Nitrogen fixation rate of Acacia mangium Wild at mid rotation in Brazil is higher in mixed plantations with Eucalyptus grandis Hill ex Maiden than in monocultures

  • Ranieri R. PaulaEmail author
  • Jean-Pierre Bouillet
  • José L. de M. Gonçalves
  • Paulo C. O. Trivelin
  • Fabiano de C. Balieiro
  • Yann Nouvellon
  • Julianne de C. Oliveira
  • José C. de Deus Júnior
  • Bruno Bordron
  • Jean-Paul Laclau
Original Paper


Key message

Inter-specific interactions with eucalypts in mixed plantations increased N 2 fixation rate of acacia trees compared to monocultures. N 2 fixation was higher during the wet summer than during the dry winter both in acacia monocultures and in mixed plantations.


Introducing N-fixing trees in fast-growing tropical plantations may contribute to reducing the long-term requirements of N fertilizers. Management practices established in forest monocultures should be revisited in mixed-species plantations.


This field experiment aimed to compare N2 fixation rates of Acacia mangium Wild in monospecific stands and in mixed-species stands with Eucalyptus grandis W. Hill ex Maiden. A secondary objective was to gain insight into the seasonal variations of N2 fixation.


15N was applied to acacia and eucalypt monocultures and mixed-species with a 1:1 ratio at mid rotation. Leaves were collected in autumn, winter, spring, and summer to determine the foliar N concentrations and 15N atom fraction. The N content in the above-ground biomass was estimated as well as the percentage of N derived from atmospheric N2 (%Ndfa) using eucalypts in monoculture as reference plants.


%Ndfa values averaged over the year were 14% in monoculture and 44% in mixed-species stands. While the stocking density of acacia trees was twice as high in monoculture as in mixture, the amounts of N fixed in above-ground biomass of acacia trees were close (35–39 kg N ha−1) at 39 months after planting. %Ndfa values were higher during the wet summer than the dry winter both in acacia monocultures and in mixed plantations.


The stocking density of acacia trees can be reduced in mixed plantations with eucalypts in comparison to acacia monocultures with a low influence on the input of N to soil through biological fixation.


Symbiotic N2 fixation Seasons Mixed-species plantations Competition Forest rotation 

1 Introduction

In the tropics, nitrogen-fixing trees (NFT) may fix large amounts of atmospheric N2 (Binkley and Giardina 1997; Nygren et al. 2012). Associating NFTs with non-nitrogen fixing trees can increase biomass production of plantations (Piotto 2008; Bouillet et al. 2013; Santos et al. 2016), soil carbon sequestration (Resh et al. 2002), microbial diversity (Rachid et al. 2013), and soil nutrient availability (Voigtlaender et al. 2012; Koutika et al. 2014). NFTs are likely to improve the N status of companion species rapidly, through below-ground pathways (Nygren and Leblanc 2015; Paula et al. 2015), and in the long term, through decomposition of N-rich litter and pruning residues (Beer 1988; Forrester et al. 2006).

N2 fixation is regulated by numerous factors such as water availability in the soil, temperature, nutrient availability, and interactions between plants and soil microorganisms (Rastetter et al. 2001; Vitousek et al. 2002; Soussana and Tallec 2010; Diagne et al. 2013; Augusto et al. 2013). High soil temperatures and water content in autumn and spring led to an increase in nodule biomass and N fixation for Acacia dealbata Link seedlings growing in 2-year-old Eucalyptus regnans F. Muell. stands (Adams and Attiwill 1984).

The percentage of N derived from atmospheric N2 (%Ndfa) is highly dependent on plant age and species (Parrotta et al. 1996; Isaac et al. 2011). %Ndfa values ranging from 10 to 100% have been found for tropical NFTs (Binkley and Giardina 1997; Forrester et al. 2006; Nygren et al. 2012). The gradual decrease in %Ndfa that occurs after planting has been mainly associated with the ability of NFTs to regulate N2 fixation depending on their N requirements and changes in soil N availability (Vitousek et al. 2002; Barron et al. 2011; Sheffer et al. 2015). Regulation of N2 fixation has been reported for natural tropical and temperate forests (Pfautsch et al. 2009; Barron et al. 2011) and may account for the temporal changes in %Ndfa in mixed-species planted forests (Parrotta et al. 1996; Balieiro et al. 2008; Bouillet et al. 2008).

Intra- and inter-specific competition for N, light, and water may influence N2 fixation (Vitousek et al. 2002; Forrester et al. 2007; Wurzburger and Miniat 2014; Sheffer et al. 2015). Non-leguminous species with highly competitive soil N uptake may increase the %Ndfa values of the associated NFTs, as has been observed for Pseudosamanea guachapele (Kunth) Harms in mixed-species plantations with Eucalyptus grandis Hill ex Maiden (Balieiro et al. 2008) and for several acacia species growing with grasses in African savannas (Cramer et al. 2007). The competition for light and water may limit plant growth and N2 fixation rates, as reported for Pisum sativum L. associated with Hordeum vulgare L. (Jensen 1996) and A. mangium mixed with E. grandis (Bouillet et al. 2008). However, inter-specific competition for light and water by Pinus palustris Mill increased the root nodule biomass and N2 fixation of Morella cerifera (L.) Small (Hagan and Jose 2011).

N2 fixation rates depend largely on environmental factors. Understanding the factors controlling N2 fixation in tropical planted forests could help to improve soil preparation and weed control techniques as well as fertilization regimes in order to optimize the N inputs to the soil over the rotation cycle. Our study set out to assess the seasonal variability of %Ndfa for A. mangium trees grown in monoculture and in association with E. grandis trees. These two species are widely planted in tropical regions (FAO 2010) and mixed-species plantations with eucalypts and acacias may be an alternative to eucalypt monocultures in N-deficient soils (Bouillet et al. 2013; Forrester et al. 2006). We tested the hypotheses that (1) %Ndfa of A. mangium trees is higher in mixed-species plantations with E. grandis trees than in monospecific stands and (2) %Ndfa is higher during the rainy summer than during the dry winter both in mixed plantations and in A. mangium monoculture.

