralized N was equal to the N absorbed by the grass during the simulated 54-weeks-period. The increase rate of shoot N content was set equal to 200 mg N for each 12-weeks regrowth period. This value and those selected for the initial d15Ns and d15Nro (Table 2) were close to those observed in the greenhouse experiment. The values of Pu, Ptr, Psa, and Proa were estimated from the results reported by Cruz (1997) on N partitioning in frequently defoliated D. aristatum. 3. Results 3.1. Greenhouse experiment The d15N value of greenhouse grown D. aristatum shoots was 1.6470.05 (mean7standard error of mean, SEM) 12 weeks after transplanting, which was close to the value in grass in the P6 plot where the greenhouse plants originated (1.5070.13; ‘‘initial value’’ in Fig. 3). The d15N value
ARTICLE IN PRESS J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903
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Table 2 Values of the model parameters used to simulate the temporal change of the isotopic signature of soil mineral N and grass in the greenhouse experiment Parameter
Explanation
Unit
Value
Source
Soil d15No N0 14 N0 15 N0 k14 k15 b
d15N for soil organic N Mineralizable soil N at time ¼ 0 Mineralizable soil 14N at time ¼ 0 Mineralizable soil 15N at time ¼ 0 Mineralization rate constant of 14N Mineralization rate constant of 15N Fractionation coefficient for mineralization
— mg [N] kg1 mg [N] kg1 mg [N] kg1 week1 week1 —
6.0 500 498.19 1.81 2.37 103 2.3641 103 1.0025
This study This study This study Calculated from N0 and 14N0 Sierra et al. (2002) Calculated from k14 and b This study
Plant d15Ns, d15Nro U0 s Pu Ptr Ps–a Pro–a
d15N in grass shoot and root at time ¼ 0 — Increase rate of shoot N content mg [N] week1 Ratio of increase rate of root N content to increase rate of shoot N content — Root-to-shoot N retranslocation coefficient — Proportion of shoot N retained after cut — Proportion of root N retained after cut —
of the greenhouse experiment. In both cases, 95% of the mineral N was present as NH+ 4 –N.
4.0
3.2. Root biomass
3.0
δ Shoot 15N
1.5 (at time 0) This study 16.67 This study 0.30 Cruz (1997) 0.15 Cruz (1997) 0.10 Cruz (1997) 0.50 Cruz (1997)
2.0
1.0
0.0 0
9
18
27
36
Weeks after transplanting
Fig. 3. Greenhouse experiment: d15N of grass shoots as a function of the time after transplanting. Swards of Dichanthium aristatum were transplanted from P6 agroforestry plot to monoculture pots filled with soil from the same plot. Vertical bars indicate the standard error (n ¼ 5).
increased from 12 to 34 weeks in greenhouse, with a strong variation between pots at 21 weeks after transplanting (Fig. 3). The d15N values were quite uniform 34 weeks after transplanting, and appeared to reach the highest value. The mean7SEM of d15N value was 3.4470.24, which was higher than any value observed in the field (maximum 2.70). The mean of d15N in D. aristatum shoots in the last greenhouse harvest was used as the reference value without presence of a N2-fixing plant, term d15Nr in Eq. (4). Total N content in shoots was 14.370.5 mg [N] g1 [DM] at transplanting, and 9.070.2, 5.670.3, and 7.370.2 mg [N] g1 [DM] at 12, 21 and 34 weeks after transplanting, respectively. Soil mineral N content was 18.271.2 mg [N] kg1 at transplanting and 31.072.6 mg [N] kg1 at the end
The d13C values in the pure roots of D. aristatum and G. sepium were 12.1 and 26.1, respectively. This formed a sufficient contrast for application of Eq. (2) in estimation of %Mgro in the mixed root samples. Fine root density of G. sepium formed a decreasing gradient from P6 plot through OG plot to a slight increase in the P2 plot (Fig. 4a). The fine root density gradient of D. aristatum was about the reverse. Fine root biomass varied from 1.3 to 6.3 Mg ha1 in D. aristatum, and from 0.1 to 3.0 Mg ha1 in G. sepium. Fine roots densities of G. sepium and D. aristatum were negatively correlated (R2 ¼ 0:37, Po0:05). Nitrogen content in the pure G. sepium fine root sample was 21.7 mg [N] g1, and the d15N value was 0.8. 3.3. Total N and
