Plant Soil (2007) 291:199–209 DOI 10.1007/s11104-006-9186-0

ORIGINAL PAPER

Dinitrogen-fixation by three neotropical agroforestry species under semi-controlled field conditions

Humberto A. Leblanc Æ Robert L. McGraw Æ Pekka Nygren

Received: 19 October 2006 / Accepted: 15 December 2006 / Published online: 2 February 2007 Springer Science+Business Media B.V. 2007

Abstract Cultivating dinitrogen-fixing E. poeppigiana, E. fusca, and V. guatemalensis with crops in agroforestry is a relatively were planted in the same field using the existing common N management practice in the Neotrop- cylinders. The 15N application was repeated at the ics. The objective of this study was to assess the rate of 20 kg [N] ha–1 15 days after planting and –1 N2 fixation potential of three important Neotrop- 10 kg [N] ha was added three months after ical agroforestry tree species, poeppigi- planting. Trees were harvested 9 months after ana, Erythrina fusca, and Inga edulis, under planting in both years. The 15N content of semi-controlled field conditions. The study was leaves, branches, stems, and roots was deter- conducted in the humid tropical climate of the mined by mass spectrometry. The percentage

Caribbean coastal plain of Costa Rica. In 2002, of atmospheric N fixed out of total N (%Nf) seedlings of I. edulis and Vochysia guatemalensis was calculated based on 15N atom excess in were planted in one-meter-deep open-ended leaves or total biomass. The difference between plastic cylinders buried in soil within hedgerows the two calculation methods was insignificant of the same species. Overall tree spacing was for all species. Sixty percent of I. edulis trees

1 · 4 m to simulate a typical alley-cropping fixed N2;%Nf was 57% for the N2-fixing 15 design. The N was applied as (NH4)2SO4 at trees. Biomass production and N yield were 15 10% N atom excess 15 days after planting at the similar in N2-fixing and non-N2-fixing I. edulis. rate of 20 kg [N] ha–1. In 2003, seedlings of No obvious cause was found for why not all

I. edulis trees fixed N2. All E. poeppigiana and E. fusca trees fixed N ;%N was ca. 59% and & 2 f H. A. Leblanc ( ) 64%, respectively. These data were extrapolated EARTH University, 4442-1000 San Jose, Costa Rica to typical agroforestry systems using published e-mail: [email protected] data on N recycling by the studied species. Inga edulis may recycle ca. 100 kg ha–1 a–1 of N fixed R. L. McGraw from atmosphere to soil if only 60% of trees fix Department of Agronomy, University of Missouri, –1 –1 201 Waters Hall, Columbia, N2, E. poeppigiana 60–160 kg ha a , and E. MO 65211, USA fusca ca.80kgha–1 a–1.

P. Nygren Keywords Erythrina fusca Erythrina Department of Forest Ecology, University of Helsinki, P.O. Box 27, poeppigiana Inga edulis Humid tropics 00014 Helsinki, Finland 15N dilution method

123 200 Soil (2007) 291:199–209

Introduction common shade tree for perennial crops in Central and South America (Sa´nchez et al. 1993; Santana While nitrogen is seldom the limiting nutrient in and Rosand 1985). Green prunings of Erythrina natural tropical ecosystems (Martinelli et al. spp. are rich in N and form an excellent green 1999; Vitousek et al. 2002), serious N deficits manure. The amount of N supplied in prunings develop in many tropical agroecosystems because (Ramı´rez 1993; Russo and Budowski 1986)or of heavy N export in crop harvest (Lal 2004). litterfall (Escalante et al. 1984; Heuveldop et al. Cultivating dinitrogen-fixing legume trees in bio- 1988; Santana and Rosand 1985)ofErythrina spp. logical interaction with crops in tropical agrofor- is greater than the N removal in the harvest of estry systems is a relatively common N coffee berries or cacao ( L.) management practice in the Neotropics, espe- pods. cially in production of perennial plantation crops Although both Inga and Erythrina spp. are like coffee and cacao (Beer et al. 1998). Legume widely used in agroforestry practices and their trees are also used for fodder production in positive effect on soil organic matter and N association with tropical grasses, green manure reserves have been shown in various studies (Beer production for annual crops in alley cropping, et al. 1998; Haggar et al. 1993; Hands 1998; living hedgerow fences and fence posts, support Leblanc and McGraw 2006), early attempts to for shade-tolerant climber crops, and timber quantify N2 fixation in agroforestry resulted in production. The beneficial effects of legume trees low estimates of annual N2 fixation rates for both on tropical soils include increased soil and Erythrina (Fassbender 1987; Lindblad and Russo microbial C and N content in comparison to 1986) and Inga spp. (Roskoski 1982; Roskoski annual cropping (Mazzarino et al. 1993; Sierra and Van Kessel 1985). These data were based on et al. 2002), and long-term accumulation of soil closed-chamber acetylene reduction assays, which organic matter and N (Dulormne et al. 2003; significantly underestimates the N2 fixation rate Haggar et al. 1993). Trees of legume genera (Minchin et al. 1983, 1986), or N balance com- Erythrina L. (: Papilionoideae) and Inga parisons of ecologically mismatching agroforestry Mill. (Mimosoideae) are common woody compo- systems. Although these early estimates were nents in agroforestry practices throughout Neo- inadequate, they started a vigorous debate on tropics. whether the true value of legume trees in agro-

