Symbiotic Dinitrogen Fixation by Trees: an Underestimated Resource In

Nutr Cycl Agroecosyst DOI 10.1007/s10705-012-9542-9

REVIEW ARTICLE

Symbiotic dinitrogen ﬁxation by trees: an underestimated resource in agroforestry systems?

Pekka Nygren • Marı´a P. Ferna´ndez • Jean-Michel Harmand • Humberto A. Leblanc

Received: 24 March 2012 / Accepted: 24 October 2012 Ó Springer Science+Business Media Dordrecht 2012

Abstract We compiled quantitative estimates on long-term system N balances. The general aver- symbiotic N2 fixation by trees in agroforestry systems age ± standard deviation of tree dependency on N2 (AFS) in order to evaluate the critical environmental and fixation (%Ndfa) in 38 cases using N isotopic analyses management factors that affect the benefit from N2 was 59 ± 16.6 %. Under humid and sub-humid condi- fixation to system N economy. The so-called ‘‘N2-fixing tions, the percentage was higher in young (69 ± 10.7 %) tree’’ is a tripartite symbiotic system composed of the and periodically pruned trees (63 ± 11.8 %) than in free- plant, N2-fixing bacteria, and mycorrhizae-forming growing trees (54 ± 11.7 %). High variability was fungi. Almost 100 recognised rhizobial species associ- observed in drylands (range 10–84 %) indicating need ated with legumes do not form an evolutionary homol- for careful species and provenance selection in these ogous clade and are functionally diverse. The global areas. Annual N2 fixation was the highest in improved bacterial diversity is still unknown. Actinorrhizal symbi- fallow and protein bank systems, 300–650 kg [N] ha-1. oses in AFS remain almost unstudied. Dinitrogen fixation General average for 16 very variable AFS was in AFS should be quantified using N isotopic methods or 246kg[N]ha-1, which is enough for fulfilling crop N needs for sustained or increasing yield in low-input agriculture and reducing N-fertiliser use in large-scale P. Nygren agribusiness. Leaf litter and green mulch applications Department of Forest Sciences, P.O. Box 27, release N slowly to the soil and mostly benefit the crop 00014 University of Helsinki, Finland through long-term soil improvement. Root and nodule

P. Nygren (&) turnover and N rhizodeposition from N2-fixing trees are Finnish Society of Forest Science, P.O. Box 18, sources of easily available N for the crop yet they have 01301 Vantaa, Finland been largely ignored in agroforestry research. There is e-mail: pekka.nygren@metla.fi also increasing evidence on direct N transfer from N2- M. P. Fernańdez fixing trees to crops, e.g. via common mycelial networks Ecologie Microbienne, UMR5557, USC 1193, of mycorrhizal fungi or absorption of tree root exudates Universite´ Lyon1, 43 boulevard du 11 novembre 1918, by the crop. Research on the below-ground tree-crop- 69622 Villeurbanne Cedex, France microbia interactions is needed for fully understanding

J.-M. Harmand and managing N2 ﬁxation in AFS. CIRAD, UMR Eco&Sols, 2 Place Viala, 34060 Montpellier Cedex 01, France Keywords 15N Actinorrhizal trees Legume trees H. A. Leblanc Management practices Nitrogen balance Rhizobial EARTH University, 4442-1000 San Jose´, Costa Rica symbiosis 123 Nutr Cycl Agroecosyst

Introduction 1990s because of studies suggesting little benefit of N2 fixation to system level N balance in AFS (Fassbender Nitrogen is the first plant growth-limiting factor after 1987; Garrity and Mercado 1994; van Kessel and water in most ecosystems. Agroecosystems may be Roskoski 1981) or sustainability (Kass 1995). Recent even more N limited than natural ecosystems because research based on N isotopic relations in whole plant of heavy N export in crop harvest (Nair et al. 1999). (Leblanc et al. 2007; Peoples et al. 1996; Sta˚hl et al. The atmospheric N2 is the biggest pool of N in the 2002, 2005) or compilation of whole AFS N balance world but only some prokaryotic microbes are able to (Dulormne et al. 2003) indicate that symbiotic N2 reduce it, thus playing a key role in both terrestrial and fixation may have been underestimated as a N source marine ecosystems. Crops that form symbiosis with for AFS.

N2-fixing microbes, most notably legumes (Fabaceae The N2-fixing symbiosis is regulated by both super family) with certain a- and b-Proteobacteria intrinsic and environmental factors. The intrinsic (rhizobia), are an alternative to cope with N deficien- physiological and morphological factors form the cies in agroecosystems. basis of the functional plant groups as they result in Legume crops and forages respond only a part of differences in resource requirements, seasonality of human needs of plant food and fibre and, thus, vast growth, and life history (Tilman et al. 1997), i.e. agricultural areas depend on industrially-fixed N responses to the environment and interactions with fertilizers. These are often unavailable for small-scale other organisms. In spite of the recent advances in farmers in the developing world and may cause biotechnology, our ability to manage the intrinsic environmental problems such as contamination of factors of plants and N2-fixing bacteria are still quite water sources. Agroforestry systems (AFS) with ‘‘N2- limited. In practical agroforestry, these intrinsic fac- fixing trees’’1 provide alternatives to alleviate these tors may be taken into account only by selecting problems if managed properly. These systems are suitable tree species (Aronson et al. 2002) and diverse including but not restricted to cultivation of combinations of trees and bacterial strains (Acosta- cereals and other crops between rows of periodically- Duran and Martıńez-Romero 2002; Bala and Giller pruned trees in alley cropping (Akinnifesi et al. 2010; 2001; Bala et al. 2003). Kang et al. 1981; Rowe et al. 1999), shade trees with The macroenvironment is out of human control, in perennial crops (Beer et al. 1998; Soto-Pinto et al. spite of activities such as development of vegetation- 2010), improvement of fallow phase with N2-fixing based C sequestration to mitigate the global climate trees (Chikowo et al. 2004; Harmand et al. 2004; Sta˚hl change (Soto-Pinto et al. 2010). On the other hand, et al. 2002, 2005), living supports for climbing crops agroforestry offers a wide variety of tools for managing (Salas et al. 2001), and simultaneous cultivation of the microenvironment for better sustainability and fodder trees and grass (Blair et al. 1990; Dulormne productivity, starting with simple techniques like opti- et al. 2003). misation of tree spacing in alley cropping for best N Many tree species used in AFS provide multiple benefit and minimal crop shading (Akinnifesi et al. products including fuelwood, fodder, or several non- 2008). It is also important to understand the effects of the timber forest products. Dinitrogen-fixing trees are agroforestry management on the N2-fixing symbiosis; often preferred in comparison to other multi-purpose e.g., the green pruning of trees practiced for increasing species because of the assumed benefit to the whole nutrient recycling and reducing crop shading may also system N balance. Thus, symbiotic N2 fixation in AFS disturb nodulation (Nygren and Ramı´rez 1995). was enthusiastically studied in the 1970s and 1980s Although several reviews on N2 fixation in AFS but it almost disappeared from research agenda in the have been published (Bryan 2000; Giller 2001; Kass et al. 1997; Khanna 1998; Mafongoya et al. 2004; Sanginga et al. 1995), they are mostly descriptive 1 In fact, no tree fixes atmospheric N2 because all organisms compilations of relevant data. Thus, critical analysis capable of N fixation are Bacteria or Archae. However, in order 2 on issues such as the methods used in N2 fixation to avoid repeating the long correct expression ‘‘trees forming research within the agroforestry context, functional N -fixing symbiosis with bacteria’’ we use the common though 2 importance of N -fixing symbiosis for the legumes and inaccurate term ‘‘N2-fixing trees’’ for referring to these trees as a 2 group. actinorrhizal plants, and effects of the different AFS 123 Nutr Cycl Agroecosyst

management practices on symbiotic N2 fixation seems Dinitrogen-fixing organisms timely. Recent research (Andre´ et al. 2005; Cardoso and Kuyper 2006; Duponnois and Plenchette 2003; Dinitrogen-fixers have been reported among most of Ingleby et al. 2001; Lesueur and Sarr 2008) also the taxonomic divisions of Prokaryota and the meth- indicates the importance of the tripartite symbiosis anogenic Archae, thus presenting large genetic and between plants, N2-fixing bacteria, and mycorrhizae- physiological diversity. The N2-fixing bacteria are forming fungi on N2 fixation. currently divided into symbiotic and free-living N2- Our aim is to analyse symbiotic N2 fixation by fixers according to their capacity to form mutualistic agroforestry trees as a part of their functions and association with eukaryotic higher organisms, either indicate the critical environmental and management plants or animals, or their saprophytic life as compo- aspects needed to benefit from N2 fixation at AFS nents of environmental microflora. Most symbiotic N2- level. We also evaluate the research methodologies fixing bacteria also have a saprophytic stage. However, and compile quantitative data on N2 fixation in AFS some symbiotic strains that remain non-isolated could based on the most reliable methods only. The specific represent obligate symbionts or intermediate stages in objectives of the review are: revise current knowledge evolution towards a greater symbiotic dependence. on N2-fixing microbia relevant for understanding the Dinitrogen-fixers associated with plants comprise 3 functioning of AFS; evaluate the methods for estimat- main groups: (1) Cyanobacteria that establish ectosym- ing N2 fixation suitable for use in AFS; evaluate the biosis (non-intracellular location) with a large diversity of published data on symbiotic N2 fixation in AFS in the fungi and various plant groups, including mostly bryo- light of current knowledge on ecophysiology of phytes and cycads but very few higher plants; (2) legumes and actinorrhizal plants; and evaluate the a-andb-Proteobacteria that form symbiosis in specialised importance of symbiotic N2 fixation for AFS functions structures, nodules, within roots or stems of legumes and and productivity. Parasponia spp.; and (3) Frankiaceae (Actinobacteria) that nodulate plants belonging to 25 genera within eight Angiosperm families. Plants forming symbiosis with Frankiaceae are collectively called actinorrhizal plants. Biological N2 fixation and N2-fixing organisms They are mostly trees or shrubs. (Vessey et al. 2004).