15
N content in soil and plant in the field
Soil total N content was significantly higher in the sampling points in the P6 plot than in those of the P2 or OG plots (Duncan’s MRT at Po0:05; Fig. 4b). Differences in soil d15N between sampling points were not significant. The mean7SEM of soil d15N value was 6.0170.05 (Fig. 4b). Soil total N was positively correlated with tree fine root density (R2 ¼ 0:78, Po0:05), but soil d15N was not correlated with either G. sepium or D. aristatum fine root density in the adjacent transect line. Total N concentration in shoots of D. aristatum averaged 1.43 mg [N] g1 in P6, 1.14 mg [N] g1 in the open grassland, and 1.56 mg [N] g1 in P2 (Fig. 4c). It had a weak but significant positive correlation with tree fine root density (R2 ¼ 0:30, Po0:05) and soil total N content (R2 ¼ 0:30, Po0:05). The d15N of grass shoot did not present any clear spatial pattern across the transect line,
ARTICLE IN PRESS J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903
3.4. Estimation of transfer of fixed N from legume trees to grass
D. aristatum
3.5
1899
G. sepium
Fine root density (g dm-3)
3.0 2.5 2.0 1.5 1.0 0.5 0.0
(a)
0
10
30
Total N
4.0
7
δ15 N
3.5
6
3.0
5
Soil 15N
Soil total N (g kg-1)
20
2.5
(b)
4 0
10
20
30
3.5. Simulation results
Total N 20
3.0
15
δ N
2.5 15 2.0 1.5
10
Shoot 15N
Shoot total N (mg g -1)
The percentage of N fixed from atmosphere out of total N in shoots of D. aristatum, calculated according to Eq. (4), averaged 36% in P6 and in OG, and 27% in P2. Percentage of fixed N in grass shoot was not correlated with either tree fine root density or soil N content. Because total N concentration was high in P6, the concentration of fixed N was the highest in the shoots of D. aristatum growing in this plot. The averages were 0.52, 0.38 and 0.39 mg [Nf] g1 in P6, OG, and P2, respectively. Considering the average annual grass N yield of 195 kg ha1 year1 (Dulormne et al., 2003), the percentages of N fixed from atmosphere in D. aristatum correspond to 53 and 70 kg ha1 year1 of grass N of atmospheric origin in P2 and P6 plots, respectively. A significant correlation was observed between the fine root density of G. sepium and concentration of fixed N in shoots of adjacent D. aristatum samples (Fig. 5). The three points with highest fine root densities of G. sepium correspond to P6 plot, while data for P2 and OG plots is mixed within each others in the points with low fine root densities. The concentration of fixed N in grass shoot was not correlated with soil d15N.
1.0
Simulated d15Nm increased from 3.50 to 3.81 (Fig. 6a) and simulated d15N0 increased from 6.00 to 6.32 (data not shown) in 54 weeks. This was related to 15N fractionation during N mineralization. The temporal change in d15N differed slightly between the shoot and root biomass (Fig. 6a). The increase of d15Ns was faster than that of d15Nro because more soil mineral N was allocated to the shoot. However, at the end of the third regrowth period (36
5
8
0.5 0
0.0 0
10
P2
20
OG
30
P6
Transect position (m)
Fig. 4. Field experiment: (a) Fine root density of Dichanthium aristatum and Gliricidia sepium on the studied transect; (b) total N content and d15N in soil; (c) total N content and d15N in shoots of D. aristatum. Values in (b) and (c) correspond to the second transect line (see Fig. 1). Vertical dashed lines indicate tree rows.
6 Fixed N (mg [N] g -1)
(c)
4
y = 1.98 x + 3.47
2
R2 = 0.55, P < 0.05
0
except in P2 where d15N was higher in the alleys (Fig. 4c). The d15N values varied from about 2.6 in the alleys of P2 to 0.85 in the open grassland next to P6 plot. Shoot d15N was not correlated with either tree fine root density or soil d15N.