The genus Inga is composed of ca. 300 forestry is N2 fixation or beneficial modifications Neotropical tree species, many of which have of microenvironment that could be associated multiple uses on local farms (Lawrence et al. with any tree species (Beer 1988; Beer et al. 1998; 1995). Fruits of 50 Inga species are eaten and Inga Budowski et al. 1984; Kass et al. 1997). fuelwood is used domestically and marketed in Recent studies based on either natural abun- many Latin American countries. At least 33 Inga dance of the stable 15N isotope (Ladha et al. 1993; species are used as shade trees for perennial crops Nygren et al. 2000; Peoples et al. 1996) or label- in agroforestry. Inga edulis Mart. may be the most ling with 15N (Liyanage et al. 1994; Peoples et al. economically important species of this genus 1996; Sta˚hl et al. 2002, 2005) have shown consid- because it grows well on acidic soils, produces erably higher N2 fixation potential of several tree edible fruits, and is a common shade and fuel- species used in agroforestry. Several Inga spp. wood tree on Latin American farms (Hands 1998; showed N2 fixation potential according to a Le´on 1998). screening based on 15N natural abundance in a The genus Erythrina is pantropical with 70 rain forest (Roggy et al. 1999) and a freshwater species in the Neotropics (Neill 1993). Erythrina swamp forest (Koponen et al. 2003) in French poeppigiana (Walpers) O.F. Cook is the most Guiana. Percentage of N derived from atmo- popular shade tree for coffee (Coffea arabica L.) sphere out of total N in I. edulis determined by in Costa Rica (Ramı´rez 1993). Erythrina fusca 15N dilution varied from 10 to 52% in a pot Lour. (syn. E. glauca), which is the only naturally experiment, depending on growth medium pantropical species of the genus (Neill 1993), is a (Leblanc et al. 2005). Thus, it appears that N2