Dinitrogen ﬁxation process Cyanobacteria and other free-living diazotrophs

All N2-ﬁxing Prokaryota reduce atmospheric N2 using Taxonomy of Cyanobacteria is problematic (Castenholz the nitrogenase enzyme: 2001) and recent molecular studies indicate considerable biodiversity. Among plants symbiotically associated þ nitrogenase N2 þ 8H þ 8e þ 16ATP ! 2NH3 þ H2 with Cyanobacteria, only the aquatic ferns of the genus Azolla have economic importance in farming systems þ 16ADP þ 16Pi (Herridge et al. 2008) and none is important in AFS. ð1Þ Endophytic free-living and associative diazotroph-

Thus, reducing a N2 molecule to two NH3 mole- ic bacteria within the rhizosphere of Gramineae may cules requires 8 protons (H?) and electrons (e-) and it make substantial contributions to N balance of an releases a hydrogen molecule (H2). The energy for N2 agroecosystem. The best-studied case is probably ﬁxation is released by breaking 16 adenosinetriphos- sugar cane (Saccharum spp.; Baldani et al. 1997), phate (ATP) molecules to adenosinediphosphate which is never used in AFS as far as we know. Some

(ADP) and inorganic phosphate (Pi). Details of the other plants known to harbour N2-fixing endophytes N2 fixation process can be found in most textbooks of such as forage grasses (Herridge et al. 2008), coffee plant physiology (e.g. Taiz and Zeiger 2006) or soil (Coffea arabica L.; Fuentes-Ramı´rez et al. 2001; microbiology (e.g. Paul and Clark 1996) and they will Jimeńez-Salgado et al. 1997), and banana (Musa spp.; not be repeated here, except for issues needed for Martıńez et al. 2003) are often used in AFS. As far as better understanding the discussion on symbiotic N2 we know, the role of the free-living N2-fixing bacteria fixation in AFS. in crop N supply has not been estimated in any AFS. 123 Nutr Cycl Agroecosyst

Symbiotic N2-fixing organisms Methylobacterium, Ochrobactrum, Phyllobacterium, and Shinella spp.) were recently shown to be rhizobia The nodules within which the symbiotic N2 fixation by the presence of nod and nif genes, which encode occurs are a plant organ. Nodule growth is stimulated nodulation and N2 fixation. Some bacteria found in by the symbiotic N2-fixing bacteria, and it contains a nodules, such as Agrobacterium spp., were shown to bacteroid space where the actual N2 fixation occurs be devoid of nodulation and N2 fixation genes. These (Minchin 1997). The symbiotic bacteria survive as strains often form mixed populations with nodulating free-living in the soil but many fix N2 only in rhizobial strains in the nodules (reviewed by Bala- symbiosis with a host plant. The endophytic bacteria, chandar et al. 2007). An up-to-date list of rhizobial called bacteroids, lose part of their cellular organelles taxa with recommended nomenclature is maintained and, thus, become completely dependent on the host by the ‘‘ICSP Subcommittee on the taxonomy of plant. Synthesis and catalytic activity of the nitroge- Rhizobium and Agrobacterium’’ (http://edzna.ccg. nase enzyme are inhibited by oxygen. Consequently, unam.mx/rhizobial-taxonomy/node/4) or the New the bacteroid space is anaerobic, protected by an O2 Zealand rhizobia website (http://www.rhizobia.co.nz/ diffusion barrier in the inner cortex of the legume taxonomy/rhizobia.html). nodules (Minchin 1997) or by specialised structures, In July 2012, the New Zealand rhizobia website such as vesicles of Frankia spp. The bacteroids are, recognised 98 rhizobial species. Nevertheless, the however, aerobic and the transport compound leghe- global diversity is still unknown because less than moglobin supplies O2 to the bacteroids without 30 % of the 728 genera and the ca. 19,325 species of damaging the nitrogenase. Ammonia produced by the Fabaceae have been tested for nodulation (Sprent symbiotic N2 fixation is assimilated to amino acids or 2009). Although considerable effort has been made ureides in the root nodules and transferred in this form during the last 20 years to describe rhizobial diversity to the plant’s vascular system (Vessey et al. 2004). from unstudied wild legumes, the symbionts have been identified to species level only in a few studies. Many of the recently described ‘‘new rhizobia’’have Dinitrogen-fixing bacteria in agroforestry systems been isolated from tropical legume trees. They seem to be a source of large microbial diversity, particularly Legume symbioses among the fast-growing rhizobia. In an extensive numerical analysis of 115 phenotypic characteristics The bacterial species associated with legumes of ca. 130 rhizobial strains, 12 clusters composed of tree described so far are diverse and do not form an rhizobia were found among the 19 clusters obtained evolutionary homologous clade. They are classified (Zhang et al. 1991). An important diversity is found in into 13 genera belonging to two distinct phylogenetic the diversification centres of the legumes, most of which branches where rhizobia (i.e. bacteria forming N2- aretropical(Lieetal.1987); e.g. in the sub-Saharan fixing nodules with legumes) are intermingled with African centre of diversity of legume trees (The Sudan, many non-symbiotic bacteria. Earlier, rhizobia were Ethiopia, and Kenya), a wide phylogenetic bacterial assumed to belong to five genera of a-Proteobacteria diversity was found in a relatively low number of host recognized as Azorhizobium, Bradyrhizobium, Rhizo- species (Nick et al. 1999;Odeeetal.1997, 2002; bium, Mesorhizobium, and Ensifer (formerly Sinorhi- Wolde-Meskel et al. 2005; Zhang et al. 1991). These zobium) (Young and Haukka 1996; Zakhia and de studies suggest that studying symbionts of unexplored Lajudie 2001). wild legumes from new biogeographical areas will Besides these genera, several other a-Proteobacte- reveal additional diversity. ria (Allorhizobium, Devosia, Methylobacterium, Before the modern phylogenetic techniques Ochrobactrum, Phyllobacterium, and Shinella spp.) became widely available, rhizobia were commonly and b-Proteobacteria (Burkholderia, Cupriavidus (ex. classified to fast- and slow-growing strains; the latter Waustersia ex. Ralstonia), and Herbaspirillum spp.) were later classified to the Bradyrhizobium genus. It have been isolated from legume nodules (reviewed by seems that often a given tree species recognises Balachandar et al. 2007). Several of these species preferentially either slow- or fast-growing symbionts (Blastobacter, Burkholderia, Cupriavidus, Devosia, (Turk and Keyser 1992; Wolde-Meskel et al. 2005); 123 Nutr Cycl Agroecosyst e.g., strains isolated from Acacia senegal (L.) Willd. The specificity apparently varies depending on the and Prosopis chilensis (Molina) Stuntz in the Sudan level, at which it is analysed (nodulation, effective- were all fast-growing whereas strains from Acacia ness, or both), and the applied methodology. Rhizobia mangium Willd. in Thailand were slow-growing may be promiscuous for nodulation and have high (Zhang et al. 1991). The phenomenon has not been specificity for effectiveness as was demonstrated in widely studied but Wolde-Meskel et al. (2005) noted Robinia pseudoacacia L., Acacia mearnsii De Wild. that in a few cases a tree species was nodulated with (Turk and Keyser 1992), and A. mangium (Galiana both fast- and slow-growing rhizobia. However, the et al. 1990; Prin et al. 2003). Globally, there is no classification to slow- or fast-growing strains may not obvious correlation between phylogenies of rhizobia be genetically relevant; e.g., fast-growing strains from and the hosts, from which they were isolated. A given Inga edulis Mart. were genetically associated with tropical legume tree may be nodulated by a wide slow-growing bradyrhizobia (Leblanc et al. 2005). diversity of rhizobia (Bala and Giller 2001; Odee et al. Because there is no specific selective medium for 1995, 1997) and several rhizobia isolated from tropical isolating rhizobia directly from soil, most of the trees are also able to nodulate herbaceous legumes biodiversity studies have been conducted sampling (Herrera et al. 1985; Zhang et al. 1991). nodules from field-grown plants or from trap plant experiments. The natural rhizobial populations obtained Actinorrhizal symbiosis from field-collected nodules seem to be more diverse than the trapped ones (Liu et al. 2005;Wangetal.1999, All N2-fixing actinorrhizal symbionts belong to the 2002a). Indeed, the latter approach only allows studying unique genus Frankia, which forms nodules in the roots compatible nodulating strains under the specific condi- of ca. 280 nodulating non-legume species that belong to tions used and estimates depend mostly of what and how 25 plant genera. This low number of species and genera many species are used for trapping, thus resulting in an compared with legumes does not imply low genetic underestimation of the global biodiversity. diversity at the plant level because eight Angiosperm In addition to taxonomic diversity, rhizobia that families are concerned (Dawson 2008). The main species nodulate legume trees are functionally very diverse, used in tropical AFS belong to the genera Casuarina and i.e. with respect to their ecological, physiological, and Allocasuarina (Casuarinaceae), and Alnus (Betulaceae). biochemical properties (Zhang et al. 1991). Particu- Alnus spp. and Hippophae rhamnoides L. (Elaeagna- larly, their cross-nodulation patterns vary remarkably ceae) are also used in some temperate AFS. (Table 1). Fast-growers seem to be more specific in The first successful isolation of a Frankia sp. comparison to slow-growers that are generally found occurred only 30 years ago. Today at least 11 species promiscuous. Each rhizobial species has a defined host have been reliably isolated and identified, and several range, varying from very narrow to very broad others require verification. However, the majority of (Duhoux and Dommergues 1985; Graham and Hub- Frankia diversity remains to be described because bell 1975; Trinick 1982); e.g. Calliandra calothyrsus almost half of the known Frankia strains have not been Meisn. (sub-family Mimosoideae, tribe Ingeae), Leu- successfully grown ex-planta. Modern methods of caena leucocephala (Lam.) de Wit (Mimosoideae: molecular biology allow the characterisation of strains Mimoseae), and Gliricidia sepium (Jacq.) Kunth ex directly from the nodules, thus avoiding the limiting Walp. (Papilionoideae: Robineae) were able to nodu- culture step. Due to the difficulties to cultivate Frankia late in most of the soils tested indicating that their spp. and lack of reproducibility of some classical symbiotic partners are widely distributed (Bala and taxonomical methods, most of the Frankia genomic Giller 2001). These phylogenetically diverse hosts species described are not yet named. The only also shared their symbionts, because same rhizobial recognised named species is F. alni (Table 1). species were isolated from their nodules (Bala and Frankia spp. are probably distributed in all conti- Giller 2001; Moreira et al. 1998; Oyaizu et al. 1993). nents except Antarctica under very diverse soil and Sesbania sesban (L.) Merr. (Papilionoideae: Ses- environmental conditions, including areas devoid of banieae) is a common species in AFS that failed to actinorrhizal plants. The capacity of Frankia spp. to nodulate in most of the soils tested suggesting a higher fix N2 saprophytically and sporulate may explain their symbiotic specificity (Bala and Giller 2001). survival out of the normal distribution of the host 123 Nutr Cycl Agroecosyst

