15N natural abundance as a tool for assessing N2-fixation of herbaceous, shrub and tree legumes in improved fallows

Soil Biology & Biochemistry 34 (2002) 1059±1071

15

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N natural abundance as a tool for assessing N2-®xation of herbaceous, shrub and tree legumes in improved fallows S.M. Gathumbi a,b,1, G. Cadisch b,*, K.E. Giller b,c,2 a

Kenya Forestry Research Institute, P.O. Box 20412, Nairobi, Kenya Department of Agricultural Sciences, Imperial College at Wye, University of London, Wye, Ashford, Kent TN25 5AH, UK c Department of Soil Science and Agricultural Engineering, University of Zimbabwe, Box MP 167, Mount Pleasant, Harare, Zimbabwe b

Received 12 July 2001; received in revised form 24 January 2002; accepted 8 February 2002

Abstract Short-term legume±cereal rotation systems (referred to as improved fallows) with N2 ®xing leguminous species are being actively promoted to improve soil fertility in fallowed ®elds of smallholder farms in many parts of the tropics. Few estimates of N2-®xation in deep-rooted woody fallow species are available due to methodological dif®culties. We evaluated and developed the natural d 15N abundance method for assessing N2-®xation in herbaceous and woody legumes on a Kandiudal®c Eutrudox in western Kenya by (i) assessing isotopic discrimination during N2-®xation and translocation, (ii) measuring variability of 15N with depth, (iii) comparing with an independent method (ureide assay) and (iv) using several non-®xing reference plants. Most tested tree/shrub legumes showed no 15N discrimination during N2-®xation (i.e. whole plant d 15N was close to 0½). Signi®cant 15N isotopic discrimination occurred during translocation of ®xed N, which resulted in 15N depletion in shoots (up to 21.76½ in Sesbania sesban) compared with roots and nodules which were 15N enriched. Soils were highly enriched in 15N (8.2±10.8½) with little variation with depth to 2 m. d 15N signatures of plant available N measured using non-®xing reference plants were lower than those of total soil N. d 15N of the non-®xing reference species maize, Lantana camara and Tithonia diversifolia varied by 2.0½ and resulted in corresponding variation of N2-®xation estimates for respective species. 15N based estimates of N2-®xation of pigeonpea and siratro were linearly related with those obtained using the ureide method (R2 ˆ 0:80; slope ˆ 0:82) and con®rmed the utility of the 15N natural abundance method. Field observations showed that under non-PK limiting growth conditions, the proportion of N2 ®xed ranged 75±83, 63±74, 55±67, 46±59, 36±54, 35±50, and 36±51% for Crotalaria grahamiana, Tephrosia vogelii, pigeonpea (Cajanus cajan), S. sesban, Calliandra calothyrsus, siratro (Macroptilium atropurpureum) and groundnut (Arachis hypogaea). This resulted in average amounts of N2 ®xed of 142, 100, 91, 52, 24, 64 and 8 kg N ha 21, respectively, 9 months after planting. The amount of soil derived N ranged between 31 and 57 kg N ha 21 in woody species. The net N balance of woody fallows (after adjusting for N export in wood) was highest in Crotalaria due to high N2-®xation and small amount of N exported in wood. Overall, partial N balances indicated that additional N derived from N2-®xation constituted a major component of recyclable N of the system. We conclude that, in soil with suf®cient and relatively uniform background 15N abundance and using appropriate, or a range of, non-®xing reference plants, the natural d 15N abundance method is a useful tool for estimating the amount of N derived from N2®xation by ®eld grown herbaceous and woody legumes. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: d 15N signatures; Reference plants; Soil depth; Mineralizable N; Improved fallows; N balance; Western Kenya

1. Introduction Legumes in agricultural systems can contribute substantially to the overall N economy of the system by sourcing * Corresponding author. Tel.: 144-20-7594-2614; fax: 144-12-33813140. E-mail address: [email protected] (G. Cadisch). 1 Present address. MacArthur Agro-Ecology Research Centre, 300 Buck Island Ranch Road, Lake Placid, FL 33852, USA. 2 Present address: Plant Production Systems, Department of Plant Sciences, Wageningen University, P.O. Box 430, 6700 AK Wageningen, The Netherlands.

atmospheric N through symbiotic N2-®xation and through subsoil N retrieval. This is especially so in legume-crop rotations in sequential cropping systems comprising fastgrowing fallow species that are capable of accumulating large amounts of foliage rich in N (Gathumbi et al., 2002). In addition, decomposing roots and nodules can release appreciable amounts of nitrogen (Peoples and Herridge, 1990). Thus the ability to ®x N2 should be one of the criteria for selecting legumes for use in short duration fallow systems aimed at replenishing soil fertility in intensively cultivated agricultural land. However, few estimates of N2-®xation in woody fallows exist, at least in part, due to

0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(02)00038-X

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the lack of adequate methodology for measuring N2-®xation with deep-rooted trees and shrubs. Recent studies with fallows conducted in east and southern Africa have mainly focused on their potential to utilize soil nutrient resources by evaluating depletion patterns of soil mineral N to depths of up to 4 m (Hartemink et al., 1996; Kwesiga and Coe, 1994; Mekonnen et al., 1997). However, the methods employed can only provide qualitative information rather than a quantitative estimate of the N sourced from mineral N in the soil. Reliable N2-®xation estimates for these legumes would also facilitate a better quanti®cation of soil N resources accessed by the different legumes. Various methods have been developed to measure N2®xation. The choice of a particular method depends on the type and site of the experiment, the available resources and the species and/or system in question (Chalk, 1985; Giller, 2001; Shearer and Kohl, 1986; Witty, 1983). 15N isotope techniques ( 15N dilution and natural 15N abundance methods) have been widely used to obtain time-averaged estimates of the %N derived from N2-®xation during the growing period of crop or pasture legumes but these methods encounter greater problems when used with deep-rooted shrubs and trees (Boddey et al., 2000; Hairiah et al., 2000), particularly in undisturbed natural ecosystems (Handley and Scrimgeour, 1997). The application of 15N to the surface or topsoil leads to an uneven and shallow enrichment with depth (Cadisch et al., 2000) which makes its use problematic with deep-rooted trees. Thus recent attempts have focused more on the use of the natural 15N enrichment of soil as a tool to measure N2-®xation. The natural 15N abundance technique is based on the same principle as the 15 N isotope dilution technique but the small differences that occur between the atmospheric nitrogen and the natural 15N enrichments of soils due to isotopic discrimination processes during N transformations are utilized to estimate N2-®xation by legumes (Peoples et al., 1997; Shearer and Kohl, 1986). The accuracy of the estimates of N2-®xation obtained using this technique is in¯uenced by the degree and uniformity of the 15N abundance in the soil where the crop is growing. Unkovich et al. (1994) suggested a minimal natural 15N enrichment of 2½ d 15N units. Shearer and Kohl (1986) recommended a minimum soil enrichment of 5±7½ for reliable use of the natural 15N abundance method which appears to be more adequate given the potential problems with spatial variability and isotopic discrimination (Handley and Scrimgeour, 1997). The d 15N values obtained from plant materials growing on a soil may differ from the total soil 15N signature as a result of isotopic fractionation and discrimination in both soil N transformations and plant uptake (Kohl and Shearer, 1980; Shearer and Kohl, 1986). As a result of such isotopic fractionation processes and changing history of land use, soil organic matter may not be uniformly labelled with 15N and hence the plant available 15 N may have a different signature to that of the total soil N (Cadisch et al., 2000). Studies conducted in agroforestry