123 Plant Soil (2007) 291:199–209 201

fixation by important Erythrina and Inga spp. temperature is 25.1C. The soil is classified as a should be reassessed using N isotopic methods. typic dystropept. The main problem of the 15N dilution method under field conditions is achieving uniform label- N2 fixation under semi-controlled conditions ling of the soil both spatially and temporarily, and selecting a suitable non-N2-fixing reference spe- of I. edulis, E. poeppigiana,andE. fusca cies (Chalk and Ladha 1999). This problem is were collected from trees at the EARTH Uni- especially important in the case of trees that may versity campus. The fresh pulp of the seeds of I. scavenge nutrients from a horizontally extensive edulis was removed by immersing them in water area and from several soil layers. Many agrofor- at ambient temperature for 1 h. After pulp estry trees have extensive root systems and it may removal, the seeds were inoculated with brady- be impossible to find areas without the presence rhizobia by immersing for 12 h in water contain- of legume roots within tens of meters from the ing macerated root nodules taken from mature I. nearest tree (Hauser 1993; Hauser and Gichuru edulis trees together with soil taken from beneath 1994; Sierra and Nygren 2006). This means that the same trees. No pulp removal was necessary 15N label should also be applied in large areas for seeds of the Erythrina spp. but otherwise they outside the study plots in order to be sure that N2- were treated and inoculated following the same fixing and reference trees take up only procedure. 15N-labelled N from the soil. An alternative to The field experiment on I. edulis was con- overcome this problem is to apply 15N within an ducted in 2002–2003 and on Erythrina spp. in area restricted by root barriers (Sta˚hl et al. 2002, 2003–2004. Seeds of I. edulis and the non-N2- 2005). The use of barriers reduces loss of the 15N fixing control, Vochysia guatemalensis Donn. Sm, label in horizontal flow of soil solutes, growing of were planted in plastic bags and the seedlings tree roots out of the labelled area, and mixing of were grown in a greenhouse for 2 months. After root systems of N2-fixing and reference species. that, the most homogenous seedlings were se- The objective of this study was to assess the N2 lected for planting in the field in order to fixation potential by three important Neotropical minimize non-treatment variation between trees. agroforestry tree species, Erythrina poeppigiana, Seedlings of I. edulis and V. guatemalensis were E. fusca,andInga edulis, under semi-controlled planted in November 2002 in the field in rows 1 m field conditions. The growth conditions are called apart and 4 m between rows to simulate a typical semi-controlled because the trees were exposed alley-cropping spacing in five blocks. One-meter- to the natural humid tropical climate and grown deep open-ended plastic cylinders (diameter in chemically natural soil, however root growth 0.58 m) were buried within the tree rows. The was restricted to the 15N labelled soil by root cylinders were filled with 25 cm of gravel in the barriers around each individual tree. The results bottom to restrict taproot growth. The top 75 cm are discussed in an agroforestry context. of the cylinders was filled with the soil removed when the cylinders were buried. The soil layers were returned in the original order. Materials and methods The 15N was applied 15 days after transplant- ing into the cylinders. The dosage was –1 Study site 20 kg [N] ha applied as (NH4)2SO4 at 10% 15 N atom excess. The (NH4)2SO4 was diluted The study was conducted at the EARTH with distilled water and applied at a rate of 200 ml University research farm located in the Carib- m–2 to every cylinder. Fifteen liters of water were bean coastal plain of Costa Rica (1010¢ N, added to uniformly distribute the 15N in the soil. 8337¢ W, 95 m a.s.l.). The climatic zone is pre- Trees were harvested after 9 months. Tree montane wet forest basal belt transition (Bolan˜ os biomass was separated into stems and branches, and Watson 1993). Average annual rainfall is which were pooled for 15N enrichment analyses, 3,464 mm (evenly distributed) and annual mean leaves, and roots. All harvested material was