2 Material and methods

2.1 Site description

The study was carried out at the Itatinga experimental station of São Paulo University (23°02′ S, 48°38′ W). The landscape is typical of the western plateau of São Paulo state, with smoothly undulating topography. The site was on the top of a hill (slope < 3%) at an elevation of 860 m ASL. The soils were Ferralsols (FAO classification) and were typical of large areas planted with eucalypts in Brazil (Gonçalves et al. 2013). The soil texture was uniform below a depth of 1 m with clay content of 13% in the A1 layer and ranging from 20 to 25% between 1 and 3 m in depth. The main soil characteristics down to a depth of 3 m in the experiment can be found in Voigtlaender et al. (2012). The same soil type under eucalypt plantations was characterized in detail (including micro-nutrients and soil solution chemistry) down to a depth of 3 m in a separate experiment (Maquère 2008). The soil pHH20 ranged from 5.4 to 5.9 depending on the soil layer. Carbon (C) concentrations were 17.6, 6.4, 5.0, and 3.5 g kg−1 in soil layers 0–0.05, 0.05–0.15, 0.15–0.5, and 0.5–1 m, respectively, total N concentrations were 0.9, 0.3, 0.4, and 0.2 g kg−1, available phosphorus concentrations (P-resin) were 4.0, 2.5, 1.9, and 1.3 mg kg−1 and cation exchange capacities (CEC) were 1.76, 0.95, 0.75, and 0.58 cmolc kg−1 (Voigtlaender et al. 2012). Low nutrient stocks in the soil (exchangeable K+, Na+, Ca2+, Mg2+ contents < 0.2 mmolc kg−1 below a depth of 0.15 m), related to the long period of eucalypt cultivation, made the area a promising environment for studying mixed-species plantations associating NFTs and eucalypts (Laclau et al. 2008; Voigtlaender et al. 2012).

Temperature is relatively low during the dry season (from June to September). Our study was carried out from November 2011 to April 2013. During this period, the cumulative rainfall collected in an open area at 100 m from the field trial was 2237 mm and the average air temperature was 20.2 °C (Fig. 1).
Fig. 1

Rainfall (vertical bars) and mean air temperature (circle) in an open area 100 m from the field trial (a) and volumetric soil water content at a depth of 15 cm (black circle), 50 cm (gray circle), and 100 cm (triangle) in the monospecific stand (b) and the mixed-species stand (c) of Acacia mangium over the study period. Volumetric soil water content was monitored in one block of the experiment using three Campbell CS616 probes per depth. Measurements were taken every half hour and averaged over the day. Soil was15N-labeled in one block on April 15th and 16th, 2012. Leaves were sampled in June 2012, September 2012, December 2012, and March 2013 (black arrows)

2.2 Experimental design

Our experiment used three treatments and three blocks of the large-scale experiment described in Laclau et al. (2008). The treatments were 100A (A. mangium monoculture), 100E (E. grandis monoculture), and 50E:50A (a 1:1 ratio of E. grandis and A. mangium, the two species being planted alternately in the row and offset in adjacent rows). The stand density was 1111 trees ha−1 (3 m × 3 m spacing). The fertilizer doses applied at planting were 40 g P plant−1 (buried at 20 cm from the plants), as well as 9 g K plant−1, 3 g B plant−1, 6 g Fe plant−1, 3 g Zn plant−1, and 1 g Mn plant−1. No N fertilization was applied. Fertilization trials at the study site and in nearby commercial forests on the same soil type showed that, with the exception of N, the amounts of nutrients applied were non-limiting for eucalypt tree growth (Gonçalves et al. 2008; Laclau et al. 2009).

Only the boles were harvested in May 2009, and the residues were spread uniformly within each plot. Seedlings of A. mangium and E. grandis were planted in the same planting rows and the same plots on November 2009, following the same experimental design and protocol. A. mangium seeds were inoculated with Rhizobium strains (BR 3609T and BR6009 provided by EMBRAPA Agrobiologia, Seropédica-Rio de Janeiro state) selected for their high N2 fixation efficiency.

The volumetric soil water content was monitored in one block at depths of 0.15, 0.50 and 1.00 m, using 3 Campbell CS616 probes per depth in 100A and 50E:50A. Measurements were taken every half hour and averaged over the day. The volumetric soil water content in the 0–100 cm soil layer was similar in 50E:50A and 100A, on average 11.3% in autumn, 7.9% in winter, 7.8% in spring, and 11.4% in summer (Fig. 1b, c).

2.3 Tree growth, stand biomass, and N accumulation

Tree height and diameter at 1.3 m (DBH) were measured in three blocks 24, 32, 36, and 39 months after planting. For multi-stem trees, the DBH of each individual stem was measured and the individual basal area was calculated. The growth of acacia trees covering the range of diameters was also monitored monthly in one block with band dendrometers (accuracy ± 0.2 mm) in 100A (n = 15) and 50E:50A (n = 10) from September 2012 (34 months after planting) to March 2013 (41 months after planting).

The above-ground biomass was estimated in January 2013 (at 39 months) sampling 10 acacias and/or eucalypts from each of the 100A, 100E, and 50E:50A treatments (total of 40 trees). The trees were taken from all three blocks for each species and each treatment with two to four trees sampled in each block. The sampled trees were equally distributed within the range of basal areas calculated from the stand inventory (Table 1). The trees were separated into leaves, branches, stemwood, and stembark. Diameters, lengths, and weights were measured in the field. Tree foliage was collected from three sections of the crown (lower, intermediate, and upper). Sub-samples were taken from all the components, dried at 65 °C to constant weight, and ground in a Willey mill (0.8 mm mesh). The mill was carefully cleaned between each milling using a vacuum cleaner, compressed air jet and ethyl alcohol. The N concentrations of the leaves, branches, stembark, and stemwood were determined using the Kjeldahl method with wet digestion using concentrated sulfuric acid and distillation using sodium hydroxide 18 M (TE36/1and TE36/3 analyzers–Tecnal Co., Piracicaba, Brazil). Allometric models established from destructive tree sampling (Tables 2, 3) were applied to the stand inventory to assess above-ground biomass and N content of A. mangium and E. grandis trees in the monospecific stands and the mixed-species stands in three blocks.
Table 1

Ranges of basal area and height of the trees sampled for the estimation of biomass and N2 fixation in the monospecific stands of Acacia mangium and Eucalyptus grandis (100A and 100E, respectively) and the mixed-species stands (50E:50A–Acacia and 50E:50A–Eucalyptus), at 39 months of age


Trees sampled for biomass quantification

Trees sampled for N2 fixation analysis

Basal area (cm2 tree−1)

Height (m)

Basal area (cm2 tree−1)

Tree height (m)





















2.4 N2 fixation

Ammonium sulfate with 98.9 x(15N) (Coplen 2011) was applied at a rate of 0.3 kg N ha−1 on April 15th and 16th, 2012, 29 months after planting. The fertilizer was diluted in water and applied uniformly to the soil litter using a watering can. We considered that the low amount of applied N did not affect the N2 fixation of acacias (Parrotta et al. 1996; Bouillet et al. 2008). Only one block was labeled owing to operational limitations (15N fertilizer and 15N/14N analysis costs and the need to use scaffolding to sample leaves up to a height of 18 m). Tree heights and basal areas were not significantly different between the blocks (Fig. 2).
Fig. 2