systems have shown that the d 15N of plant available mineral N is apparently stable during the cropping season (Herridge et al., 1990). Rapid changes in the d 15N of the soil mineral N have been observed at some sites, particularly in tilled cropped systems (Ladha et al., 1993) where the choice of the reference plant for a particular site becomes critical if the 15N natural abundance method is to be used. The natural 15N enrichment of the plant available soil N pool may vary with depth (Ledgard et al., 1984), which could be problematic when assessing N2-®xation in deep rooting trees. Many tree and shrub legumes used in improved fallow systems can form deep rooting systems and source considerable amounts of N from depth (Mekonnen et al., 1997). Under ®eld conditions, it is dif®cult to identify a reference crop that matches the rooting pattern and N sourcing ability from the different N pools with that of the N2 ®xing plants. Pate et al. (1994) reported that the enrichment of d 15N differed considerably in reference plants growing at the same site. This could have resulted from variations in N uptake patterns, variation of 15N with depth (Ledgard et al., 1984) or discrimination during NH41 or NO32 uptake (Mariotti et al., 1980). While the use of nonnodulating varieties of crop legumes has reduced problems of matching reference plants, there are currently no nonnodulating accessions for any N2-®xing legume trees. It has been recommended that at least two reference crops should be grown alongside N2-®xing test legumes to obtain a range of d 15N values which would encompass the appropriate soil 15N signature for the legume (Boddey et al., 2000). There is evidence that isotopic discrimination occurs during the process of N2-®xation and particularly during the assimilation and transport of the resulting N. This leads to variations in the natural 15N abundance in different plant parts (Yoneyama et al., 1986; Cadisch et al., 1993) and is in¯uenced by plant water and nutrient availability (Ledgard and Peoples, 1988) as well as the degree of infection and the type of rhizobia strain involved (Cadisch et al., 1993; Steele et al., 1983). Thus the accuracy of estimates of N2-®xation obtained using the natural 15N abundance technique can be enhanced by the determination of isotopic fractionation. A careful assessment of the method in each system and site is therefore essential to avoid misinterpretation of results. To assess the viability of a particular method it should be tested against an independent alternative reliable method. The ureide method has been successfully used to obtain instantaneous estimates of N2-®xation by legumes which transport the products of N2-®xation mainly in the form of ureides and to evaluate isotope methods (Herridge et al., 1996; Peoples et al., 1989b). However, only a few leguminous trees are ureide producers and these belong to the legume tribes Phaseoleae and Desmodieae (Giller, 2001) and therefore the method has limited applications. Here we included the ureide-transporting legumes pigeonpea and siratro to compare measurements of N2-®xation

S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

against the estimates obtained by the 15N natural abundance method. We evaluated the applicability of the natural 15N abundance method in quantifying the amount of N derived from N2®xation by several legume species which are currently being tested in western Kenya for their use as fallow species. In particular we: (1) determined the isotopic fractionation that occurred during N2-®xation; (2) determined the variation in the plant available 15N pool with depth; (3) compared estimates by the natural 15N abundance with the values derived by the ureide method and (4) estimated N2-®xation by several improved fallow legume species under ®eld conditions. 2. Materials and methods 2.1. Experiment 1: 15N discrimination during N2-®xation An experiment was set up in the greenhouse at Imperial College at Wye to test 15N discrimination during N2-®xation by seven legumes: Sesbania sesban (L.) Merr. from Yala, Kenya, Tephrosia vogelii Hook F. from Kenya, T. vogelii from Malawi, Crotalaria grahamiana Wight and Arn. from Mararano, Madagascar, Crotalaria juncea L., Lablab purpurens L., pigeonpea (Cajanus cajan (L.) Millsp.) cv. ICP13211 from ICRISAT, Hyderabad, India and Mucuna pruriens (L.) DC. A complete randomized design (CRD) was adopted for this experiment replicated four times. To allow sequential subsampling, six plants of each species were planted in each pot containing 2 kg washed (N-free) silica sand media. The plants were irrigated once or twice daily (100±200 ml, depending on demand) with a N free nutrient solution containing 1.5 mM MgSO4´7H20, 0.5 mM Na2HPO4, 3 mM K2SO4, 2.5 mM CaSO4´2H20, and micro-nutrients (mg ml 21: 0.0177 Zn, 0.05 Mo, 0.064 Cu, 0.55 Mn, 0.55 B) and NaFe EDTA (Hewitt, 1966). A soil inoculant was prepared from a Kenyan soil (see Section 2.4) and a soil from Chitedze, Malawi (Sakala et al., 2000) and applied to the species collected from Kenya and Malawi, respectively. The inoculum was prepared by adding 250 ml of water to 5 g of soil and shaking for 30 min and left to settle before applying at a rate of 5 ml pot21, twice after the plants germinated. To correct for the amount of 15N contained in the seed (and any contaminants in the system), the ®rst subsampling of plants was done after germination and before nodulation occurred and a weighted mean correction procedure was applied. Subsequent harvests were done at monthly intervals. Plants were separated into shoots, roots and nodules, dried at 50 8C for 72 h, ®nely ground in a piston ball mill and analyzed for %N and d 15N using a 20-20 stable isotope mass spectrometer (PDZ formerly Europa Scienti®c, Crewe, UK) coupled to a CN auto-analyzer. 15