123 202 Plant Soil (2007) 291:199–209 weighed for fresh biomass. Sub-samples of 200 g abundance of 15N in the atmosphere, or 0.3663% were dried at 60C for 72 h for leaves, and 120 h (Mariotti 1984). The total amount of fixed N in for branches, stems and roots for determining the plant tissue was calculated by multiplying the moisture content. All biomass data is reported on total N concentration in different organs by the dry matter basis. respective %Nf value. Seedlings of E. poeppigiana, E. fusca,andV. guatemalensis were planted in November 2003 in Statistical analysis the same field setting as I. edulis and V. guatemalensis the year before. The 15N application was repeated The I. edulis experiment was analyzed as 2 · 3 to the planting cylinders by applying 20 kg [N] ha–1 factorial (tree species · organ) in five blocks for 15 days after planting and 10 kg [N] ha–1 three biomass, total N concentration, and %15N atom months after planting. Application was done as excess. The Erythrina spp. experiment was ana- 15 (NH4)2SO4 at 10% N atom excess. Trees were lyzed as 3 · 3 factorial (tree species · organ) in harvested at 9 months and biomass was determined five blocks for these variables. The percentage of following the same procedure as for I. edulis. fixed N out of total N was calculated using the The 15N content of the samples was determined V. guatemalensis growing together with either by mass spectrometry. Samples were ground to a I. edulis or the Erythrina spp. as non-N2 fixing particle size smaller than 250 lm starting each year reference, respectively. Differences in the %Nf from the lower 15N concentration ( assumed were analyzed as a completely randomized de- 15 to be N2-fixers) to the higher N concentration sign. The statistical analyses were conducted (non-N2-fixing control) treatment to avoid con- using GLM procedure in SAS Statistical Software tamination. Eight milligrams of the ground mate- v. 8.02 (SAS Institute, Inc. 1999). The means are rial were weighed and placed into tin capsules of presented with the standard errors. 5 mm diam. · 8 mm height. The tin capsules were crimped into small pellets. The pellets were placed into a well plate, covered with parafilm, and closed. Results The 15N content in the plant material was deter- mined by an Isotope ratio mass spectrometer Extractable acidity in all blocks was moderate (Europa Integra) at the Stable Isotope Facility of (Table 1) and typical to natural soils in the study the University of California-Davis. area (Bertsch 1987). Differences between blocks were unlikely to have ecological significance, although they were statistically significant. Soil 15 N calculations of the study site was acidic with relatively high iron and manganese concentrations. Because of The percentage of fixed N out of total N in plant the high acidity, these metals may be available for tissue (%Nf) was calculated (Fried and Mid- plants in soil solution. Concentration of Mn delboe 1977): differed significantly between blocks, and reached  15 potentially harmful levels in blocks 1 and 2. % Nfix Nf ¼ 1 100 ð1Þ Manganese concentration in block 3 did not differ %15N ref significantly from block 1 because of high within- 15 15 block variation in the latter. Soil organic matter where % Nfix and % Nref are the % atom 15 content was significantly higher in block 5 than in excess of N in the N2-fixing and non-fixing reference plant, respectively. The % atom excess blocks 2 and 3. Within-block variation in organic of 15N was calculated: matter content was high in blocks 1 and 4. Concentrations of N, P, and Fe did not vary 15 15 15 % Ni ¼ % Nsample % Nair ð2Þ significantly between blocks (P = 0.20, 0.36, and 0.70, respectively, in ANOVA). 15 15 where % Nsample is the percentage of N out of Biomass production of the reference species, 15 total N in the sample, and % Nair is the natural V. guatemalensis, was similar in both years (Fig. 1

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Table 1 Soil characteristics of each block used in the N2 fixation study under semi-controlled field conditions Block pH Extractable acidity Organic matter N P Fe Mn cmol kg–1 gkg–1 mg kg–1

1 4.70 0.66 d ± 0.15 61.1 ab ± 29.6 2.07 ± 0.06 10.4 ± 3.26 682 ± 56.1 147 ab ± 50.7 2 4.62 1.27 ab ± 0.35 43.4 b ± 12.3 2.00 ± 0.20 7.73 ± 1.46 713 ± 67.2 155 a ± 43.6 3 4.56 1.44 a ± 0.19 46.7 b ± 13.6 2.43 ± 0.76 9.53 ± 2.34 733 ± 51.5 82.3 bc ± 8.14 4 4.61 1.02 bc ± 0.12 51.6 ab ± 28.2 3.53 ± 1.57 11.4 ± 4.71 672 ± 84.0 60.7 c ± 16.6 5 4.68 0.93 cd ± 0.06 94.7 a ± 13.0 3.57 ± 0.90 13.5 ± 1.93 633 ± 123 46.3 c ± 18.9 Soil pH in water is median of three samples, other figures are mean ± standard deviation. Means within a column followed by same letter do not differ significantly (Duncan’s multiple range test at 5%)

and Table 2). No significant differences between ratio (Table 2). Thus, it may be assumed that years were observed in any biomass component differences in biomass production of the legume (Student’s t-test), total biomass, or root:shoot trees I. edulis, E. poeppigiana,andE. fusca reflect species differences and not a year effect (Fig. 1,

600 Table 2). I. edulis fix I. edulis non-fix Six out of the ten I. edulis trees fixed N2 as 15 500 V. guatemalensis measured by the N isotope dilution technique (Fig. 2). Trees that did not fix N2 were planted in ) 1 - 400 blocks 2 and 4. Both N2-fixing and non-N2-fixing

ree trees were nodulated; the nodule biomass was

s 300

sgt 10.3 ± 2.8 and 8.8 ± 1.9 g per tree (mean ± stan- m dard deviation) in N -fixing and non-N -fixing I.