Mean tree height (a) and basal area (b) of Acacia mangium and Eucalyptus grandis in the monospecific stands (100A and 100E, respectively) and the mixed stand (50E:50A–Acacia/50E:50A–Eucalyptus). Vertical bars indicate standard deviations between blocks (n = 3). No statistical differences were found between blocks (P ≥ 0.3). For a given date, different letters indicate differences between treatments (p < 0.05)

In each treatment and for each species, we selected four trees evenly distributed over the range of basal areas in April 2012 (Table 1). Leaves were sampled in June 2012 (end of autumn), September 2012 (end of winter), December 2012 (end of spring) and March 2013 (end of summer). For each tree and each sampling date, we divided the crown into three tiers of equal height and all the leaves of two pairs of opposite branches in the crown (north-south and east-west) were collected from each tier of the canopy and pooled to make a composite sample representative of the crown (35 g of leaves collected on average). The sampled leaves were then dried at 65 °C and ground. 10 mg of dry material (Barrie and Prosser, 1996) were put in tin capsules for isotopic analysis. N concentrations and x(15N) values of the leaves were determined using a Hydra 20–20 mass spectrometer coupled to an automatic N analyzer (ANCA-GSL, SERCON Co., Crewe, UK). The precision of the isotopic measurements was 0.0001 x(15N).

The percentage of N derived from N2 fixation (%Ndfa) for each acacia was calculated using the equation (Fried and Middelboe 1977):
$$ \%\mathrm{Ndfa}=\left(1-\frac{{x\mathrm{E}}_{\mathrm{Fx}}}{{x\mathrm{E}}_{\mathrm{Refx}}}\right)\times 100 $$
where xEFx was the excess atom fraction in the acacia leaves at each sampling period and xERefx was the mean value (n = 4) of xE(15N) in the eucalypt leaves sampled at the same dates in 100E treatment. At each sampling date, the excess atom fraction was calculated for each individual tree using the equation:
$$ {x}^{\mathrm{E}}=x{\left({}^{15}\mathrm{N}\right)}_{\mathrm{leaves}}\hbox{--} x{\left({}^{15}\mathrm{N}\right)}_{\mathrm{air}} $$

A previous study during the first rotation of our experiment showed that the %Ndfa values were roughly similar using the 15N labelling method sampling only the foliage or the whole tree (Bouillet et al. 2008). We considered that this equation was valid for estimating %Ndfa as the labeled stand was young and the 15N:14N ratio in the fertilizer was high (Hardarson and Danso 1993; Chalk and Ladha 1999; Bouillet et al. 2008).

2.5 Calculations and statistical analysis

Allometric equations were established for estimating the dry matter and N content of each tree component (Tables 2 and 3 in “Appendices”). The biomass and N content linear models were tested and adjusted using the packages plyr (Wickham and Francois 2015) and leaps (Lumley 2009). Differences between treatments and blocks in tree height, tree basal area, above-ground biomass, and N content were tested at each sampling date using two-way ANOVA. Differences between the DBH growth rates of acacias in 100A and in 50E:50A were tested at each sampling date using one-way ANOVA. The differences for x(15N) in the acacia and eucalypt leaves were tested over the year using ANOVA with treatment as fixed factor, sampling date as a repeated measurement factor, and interaction treatment × sampling date. Differences between the %Ndfa values of acacia trees in 100A and in 50E:50A over the year were tested using ANOVA with treatment as fixed factor, sampling dates as repeated measures, and interaction treatment × sampling date. The homogeneity of variance was tested by Levene’s test, and the normal distribution of residuals was tested using the Shapiro-Wilks test. The values were log-transformed when the variances were unequal. When ANOVA indicated that the effects were significant, the means were compared using the Tukey test. The significance level was 0.05. The statistical analyses were performed with R (R core Team 2015).

3 Results

3.1 Tree growth

Eucalypts were significantly taller than acacias regardless of the sampling date (Fig. 2a). At 24 months after planting, the height of the eucalypts was not significantly different between 100E and 50E:50A. However, from 32 months onwards, the eucalypts were significantly taller in 100E than in 50E:50A. At 24 months after planting, the acacias were significantly taller in 50E:50A than in 100A with heights of 6.0 and 5.5 m, respectively. For older trees, there was no significant difference.

At 24 months, the basal area was significantly greater for acacias than eucalypts regardless of the treatment (Fig. 2b). From 36 months onwards, the individual basal area of acacias and eucalypts was not significantly different between 100A and 100E, but acacias in 50E:50A had the lowest basal areas. At 39 months, the basal area of eucalypts was significantly higher in 50E:50A than in 100E (Fig. 2b).

The DBH growth rate of acacias was low between September and December 2012 with mean values of 0.4 cm month−1 in 100A and 0.2 cm month−1 in 50E:50A (Fig. 3) and was significantly higher in 100A than in 50E:50A from December 2012 to March 2013. The highest DBH growth rate of acacias was between January and March 2013 with mean values of 0.8 cm month−1 in 100A and 0.3 cm month−1 in 50E:50A.
Fig. 3

Mean DBH growth rate of Acacia mangium trees in monospecific stand (100A) and mixed stand (50E:50A) measured monthly with band dendrometers. Vertical bars indicate standard deviations between trees (n = 15 in 100A; n = 10 in 50E:50A). The sampling dates of acacia leaves are indicated by arrows and vertical dotted line. For a given date, different letters indicate differences between treatments (p < 0.05)

3.2 Above-ground biomass and N content

At 39 months, the above-ground biomass of eucalypts was not significantly different between 100E and 50E:50A, with mean values of 63.2 and 68.3 kg tree−1, respectively (Fig. 4a). The above-ground biomass of acacias was 43% higher in 100A than in 50E:50A, with mean values of 40.6 and 28.3 kg tree−1, respectively (Fig. 4a).
Fig. 4

Mean biomass (a) and N content (b) (± standard deviation) of above-ground compartments of Acacia mangium and Eucalyptus grandis trees in the monospecific stands (100A and 100E, respectively) and the mixed-species stand (50E:50A–Acacia and 50E:50A–Eucalyptus) at 39 months of age. No statistical differences were found between blocks (p ≥ 0.16). Different letters indicate differences between treatments (p < 0.05) in the total tree biomass and N content of the above-ground compartments. Stem biomass in 100A, 100E, 50E:50A–Acacia and 50E:50A–Eucalyptus were 19.9, 49.6, 8.0, and 22.9 Mg ha−1, respectively

At 39 months, the N content in the above-ground biomass of individual trees was not significantly different between eucalypt trees in 50E:50A (277.3 g N tree−1 on average) and acacia trees in 100A (246.4 g N tree−1) or between eucalypt trees in 100E (224.1 g N tree−1) and acacia trees in 100A. However, the above-ground N content in individual acacias was significantly lower (167.8 g N tree−1) in 50E:50A than 100A (Fig. 4b).