Table 1 Symbiotic N - 2 Host plant genus Symbionts References fixing bacteria found with legume and actinorrhizal Legume tree genera trees commonly used in Acacia spp. Sinorhizobium fredii Wolde-Meskel et al. (2005) agroforestry systems S. terangae Boivin and Giraud (1999) S. saheli Lortet et al. (1996) S. kostiense Nick et al. (1999) S. americanum Toledo et al. (2003) S. arboris Nick et al. (1999) Ensifer mexicanus Llore al. (2007) Allorhizobium undicola De Lajudie et al. (1998) Mesorhizobium plurifarium De Lajudie et al. (1998) M. huakuii Sprent (2009) Bradyrhizobium sp. Dupuy et al. (1994) Albizia spp. M. albiziae Wang FQ et al. (2007) Calliandra spp. Rhizobium tropici Martıńez-Romero et al. (1991) R. gallicum Zurdo-Pin˜eiro et al. (2004) R. mongolense Wolde-Meskel et al. (2005) Erythrina spp. Rhizobium sp. Zhang et al. (1991) B. liaoningense Wolde-Meskel et al. (2005) Faidherbia spp. Allorhizobium undicola De Lajudie et al. (1998) Ochrobactrum sp. Ngom et al. (2004) B. elkanii Wolde-Meskel et al. (2005) Gliricidia spp. R. tropici Hernańdez-Lucas et al. (1995) R. etli Hernańdez-Lucas et al. (1995) Sinorhizobium sp. Acosta-Durań and Martıńez-Romero (2002) Inga spp. B. japonicum Leblanc et al. (2005) B. liaoningense Leblanc et al. (2005) Leucena spp. R. tropici Martıńez-Romero et al. (1991) R. etli biovar phaseoli Segovia et al. (1993) R. gallicum Hernańdez-Lucas et al. (1995) R. giardinii Amarger et al. (1997) S. morelense Wang ET et al. (2002b) M. plurifarium De Lajudie et al. (1998) M. albiziae Wang FQ et al. (2007) Prosopis spp. M. plurifarium De Lajudie et al. (1998) M. chacoense Vela´squez et al. (2001) S. kostiense Sprent (2009) S. arboris Sprent (2009) Robinia pseudoacacia R. multihospitium Han et al. (2008) Sesbania spp. R. huautlense Wang ET et al. (1998), S. saheli De Lajudie et al. (1994) S. terangae De Lajudie et al. (1994) Ensifer adhaerens Casida (1982) Azorhizobium caulinodans Dreyfus et al. (1988) Azorhizobium johannense Moreira et al. (2000) S. meliloti Wolde-Meskel et al. (2005) S. fredii Wolde-Meskel et al. (2005)

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Table 1 continued Host plant genus Symbionts References

Tephrosia candida R. hainanense Chen et al. (1997) Actinorrhizal tree genera Alnus spp. 3 genomic species An et al. (1985) (3 to be confirmed) Fernańdez et al. (1989) Akimov and Dobritsa (1992) Shi and Ruan (1992) Casuarina spp. 1 genomic species Fernańdez et al. (1989) Elaeagnus spp and Hippophae spp 5 genomic species Fernańdez et al. (1989) (5 to be confirmed) Akimov and Dobritsa (1992) Lumini et al. (1996) An et al. (1985) plants (Burleigh and Dawson 1994; Maunuksela et al. or contribution of N2 fixation to plant or system N 2000, 2006; Paschke and Dawson 1992). economy. In order to really evaluate the N benefit from

Consequently, many actinorrhizal plants are known N2-fixing trees to an AFS, N2 fixation must be for their high capacity to acclimate to environments estimated. Not all legume trees fix N2, e.g. Senna different from their natural range and they are, thus, spp. that are common in AFS (Ladha et al. 1993; Sta˚hl widely used as exotics, e.g. in plantation forestry. The et al. 2005). Non-nodulating species are most common nodulation out of the natural range is generally good in among the Caesalpinoideae but exist also in Mimo- Alnus spp., Myricaceae, and Elaeagnaceae (Dawson soideae and Papilionoideae (Sprent 2009). Further, 2008). Family Casuarinaceae that originates from tree management may have important effects on

Australia and New Zealand and has been introduced nodulation and N2 fixation by trees reported as N2- in Africa, the Americas, and Asia, is a good example on fixers (Nygren and Ramı´rez 1995). how distribution history can bias the knowledge about Several excellent reviews on the methods for strain diversity and plant-strain specificity. The first quantifying N2 fixation have been published and they studies indicated a strong genetic homogeneity of will be referred to in the subchapters dealing with Casuarina-nodulating strains at both intra- and inter- relevant techniques. Unkovich et al. (2008), which is specific level all around the world (Fernańdez et al. freely available in electronic form, is a good general 1989; Nazaret et al. 1989; Rouvier et al. 1992). starting point but the authors largely deal with crop However, these studies concerned only the introduc- legumes. Boddey et al. (2000) and Domenach (1995) tion areas and often the most widely distributed species provide reviews targeting N2-fixing trees. Before Casuarina equisetifolia L. Further studies within the dealing with methods suitable for AFS, it is necessary native range indicated genetic diversity of the strains to note limitations of some old methods. and a high specificity between the plant species and the Acetylene reduction assay (ARA) is based on the symbiont genotype (Rouvier et al. 1992). Globally, the multiaffinity of nitrogenase to break triple bounds in actinorrhizal symbioses vary from very promiscuous to gas molecules (see Giller 2001 or Hunt and Layzell very specific associations even within a family, like in 1993 for a review). Nodules are incubated in acetylene the case of Myricaceae (Huguet et al. 2005). (C2H2) enriched atmosphere and the production of ethylene (C2H4) is measured. Theoretically, C2H4 1 production rate is /4 of N2 fixation rate (Giller 2001). Estimation of N2 fixation However, the ratio seems to be highly variable in natural systems and ARA requires calibration with

Need to quantify N2 fixation other methods in order to be quantitative (Hunt and Layzell 1993). Further, the incubation conditions may When preparing this review, it became evident that cause serious disturbance to nodule functions leading symbiotic N2 fixation is often cited in agroforestry to underestimation of the actual N2 fixation rate literature without actual estimates on N2 fixation rate (Giller 2001; Minchin et al. 1983; 1986). Thus, earlier

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reports on low N2 fixation rate by individual agrofor- (Chalk and Ladha 1999) is estimated as the percentage estry trees (Lindblad and Russo 1986; Roskoski and of N derived from atmosphere out of total plant N Van Kessel 1985) and extrapolations to AFS level of (%Ndfa): ca. 40 kg ha-1 year-1 (Roskoski 1982) based on ÀÁ ARA should be dealt with caution. Unfortunately, 15 15 %Ndfa ¼ 1 % Nex;fix=% Nex;ref ð2Þ they have been quite influential among agroforestry 15 15 15 researchers in Latin America (Beer et al. 1998). While where % Nex,fix and % Nex,ref are N atom excess the ARA must be dismissed as a quantitative field percentage with respect to atmosphere in the N2-fixing method, it has undeniable value as a rapid method for and non-N2-fixing reference plant, respectively. The 15 checking if nitrogenase is active (Roggy et al. 1999b; % Nex is calculated simply by subtracting the atmo- 15 15 Unkovich et al. 2008). spheric N percentage from the measured N Another method unsuitable in agroforestry research percentage in the sample, determined by an isotope is the xylem solute method (Peoples et al. 1996). It is ratio mass spectrometer. 15 used for ureide-transporting crop legumes such as The pros and cons of the N dilution method soybean (Glycine max L.): xylem sap is collected and have been extensively revised by Unkovich et al. relative amounts of ureides, amino acids, and nitrate (2008). They also present different variants of the - (NO3 ) out of total xylem N are calculated. The ureide basic method for particular experimental conditions. proportion is used as an indicator of N2 fixation, which The main advantage of the method is that it provides may be calibrated against another quantitative method both yield-independent and time-integrated estimates (Unkovich et al. 2008). However, it has little value in of %Ndfa. Main difficulties are related to temporal agroforestry research because very few trees transport and spatial non-uniformity of the distribution of the 15 ureides; only two tree species of the legume tribe N label in the substrate. When small plants in small 15 Desmodiae were found to be ureide transporters in an pots are studied, the N label may be efficiently extensive review by Giller (2001). Thus, the often- mixed in the substrate (Unkovich et al. 2008). repeated statement that temperate legumes transport Studies on N2 fixation by seedlings or saplings are fixed N in the form of amino acids and tropical of little use for agroforestry research because juve- legumes as ureides (Vessey et al. 2004) may be a false nile trees may behave differently from mature trees perception based on a few crop legumes. in many aspects, as we will show in the chapter ‘‘Symbiotic N2 fixation in whole plant physiology’’. The limits of complete mixing of 15N with substrate Controlled and semi-controlled conditions are soon met: when 200 l barrels buried in soil were used for controlling root growth and 15N distribution We call laboratory and greenhouse experiments where in the field, the top 0–15 cm of the barrel soil was the growth conditions are mostly under the research- significantly more enriched than the deeper layers ers’ control as controlled condition experiments. (Kurppa et al. 2010). Semi-controlled conditions refer to field experiments Under semi-controlled field conditions, the only where some environmental factors are restricted, possibility to deal with the problems caused by the typically root growing space by physical barriers uneven distribution of the 15N tracer is to use an (Kurppa et al. 2010; Leblanc et al. 2007; Sta˚hl et al. adequate reference plant. Unkovich et al. (2008) list 2005). Under these conditions, using the stable heavy characteristics, which the reference plant should have 15 N isotope as a tracer may be the most adequate in common with the N2-fixing plant: (1) same life form method. The method is based on the stable proportion (i.e. only trees should be used as references for trees); of 15N out of atmospheric N, with mean 0.3663 % and (2) adaptation to similar environmental conditions; (3) standard deviation 0.0002 % (Mariotti 1983). If 15N rooting zone and relative N uptake pattern within the enriched fertilizer is applied to the substrate, a root system; (4) mycorrhizal status; and (5) growth 15 N2-fixing plant becomes less enriched with N than habit and phenology. Further, the N2-fixing and non- a non-N2-fixing plant because the N taken up from the N2-fixing reference plant should prefer the same substrate is diluted with atmospheric N2 in the former. inorganic N form for uptake (Schimann et al. 2008). The symbiotic dependence of the N2-fixing plant In natural ecosystems, plants tend to differentiate with 123 Nutr Cycl Agroecosyst respect to these factors in order to share soil resources In the basic application of the 15N natural abun- within the same site, which results in niche differen- dance method, the deviation of the sample 15N content 15 tiation (McKane et al. 2002). Ecologically, the N2- from that of atmosphere (d N) is calculated (Shearer fixing and the reference plant should share the same and Kohl 1986): niche except for N2 fixation. Following to that, the crop species associated with a N -fixing tree in an AFS 15N=14N 15N=15N 2 15 sa at & d N ¼ 15 14 1; 000 ð3Þ is seldom a good reference because species selection N= Nat in agroforestry targets to complementary resource use 15 14 15 14 (Ong et al. 2004) and, thus, niche differentiation. where N/ N is the ratio of Nto N and subscripts sa and at refer to the sample and atmosphere, 15 Field studies respectively. In the N natural abundance method, %Ndfa is estimated by comparing the d15N values in a 15 N2-fixing plant growing in the field (d Nf), a non-N2- When N2 fixation is estimated under field conditions, 15 the problems associated with the uniformity of 15N fixing reference plant growing in the same soil (d Nr), and the N2-fixing plant growing in N-free environment labelling and match between the characteristics of the 15 (d N0), i.e. depending only on N2 fixation for N N2-fixing and reference tree become manifold in comparison to controlled-environment studies. It is supply (Shearer and Kohl 1986): 15 15 15 almost impossible to distribute the N tracer uni- d Nr d Nf 15 formly in a soil profile. Injecting the N tracer to %Ndfa ¼ 15 15 100% ð4Þ d Nr d N0 different soil depths has been used for studying rooting 15 depths and N uptake in AFS (Lehmann et al. 2001; While the N natural abundance in the atmosphere is Rowe et al. 1999; 2001). The injection is always very stable and uniform around the world (Mariotti punctual and extensive sampling is required for 1983), it varies in the soils and plants because plant assuring a reliable recovery of the 15N signal in the metabolism and several microbiological processes in plants (Rowe and Cadisch 2002). Although the deep the soil cause isotopic fractionation (Handley and Raven injection labelling has some promise for agroforestry 1992). Most soil microbiological processes discriminate 15 research, it has not yet been used for estimations of the against N(Ho¨gberg 1997), thus, enriching soil with 15 contribution of N2 fixation to N economy of the N2- this isotope. This causes a slight difference in the d Nof fixing trees or the whole AFS. It seems that selection plants depending on soil N and plants partially fixing of a ‘‘correct’’ reference tree species that uses the same atmospheric N2. The difference can be detected with form of N from same depth and horizontal area as the modern isotope ratio mass spectrometers but all work