y( 2 2 200 Dr a edulis, respectively. The N2-fixing and non-N2- I. edulis 100 fixing trees were entered as pseudospe- cies to ANOVAs for testing the differences in

0 biomass production, total tissue N concentration, leaf stem roots 15N atom excess, and N yield in year 2003. Block,

600 species, organ, and species · organ interaction E. poeppigiana (P < 0.0001 for all) had significant effect on total E. fusca 500 V. guatemalensis N concentration in tree tissues (Fig. 2). Block and species significantly affected the 15N atom excess ) 1 - 400 in 2003 (P < 0.0001) with N2-fixing I. edulis

t 15 ree having the significantly lowest N enrichment.

s 300 sg Vochysia guatemalensis also had significantly a 15 m lower N enrichment than non-N -fixing I. edulis

y( 2 200 Dr (Duncan’s MRT at 5%). In 2004, the 15N atom excess was about ten 100 times greater than in 2003 (Figs. 2 and 3). Species,

0 organ, and species · organ interaction leaf stem roots (P < 0.0001) had a significant effect on total N Organ concentration in tree tissues in 2004 (Fig. 3), with Fig. 1 Biomass production by organ in 9 months by Inga the two Erythrina spp. having higher N concen- edulis and Vochysia guatemalensis in 2003 (top), and tration than V. guatemalensis. Block (P = 0.0063), Erythrina poeppigiana, E. fusca, and V. guatemalensis in species (P < 0.0001) and species · organ interac- 2004 (bottom) under semi-controlled conditions, in the 15 humid Caribbean lowlands of Costa Rica. The error bars tion (P = 0.0074) had significant effect on N indicate standard error of mean atom excess in 2004, with the two Erythrina spp.

123 204 Plant Soil (2007) 291:199–209

Table 2 Total biomass and root:shoot ratio (mean ± SEM) of the test trees after 9 months growth under semi-controlled humid tropical conditions Tree species Year n Total biomass g Root:shoot ratio

Vochysia guatemalensis 2003 9 427 ± 84.2 b 0.18 ± 0.01 b Inga edulis, non-N2-fixing 2003 4 1,348 ± 87.4 a 0.29 ± 0.03 a Inga edulis,N2-fixing 2003 6 1,047 ± 141 a 0.28 ± 0.02 a Vochysia guatemalensis 2004 7 558 ± 90.4 ab 0.20 ± 0.02 b Erythrina poeppigiana 2004 7 453 ± 113 b 0.38 ± 0.06 a Erythrina fusca 2004 9 853 ± 162 a 0.32 ± 0.03 ab The means followed by same letter within a column and year (2003 or 2004) do not differ significantly according to Duncan’s multiple range test at 5%. The differences between years for the reference species V. guatemalensis were not significant according to Student’s t-test at 5%

40 40 D 35 D 35 [M]) [M]) 1 1 - -

30 30

gN]g 25 gN]g 25

n n om[ 20 om[ 20 n n erati( 15 erati( 15 n n ot 10 ot 10 c c

lc 5 lc 5 Tota N Tota N 0 0 leaf stem roots leaf stem roots

0.09 0.9

0.08 0.8

0.07 0.7

0.06 s 0.6 s s e c c

xs 0.05

x 0.5 e

Ne Ne

5 0.04

5 0.4 1 1 % 0.03 % 0.3

0.02 0.2

0.01 0.1

0.00 0.0 leaf stem roots leaf stem roots Organ Organ I. edulis fix I. edulis non-fix V. guatemalensis E. poeppigiana E. fusca V. guatemalensis

15 Fig. 2 Total N concentration (top) and % N atom excess Fig. 3 Total N concentration (top) and %15N atom excess (bottom) by organ in N2-fixing Inga edulis, non-N2-fixing (bottom) by organ in Erythrina poeppigiana, E. fusca, and I. edulis, and non-N -fixing reference tree Vochysia 2 non-N2-fixing reference tree Vochysia guatemalensis under guatemalensis under semi-controlled conditions, in the semi-controlled conditions, in the humid Caribbean low- humid Caribbean lowlands of Costa Rica. The error bars lands of Costa Rica. The error bars indicate standard error indicate standard error of mean of mean having lower 15N enrichment than V. guatemal- The percentage of N fixed from atmosphere ensis. Differences between the Erythrina spp. out of total N was calculated based on 15N atom were not significant (Duncan’s MRT at 5%). excess in leaves or total biomass (Fig. 4). The