2.2. Experiment 2: N isotopic signatures of plant available N with soil depth To evaluate the variability of plant available soil 15N with

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depth, an experiment was conducted in Maseno, western Kenya between September 1998 and February 1999. Pregerminated seeds of Senna spectabilis (DC.) H.S. Irwin and Barneby, Tithonia diversifolia (Hemsl.) A. Gray and maize (Zea mays L.) were sown in pots ®lled with 3 kg of soil taken from 0±0.20, 0.50±1.00, 1.00±1.50 and 1.50± 2.00 m soil pro®le layers of a Kandiudal®c Eutrudox (see Section 2.4). Each pot was planted with four plants, which were later thinned to two. Pots were maintained close to ®eld capacity. The choice of the species was made based on the fact that Senna was a non-nodulating legume tree species, Tithonia a non-leguminous shrub and maize a reference plant commonly available. The experiment was laid out in a randomized complete block design with four replicates. The plants were harvested 60 days after planting, oven dried at 50 8C for 72 h before ®ne grinding using a piston ball mill and analysed for d 15N and %N as above. 2.3. Experiment 3: relationship between relative ureide abundance and N2-®xation To assess N2-®xation estimates obtained by the natural N abundance method with an independent method their relationship with ureide indices was established. The ®rst part of this experiment was conducted in the greenhouse at Maseno, western Kenya between November 1998 and March 1999 in order to evaluate the applicability of the ureide method for the ureide producing legumes pigeonpea (C. cajan (L.) Millsp.) ICP13211 from ICRISAT, Hyderabad, India and siratro (Macroptilium atropurpureum (DC.) Urb. accession GBK12102 from ILRI, Addis Ababa, Ethiopia). Seven pre-germinated seeds of pigeonpea, siratro or the non-®xing reference legume Senna were planted in each pot containing 3 kg washed river sand and later thinned to ®ve plants per pot. Legumes were inoculated with a soil inoculum as described in Section 2.2. Throughout the growth period, the plants were supplied with a modi®ed Hewitt's nutrient solution (Hewitt, 1966) supplemented with increasing levels of N (0, 12.5, 25, 50, 100 mg N pot 21 week 21) in form of K 15NO3 at 1.49 at.% 15N. A completely randomized design replicated four times was used. Plants were harvested after 4 months and separated into roots, nodules, leaves, stems 1 petioles, litter, oven dried at 50 8C, ground using a ball piston mill and analyzed for %N and 15N as described earlier. The %N derived from N2 ®xation was calculated as " # at:% 15 N excesslegume %N derived from N2 ˆ 1 2 100 at:% 15 N excesssolution 15

…1† 15

15

where at.% N excess is at.% N 2 0.3663. For determination of nitrate and ureides 0.5 g of ground stems 1 petioles were extracted in 20 ml hot water for 3 min and then ®ltered gravimetrically through Whatman ®lter paper No. 5 into 25 ml volumetric ¯asks. After cooling

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at room temperature, the suspension was made up to 25 ml with distilled water. The total concentration of ureides (allantoin and allantoic acid) was determined in 0.2 ml aliquots of the hot water tissue extracts using the procedure of Peoples et al. (1989a). Plant tissue nitrate concentration was determined in 0.05 ml aliquots of the hot water extracts by the salicylic method (Cataldo et al., 1975). The relative ureide abundance (RUA) in the tissue extract was calculated using the formula of Herridge (1984) as expressed below   4 £ ureides RUA…%† ˆ 100 …2† 4 £ ureides 1 nitrates where the concentration of the ureides and nitrates in the stem tissue extracts are both expressed in mmol. The relationship between RUA and N2-®xation estimates by the 15N dilution method was then established. The second part of the experiment involved ®eld sampling of pigeonpea and siratro plant material in a ®eld experiment (Gathumbi et al., 2002) to allow comparison of N2-®xation estimates by the natural 15N abundance with the independent ureide method. The two legume species were established as single species or mixed (with S. sesban) species fallows through direct sowing within maize rows (relay cropping) after the ®rst maize weeding operation in June 1998 at a row spacing of 0.75 m £ 0.75 m. After harvesting the maize in September 1998, the plots were weeded and the legumes were left to grow and accumulate more biomass throughout the short rains cropping season, which runs from October±March of the next year. Eight and nine months after fallow establishment plant samples were taken from all the species in sole and mixed fallows. For each species young shoots (leaves and branches ,5 mm thick) were destructively sampled from the main leader branches and then bulked. The samples were then oven dried at 50 8C for 72 h before ®ne grinding using a piston ball mill and analyzed for d 15N as described earlier. The %N derived from N2-®xation was calculated according to Shearer and Kohl (1986) using natural fallow, maize and Tithonia as reference plants (averaged). For ureides, nitrates and RUA evaluation, separate samples were taken from the same plants and separated into leaves, petioles and twigs. The petioles and twigs were then bulked and dried at 50 8C for 72 h before ®ne grinding to pass through a 1 mm sieve and analyzed for ureides, nitrates, RUA as described earlier. 2.4. Experiment 4: estimation of N2-®xation using the natural d 15N abundance under ®eld conditions This experiment was conducted on two farmer's ®elds in western Kenya (0806 0 N, 34834 0 E) (Gathumbi et al., 2002). The area is at an altitude of about 1330 m above sea level. Soils are highly weathered and are generally classi®ed as very ®ne, kaolinitic, isohyperthermic Kandiudal®c Eutrudox. The rainfall in the study area is distributed in two crop growing seasons per year with an annual mean of 1800 mm. The long rains cropping season extends from