N2-fixing tree is the only method to overcome the phases must be conducted with great care because the problem of uneven distribution of the 15N tracer in soil differences are only a few %-units. (Chalk and Ladha 1999). However, often too little pre- The general trend of the soil becoming naturally 15 information is available on these tree characteristics. N enriched does not hold in all soils. If the non-N2- 15 Thus, using several reference species is recommended fixing trees have a low soil-derived d N that cannot 15 (Unkovich et al. 2008). be distinguished from the d NofN2-fixing trees, 15 Given the problems of the 15N dilution method application of the N natural abundance method is under field conditions, the 15N natural abundance impossible (Gehring and Vlek 2004; Roggy et al. method (Shearer and Kohl 1986) is often recom- 1999a). In some cases, the soil may be so depleted of 15 mended for quantifying N2 fixation in AFS. Handley N and, consequently, the reference trees have so 15 15 and Raven (1992), Ho¨gberg (1997), and Martinelli much lower d N than the N2-fixing trees that the N et al. (1999) reviewed the behaviour of 15N in plant natural abundance method is applicable because active 15 metabolism, soil–plant systems, and natural forests, N2-fixers tend to have d N close to 0, in this case respectively. Boddey et al. (2000), Domenach (1995), significantly higher than the non-N2-fixing reference and Gehring and Vlek (2004) reviewed the application (Augusto et al. 2005; Domenach 1995; Domenach 15 of the N natural abundance method for estimating N2 et al. 1989). fixation by trees. Here we revise the basic idea and Further, it was shown among non-N2-fixing cae- some agroforestry applications. salpinoids that Eperua falcata Aubl., which prefers 123 Nutr Cycl Agroecosyst

- NO3 as the inorganic N source, had significantly Use of several reference tree species is recom- lower d15N than Dicorynia guianensis Amshoff, which mendable also in 15N natural abundance method ? uses ammonium (NH4 ) for N supply in the same soil because often too little is known about the character- (Schimann et al. 2008). This suggests that the N2-fixing istics described above for selecting an ecologically tree and the reference tree should use the same and physiologically similar reference to a N2-fixing inorganic N form. Most legumes form symbiosis, in tree (Boddey et al. 2000; Unkovich et al. 2008). addition to rhizobia, with arbuscular mycorrhizae- Several candidate references can also be screened in a forming fungi (AMF) and some with ectomycorrhizae- pre-trial (Nygren and Leblanc 2009). This will allow forming fungi (ECM; Sprent and James 2007). the removal of apparent outliers; e.g. in a comparison

According to a global analysis, AMF deplete the host of five non-N2-fixing tree species, Nygren and Leblanc plant with 15N and ECM even more (Craine et al. 2009) (2009) found that one species had much lower d15N yet the difference between AMF and ECM plants than the four other species, which had little variation decreases when they grow in the same site (Hobbie and among themselves. Non-N2-fixing legume trees may Ho¨gberg 2012). It seems that the effect of mycorrhizae be the best references for legume trees if available on plant d15N depends on an interaction between (Roggy et al. 1999b). Further, if the research involves fungal strain, environment, and host plant (Boddey comparisons over different sites, some of the reference et al. 2000). The mycorrhizal effects are further species should be used in all sites if possible (Roggy complicated by the fungal uptake of organic N et al. 1999b). (Boddey et al. 2000;Na¨sholm et al. 2009). However, In some cases, it may also be possible to use a non- before a better understanding on these effects arises, nodulating phenophase of the studied species as the we recommend using reference trees that have the reference (Salas et al. 2001). This method requires that same mycorrhizal type and preference for soil N as the the isotopic signature of the soil N is stable over time, 15 studied N2-fixing tree. As in the case of N dilution sufficient variation in nodulation exists between the method, the reference plant should have similar rooting phenophases, and the temporal variation in N2 fixation pattern and phenology as the N2-fixing plant. of the nodules is small. The approach was successfully The question whether to use the associated non-N2- applied for estimating rainy season N2 fixation in fixing plant or an independent reference growing in Erythrina lanceolata Standl. using the dry season non- similar soil but without contact with the N2-fixing nodulating phenophase as the reference (Salas et al. plant is more important in the case of 15N natural 2001). abundance than the 15N dilution method. In the latter case, the selection is an issue of ecological and Sampling procedures physiological similarity, which is seldom met between the trees and crops in AFS. In the field, a N2-fixing tree Whole tree harvesting is recommended for seedlings may slowly decrease the soil d15N by recycling litter and saplings because the isotopic signal may vary in and root exudates depleted of 15N. Consequently, the different parts of a tree (Kurppa et al. 2010; Leblanc d15N of the associated plants also decreases over time et al. 2007). In the case of big trees, this is often not

(van Kessel et al. 1994; Sierra and Nygren 2006). possible. Typically, both N2-fixing and non-N2-fixing Leucaena leucocephala altered so much the soil trees tend to have the highest d15N in leaves and the isotopic signature in six years that by the end of the lowest in stem and coarse roots (Boddey et al. 2000). study period, associated weeds had almost the same Leaves are often the preferred sink for recently fixed N isotopic signature as the tree itself (van Kessel et al. (Domenach 1995) and they are, thus, the best choice 1994). The d15N gradually increased in Dichanthium for sampling if the objective is to follow seasonal or aristatum (Poir.) C.E. Hubb. grass brought from a field other variation in N2 fixation in time scale of weeks or site with Gliricidia sepium trees to a greenhouse, few months. Large structural parts of a tree, stem and probably because of removal of N transferred from the coarse roots, may be envisioned to integrate the tree by successive grass cuttings (Sierra and Nygren isotopic signature of N sources over time. They may 2006). Thus, the reference trees should grow without be the choice if the objective is to understand the long- direct contact with the N2-fixing trees but in a similar term role of N2 fixation in trees’ N economy (Nygren soil. and Leblanc 2009). Fine roots have seldom been 123 Nutr Cycl Agroecosyst

15 sampled. In a study using N enrichment, higher affected by isotopic fractionation, indicating active N2 proportion of 15N than total N was retained in the fine fixation in I. edulis (Kurppa et al. 2010; Leblanc et al. roots of Gliricidia sepium suggesting that soil N may 2007). be preferentially used for fine roots and fixed N is transported to the shoot. However, the root:shoot 15 sharing of N and total N was the same in the legume Symbiotic N2 fixation in whole tree physiology tree Inga edulis and non-N2-fixing crop Theobroma cacao L. (Kurppa et al. 2010). Thus, dynamics of fine Conceptual model on nitrogen acquisition roots require further study for understanding the whole in the tripartite plant-bacteria-mycorrhizae plant isotopic relationships. symbiotic system Sanginga et al. (1995) claimed that the 15N natural abundances in different organs tend to be proportion- It may be assumed that legumes (Bethlenfalvay 1992; ally equal in N2-fixing and non-N2-fixing trees and, Bethlenfalvay and Newton 1991) and actinorrhizal consequently, estimates of the %Ndfa are about the plants (Gardner and Barrueco 1999) are mycorrhizal, same if the same organ of both trees is used. Thus, they except Lupinus spp. (Sprent and James 2007). Thus, proposed that leaf sampling would result in reliable ecophysiology of N2 fixation must be evaluated within %Ndfa estimates. This hypothesis held between the context of the tripartite symbiosis formed by