123 Plant Soil (2007) 291:199–209 205

80 24

70 21 e

h 60

pe 18 ) 1 50 - 15 amos r e re

ot 40

12 d d l erm 30 egt

i( 9 Ny f

o 20 6 %Nfixf 10 3 0 leaves total biomass 0 Calculation basis I. edulis fix I. edulis non-fix V. guatemalensis

I. edulis E. poeppigiana E. fusca 16

Fig. 4 Percentage of N fixed from atmosphere out of total 14 N in leaves and total biomass of N2-fixing Inga edulis, 12

Erythrina poeppigiana, and E. fusca under semi-controlled ) 1 - 10 conditions, in the humid Caribbean lowlands of Costa e

Rica. The error bars indicate standard error of mean re

8 d l egt i(

6 difference between the two calculation methods Ny was not significant for any of the species (Stu- 4 dent’s t-test at 5%). The leaf-based percentage of 2 fixed N in the N -fixing I. edulis trees was ca.57 2 0 %. All E. poeppigiana and E. fusca trees fixed N2 E. poeppigiana E. fusca V. guatemalensis and the leaf-based percentages of fixed N were ca. Tree species 59 and 64%, respectively. Soil Atmosphere

The higher N concentration in the N2-fixing Fig. 5 Total N yield from soil and atmosphere in 9 months compared to the non-N2-fixing I. edulis trees by N2-fixing Inga edulis, non-N2-fixing I. edulis, and non- compensated for smaller biomass production in N2-fixing reference tree Vochysia guatemalensis in 2003 the former resulting in almost equal total N yield (top); and Erythrina poeppigiana, E. fusca, and (Fig. 5). Vochysia guatemalensis had significantly V. guatemalensis in 2004 (bottom) under semi-controlled conditions, in the humid Caribbean lowlands of Costa lower total N yield than N -fixing or non-N -fixing 2 2 Rica. The error bars indicate standard error of mean I. edulis in 2003, and E. fusca in 2004. The difference in total N yield was not significant between V. guatemalensis and E. poeppigiana in likely to have little biological importance. The

2004 (Duncan’s MRT at 5%). When the N2-fixing most important difference was observed in Mn trees were compared (Duncan’s MRT at 5%), I. concentration with potentially toxic levels (Izag- edulis accumulated significantly more N from uirre Mayoral and Sinclair 2005) in blocks 1 and fixation (12.1 g [N] tree–1) than E. poeppigiana 2. However, within-block variation was quite (5.9 g [N] tree–1). Erythrina fusca was intermedi- high, which may mean that high Mn concentra- –1 ate with 10.0 g [N] tree from N2 fixation. tions were shared by parts of these two blocks. If that was the case, general whole-block high concentrations may not have existed. Discussion We tested the reference species V. guatemal- ensis prior to this experiment and determined that Soils were quite uniform in all blocks of the field it performed well in both soil and sand media site with no significant differences in N, P, and Fe (Leblanc et al. 2005). The good contrast in 15N concentrations (Table 1). Some variation was enrichment between the reference and the three observed in soil organic matter content but it is legume species in this study indicates that it