March to August and the short rains season runs from September to January. The initial soil physical and chemical characteristics were: pH (H20) of 5.60 (in a 1:1 soil/H20 suspension); 14.0 g kg 21 organic C (by wet oxidation with heated acidi®ed dichromate followed by colorimetric determination of Cr 31 (Anderson and Ingram, 1993)); 1.3 mg kg 21 extractable P and 0.31 cmolc kg 21 exchangeable K (by extraction with 0.5 M NaHCO3 1 0.01 M ethylenediaminetetraacetic acid, pH 8.5) exchangeable Ca 5.4 cmolc kg 21, Mg 1.70 cmolc kg 21 and exchangeable acidity 0.45 cmolc kg 21 (by 1 M KCl extraction). Soil texture was sand 27%, clay 52%; silt 21% and a bulk density of 1.3 g cm 23. Nursery raised legume seedlings were transplanted to the experimental plots (6.0 £ 5.25 m 2) on 20 October 1997. Plant spacing was 0.75 £ 0.75 m 2 for all woody legumes. Under-storey species siratro and groundnuts were planted at a spacing of 0.375 £ 0.75 m 2 and 0.20 £ 0.375 m 2, respectively. During planting, all plots received 100 kg ha 21 of each P and K. Plant samples were taken for analysis to estimate the proportion of plant N derived from ®xation by the legume species which included S. sesban (L.) Merr. from Yala, Kenya, pigeonpea (C. cajan (L.) Millsp.) ICP13211 from ICRISAT, T. vogelii Hook F. from Kenya, C. grahamiana Wight and Arn. from Mararano, Madagascar, siratro (M. atropurpureum (DC.) Urb. accession GBK12102 from ILRI, Addis Ababa, Ethiopia), Calliandra calothyrsus Messn. from Kakamega, Kenya and groundnut (Arachis hypogaea L.) in January 1998. Tithonia, maize, natural weeds (natural fallow plot) and Lantana camara L. from plot borders) were used as non-®xing reference plants. Newly developed leaves and young (succulent with diameter ,5 mm thick) and old (diameter .5 mm thick) shoot materials were taken separately, placed in paper bags and oven dried at 50 8C for 72 h. The samples were then ®nely ground to pass through 1 mm sieve and then analyzed for %N and d 15N as described earlier. The %N derived from N2-®xation was calculated as %N derived from N2 -fixation " 15 # d Nreference crop 2 d15 NLegume ˆ 100 d15 Nreference crop 2 B

…3†

where B is a measure of isotopic fractionation during N2®xation (see Section 2.1) and the reference crop is a non-N2®xing plant. 3. Results 3.1. Experiment 1: 15N discrimination during active N2®xation Seed derived d 15N values determined in 21 d old nonnodulated plants varied signi®cantly (21.13 to 2.63½)

0.003** 0.040**

0.045 0.039 0.035 0.034 nd 0.045 0.040 0.031

0.007 b

1.75 1.57 1.55 1.56 nd 1.27 1.56 1.17

0.227 b

0.27*

1.21**

20.14 0.17 20.31 20.14 1.25 20.60 0.05 0.08 0.105 0.163 0.205 0.185 0.096 0.169 0.087 0.065

N yield (g plant )

Biomass accumulation 2 months after planting was signi®cantly higher …P , 0:05† for maize for all soil depths compared with both Senna and Tithonia (Fig. 1). Yields of maize and Tithonia declined signi®cantly with increasing soil depth. The d 15N signatures of all plant species tested were signi®cantly lower than that of the total soil N in which they were growing (Table 3). Shoot materials of maize and Senna were not signi®cantly affected by the soil depth while Tithonia grown in soils obtained from 1 to 1.5 m depth had a lower d 15N signature than when grown in topsoil. Shoot d 15N values of maize were signi®cantly lower than those of either Senna or Tithonia except for the 1±1.5 m depth treatment.

21

DM (g plant )

20.27 20.21 20.60 20.25 nd 21.04 20.30 20.27

3.88 4.95 6.63 5.60 3.68 5.73 3.45 2.37

d 15N a (½) N yield (g plant 21) DM (g plant 21)

d N (½)

90 days

a 15

3.2. Experiment 2: 15N isotopic signatures of plant available N with soil depth

0.60***

d N (½) N yield (g plant )

2.63 0.79 20.42 0.07 1.64 21.13 0.55 20.03

21

60 days

15

3.3. Experiment 3: relationship between relative ureide abundance and N2-®xation

Weighted mean values uncorrected for initial seed 15N. not signi®cant. a

0.001 b 0.028 b SED

b

0.003 0.004 0.002 0.003 0.003 0.003 0.002 0.003 0.15 0.14 0.11 0.15 0.11 0.09 0.15 0.14 Pigeonpea C. grahamiana C. juncea L. purpurens M. pruriens S. sesban T. vogelii (Kenya) T. vogelii (Malawi)

DM (g plant )

1063

between species (Table 1). The harvest at 60 d revealed that plants were nodulated and actively ®xing N2. N yield was similar for all the plants analyzed and the d 15N had declined in most species compared with the ®rst harvest. At 90 d after planting, plants were much larger and signi®cant differences in biomass production between species were observed. The largest N yield of 0.21 g plant 21 was obtained in C. juncea and the smallest (0.07±0.09 g plant 21) in the two Tephrosia varieties. Uncorrected whole plant weighted d 15N values ranged between 20.60 and 1.25½. At the third harvest, most plants had depleted shoot d 15N values as opposed to 15 N enriched roots and nodules (Table 2). Nodules from all species were highly enriched (4±6½) in 15N. Whole plant weighted d 15N values corrected for initial seed 15N were in most cases close to zero. However, Sesbania was 15N depleted while Mucuna was signi®cantly enriched compared with atmospheric N2.

21 21

21 days Species

Table 1 Whole plant biomass yield, N accumulation and d 15N signature of eight legume species grown in pure sand and irrigated with a N-free nutrient solution under greenhouse conditions (*P , 0.05; **P , 0.01; ***P , 0.001; nd ˆ not determined; DM ˆ dry matter; SED ˆ standard error of the difference between treatment means)

S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

Shoot biomass and N yield increased with increasing amounts of NO3 ±N supply for all species (Table 4). There was no clear relationship between NO3 ±N supply and nodule weight for pigeonpea but nodule weight of siratro was observed to have an inverse linear relationship with NO3 ±N supply …R2 ˆ 0:91†: For both pigeonpea and siratro, the amount of N derived from N2-®xation initially increased with increasing NO3 ±N but the proportion of N obtained through ®xation decreased as expected. Similarly, plant tissue ureide concentration and the derived relative ureide indices were found to decrease with increasing NO3 ±N supply. The RUAs were observed to be linearly related with the %N derived from ®xation …R2 ˆ 0:78 2 0:87†: NO3 ±N concentrations in plant tissues on the other hand were not affected by the amounts of fertilizer supplied although the NO3 ±N concentration was relatively higher for pigeonpea than siratro. Senna had very small shoot biomass and N yield compared with the two legumes, which can be explained by the fact that it had only one