G. sepium and five different reference tree species in plants, N2-fixing bacteria, and mycorrhizae in all a cacao plantation but not with I. edulis (Nygren and natural and man-made ecosystems (Barea et al. 1992; Leblanc 2009). The estimates of %Ndfa based on 15N Bethlenfalvay and Newton 1991; Cardoso and Kuyper enrichment (Eq. 2) were about the same using leaves 2006; Kuyper et al. 2004). Most legumes seem to form only and whole sapling harvesting for three agrofor- symbiosis with AMF (Bethlenfalvay 1992; Cardoso estry tree species, including I. edulis (Leblanc et al. and Kuyper 2006; Kuyper et al. 2004; Sprent and 2007). Leaves were recommended for 15N sampling in James 2007). A minority of legume species, notably trees but not in herbaceous legumes by Unkovich et al. Acacia spp. and caesalpinoids, may form symbiosis (2008). with ECM or both AMF and ECM (Haselwandter Isotopic fractionation within a tree resulting in and Bowen 1996; Sprent and James 2007). More differences in N isotopic relationships between tree than 40 actinorrhizal species have been recorded to organs may affect the usability of the 15N natural support ECM, AMF, or both symbioses (Gardner and abundance method in some cases. Some nodulated Barrueco 1999). Inga spp., in which ARA indicated nitrogenase A simplified scheme of potential N acquisition activity, had high leaf d15N values typical for non- strategies by the tripartite symbiosis is presented in

N2-fixing trees in a rain forest (Roggy et al. 1999b) and Fig. 1. The soil N forms available for the mycorrhizal ? - a tropical fresh water swamp forest (Koponen et al. fungi vary: AMF may take up soil NH4 ,NO3 ,and - 2003) in French Guiana. In both cases, most Inga spp. amino acids, while some ECM cannot use NO3 but 15 had leaf d N values typical for N2-fixing trees. In a they are able to use, in addition to amino acids, some Costa Rican cacao plantation, I. edulis shade trees had other organic N forms in the soil such as simple proteins a high d15N in leaves but low stem and coarse root and oligopeptides (Lambers et al. 2008; Smith and Read 15 d N values were typical for N2-fixers (Nygren and 2008). Mycorrhizae also enhance plant N nutrition by Leblanc 2009). These data may indicate strong largely increasing the soil volume available to uptake of isotopic fractionation in the N transport and metabo- N and other nutrients because of the extensive extra- lism of some Inga spp. Similar observations have been radical mycelium. Fungal hyphae also access smaller made in Acacia mangium (Bouillet et al. 2008) and soil pores than plant roots (Smith and Read 2008). The

A. senegal (Isaac et al. 2011a). Although the strong N2 ﬁxation process by symbiotic bacteria within the root within-tree isotopic fractionation may be a rare nodules has been described above. phenomenon, its possible occurrence should be taken Soil inorganic N taken up by the mycorrhizal into account when analysing 15N natural abundance fungus is processed in the fungal hyphae (Smith and ? data and nodulation must be revised in all trees Read 2008). Nitrate is reduced to NH4 , which is 15 ? sampled. The N dilution method does not seem to be further assimilated to amino acids together with NH4 123 Nutr Cycl Agroecosyst

Fig. 1 A simplified scheme of nitrogen acquisition strategies of flows of organic N by thick arrows, flows of organic carbon (Co) a plant, which forms symbiosis with N2-fixing bacteria and by double arrows, feedback loops by dashed arrows, metabolic ? - mycorrhiza-forming fungi. The potential N sources (clouds) are processes by valves, and NH4 and NO3 transporters with soil ammonium and nitrate, which the plant may take up directly ellipsoids. The metabolic processes are breakdown of AA to ? - ? ? via its own fine roots or via mycorrhizal symbiosis, soil amino NH4 ,NO3 reduction to NH4 , assimilation of NH4 to AA, acids (AA), which may be taken up by both arbuscular- and biological N2 fixation. There is evidence on N transfer from ? mycorrhizal and ectomycorrhizal fungi, other soil organic N the plant to mycorrhizal fungus as either NH4 or AA, thus (No), which may be taken up by the ectomycorrhizal fungi, and double-ended arrows. Details of leaf N metabolism and atmospheric N2, which is fixed by the symbiotic bacteria in the photochemistry that also affect plant N status are beyond the root nodules. Flows of inorganic N are indicated by thin arrows, scope of this review

- taken up from the soil. The plant may reduce NO3 in reduced N in the phloem flow. Nitrogen is transported - - the roots or transport it to the leaves where NO3 is to leaves as NO3 or amino acids in the xylem, and reduced using NADH and NADPH produced in amino acids may be transported from leaves back to photosynthesis (Oaks 1992; Zerihun et al. 1998). Free roots in phloem (Fig. 1). Comparison of nodulation ? NH4 is toxic to plants and must be assimilated in the and canopy N flows in the agroforestry tree Erythrina site of reduction. Carbon transported in phloem from poeppigiana (Walp.) O.F. Cook suggested that nod- ? the leaves is needed for NH4 assimilation in the roots ulation was regulated by N needs of the canopy with or mycorrhizal mycelium. There is also evidence on N reduced nodulation when net N flow turned downward transfer from the plant to mycorrhizal fungus, espe- from canopy to roots (Nygren 1995). Accumulation of ? - cially from N-rich roots of N2-fixing plants (Arnebrant amino acids in the roots controls the NH4 and NO3 et al. 1993; He et al. 2003; Simard et al. 2002). transporters in all plants (Amtmann and Blatt 2009) Parsons et al. (1993) suggested that nodulation and and the blockage of the transporters by high root

N2 fixation may be regulated by the concentration of amino acid concentration may result in N exudation 123 Nutr Cycl Agroecosyst from roots to soil observed in many plants (Fustec requirements (Eq. 1). For evaluating the importance et al. 2010; Wichern et al. 2008). Thus, root amino of the P on N2 fixation, we searched the CAB Abstracts acid pool may be envisioned to play a key role in database for articles published 2000–2009 on the regulating the N acquisition of the whole tripartite effects of P fertilisation on nodulation and N2 fixation symbiotic system (Fig. 1). It may be hypothesised that in trees. All studies dealing with tropical tree seedlings the root pool size is maintained by the balance between under greenhouse or nursery conditions (Binkley et al. the influxes from the tripartite symbiosis and efflux to 2003; Diouf et al. 2008; Pons et al. 2007; Uddin et al. foliage. Thus, N acquisition may be ultimately regu- 2008) indicated positive effects of P fertilisation on N2 lated by N needs in the plant canopy. Not all evidence fixation. All field studies were conducted on actinor- to support this hypothesis is available but we feel that it rhizal trees in temperate forests. These studies indi- is a fruitful starting point for further research. cated no direct P effect on nitrogenase activity (Uliassi

and Ruess 2002)orN2 ﬁxation rate (Augusto et al. Energetics of N2 ﬁxation 2005; Cavard et al. 2007; Gokkaya et al. 2006) but enhanced nodulation was observed in one case (Uliassi

Symbiotic N2 ﬁxation is quite an energy intensive and Ruess 2002). strategy for acquiring N (Vance and Heichel 1991; In a recent study, however, Isaac et al. (2011a)

Vitousek et al. 2002). Following the biochemically- found that P supply rates did not markedly affect N2 based estimation method of Thornley and Johnson fixation rates of Acacia senegal seedlings under non- (1990), 4.29 g of glucose is needed to reduce 1 g of N limiting N supply, but higher P supply stimulated ? - to NH4 from NO3 in the roots and 5.00 g of glucose growth, which resulted in greater mineral N uptake for fixing 1 g of N from the atmosphere. Thus, the C from soil solution. On the other hand, the N2 fixation cost of N2 fixation is only about 17 % higher than that rate of A. senegal increased with increasing soil P - of NO3 reduction in roots. However, glucose con- availability in natural stands of the Rift Valley in sumption per gram of protein produced is considerably Kenya (Isaac et al. 2011b). Application of rock lower when amino acids (on average 0.89 g glucose) or phosphate did not significantly affect N2 fixation by ? NH4 (1.77 g) are available as the N source (Zerihun Inga edulis in a P-poor soil in the humid Atlantic et al. 1998). At whole plant level, acquisition of soil N lowlands of Costa Rica (Leblanc 2004). also requires C for growth and maintenance of the root Severe P deficiency markedly impaired symbiotic system. Legumes probably evolved in the humid N2 fixation in the tropical Casuarinaceae species ? tropical forests (McKey 1994) where NH4 is often a (Sanginga et al. 1989) and other actinorrhizal plants - much more abundant inorganic N form than NO3 (Gardner and Barrueco 1999). Several actinorrhizal (Boddey et al. 2000). Under these conditions, N2-fixing genera such as Comptonia and Myrica (Myricaceae) symbiosis may have been energetically more appro- develop cluster (proteoid-like) roots, which could priate N acquisition strategy than growing roots for enhance P uptake in P poor soils (Berliner and Torrey - competing for the scarce soil NO3 resources. 1989). Carbon consumption to N2 fixation may be compen- Both plant roots and hyphae of mycorrhizal fungi sated by increased photosynthetic rate; photosynthetic secrete extracellular phosphatases to the rhizosphere rate of several nodulated herbaceous crop legumes was (Grierson et al. 2004; Louche et al. 2010; Treseder and increased on average by 28 % in comparison to non-N2- Vitousek 2001). Phosphatases hydrolyse the ester- fixing plants (Kaschuk et al. 2009). In most cases, phosphate bonds in soil organic P, thus releasing increase in photosynthetic production was higher than phosphate to soil solution where it may be taken by the C allocation to the rhizobial symbiont (4–16 % of C plant roots or hyphae of mycorrhizal fungi. Extracel- fixed in photosynthesis). We are not aware of any lular phosphatases in the rhizosphere assure the respective studies on N2-fixing trees. breakdown of organic P compounds in the proximity of roots. Phosphatases contain 8–32 % of N (Treseder

Phosphorus in the N2-fixing trees and Vitousek 2001), and it has been proposed that N2- fixing plants have an advantage in producing these Phosphorus has been considered a limiting nutrient for N-rich compounds (Wang et al. 2001). Houlton et al. symbiotic N2 fixation process because of the high ATP (2008) presented a hypothesis that improved P 123 Nutr Cycl Agroecosyst nutrition because of a higher production rate of nodulation of Leucaena leucocephala seedlings and extracellular phosphatases may explain the abundance dual inoculation was recommended (Arau´jo et al. of legume trees in P-poor humid tropical forests. 2001). Dual rhizobia-AMF inoculation also enhanced Higher phosphatase activity has been observed in the initial growth of C. calothyrsus under field conditions soil below both actinorrhizal (Giardina et al. 1995; but statistically significant differences between vari- Zou et al. 1995) and legume trees (Allison et al. 2006; ous inoculation treatments disappeared after 24 months

Zou et al. 1995) than below non-N2-ﬁxing trees. As far (Lesueur and Sarr 2008). It has also been shown that the as we know, no studies on the phosphatase production percentage of roots infected by mycorrhizae is mark- in AFS exist. However, it may be an important yet at edly higher in nodulated than non-nodulated actinor- the moment unstudied positive interaction between rhizal plants (Chatarpaul et al. 1989).