123 206 Plant Soil (2007) 291:199–209 performs well also under field conditions. Both thyrsus Meisn 50–70% and Sesbania sesban (L.) V. guatemalensis and the legume species are Fawc. & Rendle 70–90% using both 15N natural native to acidic soils of humid tropics, and they abundance and 15N dilution methods (Sta˚hl et al. may have similar soil N uptake patterns. 2002). Both Erythrina spp. also fall within this 15 The N atom excess was almost ten times range. The differences in N2 fixation percentage higher in 2004 than in 2003. The phenomenon was were small between the three species studied consistent for all biomass components, and also here, except that four out of ten I. edulis did not included the non-N2-fixing V. guatemalensis ref- fix N2 at all. erence. There are two possible reasons for this We are not sure why only six out of the ten I. 15 observation. First, the vertical distribution of N edulis trees fixed N2. In a separate study (Leblanc within the cylinders may have become more et al. 2005), all I. edulis trees were inoculated with uniform over the course of the year since appli- pure rhizobia cultures and grown under con- cation, while during the first experiment only the trolled conditions in a glasshouse. All of the trees topsoil was labeled. Thus, only the upper part of fixed N2 in the glasshouse study. In this study, the the tree root systems might have absorbed 15N trees were inoculated with a mix of native enriched N from soil in 2003. Second, during the rhizobia from macerated nodules collected from 2004 experiment an additional 10 kg [N] ha–1 as several I. edulis trees. It could be that some of the 15 (NH4)2SO4 at 10% N atom excess were applied strains were not effective in fixing N2. Sylvester- three months after the first application at planting Bradley et al. (1991) evaluated the effectiveness –1 15 (20 kg [N] ha applied as (NH4)2SO4 at 10% N of 21 rhizobia strains isolated from Pueraria atom excess). The additional 15N enriched N was phaseoloides (Roxb.) Benth. Fourteen of the 21 added at that time because we were not sure if the strains evaluated were ineffective. Purcino et al. lack of N2 fixation of four I. edulis trees was due (2000) tested the effectiveness of 230 authenti- to a low 15N trace. However, because of the cated Bradyrhizobium strains isolated mainly consistency of these between-year differences in from the centers of diversity of the genus Arachis. all species and biomass components, they did not All of the strains produced nodules in Arachis affect the estimates of N2 fixation percentage. pintoi Krapov. & W.C. Gregory, but only 48 were The similar 15N atom excess among I. edulis, E. classified as effective. The rest of the strains were poeppigiana and E. fusca organs has practical classified as either lowly effective or ineffective. application. For instance the leaves, which are The second possible explanation for the lack of easily harvested and prepared for analysis com- N2 fixation could be that I. edulis trees vary in pared to other tree organs, can be used to their capacity to fix N2 and to establish an estimate the percentage of N2 fixation for the appropriate symbiosis with rhizobia. Plant culti- whole tree (Sanginga et al. 1995). Using leaves var-rhizobia interactions for N2 fixation have can save time and requires fewer samples, thus been demonstrated in several species such as reducing the cost of mass spectrometry analysis. soybean (Glycine max (L.) Merr.) (Aibaidao

However, if the total N accumulation from N2- et al. 1999; Sanginga et al. 2000), peanut (A. fixation is to be calculated, the whole tree must be hypogaea L.) (Arrendell et al. 1986), Medicago harvested to determine dry matter yield. rigidula L. (Brockwell et al. 1988) and A. pintoi

The I. edulis trees that fixed N2 in this study, (Purcino et al. 2000). averaged 57% fixed N2 out of total N in a tree, The horizontal spread and vertical distribution which is within the range reported for other of roots is a big concern in 15N field experiments species used in tropical agroforestry: Gliricidia (Baker et al. 1990; Sta˚hl et al. 2002). The I. edulis sepium 50–51% using 15N dilution method (Kadi- trees were grown in 1-m deep plastic cylinders ata et al. 1998; Hairiah et al. 2000); Acacia with 25 cm of compacted gravel in the bottom to mangium Willd 61% using 15N natural abundance restrict root growth. In addition, a 50-cm deep method (Galiana et al. 1998); Leucaena leuco- ditch surrounded each block. Kadiata et al. cephala (Lam) De Wit 73% using 15N dilution (1998) successfully used a similar methodology method (Kadiata et al. 1998); Calliandra calo- in an experiment to determine biological N2

123 Plant Soil (2007) 291:199–209 207

fixation of G. sepium and L. leucocephala. Soil Extrapolation of our results to field conditions characteristics were similar for all treatments is difficult because foliage pruning causes turn-

(Table 1). The non-N2-fixing trees grew in blocks over of N2-fixing nodules of E. poeppigiana 2 and 4. Block 2 had high soil Mn concentration, (Nygren and Ramı´rez 1995). It can be estimated which may reduce or impede nodulation (Izagu- from Nygren and Ramı´rez (1995) that about 1/3 irre Mayoral and Sinclair 2005). However, Mn is of foliar N is accumulated before N2-fixing unlikely to play any impeding role in block 4. symbiosis is reestablished following foliage prun-