0.29**

20.22 0.17 20.31 20.15 1.26 20.59 0.03 0.08 5.35 6.10 5.18 4.46 3.66 5.89 5.35 6.20

nd 0.33*** a

d 15N for shoot and whole plant corrected for the initial 15N content in seed.

nd 0.53*** nd SED

10 5 4 9 13 13 10 11 0.26 0.03 0.23 0.08 1.64 1.07 0.02 0.06 14 13 15 10 14 15 15 13 21.06 20.13 20.57 20.82 0.91 21.76 20.66 20.74 76 82 81 81 73 72 75 76

Proportion of N in plant part (%)

1.05**

d 15N a (½) d N (½) d N (½) Proportion of N in plant part (%)

Proportion of N in plant part (%)

Fig. 1. Biomass accumulation (mg pot 21) of maize, Senna and Tithonia plants grown in soils taken from different pro®le depths 2 months after planting.

Pigeonpea C. grahamiana C. juncea L. purpurens M. pruriens S. sesban T. vogelii (Kenya) T. vogelii (Malawi)

Species

Shoots

15

a

d N (½)

Roots

15

Nodules

15

Whole plant

S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071 Table 2 Allocation of N in shoots, roots and nodules and the corresponding d 15N signatures of improved fallow species grown in N-free nutrient solution under greenhouse conditions at 90 d after planting (**P , 0.01; ***P , 0.001; nd ˆ not determined; SED ˆ standard error of the difference between treatment means)

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source of N supply and is also a slow growing species at seedling stage. Concentrations of NO3 ±N in plant petioles obtained from the ®eld did not differ signi®cantly …P . 0:05† between pigeonpea and siratro in sole and in mixed species fallows at both 8 and 9 months after planting (Table 5). Ureide±N was slightly higher for siratro than pigeonpea for both sampling times. The RUA was generally less in pigeonpea than in siratro for both sole and mixed fallows. From the relationship between RUA and the 15N based N2-®xation estimates established in the pot experiment corresponding ureide based ®xation estimates were obtained. Estimations of the proportion of N derived from N2-®xation compared reasonably well between the ureide and the natural 15 N abundance method (Fig. 2) although the data were somewhat clustered. The ureide method gave slightly larger estimates of N2-®xation than the isotope dilution method. 3.4. Experiment 4: estimation of N2-®xation using the natural d 15N abundance under ®eld conditions Initial soil samples taken from the study sites indicated that the d 15N of the total soil N ranged between 8.2 and Table 3 Effect of soil depth on the d 15N of shoots of maize, Senna and Tithonia (*P , 0.05; **P , 0.01; ***P , 0.001; SED ˆ standard error of the difference between treatment means) Depth (m)

0±0.20 0.50±1.00 1.00±1.50 1.50±2.00

Soil

8.7 9.8 9.8 9.0

SED a

not signi®cant.

d 15N (½)

SED

Maize

Senna

Tithonia

4.59 5.18 5.29 4.41

5.87 6.57 6.00 5.86

7.12 6.54 5.77 6.30

0.43 a

0.25 a

0.40*

0.45** 0.32** 0.35 a 0.25***

Table 4 Effect of nitrate supply on shoot dry matter (DM) yield, total N, nodule biomass, N derived from N2-®xation and concentration of N solutes in tissue extracts of pigeonpea and siratro under greenhouse conditions (*P , 0.05; SED ˆ standard error of the difference between treatment means; RUA ˆ relative ureide abundance) Shoot yield (g plant 21)

Total N (mg plant 21)

Nodule DM (mg plant 21)

(%N ®xed a (%)

N2 ®xed a (mg plant 21)

Ureides (mmol ml 21)

NO3 ±N (mmol ml 21)

RUA

Pigeonpea 0 mg N 12.5 mg N 25 mg N 50 mg N 100 mg N

1.8 2.4 2.5 2.6 2.9

29.4 44.6 43.8 52.6 65.5

35 61 62 59 58

100 96 92 86 77

29 43 40 45 50

0.55 0.44 0.27 0.23 0.05

0.38 0.37 0.40 0.35 0.37

85 82 69 71 32

SED

0.22*

0.06*

0.03 b

Siratro 0 mg N 12.5 mg N 25 mg N 50 mg N 100 mg N

0.6 0.9 1.1 1.1 1.3

0.19 0.16 0.13 0.10 0.10

0.28 0.26 0.27 0.24 0.24

SED

0.06*

0.02*

0.03 b

S. spectabilis 0 mg N 12.5 mg N 25 mg N 50 mg N 100 mg N

0.05 0.13 0.16 0.30 0.53

SED

0.031*

6.29*

18.3 25.1 36.1 36.6 41.9 3.93*

0.8 1.9 3.0 6.2 14.0

23 b

64 45 44 38 13 14*

1.03*

100 95 91 84 69 1.11*

5.38*

18 24 33 31 29 2.81*

8.5*

72 70 64 63 61 2.5*

S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

NO3 ±N supply

0.001*

a 15 b

N dilution method. not signi®cant.

1065

1066

S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

Table 5 Plant tissue NO3 ±N and ureide±N concentration and the calculated RUA for pigeonpea and siratro grown alone and in mixtures with Sesbania. RUA was obtained by analyzing ureide concentration in plant petioles sampled 8 and 9 months after fallow establishment (CV ˆ coef®cient of variation; RUA ˆ relative ureide abundance; means in a column followed by the same letter are not signi®cantly different by Tukey test …P . 0:05†) Species

Eight months after planting 21

Nine months after planting 21

NO3 ±N (mmol ml )

Ureide±N (mmol ml )

RUA

NO3 ±N (mmol ml 21)

Ureide±N (mmol ml 21)

RUA

Pigeonpea with Sesbania Pigeonpea Siratro with Sesbania Siratro

0.223a 0.179a 0.220a 0.270a

0.030b 0.021b 0.152a 0.120a

36bc 33c 72a 63ab

0.237a 0.228a 0.248a 0.468a

0.072b 0.043b 0.219a 0.176a

52ab 44a 75b 55ab

CV (%)