0.0
0.4
0.8
1.2
1.6
-3
Fine root density of G. sepium ( g dm )
Fig. 5. Concentration of N fixed from atmosphere in the shoots of the grass Dichanthium aristatum as a function of fine root density of the associated legume tree Gliricidia sepium.
ARTICLE IN PRESS J. Sierra, P. Nygren / Soil Biology & Biochemistry 38 (2006) 1893–1903 4.0
60
3.5
50
2.5
δ15Nm
2.0
δ15Nro
δ15Ns
Estimate with δ15Nm at t = 0
10 0
0
12
(a)
24
36
48
0.5
4.0
3.5
3.5 δ15Nm 15
δ Ns (initial simulation)
δ15N
4.0
2.5
δ15Ns (Ps-a = 0.2) δ15Ns (Ptr = 0.3)
2.0
1
(b)
Time (weeks)
3.0 δ15N
30 20
1.5
1.5 2 δ15Ns
2.5
3
3.0 2.5 δ15Nm
2.0
1.5
δ15Ns
1.5 0
(c)
Estimate with δ15Ns at t = 36 weeks
40
3.0
%Nf
δ15N
1900
12
24
36
48
Time (weeks)
0
12
(d)
24
36
48
Time (weeks)
Fig. 6. Simulated temporal change in soil and plant d15N in the greenhouse experiment: (a) grass and soil mineral d15N as a function of time. Parameter values in Table 2 were used. (b) Percentage of fixed N in the grass (%Nf) as a function of grass d15N. The %Nf was estimated with two different values of d15N for the reference grass plant: d15Nm at time 0 or d15Ns at time 36 weeks. Differences between the estimates were o3%. (c) Effect of a change in the values of the model parameters Psa and Ptr (Table 2) on the d15N of the grass. Value of d15Ns (initial simulation) corresponds to d15Ns in (a). (d) Effect of a four-time reduction in plant N uptake in the first regrowth period (0–12 weeks) on the d15N of the grass. Subscripts m, s, and ro refer to soil mineral N, grass shoot, and grass root, respectively. Vertical bars in (a) and (d) indicate the time of grass harvest (every 12 weeks).
weeks) both d15N values were similar and close to the d15Nm value; 3.60 for d15Ns, 3.59 for d15Nro, and 3.66 for d15Nm. At this time, the fixed N represented only 0.1% of the grass shoot N content. To calculate the error involved for using d15Nr such as it was determined in this study, the estimate of %Nf (Eq. (4)) was performed with two values of d15Nr: (i) d15Nr was set equal to d15Nm at time 0, and (ii) d15Nr was set equal to d15Ns at time 36 weeks; e.g. the time corresponding to the end of the greenhouse experiment. The first estimate corresponds to the theoretical value of %Nf because no 15 N fractionation was assumed for N uptake; that is, d15Nm at time 0 is equal to the d15Ns of a plant without contact with legume roots and without any effect of soil 15N fractionation. The second estimate corresponds to the experimental procedure proposed in this study. The theoretical results indicated that even if 15N fractionation during N mineralization induces an overestimate of the fixed N transferred to the grass, the error is small for a large range of d15Ns (Fig. 6b). For example, the overestimate was 1.0 and 1.5% for N transfer of 50 and 10%, respectively. The error of the estimate was not affected by the plant parameter values used in the model. Fig. 6c shows that changes in Psa and Ptr caused the initial increase in d15Ns, but it was very similar for all the simulations at 36 weeks.
4. Discussion 4.1. Soil N and
15
N content
Soil N content has increased 2.5 Mg [N] ha1 in P6, and about 1.8 Mg [N] ha1 in P2 and OG in 12 years after planting G. sepium in the current study plots (Sierra and Nygren, 2005). Because tree pruning residues were removed from the site and litterfall was small because of frequent pruning, it may be concluded that N released from tree roots was the most important N input in the three plots. In the present study, no correlation was observed between soil d15N and tree root density. Considering the typically rapid fine root dynamics of periodically pruned trees (Mun˜oz and Beer, 2001), there is no reason to assume that soil d15N would be correlated with the current tree fine root density in a point in the field. The isotopic signature of the soil rather reflects the cumulative effect of presence and absence of tree roots during the 12-years experimentation. Soil d15N may be expected to stabilize in 4–6 years after introducing a dense legume tree plantation (van Kessel et al., 1994). The change in soil d15N is a result of long-term accumulation of tree litter depleted in 15N, and counteracting soil microbial processes that tend to enrich the soil in 15N (Ho¨gberg, 1997).