3.3 Seasonal variations of x(15N) and %Ndfa

The x(15N) values of the acacia and eucalyptus leaves did not change significantly throughout the year (Fig. 5). The x(15N) of the acacia leaves was significantly lower in 50E:50A than in 100A with mean values across the sampling dates of 0.3751 and 0.3816, respectively. The x(15N) of the eucalypt leaves was significantly higher in 50E:50A than in 100E with mean values across the sampling dates of 0.3958 and 0.3823, respectively. Acacia and eucalypt leaves in monospecific stands had similar x(15N) values in all seasons except in summer (March 2013) when x(15N) values were on average 0.3847 in eucalypt leaves and 0.3773 in acacia leaves.
Fig. 5

Seasonal variation of x(15N) in the leaves of Acacia mangium and Eucalyptus grandis trees in monospecific stands (100A and 100E, respectively) and mixed-species stand (50E:50A Acacia and 50E:50A–Eucalyptus). Vertical bars indicate standard deviations between trees (n = 4). Different lower case letters indicate significant differences (p < 0.05) between treatments at each sampling date

The N2 fixation rate of acacias varied significantly throughout the year with the highest %Ndfa values found in March 2013 (end of summer) (Fig. 6). The N2 fixation rate of acacias was significantly higher in 50E:50A than in 100A, and the seasonal behavior was similar in the two treatments (non-significant interaction treatment × sampling date). %Ndfa in 100A ranged from 0 to 14% in June, September, and December. Over the same period, the %Ndfa in 50E:50A ranged from 30 to 52% (Fig. 6). %Ndfa values averaged over the study period were ~ 3 times higher in 50E:50A than in 100A (Fig. 6).
Fig. 6

Seasonal variation of the percentage of N derived from fixation (%Ndfa) in Acacia mangium trees growing in monospecific stands (100A) and mixed-species stand with Eucalyptus grandis trees (50E:50A–Acacia). Vertical bars indicate standard deviations between trees (n = 4). Different letters indicate significant differences in %Ndfa (p < 0.05) between treatments at each sampling date. The %Ndfa values of acacias changed significantly throughout the year (p < 0.05). %Ndfa was taken to be 0 when the x(15N) value was higher in individual acacias than the mean x(15N) value in the eucalypts

The total N derived from N2 fixation in the above-ground biomass (i.e., leaves, branches, bark, and wood) of acacia trees sampled 39 months after planting was estimated at 35.1 kg N ha−1 in 100A and 39.1 kg N ha−1 in 50E:50A, whereas the stocking density of acacia trees was twice as high in 100A as in 50E:50A.

Statement on data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

4 Discussion

4.1 Above and below-ground growth of acacia and eucalypt trees

Tree height and above-ground biomass were higher for eucalypt trees than for acacia trees both in monospecific and mixed-species stands, as observed in the first rotation of this experiment which was set up in 2003 after 60 years of eucalypt plantation management without fertilizer amendment (Laclau et al. 2008). Climatic conditions limit A. mangium growth in the study region leading to rapid overtopping by E. grandis in mixed-species stands (Nouvellon et al. 2012). The individual basal area of eucalypt trees was higher in 50E:50A than in 100E, as a result of less competition for light by acacia trees than by eucalypt trees in the mixed stands (le Maire et al. 2013), as well as the increase in N availability for the eucalypts (Paula et al. 2015).

The similar foliar x(15N) values in 100A and 100E in June, September, and December 2012 might be explained by a high capacity of both species for taking up soil N. During the first rotation of this experiment, the fine root biomass (diameter < 1 mm) in the 0–0.5-m-soil layer was lower in 100E than in 100A (Silva et al. 2009; Laclau et al. 2013), with values of 100 g m−2 in 100E and 144 g m−2 in 100A at 5 years after planting. High fine root densities for acacias in the upper soil layer of the mixed stands (50 g m−2 compared with the 100 g m−2 for eucalypt trees in 100E with double the stocking density) strongly suggest that both species had access to the 15N applied in 50E:50A. While nitrogen fixation by A. mangium trees probably led to x(15N) values in the leaves close to that of the air (Bouillet et al. 2008) and could consequently decrease the x(15N) signature of E. grandis trees in mixed stands, foliar x(15N) values of eucalypt trees were higher in 50E:50A than in 100E at each sampling date. This pattern suggests that, for an individual E. grandis tree, a higher proportion of the 15N applied in fertilization was taken up in 50E:50A than in 100E. A reduction of competition for soil N for each E. grandis tree in 50E:50A relative to 100E might result from the N2 fixation of A. mangium trees, which decreased the demand of soil N for A. mangium trees and therefore increased the availability of 15N applied for E. grandis trees. Moreover, E. grandis trees could be more competitive than A. mangium trees in capturing the 15N applied as fertilizer, which could lead to higher accumulation rates of x(15N) in each E. grandis tree in 50E:50A than in 100E. Specific root length and specific root area were greatly enhanced for both species in mixed stands relative to the monocultures in our experiment, which probably contributed to increasing the capacity of the trees to take up soil N (Germon et al. 2017). Sap flow measurements in the same plots and stand age showed that transpiration of eucalypts was on average 20% higher in 50E:50A than in 100E (unpublished data), which suggests a higher capacity of eucalypts to take up soil resources in mixed stands than in monocultures. A glasshouse experiment showed that Acacia mangium and Eucalyptus seedlings take up the same form of mineral N (Epron et al. 2016) and the 15N natural abundance was similar in acacia and eucalyptus leaves at age 30 months in the first rotation of our experiment (Bouillet et al. 2008), which suggests that different N nutrition patterns for eucalypt and acacia were unlikely to change foliar x(15N) values after labelling. Therefore, higher foliar x(15N) values for E. grandis trees in 50E:50A than in 100E probably reflected both enhanced capture of soil resources and differences in availability of 15N in the soil. The enhanced uptake of soil N for E. grandis trees in 50E:50A relative to 100E did not increase the above-ground biomass, which results in particular from changes in C partition between above- and below-ground tree components (Nouvellon et al. 2012; Epron et al. 2013).