20.2

44.2

24.9

57.9

59.2

26.1

10.8½ (Table 6). d 15N values showed little variation with soil pro®le depth apart from the topsoil which was least enriched. The d 15N of non-N2-®xing controls ranged from 4.22 to 6.21½ for Lantana and Tithonia, respectively (Table 7). For the calculation of N2-®xation, B values (Eq. (3)) for the different species were obtained from the shoot d 15N of plants grown in the greenhouse experiment (Table 2), except for siratro, groundnuts and Calliandra for which B values were obtained from literature. All legumes had signi®cantly lower d 15N values than the non-®xing controls. Calliandra had the highest d 15N signature as compared with the other legume species. Differences in young and older shoot materials were smaller than 0.5½ except for C. grahamiana and Calliandra. Because of the uncertainty of which reference crop was most appropriate, the estimates for the %N derived from N2-®xation for each species were calculated using the d 15N values of the four reference crops and hence a range of estimates was obtained. We believe the correct estimate for the %N derived from N2-

®xation for each species falls within the respective range. Calliandra, siratro and groundnuts were observed to ®x similar proportions (35±54%) of their N. C. grahamiana had the largest %N from N2-®xation (75±83%) (Table 7). Apart from Calliandra, the legume trees appeared to obtain a higher proportion of their N from the atmosphere compared with the herbaceous legumes siratro and g roundnuts. The estimated net N contribution to the different systems by these legumes was calculated using the average of the estimated %N from N2-®xation (Table 7) combined with respective fallow N yield data obtained from Gathumbi et al. (2002). Total ®xed N2 contained in the above-ground biomass was highest for C. grahamiana with 142 kg N ha 21 during the 9 month fallow period. Tephrosia and pigeonpea ®xed nearly 100 kg N ha 21 while Calliandra and groundnuts ®xed only 24 and 8 kg N ha 21, respectively (Table 8). Siratro was observed to obtain the highest amount of soil N (81 kg ha 21) which was about 56% of its total above-ground N yield. All the other woody species acquired amounts of soil N ranging between 31 and 57 kg N ha 21. Net N contributions from above-ground biomass after Table 6 Initial d 15N and %N measured in soils taken from different depths for the two ®eld sites at Nyabeda in western Kenya

Fig. 2. Relationship between the estimates of N2-®xation obtained using the natural d 15N abundance method and those obtained while using the ureide method for ®eld grown ureide-transporting legume species.

Site

Depth (m)

d 15N (½)

%N

1 (Dickson Odhiambo)

0±0.20 0.20±0.40 0.40±0.60 0.60±0.80 0.80±1.00 1.00±1.50 1.50±2.00

8.8 10.4 10.8 10.3 9.9 10.3 10.2

0.16 0.11 0.07 0.04 0.03 0.03 0.02

2 (James Ndindi)

0±0.20 0.20±0.40 0.40±0.60 0.60±0.80 0.80±1.00 1.00±1.50 1.50±2.00

8.2 9.6 10.2 10.3 10.0 9.2 9.3

0.13 0.10 0.08 0.06 0.05 0.04 0.03

S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

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Table 7 Estimates of %N derived from N2-®xation in young and old shoot materials by the different legume species under ®eld conditions (CV ˆ coef®cient of variation; SED ˆ standard error of the difference between treatment means; *P , 0.05, ***P , 0.001) B-value a

Legume species

Pigeonpea C. grahamiana S. sesban T. vogelii C. calothyrsus Siratro Groundnut

21.06 20.13 21.76 20.66 20.50 c 22.34 d 22.60 e

SED CV (%)

Shoot material d 15N (½)

N2-®xation estimate (%)

Old

Average

Range

62 79 53 68 46 43 44

(55±67) b (75±83) (46±59) (63±74) (36±54) (35±50) (36±51)

1.36 0.57 1.62 0.94 1.39 2.14 1.74

1.22 1.31 1.30 1.38 3.67 1.67 1.74

0.53***

0.43***

35.8

Non-N2 ®xing controls: Digitaria scalarum (couch grass) L. camara Maize T. diversifolia

4.22 5.05 6.21

SED

0.38*

a c d e

6.5***

25.0

16.2

5.26

CV (%) b

Young

10.3

d Value obtained from the same plant grown on N-free sand-nutrient solution mixture and hence relaying solely on N from atmospheric N2-®xation. Range obtained by taking the lowest and the highest N2-®xation estimates of a speci®c species as derived from the four non-N2 ®xing reference crops. From Cadisch (1999) personal communication. From Yoneyama et al. (1986). From Cadisch et al. (2000).

fallow ranged between 46 and 149 kg ha 21 for woody legumes (Table 8). All N contained in above-ground foliage biomass for siratro (145 kg ha 21) is potentially recycled to crops since this species has no woody component. Total amount of N exported from the fallow in wood ranged between 28 and 38 kg ha 21 for all other species excluding the poor yielding Calliandra which contained only 9 kg N ha 21 in wood.

4. Discussion 4.1. 15N natural abundance as a tool for estimating N2®xation of woody fallow species Estimates of N2-®xation using the natural 15N abundance method revealed that considerable amounts of N can be ®xed by most of the fallow legumes under investigation in

Table 8 Net N contribution of 9 month-old monoculture legume fallows to the overall soil N economy (***P ˆ 0.001; SED ˆ standard error of the difference between treatment means; CV ˆ coef®cient of variation; nd ˆ not determined) Fallow species

C. grahamiana T. vogelii Pigeonpea S. sesban Siratro C. calothyrsus Groundnut

Above-ground biomass N a

177 150 148 100 145 55 18

N source (kg ha 21) N2-®xation

Soil

142 100 91 52 64 24 8

35 50 57 48 81 31 10

SED

25.0***

20.1***

12.2***

CV (%)

31.3

41.4

38.9

a b

Gathumbi et al. (2002). Amount of N2 ®xed above-groundÐN off-take in wood.