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In spite of the gradient of soil N content (Fig. 4b), soil d15N was quite homogeneous throughout the experimental site, and no differences were detected between sampling sites. This was an essential condition for using the 15N natural abundance method for estimating in situ N transfer across the tree fine root density gradient. Further, the d15N value of the P6 soil used in greenhouse was representative for the homogeneous experimental site. The difference between the d15N values of the reference plant and N2-fixer in N-free environment should be at least 5%-units in order to obtain a reliable estimate of N2 fixation by the 15N natural abundance method (Ho¨gberg, 1997). If the same condition is applied for N transfer studies using 15N natural abundance, the d15N values of the potted D. aristatum reference and G. sepium in N-free medium, +3.44 and 2.07 (Nygren et al., 2000), respectively, provided sufficient contrast in this study. 4.2. Estimate of d15N of the reference plant Using of a field-grown reference plant was not suitable in this study because tree roots were present everywhere. Wide horizontal extension of tree root systems has been observed in several agroforestry systems (Hauser, 1993; Schroth and Zech, 1995). In plots cultivated with D. aristatum monocrop for at least 20 years in the Godet Experimental Station, the soil d15N was 9.570.6 (mean7SEM, n ¼ 5) (J. Sierra, unpublished data). Because the reference plant should grow in a soil with similar 15N signature as the plant intercropped with N2-fixers, D. aristatum from a monocrop plot was not a suitable reference. Therefore, we proposed a method based on the determination of d15N of the grass in a pot filled with the soil of the agroforestry site. In such pot system with no external N input, the d15N of the grass is expected to increase over time for two reasons: (i) frequent grass cut causes a dilution of the fixed N transferred from the tree, which has a d15N much lower than that of the soil N, and (ii) 15N fractionation during mineralization causes increase in the d15N of the soil mineral N. A model was developed for separating the effects of both processes on the d15Nr value estimate. Simulations indicated that 3% of the calculated increase in grass d15N was associated to 15N fractionation during mineralization, and 97% was due to the dilution of the initial fixed N present in the grass. Thus, it may be concluded that the increase of d15Ns observed in the greenhouse experiment (Fig. 3) was mainly due to the decrease in the proportion of N derived from G. sepium because of N export in grass harvest. Simulations also indicated that the amount of fixed N in the grass was negligible at 36 weeks after transplanting. The increase of d15Ns in pots also indicates that rhizospheric diazotrophs were not the source of fixed N in grass, because diazotrophs would have been transferred to the pots in the field soil and mostly intact grass root systems. Therefore, both experimental and theoretical
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results suggest that the procedure proposed in this study provided a suitable estimate of d15Nr for estimating the transfer of fixed N from the tree to grass (Eq. (4)). Differences between the greenhouse-observed and simulated trend of d15Ns during the first regrowth period (Figs. 3 and 6a) were probably due to grass transplanting stress. Poor grass growth during the first regrowth period probably reduced N uptake and dilution of the fixed N content causing the small increase in the d15Ns value (Fig. 3). The simulation, in which N uptake rate for this period was set four times lower than the initial value (U0 s in Table 2), reproduced well the small increase in d15Ns during that period (Fig. 6d). The estimate error was similar to that calculated in the basic simulation (Fig. 6b) because the differences in d15Ns at 36 weeks were negligible between simulations. Denitrification and volatilization discriminate against 15 N causing soil mineral 15N enrichment, and NH+ 4 becomes relatively enriched by 15N in comparison to 15 NO N fractionation during nitrification 3 because of (Ho¨gberg, 1997). These potential changes in the isotopic signature of available soil N may affect plant