N2-fixing trees and crops in AFS. Based on their own observations and earlier work The fact that P fertilisation enhanced growth of tree (Habte 1995; Habte and Turk 1991; Manjunath and seedlings in pot experiments (Diouf et al. 2008; Pons Habte 1992), Ingleby et al. (2001) concluded that et al. 2007; Uddin et al. 2008) but results on mature among the common agroforestry trees, N2-fixing C. trees in the field were more variable (Gokkaya et al. calothyrsus, Gliricidia sepium, and L. leucocephala,

2006; Uliassi and Ruess 2002) suggest that caution and the non-N2-ﬁxing Senna siamea (Lam.) H.S. Irwin should be applied for extrapolating the pot results to the & Barneby are highly responsive to mycorrhizae while

field. In one case, the same tree species, Falcataria the non-N2-fixing Senna reticulata (Willd.) H.S. Irwin moluccana (Miq.) Barnaby & J.W. Grimes, was & Barneby and N2-fixing Sesbania pachycarpa DC. studied both in a pot culture and in a forest. Phosphorus are less responsive. fertilisation enhanced N2 fixation by seedlings in the Most studies revised indicate beneficial effects of pot study (Binkley et al. 2003), while higher phospha- the tripartite symbiosis on plant development and N2 tase activity was observed in soils under F. moluccana fixation. The beneficial effect of mycorrhizae seems to than non-N2-fixing trees in Hawaii (Allison et al. be most important under P limitation supporting 2006). It is possible that mature trees and associated Bethlenfalvay’s (1992) suggestion that the main func- mycorrhizae produce more phosphatases than seed- tion of the mycorrhizal symbiont is P supply to the lings and, thus, better cope with soil P deficit. tripartite system. The observations that AMF colonisation was more beneficial than P fertilisation (Arau´jo

Interaction between N2-fixing bacteria et al. 2001) and that dual inoculation enhanced biomass and mycorrhizae production independently of P level (Weber et al. 2005) suggest, however, that mycorrhizal colonisation Mycorrhizae may also alleviate the effects of soil P may enhance the functioning of the tripartite symbiotic deficit on N2 fixation through enhanced P supply to the system also in ways other than just increased P supply. tripartite symbiotic system. Best growth and highest It should also be noted that mature trees might function shoot nutrient concentrations were observed in P differently as host than seedlings or saplings. fertilised Acacia senegal seedlings inoculated with rhizobia and dual inoculation with rhizobia and AMF was the second best treatment (Colonna et al. 1991). In Estimates of N2 fixation in agroforestry systems a sterile, low P substrate, AMF colonisation in roots of Calliandra calothyrsus seedlings decreased with Field estimates on the dependence of agroforestry increasing P addition (Ingleby et al. 2001). Biomass trees on symbiotic N2 fixation production was not affected by AMF colonisation or P level but higher nodulation was observed in the AMF- When compiling quantitative data on N2 fixation in colonised plants. Rhizobia-ECM dual inoculated AFS, we excluded seedling studies, because we have Acacia holosericea A. Cunn. ex. G. Don seedlings shown in the previous chapter that seedlings may had the highest biomass production (Andre´ et al. behave quite differently from mature trees. On the 2005). Both AMF and ECM colonisation enhanced other hand, many legume trees used in AFS are fast- nodulation in the same tree species (Duponnois and growing and some management practices, notably Plenchette 2003). Inoculation with AMF enhanced green pruning, maintain them in a physiological state 123 Nutr Cycl Agroecosyst resembling juvenile trees, e.g. by impeding flowering review by Andrews et al. (2011), 42 % (SD 5.4 %).

(Nygren et al. 2000). Thus, we accepted data from Because competition with non-N2-fixing plants seems experiments with at least 1-year-old saplings. We also to increase the legume dependence on N2 fixation included data on tree plantations if the tree species is (Andrews et al. 2011) competition with crops may known to be used in agroforestry. Data were most enhance N2 fixation by trees in AFS. Soil N depletion abundant on the %Ndfa including 38 data points due to N export in crop harvest may also partially

(Table 2). We classified the data according to man- explain the higher legume dependence on N2 fixation agement practices and climate. Climate was classified in AFS than natural ecosystems. into three broad categories based on our interpretation Because of the high within-group variation and low of the data given in original articles: dry (water deficit number of studies under dry conditions, we compared prevails most of the year), subhumid (seasonal climate statistically only the three distinct cases under humid with water deficit for six months or less), and humid or subhumid conditions: young trees (1–2 years) and (short water deficit periods or none at all). Two irrigated older systems ([2 years) further divided into pruned experiments (Gauthier et al. 1985; Kadiata et al. 1997) and unpruned practices. Juvenile trees had a signifi- were pooled with humid climate in the following analyses. cantly higher %Ndfa than mature trees in unpruned We pooled data on 15N dilution (Eq. 2) and natural systems while mature trees in pruned systems did not abundance (Eq. 4) methods. The general mean of the differ significantly from either juvenile or unpruned %Ndfa of the compiled data (Table 2) was 59 % mature trees (analysis of variance followed by Dun- (Standard deviation, SD 16.6 %) and the range was can’s Multiple Range Test at 5 %). The ANOVA had a from 10 to 85 %. We used the mean of minimum and low r2 value, 0.258 with significant P value (0.0176). maximum in the calculation of the general mean when Thus, no strong conclusions may be drawn but the a range was given in Table 2. Five studies were differences reveal some possible trends that may be conducted in dry environments (Aronson et al. 2002; fruitful for further research together with causes for Gueye et al. 1997; Isaac et al. 2011b; Raddad et al. the ranges observed in several cases included in 2005; Unkovich et al. 2000) with the mean %Ndfa of Table 2. Especially a meta-analysis on these effects 49 % (SD 25.3 %, N = 8). The data compiled also will be interesting when more data become available. included ten cases of young (1–2 years) trees under The ranges reported in Table 2 were caused by five humid or sub-humid conditions with mean %Ndfa of factors. Differences because of reference species 69 % (SD 10.7 %). The remaining 20 studies were (Sta˚hl et al. 2005) or tree organ (Chikowo et al. conducted with at least 2-year-old trees under humid 2004) are methodological issues. A range of N2 or subhumid conditions with mean %Ndfa of 57 % fixation estimates based on various reference species (SD 12.1 %). However, only three of these studies would be more reliable than single value because the were conducted in systems that had been subjected to range also gives an estimate of the reliability of the the same management for several years, i.e. a 15-year- estimates. However, the range was reported only by old shaded cacao plantation (Nygren and Leblanc Sta˚hl et al. (2005). Interestingly, the same reference

2009), a 12-year-old improved fallow of Acacia species gave very similar N2 fixation estimates in mangium (Mercado et al. 2011), and an 8-year-old another study and the seasonality had a much stronger cut-and-carry forage production system under partial effect on the N2 fixation estimates (Sta˚hl et al. 2002). pruning regime (Nygren et al. 2000). The studies on at An estimate based on the average d15N of several non- least 2-year-old systems under humid or subhumid N2-fixing references was reported in three other conditions were further divided into pruned and articles (Aronson et al. 2002; Lesueur and Sarr 2008; unpruned systems, with the mean %Ndfa of 63 % Nygren and Leblanc 2009). The estimates based on (SD 12.0 %, N = 7) and 54 % (SD 11.7 %, N = 13), one or several herbaceous references (Chikowo et al. respectively. The popular agroforestry tree Gliricidia 2004; Mercado et al. 2011; Unkovich et al. 2000) sepium appeared eight times in Table 2 being the only should be dealt with caution; we agree with Unkovich species, for which we calculated the species mean for et al. (2008) that the reference should be of the same

%Ndfa, 67 % (SD 13.0 %). life form as the N2-ﬁxer. The wide range of estimates All these means are higher than the general mean for different tree organs (Chikowo et al. 2004) may be for legume trees in natural ecosystems according to the caused by the use of the Hyparrhenia rufa (Nees) 123 123 Table 2 Percentage of nitrogen derived from atmosphere out of total nitrogen (%Ndfa) in several tree species used in agroforestry

N2-ﬁxing Associated species Reference species Climate System Management Estimation method %Ndfa References species information

Acacia Zea mays Hyparrhenia rufa Subhumid Improved fallow Unpruned 15N natural abundance; 48–79a Chikowo angustisima whole tree et al. (2004) Acacia caven None Schinus polyganus and Dry Experimental Free growth 15N natural abundance; 50 Aronson et al. Fraxinus excelsior plantation leaf biomass (2002) Acacia Eucalyptus grandis Eucalyptus grandis Subhumid Mixed plantation Free growth 15N enrichment; whole 59 Bouillet et al. mangium tree biomass (2008) Acacia None Eucalyptus urophylla Humid Experimental Free growth 15N natural abundance; 42–62b Galiana et al. mangium plantation sampling not reported (1998) Acacia None Understorey weeds Humid Improved fallow, Free growth 15N natural abundance; 57 Mercado et al. mangium 12 years whole tree (2011) Acacia senegal None Balanites aegyptiaca Dry Experimental arabic Free growth 15N natural abundance; 24–61c Raddad et al. gum production leaf biomass (2005) Acacia senegal Grass Balanites aegyptiaca Dry Tree-grass savanna Free growth 15N natural abundance; 33–39d Isaac et al. leaf biomass (2011b) Albizia lebbeck None Senna siamea Irrigated 16-month Variable pruning 15N enrichment; shoot 74–83e Kadiata et al. greenhouse frequency biomass (1997) experiment Calliandra None Eucalyptus deglupta and Subhumid Improved fallow Free growth 15N enrichment; whole 5–54f Sta˚hl et al. calothyrsus Grevillea robusta tree biomass (2002) Calliandra None Senna spectabilis Subhumid Protein bank 7 prunings in 2 years 15N natural abundance; 24–84f Peoples et al. calothyrsus whole tree (1996) Calliandra Khaya senegalensis, Ziziphus Mean of associated tree Not reported Mixed experimental Free growth 15N natural abundance; 30–60b Lesueur and calothyrsus mauritaniana and Anacardium species (subhumid plantation; 2 years leaf biomass Sarr (2008) occidentale assumed) Casuarina Eucalyptus x robusta Eucalyptus x robusta Sudhumid-humid Mixed tree Free growth 15N enrichment; whole 59 Parrotta et al. equisetifolia plantation tree biomass (1996) Casuarina None None Irrigated 1-year experiment Free growth 15N dilution; shoot 55 Gauthier et al. equisetifolia biomass (1985) Chamaecytisus None Schinus polyganus and Dry Experimental Free growth 15N natural abundance; 84 Aronson et al. proliferus Fraxinus excelsior plantation; 6 years leaf biomass (2002) 15 Chamaecytisus Sequential Lupinus angustifolius and Mean of Ptilotus Dry Experimental alley Pruned N natural abundance; 83 Unkovich Agroecosyst Cycl Nutr proliferus Avena sativa polystachus and cropping, 4 years coppice biomass et al. (2000) annual weeds Codariocalyx None Senna spectabilis Subhumid Protein bank 7 prunings in 2 years 15N natural abundance; 48–86f Peoples et al. gyroides whole tree (1996) Erythrina fusca None Vochysia guatemalensis Humid 14-month-old Free growth 15N enrichment; whole 64 Leblanc et al. saplings tree (2007) urCc Agroecosyst Cycl Nutr Table 2 continued