N off-take (wood) a

N recycled

N balance b

28 33 38 36 0 9 nd

149 117 110 64 145 46 18

114 67 53 16 64 16 nd

4.9*** 33.7

25.7***

21.8***

39.3

43.9

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S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

this N de®cient soil in western Kenya. C. grahamiana ®xed between 135 and 150 kg N ha 21 during a fallow period of 9 months while Calliandra ®xed considerably less both due to being a slowly growing species and obtaining a lower proportion of its N from N2-®xation (Table 7). Woody legumes tested ®xed .50% of their total above-ground N yield except Calliandra which only ®xed 24 kg N ha 21. Siratro ®xed between 51 and 73 kg N ha 21 of its total N production (145 kg ha 21) but also extracted a considerable amount of N from the soil. Although recently certain limitations of the 15N natural abundance method for measuring N2-®xation have been highlighted (Boddey et al., 2000; Handley and Scrimgeour, 1997), our results would suggest that for the soil and legumes under investigation the method can be used to obtained estimates of N2-®xation within acceptable con®dence limits. This was greatly facilitated by the relative uniformity of the natural soil 15N enrichment with depth (Tables 3 and 6). Although the enrichment of plant available N estimated using the reference plants was less than that recorded for the total soil N (Table 3) only the d 15N values of Tithonia changed signi®cantly with depth having a maximum variation of 1.3½ while in maize and Senna variation was less than 1½ to a depth of 2 m. Uniformity in 15N signatures is particularly important with leguminous trees with deep rooting systems. The second reason for a successful application of the 15N natural abundance method for estimation of N2-®xation was the considerable 15N enrichment of the soil N pool. Soil d 15N values ranged between 8.2 and 10.8½ (Table 4). Ledgard and Peoples (1988) and Shearer and Kohl (1986) suggested that enrichments of soil d 15N of 6.00½ were required for the application of the 15N natural abundance method and our results support these conclusions. Unkovich et al. (1994) suggested that d 15N values as low as 2½ could be used in pastures using carefully matched reference plants and paired sampling procedures. They also suggested that N2-®xation estimates were more dependent on the d 15N values of the legume than on the reference crop. However, this was not the case in our study given that d 15N values differed among reference plants and these differences in¯uenced the estimates of N2-®xation substantially (Table 7). Differences in d 15N values among reference plants were less in¯uenced by rooting depth than by differences between species (Table 3). Thus the choice of an appropriate reference plant was critical even with a relatively uniform 15N distribution with depth. This further justi®es the approach of using several reference plants (Boddey et al., 2000). Unlike in mixed pastures, reference plants cannot be grown in association with ®xing legumes in leguminous fallows to reduce spatial variability as this would in¯uence their N2-®xation. Furthermore, the lower the enrichment the greater the in¯uence of uncertainties due to either differences in d 15N signature of reference plants, accuracy of estimations of B values and other potential in¯uences on isotopic fractionation such as the type of mycorrhizal infection (HoÈgberg et al., 1994, 1996). Thus we suggest that plant available 15N signatures

should preferentially be .5½ for use in tree based fallow systems. Validation of the N2-®xation results of the natural 15N abundance with an independent method at certain stages in the evaluation is crucial to assess the viability of the method. The comparisons with the results of the ureide method showed a linear relationship between the two methods (see further discussion below). While instantaneous ureide measurements are not equivalent to integrated N2-®xation estimates obtained using the 15N method the good relationship between the two methods gives con®dence in the ®eld measurements of N2-®xation using natural 15 N abundance. 4.2. Isotopic fractionation during N mineralization, translocation and N2-®xation Isotopic fractionation of 14N and 15N may occur during the process of N2-®xation (Handley and Scrimgeour, 1997; Shearer and Kohl, 1986) and is an important factor to consider when using the natural 15N abundance method. Whole plant d 15N values indicate potential fractionation during N2-®xation and assimilation whereas d 15N values in different plant parts indicate the degree of fractionation that occurs during translocation of 15N from nodules to shoots. Our data suggest that there was no signi®cant isotopic fractionation during N2-®xation (i.e. whole plant d 15N for most plants varied between 20.31 and 0.17, Table 2) except for Sesbania (20.59½ for whole plant) and Mucuna (1.26½). During the experiment, Mucuna grew well immediately after germination but then its growth rate declined so that it was not sampled at the second harvest. The growth rate, however, increased sharply during the third month. This time lapse between the depletion of seed N reserve and the late onset of N2-®xation could have led to loss of 14 N (through root exudates, NH3 volatilization) which may explain the high d 15N values for Mucuna. There was signi®cant variation in d 15N signatures of different plant parts for most of the legumes indicating that there was more isotope discrimination during the assimilation and translocation of the ®xed N than during N2-®xation. At 90 d after planting, the d 15N values determined from different plant parts showed a depletion of 15N abundance in the order of shoots . roots . nodules. A similar phenomenon was observed with other legume species depending entirely on N2-®xation in previous studies (Cadisch et al., 1993; Steele et al., 1983; Turner and Bergersen, 1983; Yoneyama et al., 1986). The d 15N obtained for legume shoots for all species in this study tended to be negative except for Mucuna. Earlier studies have shown that negative d 15N values are common in most legume shoots when fully dependent on N2-®xation for growth indicating preference of 14N over 15N (Unkovich et al., 1994; Yoneyama et al., 1986). However, the d 15N values measured in the greenhouse discrimination experiment are the net result of integrated changes in 15N abundance in all

S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

plant parts during plant growth. As such, the B values of the various legumes should be determined on mature wellgrown plants. d 15N signatures in different legume plant parts have also been shown to be in¯uenced by rhizobial strain, nutritional and water status of the growing media (Cadisch et al., 1993; Ledgard, 1989; Steele et al., 1983; Yoneyama et al., 1986). In this experiment, a mixed inoculant obtained from the soils of the sites where the ®eld experiment was to be set up was used, which was believed to contain a wide range of rhizobia strains, hence ensuring nodulation by a similar range of strains as in the ®eld as suggested by Unkovich et al. (1994). According to Shearer and Kohl (1986), if NH41 assimilation and nitri®cation processes occur simultaneously in the soil, with the latter having greater isotopic fractionation, the resulting nitrate is normally depleted in 15N and the N assimilated by the soil microbes is enriched. The microbial N is later deposited as recalcitrant organic N, which leads to overall enrichment of the total soil N. As such the dilution observed in the plant materials of our reference plants could have resulted from the plants absorbing predominantly 15N depleted nitrate from the soil. The magnitude of isotopic discrimination could be different among plants during the N uptake and assimilation, resulting in variation in d 15N signatures. Alternatively there could be a direct interaction of species with N transformation processes and hence discrimination. The strong agreement between the d 15N values measured for maize and Tithonia for both pot and ®eld grown plants increased our con®dence in the reproducibility of the method and con®rmed a relatively good uniformity of plant available 15N at the ®eld site. The two species can thus be used as reference plants for N2-®xation studies although it is not possible from the data available to identify which is the more appropriate reference plant. Hence, usage of more than one reference plant is necessary to encompass the potential range of N2-®xation. 4.3. Comparison of N2-®xation estimates using the ureide and natural 15N abundance methods The %N from N2-®xation estimates obtained using the tissue ureide method were generally larger than those obtained using the natural 15N abundance method with both pigeonpea and siratro. Treatments which gave higher estimates of N2-®xation using the natural 15N abundance method also had higher estimates from the ureide method except for siratro mixed with Sesbania. This was demonstrated by the linear relationship (R2 ˆ 0:80; slope ˆ 0:82) between N2-®xation estimates obtained using the natural 15 N abundance and those obtained with the ureide method. The ureide method provides point time estimates while the natural 15N abundance method gives time-averaged estimates over the growing period (Herridge et al., 1990, 1996; Peoples et al., 1989b). Hence, if a ureide-transporting species is sampled during active nitrogen ®xation process, the abundance of the ureides in tissues is subject to be higher