d15N and must be considered when interpreting the observed d15N increase in shoots of potted grasses. About 95–98% of mineral N was present as NH+ 4 in our soil (Sierra et al., 2002; this study). Some tropical grasses release compounds from roots, which reduce population or activity of nitrifying bacteria (Ishikawa et al., 2003), and this was also observed in D. aristatum (J. Sierra, unpublished data). For this reason, neither nitrification nor denitrification was included in the model. Volatilization of NH3 has been observed to be negligible in a non-N-fertilized Vertisol similar to that used in this study (Courtaillac et al., 1998). Thus, soil mineral 15N enrichment by these processes may be discarded as a source of 15N enrichment in potted grass. 4.3. Transfer of fixed N in the field The greenhouse experiment provided indirect evidence of the transfer of fixed N from the tree to grass. In the field, all the shoot samples of D. aristatum appeared to be depleted in 15N in relation to the reference value obtained in greenhouse. This implies that N transfer occurred along the entire grass transect. The percentage of N derived from atmosphere in D. aristatum, 26–35% along the gradient of G. sepium fine root density, was within the range of values obtained by 15N-labeling of soil or plant (e.g. Rao and Giller, 1993; Farnham and George, 1994), and 15N natural abundance (Snoeck et al., 2000). Higher percentage, ca. 68%, has been observed in grass associated with alfalfa (Medicago sativa L.; Brophy et al., 1987). These data indicate that N2-fixing legumes may make important contributions to N nutrition of associated non-legumes. Nitrogen content in the grass shoot biomass was higher under the trees than in the open grassland (Fig. 4c). In an earlier long-term study this phenomenon was attributed to (i) higher shoot N/root N ratio in the grass under the trees
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because of morphogenetic effects induced by shade, and (ii) higher N mineralization and better soil N availability in P6 because of a higher N content in soil (Dulormne et al., 2003; cf. Fig. 4b). The significant correlation between the concentration of fixed N in grass shoots and tree fine root density (Fig. 5), together with the lack of correlation between these variables and soil d15N, indicate that transfer of fixed N may have involved a more direct mechanism than a transfer via the complete N mineralization cycle in soil. This hypotheses was supported by direct measurements of soil mineral d15N performed in a later work carried out in the same OG plot. Soil mineral d15N averaged 2.7170.08 in this plot, and no correlation was observed between soil mineral d15N and the distance from the nearest tree line (J. Sierra, unpublished). The value was higher than that observed in the present study for the grass shoots in the OG plot (1.3570.16; Fig. 4c), which also suggests that a part of N transfer occurred directly. Soil mineral d15N was also lower than the d15N of the reference plant, which may be associated to tree root exudates released to the soil solution of the OG plot. About 95% of N in root exudates of white clover (Trifolium repens L.) is in the form NH+ 4 (Paynel et al., 2001). If this was the case of G. sepium, NH+ 4 could be easily absorbed by the grass because it is the current N source in our soil. Nitrogen may also have been transferred from trees to grass via common mycorrhizal networks (He et al., 2003). G. sepium forms symbioses with arbuscular mycorrhizae (Okon et al., 1996). We could not find any information on mycorrhizae in D. aristatum, a little studied yet important fodder grass in the Caribbean basin, but most grasses have arbuscular mycorrhizae. In most studies, the N transfer from a N2-fixer is expressed as percentage of fixed N out of the total N in the associated plant (e.g. Brophy et al., 1987; Snoeck et al., 2000). Our results indicated that fixed N by unit of dry matter is a more suitable indicator for analyzing N transfer. For example, the amount of fixed N and N taken up from the soil were highest in P6, but grass d15N and the percentage of fixed N were similar to those observed in the OG plot. The change in soil available N as a function of the distance from the tree (Sierra et al., 2002) may explain the lack of correlation between tree fine root density and the percentage of fixed N in the grass. However, soil d15N as measured in this study is an indicator of the 15N natural abundance in the bulk soil and not in the tree rhizosphere. Further work is necessary to discriminate between direct and indirect pathways of N transfer, including the study of N recycling in the rhizospheric soil of the legume tree, and the role of mycorrhizal networks. Acknowledgements We thank Saint-Ange Sophie for skilful technical assistance and for the management of the experimental plots since 1989, and Roxane Fagan (Stable Isotope