N2-ﬁxing Associated species Reference species Climate System Management Estimation method %Ndfa References species information

Erythrina Vanilla planifolia Non-nodulated Humid Living support Free growth 15N natural abundance; 53 g Salas et al. lanceolata phenophase leaf biomass (2001) Erythrina None Vochysia guatemalensis Humid 14-month-old Free growth 15N enrichment; whole 59 Leblanc et al. poeppigiana saplings tree (2007) Faidherbia None Parkia biglobosa Dry 15-month-old Free growth 15N enrichment; whole 54 Gueye et al. albida saplings tree (1997) Gliricidia Dichanthium aristatum Gmelina arborea Subhumid Cut-and-carry fodder 100 % pruning every 15N natural abundance; 54–92f Nygren et al. sepium production 6 months shoot biomass (2000) Gliricidia Dichanthium aristatum Gmelina arborea Subhumid Cut-and-carry fodder 50 % pruning every 15N natural abundance; 60–87f Nygren et al. sepium production; 2 months shoot biomass (2000) 8 years Gliricidia None Senna spectabilis Subhumid Protein bank 7 prunings in 2 years 15N natural abundance; 58–89f Peoples et al. sepium whole tree (1996) Gliricidia Sequential Zea mays and Oryza Senna spectabilis Humid Alley cropping 4 prunings per year at 15N natural abundance; 35–59f Ladha et al. sepium sativa 50 cm shoot biomass (1993) Gliricidia Arachis pintoi (?) Peltophorum Subhumid Alley cropping Complete pruning, 15N enrichment; 50 Rowe et al. sepium dasyrrachis frequency not prunings (1999) reported Gliricidia Theobroma cacao Theobroma cacao Humid 14-month-old Free growth 15N enrichment; whole 85 Kurppa et al. sepium saplings tree (2010) Gliricidia Theobroma cacao, Bactris gasipaes, Mean of 4 non-legume Humid Shaded cacao Free growth 15N natural abundance; 57–67f Nygren and sepium non-leg. trees tree spp. plantation; leaf biomass Leblanc 12 years (2009) Gliricidia None Senna siamea Irrigated 16-month Variable pruning 15N enrichment; shoot 69–75d Kadiata et al. sepium greenhouse frequency biomass (1997) experiment Inga edulis None Vochysia guatemalensis Humid 14-month-old Free growth 15N enrichment; whole 57 Leblanc et al. saplings tree (2007) Inga edulis Theobroma cacao Theobroma cacao Humid 14-month-old Free growth 15N enrichment; whole 74 Kurppa et al. saplings tree (2010) Inga edulis Theobroma cacao, Cordia alliodora Cordia alliodora Humid Shaded cacao Free growth 15N natural abundance; 50–63f Nygren and plantation; stem biomass Leblanc 15 years (2009) Leucaena Eucalyptus x robusta Eucalyptus x robusta Subhumid Mixed tree Free growth 15N enrichment; whole 38 Parrotta et al. leucocephala plantation tree biomass (1996) 123 Leucaena None Senna siamea Irrigated 16-month Variable pruning 15N enrichment; shoot 80–84e Kadiata et al. leucocephala greenhouse frequency biomass (1997) experiment 123 Table 2 continued

N2-ﬁxing Associated species Reference species Climate System Management Estimation method %Ndfa References species information

Prosopis alba None Schinus polyganus and Dry Experimental Free growth 15N natural abundance; 10 Aronson et al. Fraxinus excelsior plantation; 6 years leaf biomass (2002) Prosopis None Schinus polyganus and Dry Experimental Free growth 15N natural abundance; 30 Aronson et al. chilensis Fraxinus excelsior plantation; 6 years leaf biomass (2002) Sesbania Zea mays (sequential) Hyparrhenia rufa Subhumid Improved fallow Free growth 15N natural abundance; 42–73a Chikowo sesban whole tree et al. (2004) Sesbania None Eucalyptus deglupta and Subhumid Improved fallow Free growth 15N enrichment; whole 70–81f Sta˚hl et al. sesban Grevillea robusta tree biomass (2002) Sesbania None Eucalyptus deglupta and Subhumid Improved fallow Free growth 15N enrichment; whole 61–71 h Sta˚hl et al. sesban Grevillea robusta tree biomass (2005)

a Depending on organ b Depending on rhizobial strain c Depending on tree provenance d Depending on tree age e Depending on pruning frequency f Depending on sampling time g Rainy season (non-nodulated in dry season) h Depending on reference species urCc Agroecosyst Cycl Nutr Nutr Cycl Agroecosyst

Stapf grass as the reference; thus, organs of the N2 ﬁxation was observed in mature trees over a rainfall reference are not comparable with tree organs. gradient 30–400 mm year-1 in Namibia (Schulze Sampling time (Ladha et al. 1993; Nygren and et al. 1991). The former result is based on a

Leblanc 2009; Nygren et al. 2000; Peoples et al. 1996; provenance that was a superior N2-fixer in a pot study Sta˚hl et al. 2002) refers to the seasonality of N2 and the trees were apparently very small, with total N fixation, which is mostly out of the control of accumulation of only 1.41 g [N] per seedling (Gueye agronomic management. The influence of tree prov- et al. 1997). Functional nodules seem to be present in enance (Raddad et al. 2005) and rhizobial strain F. albida only during 2-3 months each year when the (Galiana et al. 1998; Lesueur and Sarr 2008) imply soil is superficially humid at the end of the rainy intrinsic factors that regulate N2 fixation. These season and beginning of the dry season when tree influences may be managed by proper selection of foliage grows (Roupsard 1997). High variation has tree sources, strains of N2-fixing bacteria, and their been observed in N2 fixation of F. albida seedlings in combinations. Finally, pruning frequency (Kadiata controlled environments (Gueye et al. 1997; Ndoye et al. 1997; Nygren et al. 2000) is essentially a et al. 1995; Sellstedt et al. 1993). Thus, field estimates management issue under the control of the farmers. on N2 fixation in different environments and AFS are needed for this important tree species. Under field Dryland agroforestry conditions, vigorous provenances from Burundi fixed

more N2 than provenances from Niger and Burkina It is obvious from the data collected in Table 2 that dry Faso (Roupsard 1997). environment poses challenges for the use of N2-fixing Among actinorrhizal plants, Casuarinaceae species, trees in AFS. Although the dry environment seems to mostly used in AFS in arid and semi-arid regions, are reduce the average dependence on N2 fixation, the known for their drought and temperature resistance high within-group variation in the data indicates that and Frankia spp. isolates obtained from these species some woody legumes may form N2-fixing symbiosis have high optimal growth temperatures (Dawson well adapted to dry habitats. In fact, the mean for dry 2008). environments was biased upwards because of high These results indicate that it is possible to find tree

%Ndfa observed in Chamaecytisus proliferus (L. f.) species and provenances that are active N2-fixers in Link in dry Central Chile (84 %; Aronson et al. 2002) dry environments. Thus, we recommend screening and Australia (83 %; Unkovich et al. 2000). These trials of N2-fixing tree species and provenances for numbers were comparable only with juvenile G. selecting suitable germplasm for dryland agroforestry. sepium under constantly humid conditions in Costa No potentially ‘‘N2-fixing tree’’ should be recom- Rica (85 %; Kurppa et al. 2010). The adverse effect of mended for dryland agroforestry before its N2 fixation dry environment on N2 fixation was also observed in a capacity is verified. study of 11 Mimosoideae species in natural ecosystems of Namibia with %Ndfa typically varying Dinitrogen-fixing trees in acidic soils 10–30 % (Schulze et al. 1991). They also observed considerable variation between species with %Ndfa of Acidic soils of the humid tropics are often cited as a

49 % in two species, the maximum of 71 %, and the major constraint to N2 ﬁxation (Kass 1995) because lowest indicating no N2 ﬁxation. High variation most rhizobia and many Frankia strains do not (%Ndfa 24–61 %) was also observed between Acacia tolerate high acidity and consequent solubilisation of senegal provenances in the Sudan (Raddad et al. aluminium. However, stains tolerant to acidity exist, 2005). especially among Bradyrhizobium spp. (Graham

Faidherbia albida (Delile) A. Chev., a tree species 1992) and N2-ﬁxing trees are abundant in the humid now strongly recommended for AFS in semi-arid tropical forests with acidic soils (McKey 1994; Roggy

Africa (Garrity et al. 2010), was cited as a N2-fixer in et al. 1999a, b). Although some Frankia strains 39 references found in CAB Abstracts data base but survive in various acidic soils, a negative correlation only twice N2 fixation was estimated in the field. was found between soil acidity and nodulation in Active N2 fixation with %Ndfa 54 % at 15-month-age Alnus spp. (Smolander and Sundman 1987) and was observed in Senegal (Gueye et al. 1997), while no Elaeagnus angustifolia L. (Jamann et al. 1992; Zitzer 123 Nutr Cycl Agroecosyst

Table 3 Nitrogen ﬁxation per hectare and year in some agroforestry systems with legume or actinorrhizal trees based on mea- surement of whole plant N isotopic relations or whole system N balance

N2-ﬁxing Associated species Reference species Climate System Management Estimation N2 ﬁxation References species information method kg ha-1 year-1