4.2 Factors accounting for higher N2 fixation of acacia trees in mixture than in monoculture

Two potentially interacting factors might account for higher N2 fixation of acacia trees in the mixture than in the monoculture: large differences in mineral N availability in the soil between treatments and/or higher competition with eucalypt trees in the mixture to take up soil N than between acacia trees in monoculture. Changes in soil N mineralization might contribute to explaining the temporal variability in %Ndfa observed in 100A throughout the year. %Ndfa values averaged over the year of 14% for 3-year-old acacia trees in the second rotation was lower than for the same age trees in the first rotation. A comparison of isotopic methods in another treatment at the same experimental site suggested that %Ndfa would have been > 60% in the first rotation in 100A if the 15N dilution method had been used instead of the natural abundance method (Bouillet et al. 2008). The lower %Ndfa in 100A for the second rotation than in the first rotation is consistent with a sharp increase in soil N availability. Soil N mineralization was twice as high in 100A as in 100E at the end of the first rotation (Voigtlaender et al. 2012) and input-output budgets showed that the harvest of the first rotation led to an increase in soil N stock of 460 kg N ha−1 in 100A relative to 100E (unpublished data). Soil N mineralization rates are commonly higher in Acacia monoculture and in mixed plantations than in Eucalyptus monoculture, with strong seasonal variations (Wang et al. 2013; Mo et al. 2016). A decline in %Ndfa for Leucaena leucocephala (Lam.) mixed with Eucalyptus robusta L.E. Smith from 100 to 40% between 1 and 3.5 years after planting could be a result of the incorporation into the soil of large amounts of N fixed over this period by L. leucocephala plants (Parrotta et al. 1996). A down-regulation of N2 fixation depending on soil N availability has been reported for tropical legume trees growing in natural conditions (Barron et al. 2011). An increase in soil N content was associated with a decrease in N2 fixation rates of acacias growing with Eucalyptus regnans in natural mixed-species stands (Pfautsch et al. 2009). A similar pattern was reported in a field experiment in the Ivory Coast where the %Ndfa of A. mangium trees was 2.4 times lower in the most fertile block than in the least fertile block (Galiana et al. 2002). The relatively low maximum values of %Ndfa in this experiment during the summer (59.9% in 50E:50A and 40.4% in 100A) might reflect the increase in soil N from the establishment of the experiment in 2003 (Voigtlaender et al. 2012).

The high N requirements of the eucalypts in 50E:50A led to strong competition for soil N with the acacias, which probably increased the N2 fixation rates. The mean N content in the above-ground biomass of the eucalypts was 20% higher in 50E:50A than in 100E and 11% higher than that of the acacias in 100A. The higher N accumulation in eucalypts in 50E:50A than in 100E could be explained by the higher soil N mineralization rates in 50E:50A than in 100E for the two first years after replanting with mean values of 91 and 68 kg N ha−1 year−1, respectively (Voigtlaender 2012). Soil N mineralization was 136 kg N ha−1 year−1 in 100A over the same period, which was 49% higher than in 50E:50A (Voigtlaender 2012). The competition for soil N acquisition was, therefore, probably higher in 50E:50A than in 100A for acacias, contributing to the reduction in the above-ground biomass and N content of the acacias in 50E:50A. The higher %Ndfa of acacias in 50E:50A than in 100A was, therefore, consistent with an upregulation of N2 fixation as soil N availability decreases as commonly reported for NFTs (Vitousek et al. 2002; Barron et al. 2011; Sheffer et al. 2015). This could explain why the %Ndfa of P. guachapele plants, growing as understory in E. grandis stands, was much higher than in P. guachapele monocultures (47 vs 26%) (Balieiro et al. 2008). However, a recent study showed that competition for a limited N pool is not always the sole mechanism increasing nodulation and N2 biological fixation, and that facilitative root-root interactions might also be involved. Maize root exudates increased nodulation and the expression of genes stimulating N2 fixation in faba beans (Li et al. 2016). Further studies are needed to assess whether root exudates in mixed-species forests are likely to increase nodulation and N2 fixation of leguminous tree species.

Studies have shown the importance of sharing soil N pools with similar 15N/14N ratios for the two species when estimating %Ndfa in forest ecosystems (Parrotta et al. 1996; Chalk and Ladha 1999). Using eucalypts in 100E as reference plant fulfilled this condition as the total N concentration and the x(15N) values in the upper 15 cm of soil were not statistically different at the end of the first rotation between 100A, 100E, and 50E:50A (Voigtlaender et al. 2012). Using eucalypts growing in 100E instead of 50E:50A may have also prevented an underestimation of %Ndfa owing to the direct transfer of unlabeled N of atmospheric origin from A. mangium tree to E. grandis tree as recently observed in the same experiment (Paula et al. 2015). However, using eucalypts growing in 50E:50A could have prevented an overestimation of %Ndfa if there was multidirectional N transfer between acacias and eucalypts, as has been shown for mixed herbaceous species (Carlsson and Huss-Dunell 2014). We checked that using eucalypts in 50E:50A instead of 100E as reference plant did not change the main findings of our study (seasonal changes of %Ndfa and large differences between monoculture and mixture). %Ndfa values averaged over the study period were ~ 1.5 and ~ 3 times higher in 50E:50A than in 100A, using reference plants in 50E:50A and 100E, respectively. Our results show that monitoring seasonal changes of foliar x(15N) provide more realistic estimates of annual N2 fixation rates than only one sampling made several months after 15N soil labelling.

4.3 Seasonal variation in N2 fixation by acacia trees in monospecific and mixed stands

In agreement with our second hypothesis, there were noticeable variations in %Ndfa through the year depending on the climatic conditions and the changes in N demand of the acacia trees (Fig. 6). Leaf life span in the early growth stages is around 240 days for E. grandis (Epron et al. 2012) and 270 days for A. mangium (Nouvellon et al. 2012) at our study site. As all the leaves of six branches per tree were collected at each sampling date, the average age of the sampled leaves was approx. 120–140 days for both species. Foliar x(15N) values, therefore, depended on the amounts of 15N taken up from the soil by eucalypts and acacias over the 4–5-month period before sampling. However, N from tree reserves may also be used during leaf expansion (Proe et al. 2000; Weatherall et al. 2006) and most of the 15N taken up in the soil was probably accumulated in the early phase of leaf growth (Jordan et al. 2001). Sampling all tree compartments, and not only the leaves, would have made it possible to disentangle the effects of the internal N recycling and of the seasons on the temporal change in x(15N) of acacias leaves. However, the short duration of our study (only 1 year after 15N application) and the low difference between N2 fixation rates estimated sampling only leaves or the whole trees during the first rotation of the same experiment (Bouillet et al. 2008) suggest that 15N recycling within trees was unlikely to modify the seasonal patterns of %Ndfa. The %Ndfa values of acacias in both monospecific and mixed stands were lower in the leaves sampled in September and December, which were produced during the dry season. Tree growth was low from July to December as found for the first rotation of the same experiment (Laclau et al. 2008; Bouillet et al. 2013). Tree demand for N was probably low during the periods of low growth, which might lead to low %Ndfa. Low soil water content from July to December during the experiment might also help to explain the low %Ndfa in leaves sampled in September and December in both 50E:50A and 100A. The specific activity of nodules has been reported as lower during drought for P. sativum (Prudent et al. 2016) and Casuarina equisetifolia J. R. and G. Forst (Srivastava and Abasht 1994). The %Ndfa values were highest in summer both in 100A and 50E:50A which might be a result of the high N demand to achieve high growth rates. The N2 fixation was also maxima during the hot, rainy season for Inga jinicuil G. Don (Roskoski and van Kessel 1985), Acacia spp. (Adams and Attiwill 1984) and C. equisetifolia (Srivastava and Abasht 1994).