Laboratory of the Kansas State University, USA) for the mass spectrometry of 15N and 13C samples. This work was funded by the University of Missouri Life Sciences Mission Enhancement Programme (PN) and the De´partement Environnement et Agronomie of the Institut National de la Recherche Agronomique, France (JS). References Arnebrandt, K., Ek, H., Finlay, R.D., So¨derstro¨m, B., 1993. Nitrogen translocation between Alnus glutinosa (L.) Gaertn. seedlings inoculated with Frankia sp. and Pinus contorta Doug. ex Loud seedlings connected by a common ectomycorrhizal mycelium. New Phytologist 124, 231–242. Balesdent, J., 1996. The significance of organic separates to carbon dynamics and its modelling in some cultivated soils. European Journal of Soil Science 47, 485–493. Beer, J., Muschler, R., Kass, D., Somarriba, E., 1998. Shade management in coffee and cacao plantations. Agroforestry Systems 38, 139–164. Binkley, D., Sollins, P., McGill, W.B., 1985. Natural abundance of nitrogen-15 as a tool for tracing alder-fixed nitrogen. Soil Science Society of America Journal 49, 444–447. Brophy, L.S., Heichel, G.H., Russelle, M.P., 1987. Nitrogen transfer from forage legumes to grass in a systematic planting design. Crop Science 27, 753–758. Catchpoole, D.W., Blair, G., 1990. Forage tree legumes I. Productivity and N economy of leucaena, gliricidia, calliandra and sesbania and tree/green panic mixtures. Australian Journal of Agricultural Research 41, 521–530. Courtaillac, N., Baran, R., Oliver, R., Casabianca, H., Ganry, F., 1998. Efficiency of nitrogen fertilizer in the sugarcane-vertisol system in Guadeloupe according to growth and ratoon age of the cane. Nutrient Cycling in Agroecosystems 52, 9–17. Cruz, P., 1997. Effect of shade on the carbon and nitrogen allocation in a perennial tropical grass, Dichanthium aristatum. Journal of Experimental Botany 48, 15–24. Dulormne, M., Sierra, J., Nygren, P., Cruz, P., 2003. Nitrogen-fixation dynamics in a cut-and-carry silvopastoral system in the subhumid conditions of Guadeloupe, French Antilles. Agroforestry Systems 59, 121–129. Ekbland, A., Huss-Danell, K., 1995. Nitrogen fixation by Alnus incana and nitrogen transfer from A. incana to Pinus sylvestris influenced by macronutrients and ectomycorrhiza. New Phytologist 131, 453–459. Farnham, D.E., George, J.R., 1994. Dinitrogen fixation and nitrogen transfer in birdsfoot trefoil-orchardgrass communities. Agronomy Journal 86, 690–694. Handley, L.L., Austin, A.T., Robinson, D., Scrimgeour, C.M., Raven, J.A., Heaton, T.H.E., Schmidt, S., Stewart, G.R., 1999. The 15N natural abundance (d15N) of ecosystem samples reflects measures of water availability. Australian Journal of Plant Physiology 26, 185–199. Hauser, S., 1993. Root distribution of Dactyladenia (Acioa) barteri and Senna (Cassia) siamea in alley cropping on Ultisol. I. Implications for field experimentation. Agroforestry Systems 24, 111–121. He, X.H., Critchley, C., Bledsoe, C., 2003. Nitrogen transfer within and between plants through common mycorrhizal networks (CMNs). Critical Reviews in Plant Science 22, 531–567. Ho¨gberg, P., 1997. 15N natural abundance in soil–plant systems. New Phytologist 137, 179–203. Ishikawa, T., Subbarao, G.V., Ito, O., Okada, K., 2003. Suppression of nitrification and nitrous oxide emission by the tropical grass Brachiaria humidicola. Plant and Soil 255, 413–419. Kampshake, L.J., Hannah, S.J., Cohen, J.M., 1967. Automated analysis for nitrate by hydrazine reduction. Water Resources Research 1, 205–216. Kaplan, A., 1965. Standard Methods of Clinical Chemistry. Academic Press, New York, USA 249p.
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