1069

relative to other N solutes. Ureide measurements were only done at later stages of plant development when N2-®xation was presumably higher (due to exhaustion of soil mineral N resources). This would result in an overestimation of the %N derived from N2-®xation compared to the integrated estimates of the 15N natural abundance method and hence could account for the differences observed between the two methods. 4.4. N2-®xation of fallow species in western Kenya C. grahamiana was highly dependent on N2-®xation (75± 83%) compared with other species while about 54% of the total N from Calliandra was derived from soil. In Indonesia Sesbania was estimated to ®x about 84% (Peoples and Craswell, 1992) as compared with our estimated range of 46±59% (Table 7). In the same review, pigeonpea in Australia, Calliandra in Australia and siratro in India were estimated to ®x 65, 14 and 92% as compared with our estimates of 62, 46 and 43%, respectively. Although siratro had similar d 15N values as the other legume species except Calliandra, the proportion of N derived from N2®xation was less due to the low B value (22.4½) used, which was obtained from the literature using a different rhizobium strain and hence could have resulted in underestimation of N2-®xation. As the d 15N values obtained for the different plant legume species showed little differences except for Calliandra, the large differences observed in the estimated proportions of N2-®xation could also be attributed to the B value used in the calculation of %N derived from N2-®xation. The amounts of ®xed N contained in above-ground biomass ranged from 8 to 142 kg ha 21 (Table 8). Total recyclable N was signi®cantly different …P , 0:001† among the species and the highest estimated net N contribution after the fallow was found with C. grahamiana (149 kg N ha 21) which had the greatest biomass yield. The small amounts of total and ®xed N in groundnuts and Calliandra were mainly due to the poor biomass yield at harvesting and to a lesser extent due to low %N ®xed. The fallow duration was too short for the slow growing Calliandra. Soil derived N observed in woody species ranged between 31 and 57 kg ha 21 (Table 7). Because the available soil N source was similar with all the species, it can be argued that different species ®xed different amounts of N depending on their N demands. Siratro took up the largest amount of soil N (81 kg ha 21); about 56% of its total N contribution. This could have been due to the underestimation of N2-®xation as discussed earlier, or due to an extensive rooting system in the surface soil horizon. The lack of large differences in soil N acquizition between the improved woody fallow species suggested that they had access to the same soil N pool. This indicates that there were no signi®cant differences in rooting patterns, or that there was no substantial mineral N pool in the subsoil. Large subsoil N pools in this area (Hartemink et

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S.M. Gathumbi et al. / Soil Biology & Biochemistry 34 (2002) 1059±1071

al., 1996) occur particularly after previous crop failures, which was not the case in our study. The contribution of the different fallows to the overall soil N economy in the study area was considered as the amount of recycled N, assuming that all the above-ground foliage biomass is retained in the soil and the wood is exported from the system (Niang et al., 1996). Above-ground biomass obtained from the tested legume fallows can potentially recycle between 46 and 149 kg N ha 21 to crops after the fallow. The contribution of N2-®xation to the overall N balance of a soil can be summarized in the following simple expression (modi®ed from Peoples and Craswell (1992)) as follows

Acknowledgements

Net N-balance ˆ Nf 2 Nw

References

…4†

where Nf ˆ proportion of N from N2-®xation £ Total fallow N and Nw ˆ Wood N exported. All the fallows tested showed a positive N balance providing additional N inputs into the fallow system of 16±114 kg N ha 21. However, N exported in wood can amount up to 70% of N2 ®xed in the case of Sesbania although this has to be balanced with the need of fallows to provide additional bene®ts (e.g. ®rewood) rather than just improvements in soil fertility (Gathumbi et al., 2002). This study was conducted on P and K de®cient sites and hence mineral P and K fertilizer at a rate of 100 kg ha 21 was applied to the legume fallow plots to ensure optimum growth performance of the legumes and hence to explore the potential inputs from N2-®xation. It is evident that both %N derived from N2-®xation and the total amounts of N ®xed may be reduced by nutrient de®ciencies in nutrient depleted sites (Giller and Cadisch, 1995; Giller, 2001). Addition of fertilizer inputs to fallow plots by resource poor farmers may not be feasible largely because of economic constraints. However, results from this study have adequately demonstrated the potential contribution of the legumes in biological N2-®xation, but not perhaps the actual contributions if P and K are not added. 4.5. Conclusions Results from our greenhouse and ®eld experiments suggested that there is good potential of using the natural d 15N abundance method for evaluating the N2 ®xing ability of herbaceous/shrub/tree legumes in our ®eld sites. This was due to the relatively uniform plant available soil N pool with depth and of suf®ciently high 15N enrichment (around 5±6½). The legumes had the potential to contribute enormously to the N economy of the cropping system by sourcing N through biological N2-®xation in addition to soil N. In soils with severe N de®ciency such as those in the study area, the N contribution will be favoured because the legumes will ®x more in response to their N demand if not constrained by other nutrients such as P.

We gratefully acknowledge the assistance of both the ®eld and laboratory research teams at the ICRAF/KEFRI/ KARI Maseno Agroforestry Research Centre and Nyabeda ®eld site. This publication is an output from two projects: Agroforestry Research Network for Africa (AFRENA) funded by the European Union and NRSP R7056 funded by the UK Department for International Development (DFID) for the bene®t of developing countries. The views expressed are not necessarily those of the funding agencies.

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