5 Conclusion

Our study based on 15N labelling at mid rotation in Brazilian forest plantations suggests that soil N availability as well as the trees’ N demand are likely to influence N2 fixation of A. mangium. Lower %Ndfa values in the second rotation than in the first rotation for A. mangium monocultures were consistent with an increase in soil N mineralization. The competition for soil mineral N likely led to higher rates of N2 fixation by acacias growing in association with eucalypts than in monoculture. %Ndfa was low during the dry season when the A. mangium tree growth was limited by the climatic conditions. The management of A. mangium trees to improve the soil N status should take account of the variations in N2 fixation over successive rotations that are different between monocultures and mixed-species stands.



We should like to thank Rildo Moreira e Moreira (USP-Esalq), Eder Araújo da Silva (, the staff at the Itatinga experimental station, laboratory of stable isotope of CENA-USP, and the Applied Ecology Laboratory of ESALQ-USP for their technical support. We are also grateful Tony Tebby for the revision of the English.

Funding information

We should like to thank the São Paulo Research Foundation-FAPESP (grant 2011/20510-8), FAPESP Thematic Project (grant 2010/16623-9), Intens & Fix project (ANR-2010-STRA-004-03), ANAEE, and ATP Neucapalm (CIRAD).

Compliance with ethical standards

Declaration on conflicts of interest

The authors declare no conflicts of interest.