Alumiun toxicity to tree roots or bradyrhizobia ing of E. poeppigiana. Assuming N2 fixation of was unlikely in the site because the extractable 59% (Fig. 4) for 2/3 of above estimates on N acidity was moderate in all blocks. All trees were recycling in Costa Rican field sites results in 55 traced the same day with the same uniformly and 60 kg [N] ha–1 a–1 of fixed N in litter and distributed 15N solution. pruning residues, respectively, of the cacao plan- In our study, I. edulis was grown for 9 months tation (Beer et al. 1990), 60–160 kg [N] ha–1 a–1 at a planting density of 2,500 trees ha–1. If 60% of in the alley cropping (Sa´nchez 1989), and 70– –1 –1 the trees fixed N2, as occurred in the trees growing 90 kg [N] ha a in the coffee plantation (Russo – in cylinders, the total N2 fixed would be 16 kg ha and Budowski 1986). All these estimates are 1 . This does not appear to be much N. However, higher than early N2 fixation estimates of the trees were only grown in pots for 9 months 60 kg [N] ha–1 a–1 by E. poeppigiana (Fassbender Mature trees should accumulate more N. In 1987). The only data available on E. fusca are another experiment (Leblanc and McGraw from a cacao plantation in Brazil without pruning 2006), green mulch (leaf and twig only) produc- (Santana and Rosand 1985). The annual N tion was measured in a six-month regrowth period recycling in leaf litter was 124 kg [N] ha–1 y–1, by 3-year-old trees planted at a density of 5,000 which would mean 80 kg [N] ha–1 a–1 of N fixed trees ha–1 in an alley-cropping experiment. The from atmosphere if the proportion of fixed N was average green mulch produced was 5 Mg ha–1, 64% (Fig. 4). containing 145 kg [N] ha–1. Assuming that 60% Acknowledgements This research was supported by the of these trees fixed N2 and 57% of the N in the I. edulis leaves came from N fixation, as occurred Missouri Agricultural Experiment Station and the 2 EARTH University Research committee. in our study, 50 kg [N] ha–1 of fixed N would have been recycled to soil in one pruning cycle, or –1 –1 100 kg ha a . References Erythrina poeppigiana is used as a shade tree for coffee and cacao. Beer et al. (1990) estimated Abaidoo RC, Dashiell KE, Sanginga N, Keyser HH, that E. poeppigiana shade trees produced Singleton PW (1999) Timecourse of Dinitrogen fixa- –1 –1 –1 –1 tion of promiscuous soybeans cultivars measured by 4.62 Mg ha y leaf litter and 3.73 Mg ha y the isotope dilution method. Biol fert soils 30(3):187– pruning residues in a cacao plantation on an 192 acidic soil under humid tropical conditions in Arrendell S, Wynne JC, Elkan GH, Schneeweis TJ (1986) Costa Rica. Assuming that leaf litter contained Bidirectional selection for nitrogen activity and shoot dry weight among late generation progenies of Vir- 3% of N and pruning residues 4%—a conserva- ginia x Spanish peanut cross. Peanut Sci 13(2):86–89 tive estimate for their study area (Nygren and Baker DD, Wheeler RA, Fried M (1990) Estimation of Ramı´rez 1995;Pe´rez Castellon 1990;Sa´nchez biological dinitrogen fixation by 15N-dilution in tree 1989)—this would result in the recycling of 139 plantations. 1. Sampling strategies and first harvest –1 –1 results for three legumes species. p 24–28. In Fourth and 149 kg [N] ha a to soil in litter and meeting African association for biological nitrogen pruning residues, respectively. Under the same fixation. International Institute of Tropical agricul- conditions, 173–237 kg [N] ha–1 a–1 of N was ture. Ibadan, Nigeria recycled to soil in pruning residues of E. poepp- Beer J (1988) Litter production and nutrient cycling in coffee (Coffea arabica) and cacao (Theobroma cacao) igiana in a coffee plantation (Russo and Budow- –1 –1 plantations with shade trees. Agrofor Syst 7:103–114 ski 1986) and 144–420 kg ha a in an Beer J, Bonnemann A, Cha´vez W, Fassbender HW, experimental alley cropping (Sa´nchez 1989). Imbach AC, Martel I (1990) Modelling agroforestry

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