  1. Adams MA, Attwill PM (1984) Role of Acacia spp. in nutrient balance and cycling in regenerating Eucalyptus regnans F. Muell. Forests. II. Field studies of acetylene reduction. Aust J Bot 32:217–223. CrossRefGoogle Scholar
  2. Augusto L, Delerue F, Gallet-Budynek A, Achat DL (2013) Global assessment of limitation to symbiotic nitrogen fixation by phosphorus availability in terrestrial ecosystems using a meta-analysis approach. Global Biogeochem Cy 27:804–815. CrossRefGoogle Scholar
  3. Balieiro FB, Alves BJR, Pereira MG, Faria SM, Franco AA, Campello EFC (2008) Biological nitrogen fixation and nutrient release from litter of the Guachapele leguminous tree under pure and mixed plantation with Eucalyptus. Cerne 14:185–193Google Scholar
  4. Barron AR, Purves DW, Hedin LO (2011) Facultative nitrogen fixation by canopy legumes in a lowland tropical forest. Oecologia 165:511–520. CrossRefPubMedGoogle Scholar
  5. Beer J (1988) Litter production and nutrient cycling in coffee (Coffea arabica) or cacao (Theobroma cacao) plantations with shade trees. Agrofor Syst 7:103–114. CrossRefGoogle Scholar
  6. Binkley D, Giardina C (1997) Nitrogen fixation in tropical forest plantations. In: Nambiar EKS, Brown AG (eds) Management of Soil. Nutrients and Water in Tropical Plantation Forests, ACIAR Monograph, Canberra, pp 297–337Google Scholar
  7. Bouillet J-P, Laclau J-P, Gonçalves JLM, Moreira MZ, Trivelin PCO, Jourdan C, Silva EV, Piccolo MC, Tsai SM, Galiana A (2008) Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 2. Nitrogen accumulation in the stands and biological N2 fixation. Forest Ecol Manag 255:3918–3930. CrossRefGoogle Scholar
  8. Bouillet J-P, Laclau J-P, Gonçalves JLM, Voigtlaender M, Gava JL, Leite FP, Hakamada R, Mareschal L, Mabiala A, Tardy F, Levillain J, Deleporte P, Epron D, Nouvellon Y (2013) Eucalyptus and Acacia tree growth over entire rotation in single- and mixed-species plantations across five sites in Brazil and Congo. Forest Ecol Manag 301:89–101. CrossRefGoogle Scholar
  9. Carlsson G, Huss-Danell K (2014) Does nitrogen transfer between plants confound 15N-based quantifications of N2 fixation? Plant Soil 374:345–358. CrossRefGoogle Scholar
  10. Chalk PM, Ladha JK (1999) Estimation of legume symbiotic dependence: an evaluation of techniques based on 15N diluition. Soil Biol Biochem 31:1901–1917. CrossRefGoogle Scholar
  11. Coplen TB (2011) Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results. Rapid Commun Mass Spectrom 25:2538–2560. CrossRefPubMedGoogle Scholar
  12. Cramer MD, Chimpahango SBM, van Cauter A, Waldram MS, Bond WJ (2007) Grass competition induces N2 fixation in some species African Acacia. J Ecol 95:1123–1133. CrossRefGoogle Scholar
  13. Diagne N, Thioulouse J, Sanguin H, Prin Y, Krasova-Wade T, Sylla S, Galiana A, Baudoin E, Neyra M, Svistoonoff S, Lebrun M, Duponnois R (2013) Ectomycorrhizal diversity enhances growth and nitrogen fixation of Acacia mangium seedlings. Soil Biol Biochem 57:468–476. CrossRefGoogle Scholar
  14. Epron D, Laclau J-P, Almeida JCR, Gonçalves JLM, Ponton S, Sette C Jr, Delgado-Rojas J, Bouillet JP, Nouvellon Y (2012) Do changes in carbon allocation account for the growth response to potassium and sodium applications in tropical Eucalyptus plantations? Tree Physiol 32:667–679. CrossRefPubMedGoogle Scholar
  15. Epron D, Nouvellon Y, Mareschal L, Moreira RM, Koutika L-S, Geneste B, Delgado-Rojas JS, Laclau J-P, Sola G, Gonçalves JLM, Bouillet J-P (2013) Partitioning of net primary production in Eucalyptus and Acacia stands and in mixed-species plantations: a comparison in two contrasting tropical environments. Forest Ecol Manag 301:102–111. CrossRefGoogle Scholar
  16. Epron D, Koutika LS, Tchiclelle SV, Bouillet J-P, Mareschal L (2016) Uptake of soil mineral nitrogen by Acacia mangium and Eucalyptus urophylla x grandis: no difference in N form preference. J Plant Nutr Soil Sci 179:726–732. CrossRefGoogle Scholar
  17. FAO (2010) Global Forest Resources Assessment. Food and Agriculture Organization of the United Nations. Accessed 15 June 2016
  18. Forrester DI, Bauhus J, Cowie AL, Vanclay JK (2006) Mixed-species plantations of Eucalyptus with nitrogen-fixing trees: a review. Forest Ecol Manag 233:211–230. CrossRefGoogle Scholar
  19. Forrester DI, Schortemeyer M, Stock WD, Bauhus J, Khanna PK, Cowie AL (2007) Assessing nitrogen fixation in mixed- and single-species plantations of Eucalyptus globulus and Acacia mearnsii. Tree Physiol 27:1319–1328. CrossRefPubMedGoogle Scholar
  20. Fried M, Middelboe V (1977) Measurement of amount of nitrogen fixed by a legume crop. Plant Soil 47:713–715. CrossRefGoogle Scholar
  21. Galiana A, Balle P, N’Guessan Kanga A, Domenach AM (2002) Nitrogen fixation estimated by the 15N natural abundance method in Acacia mangium Willd inoculated with Bradyrhizobium sp. and grown in silvicultural conditions. Soil Biol Biochem 34:251–262. CrossRefGoogle Scholar
  22. Germon A, Guerrini IA, Bordron B, Bouillet J-P, Nouvellon Y, Gonçalves JLM, Jourdan C, Paula RR, Laclau J-P (2017) Consequences of mixing Acacia mangium and Eucalyptus grandis trees on soil exploration by fine-roots down to a depth of 17 m. Plant Soil doi:
  23. Gonçalves JLM, Stape JL, Laclau J-P, Bouillet J-P, Ranger J (2008) Assessing the effects of early management on long-term site productivity of fast-growing eucalypt plantations: the Brazilian experience. South Forests 70:105–118. CrossRefGoogle Scholar
  24. Gonçalves JLM, Alvares CA, Higa AR, Silva LD, Alfenas AC, Stahl J, Ferraz SFB, Lima WP Brancalioni PHS, Hubner A, Bouillet J-P, Laclau J-P, Nouvellon Y, Epron D (2013) Integrating genetic and silvicultural strategies to minimize abiotic and biotic constraints in Brazilian eucalypt plantations. Forest Ecol Manag 301:6–27. CrossRefGoogle Scholar
  25. Hagan DL, Jose S (2011) Interspecific competition enhances nitrogen fixation in an actinorhizal shrub. Plant Ecol 212:63–68. CrossRefGoogle Scholar
  26. Hardarson G, Danso SKA (1993) Methods for measuring biological nitrogen fixation in grain legumes. Plant Soil 152:19–23. CrossRefGoogle Scholar
  27. Isaac ME, Harmand J-M, Lesueur D, Lelon J (2011) Tree age and soil phosphorus conditions influence N2-fixation rates and soil N dynamics in natural populations of Acacia senegal. Forest Ecol Manag 261:582–588. CrossRefGoogle Scholar
  28. Jensen ES (1996) Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea-barley intercrops. Plant Soil 182:25–38. CrossRefGoogle Scholar
  29. Jordan MO, Gomez L, Mediene S (2001) Regulation of N uptake in young peach trees in relation to the management of carbon and nitrogen stores. Acta Hortic 564:63–69. CrossRefGoogle Scholar
  30. Koutika L-S, Epron D, Bouillet J-P, Mareschal L (2014) Changes in N and C concentrations, soil acidity and P availability in tropical mixed acacia and eucalypt plantations on a nutrient-poor sandy soil. Plant Soil 379:205–216. CrossRefGoogle Scholar
  31. Laclau J-P, Almeida JCR, Gonçalves JLM, Saint-André L, Ventura M, Ranger J, Moreira RM, Nouvellon Y (2009) Influence of nitrogen and potassium fertilization on leaf lifespan and allocation of above-ground growth in Eucalyptus plantations. Tree Physiol 29:111–124. CrossRefPubMedGoogle Scholar
  32. Laclau J-P, Bouillet J-P, Gonçalves JLM, Silva EV, Jourdan C, Cunha MCS, Moreira RM, Saint-André L, Maquère V, Nouvellon Y, Ranger J (2008) Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 1. Growth dynamics and aboveground net primary production. Forest Ecol Manag 255:3905–3917. CrossRefGoogle Scholar
  33. Laclau J-P, Nouvellon Y, Reine C, Gonçalves JLM, Krushe AV, Jourdan C, le Maire G, Bouillet J-P (2013) Mixing Eucalyptus and Acacia trees leads to fine root over-yielding and vertical segregation between species. Oecologia 172:903–913. CrossRefPubMedGoogle Scholar
  34. le Maire G, Nouvellon Y, Christina M, Ponzoni FJ, Gonçalves JLM, Bouillet J-P, Laclau J-P (2013) Tree and stand light use efficiencies over a full rotation of single- and mixed-species Eucalyptus grandis and Acacia mangium plantations. Forest Ecol Manag 288:31–42. CrossRefGoogle Scholar
  35. Li B, Li Y-Y, Wua H-M, Zhanga FF, Li CJ, Li X-X, Lambers H, Li L (2016) Root exudates drive interspecific facilitation by enhancing nodulation and N2 fixation. PNAS 113:6496–6501. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lumley T using Fortran code by Alan Miller (2009) leaps: regression sub set selection. R package version 2.9. Accessed 24 May 2016
  37. Maquère V (2008) Dynamics of mineral elements under a fast-growing eucalyptus plantation in Brazil. Implication for soil sustainability. Thesis, Agroparitech, ParisGoogle Scholar
  38. Mo Q, Li Z, Zhu W, Zou B, Li Y, Yu S, Ding Y, Chen Y, Li X, Wang F (2016) Reforestation in southern China: revisiting soil N mineralization and nitrification after 8 years restoration. Sci Rep 6:19770. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Nouvellon Y, Laclau J-P, Epron D, le Maire G, Bonnefond J-M, Gonçalves JLM, Bouillet J-P (2012) Production and carbon allocation in monocultures and mixed-species plantations of Eucalyptus grandis and Acacia mangium in Brazil. Tree Physiol 32:680–695. CrossRefPubMedGoogle Scholar
  40. Nygren P, Fernández M, Harmand J-M, Leblanc HA (2012) Symbiotic dinitrogen fixation by trees: an underestimated resource in agroforestry systems? Nutr Cycl Agroecosys 94:123–160. CrossRefGoogle Scholar
  41. Nygren P, Leblanc HA (2015) Dinitrogen fixation by legume shade trees and direct transfer of fixed N to associated cacao in a tropical agroforestry system. Tree Physiol 35:134–147. CrossRefPubMedGoogle Scholar
  42. Parrotta JA, Baker DD, Fried M (1996) Changes in dinitrogen fixation in maturing stands of Casuarina equisetifolia and Leucaena leucocephala. Can J For Res 26:1684–1691. CrossRefGoogle Scholar
  43. Paula RR, Bouillet J-P, Trivelin PCO, Zeller B, Gonçalves JLM, Nouvellon Y, Bouvet J-M, Plassard C, Laclau J-P (2015) Evidence of short-term belowground transfer of nitrogen from Acacia mangium to Eucalyptus grandis trees in a tropical planted forest. Soil Biol Biochem 91:99–108. CrossRefGoogle Scholar
  44. Pfautsch S, Rennenberg H, Bell TL, Adams MA (2009) Nitrogen uptake by Eucalyptus regnans and Acacia spp.-preferences, resource overlap and energetic costs. Tree Physiol 29:389–399. CrossRefPubMedGoogle Scholar
  45. Piotto D (2008) A meta-analysis comparing tree growth in monocultures and mixed plantations. Forest Ecol Manag 255:781–786. CrossRefGoogle Scholar
  46. Proe MF, Midwood AJ, Craig J (2000) Use of stable isotopes to quantify nitrogen, potassium and magnesium dynamics in young Scots pine (Pinus sylvestris). New Phytol 146:461–469. CrossRefGoogle Scholar
  47. Prudent M, Vernoud V, Girodet S, Salon C (2016) How nitrogen fixation is modulated in response to different water availability levels and during recovery: a structural and functional study at the whole plant level. Plant Soil 399:1–12. CrossRefGoogle Scholar
  48. Rachid CTCC, Balieiro FC, Peixoto RS, Pinheiro YAS, Piccolo MC, Chaer GM, Rosado AS (2013) Mixed plantations can promote microbial integration and soil nitrate increases with changes in the N cycling genes. Soil Biol Biochem 66:146–153. CrossRefGoogle Scholar
  49. Rastetter EB, Vitousek PM, Field C, Shaver GR, Herbert D, Gren GI (2001) Resource optimization and symbiotic nitrogen fixation. Ecosystems 4:369–388. CrossRefGoogle Scholar
  50. R Core Team (2015) R: a language and environment for statistical computing. R Foundation for Statistical Computing. Accessed 10 May 2016
  51. Resh SC, Binkley D, Parrotta JA (2002) Greater soil carbon sequestration under nitrogen-fixing trees compared with Eucalyptus species. Ecosystems 5:217–231. CrossRefGoogle Scholar
  52. Roskoski JP, van Kessel C (1985) Annual, seasonal and diel variation in nitrogen fixing activity by Inga jinicuil, a tropical leguminous tree. Oikos 44:306–312. CrossRefGoogle Scholar
  53. Santos FM, Balieiro FC, Ataíde DHS, Diniz AR, Chaer GM (2016) Dynamics of aboveground biomass accumulation in monospecific and mixed-species plantations of Eucalyptus and Acacia on a Brazilian sandy soil. Forest Ecol Manag 363:86–97. CrossRefGoogle Scholar
  54. Sheffer E, Batterman SA, Levin SA, Hedin LO (2015) Biome-scale nitrogen fixation strategies selected by climatic constraints on nitrogen cycle. Nature Plants 1:15182. CrossRefPubMedGoogle Scholar
  55. Silva EV, Gonçalves JLM, Coelho SRF, Moreira RM, Mello SLM, Bouillet J-P, Jourdan C, Laclau J-P (2009) Dynamics of fine root distribution after establishment of monospecific and mixed-species plantations of Eucalyptus grandis and Acacia mangium. Plant Soil 325:305–318. CrossRefGoogle Scholar
  56. Soussana J-F, Tallec T (2010) Can we understand and predict the regulation of biological N2 fixation in grassland ecosystems? Nutr Cycl Agroecosys 88:197–213. CrossRefGoogle Scholar
  57. Srivastava AK, Ambasht RS (1994) Soil moisture control of nitrogen fixation activity in dry tropical Casuarina plantation forest. J Environ Manag 42:49–54. CrossRefGoogle Scholar
  58. Vitousek PM, Cassman K, Cleveland C, Crews T, Field CB, Grimm NB, Howarth RW, Marino R, Martineli L, Rastetter EB, Sprent JI (2002) Towards an ecological understanding of biological nitrogen fixation. Biogeochemistry 57:1–45. CrossRefGoogle Scholar
  59. Voigtlaender M (2012) Produção de biomassa aérea e ciclagem de nitrogênio em consórcio de genótipos de Eucalyptus com Acacia mangium. University of São Paulo, ThesisCrossRefGoogle Scholar
  60. Voigtlaender M, Laclau J-P, Gonçalves JLM, Piccolo MC, Moreira MZ, Nouvellon Y, Ranger J, Bouillet J-P (2012) Introducing Acacia mangium trees in Eucalyptus grandis plantations: consequences for soil organic matter stocks and nitrogen mineralization. Plant Soil 352:99–111. CrossRefGoogle Scholar
  61. Wang F, Zhu W, Zou B, Neher DA, Fu S, Xia H, Li Z (2013) Seedling growth and soil nutrient availability in exotic and native tree species: implications for afforestation in southern China. Plant Soil 364:207–218. CrossRefGoogle Scholar
  62. Weatherall A, Proe MF, Craig J, Cameron A-D, Midwood AJ (2006) Internal cycling of nitrogen, potassium and magnesium in young Sitka spruce. Tree Physiol 26:673–680. CrossRefPubMedGoogle Scholar
  63. Wickham H, Francois R (2015) dplyr: a grammar of data manipulation. R package version 0.4.3. 15 May 2016
  64. Wurzburger N, Miniat CF (2014) Drought enhances symbiotic dinitrogen fixation and competitive ability of a temperate forest tree. Oecologia 174:1117–1126. CrossRefPubMedGoogle Scholar

Copyright information

© INRA and Springer-Verlag France SAS, part of Springer Nature 2018

Authors and Affiliations

  • Ranieri R. Paula
    • 1
    • 2
    Email author
  • Jean-Pierre Bouillet
    • 1
    • 3
  • José L. de M. Gonçalves
    • 1
  • Paulo C. O. Trivelin
    • 4
  • Fabiano de C. Balieiro
    • 5
  • Yann Nouvellon
    • 1
    • 3
  • Julianne de C. Oliveira
    • 1
  • José C. de Deus Júnior
    • 1
    • 6
  • Bruno Bordron
    • 1
  • Jean-Paul Laclau
    • 3
    • 6
  1. 1.USP, ESALQDepartamento de Ciências FlorestaisSão PauloBrazil
  2. 2.UFESDepartamento de Ciências Florestais e da MadeiraEspírito SantoBrazil
  3. 3.Eco&Sols, INRA, CIRAD, IRD, Montpellier SupAgroUniversity of MontpellierMontpellierFrance
  4. 4.USP, CENADivisão de Desenvolvimento de Técnicas Analíticas e NuclearesSão PauloBrazil
  5. 5.Embrapa SolosRio de JaneiroBrazil
  6. 6.UNESPDepartamento de Solos e Recursos AmbientaisSão PauloBrazil

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