Acacia Zea mays Hyparrhenia rufa Subhumid Improved Free growth 15N natural 91a Chikowo angustisima fallow abundance; et al. whole tree (2004) Acacia None Eucalyptus sp. Humid Improved Free growth Whole system 140 Bernhard- auriculiformis fallow, N balance Reversat 7 years (1996) Acacia None Understorey weeds Humid Improved Free growth 15N natural 129b Mercado mangium fallow, abundance et al. 12 years (2011) Acacia None Eucalyptus sp. Humid Improved Free growth Whole system 115 Bernhard- mangium fallow, N balance Reversat 7 years (1996) Acacia None Eucalyptus Subhumid Improved Free growth Whole system 90 Harmand polyacantha camaldulensis fallow, N balance (1998) 7 years Calliandra None Eucalyptus deglupta Subhumid Improved Free growth 15N 122–171c Sta˚hl et al. calothyrsus and Grevillea fallow enrichment (2002) robusta Calliandra None Cassia spectabilis Subhumid Protein bank 7 prunings in 15N natural 670d Peoples calothyrsus 2 years abundance et al. (1996) Casuarina Eucalyptus x robusta Eucalyptus x Subhumid Mixed tree Free growth 15N 73 Parrotta equisetifolia robusta plantation enrichment et al. (1996) Chamaecytisus Sequential Lupinus Mean of Ptilotus Dry Experimental Pruned 15N natural 83a Unkovich proliferus angustifolius and polystachus and alley abundance et al. Avena sativa annual weeds cropping, (2000) 4 years Chamaecytisus None Mean of Ptilotus Dry Experimental Pruned 15N natural 390a Unkovich proliferus polystachus and plantation, abundance et al. annual weeds 4 years (2000) Gliricidia Dichanthium None Subhumid Cut-and-carry 50 % pruning Whole system 555e Dulormne sepium aristatum (grass) fodder every N balance et al. production 2-4 months (2003) Gliricidia None Cassia spectabilis Subhumid Protein bank 7 prunings in 15N natural 675d Peoples sepium 2 years abundance et al. (1996) Leucaena Eucalyptus x robusta Eucalyptus x Subhumid Mixed tree Free growth 15N 74 Parrotta leucocephala robusta plantation enrichment et al. (1996) Sesbania sesban Zea mays (sequential) Hyparrhenia rufa Subhumid Improved Free growth 15N natural 56 Chikowo fallow abundance; et al. whole tree (2004) Sesbania sesban None Eucalyptus deglupta Subhumid Improved Free growth 15N 282–363c Sta˚hl et al. and Grevillea fallow enrichment (2002) robusta Sesbania sesban None Eucalyptus deglupta Subhumid Improved Free growth 15N 310–356f Sta˚hl et al. and Grevillea fallow enrichment (2005) robusta a Including ﬁxed N in litter b Fixed N in roots estimated not measured c Depending on sampling time d The authors did not show the whole tree values but provided data on N partitioning between shoot and root that we used for recalculating the estimate e All ﬁxed N in the agroecosystem, including net transfer to the crop and soil accumulation f Depending on reference species

123 Nutr Cycl Agroecosyst and Dawson 1992). We did not observe any trend transplanting (Galiana et al. 1998). The longest related to soil acidity when compiling data for persisting effect of which we are aware, is the Table 2. detection of rhizobial strains used for inoculating

In a screening of N2-fixing potential of legume Leucaena leucocephala 10 years after introduction in trees, Roggy et al. (1999b) classified 110 species as a soil with only a few native rhizobia capable of supposed N2-fixers and 33 species as supposed non- nodulating it (Sanginga et al. 1994). N2-fixers in a rain forest in French Guiana. In a Table 1 indicates high diversity of rhizobia and selection trial of 25 legume tree species for forestry Frankia spp. nodulating different tree genera. Further, and agroforestry in Costa Rica, 18 species were both controlled-environment studies (Bala and Giller inspected for nodulation and 9 for nitrogenase activity; 2006) and genetic analyses of the rhizobia infecting all of them appeared to be N2-fixers, including Acacia, legumes in the field (Diouf et al. 2007) indicate a high Albizia, Dahlbergia, Erythrina, Inga, and Pithecello- intra-specific genetic diversity among the rhizobia. bium spp. (Tilki and Fisher 1998). As is unfortunately Thus, the importance of rhizobial inoculation, at least common, no attempts to estimate N2 fixation were with selected commercial strains, is questionable in made in these studies but they indicate that a large small-holder agriculture and scarce resources may be collection of potential N2-fixers exists for acidic soils. better used for management improvements (Giller and Promising N2-fixers for AFS under these conditions Cadisch 1995). In large-scale nursery production, include Acacia mangium, Codariocalyx gyroides however, good legume-rhizobia combinations may (Roxb. ex Link) X.Y. Zhu, Erythrina fusca Lour., E. improve the seedling establishment in the field (cf. poeppigiana, Inga edulis, and G. sepium that has been Galiana et al. 1998; Lesueur and Sarr 2008). The introduced from seasonal to the humid tropics observation that Rhizobium tropici type rhizobia are (Tables 2 and 3). Another introduction from seasonal the most efficient symbionts in acidic soils, Mesorhiz- climate, C. calothyrsus, has variable performance obium strains in intermediate, and Sinorhizobium (Table 2; Peoples et al. 1996). strains in alkaline soils (Bala and Giller 2006) indicates that the host 9 rhizobia interactions may differ in different soils. Thus, only local trials may be Host-bacteria interaction valid for a particular environment. Use of selected inoculants may be necessary when

Rhizobial strain may have a significant effect on N2 N2-fixing trees are introduced to new areas. When fixation by the symbiotic system (Lesueur and Sarr exotic legume or actinorrhizal trees have been planted 2008). Many of the studies on the host 9 rhizobia in new locations where they did not occur previously, interaction have been conducted with seedlings under scarce nodulation or absence of nodulation has been controlled conditions (Andre´ et al. 2005; Bala and observed (Diem and Dommergues 1990; Sanginga Giller 2001; Makatiani and Odee 2007; Weber et al. et al. 1994; Woomer et al. 1988). It has been postulated 2005). Although they clearly indicate the importance that in the absence of a compatible host, rhizobial or of the interaction, practical conclusions are hard to Frankia strains are confronted with competition from make beyond nursery production and early establish- other soil bacteria and cannot maintain their popula- ment in the field. Lesueur and Sarr (2008) observed tion or can lose host specificity encoding genes that apparent growth differences between triple sym- (Barnet 1991). biotic systems of C. calothyrsus host and rhizobial and AMF strains disappeared after 5 months in the field. Green pruning However, plants inoculated with AMF had higher foliar N, P and K content until 12 months after Green pruning of N2-fixing trees is a common practice transplanting. According to the genetic analysis, one in many AFS for avoiding excessive shading of the of the rhizobial strains persisted in the nodules of crop (Kang et al. 1981; Akinnifesi et al. 2008), C. calothyrsus also in the field. Rhizobia-inoculated enhancing nutrient cycling (Beer et al. 1998; Kass Acacia mangium grew better than non-inoculated trees et al. 1997), or harvesting tree fodder (Peoples et al. up to 39 months after transplanting with detection of 1996). Symbiotic N2 fixation is often mentioned as an the efficient strains in nodules 42 months after important benefit in these systems because it is 123 Nutr Cycl Agroecosyst

assumed to contribute to increase in soil organic green pruning are species-specific and N2 fixation matter and N reserves (Beer et al. 1998; Haggar et al. should be estimated in all trials for selecting trees for 1993) or to produce protein-rich fodder (Peoples et al. AFS managed by pruning. 1996). Data in Table 2 indicate that pruned trees are often active N2-fixers with higher %Ndfa in pruned Whole tree N2 fixation than unpruned systems. Because green pruning may result in partial rejuvenation of the trees (Nygren et al. Much less data were available on the N2 fixation by 2000), the higher mean %Ndfa in pruned systems may agroforestry trees in terms of kg [Ndfa] ha-1 year-1, be related to the higher dependence on N2 fixation in including fixed N in the root system (Table 3) than on young trees also observable in Table 2. The placement the %Ndfa. Dulormne et al. (2003) reported the of the pruned trees as an intermediate group between estimate based on all fixed N tracked in a cut-and-carry the juvenile and unpruned mature trees in the statis- system of G. sepium and fodder grass Dichanthium tical analysis of Table 2 provides partial support for aristatum, i.e. in the trees, grass, and soil over this ‘‘rejuvenation hypothesis’’. 12 years. The whole system N balance was also used

Not all trees fix actively N2 under a periodic to estimate N2 fixation by 7-year-old Acacia mangium, pruning regime. Complete nodule turnover was Acacia auriculiformis A. Cunn. ex. Benth. (Bernhard- observed in Erythrina poeppigiana in 2 weeks after Reversat, 1996), and Acacia polyacantha Willd. complete pruning. Renodulation initiated at 10 weeks (Harmand 1998). Other data refer to fixed N in the after pruning and nodulation was abundant at trees and litter during a time range from 18 months 14 weeks after pruning (Chesney and Nygren 2002; (Sta˚hl et al. 2005) to 12 years (Mercado et al. 2011).

Nygren and Ramı´rez 1995). Leaving only 5 % of All data were converted to correspond to the annual N2 foliage in prunings twice-a-year was enough to retain fixation. Table 3 indicates a significant contribution of low nodulation in 2-year-old trees and caused stabi- N2 fixation to the N economy of the trees or whole lisation of nodule growth rather than turnover in system. Trees pruned in AFS seem to fix more N than 8-year-old trees (Chesney and Nygren 2002). The unpruned trees also in mass terms as the top three nodule turnover after complete pruning of E. poepp- systems (Dulormne et al. 2003; Peoples et al. 1996; igiana seemed to be a response to C starvation because Unkovich et al. 2000) were managed by tree pruning. of cessation of photosynthesis. Nodulation after Mafongoya et al. (2004) cite a ‘‘typical’’ range of foliage regrowth appeared to be regulated by canopy N2 fixation by trees in AFS to be 70–200 kg [Ndfa] - N needs with high nodulation when N flow was ha-1 year-1. Six out of the 16 cases compiled in unidirectional from roots to foliage and reduction in Table 3 exceeded this range, probably because we nodule biomass when N flow to roots initiated (Nygren accepted only estimates based on whole tree harvest- 1995), in line with the theoretical scheme on the ing—including root system – or whole system N regulation of N2 fixation presented in Fig. 1. balance. Schroth et al. (1995) estimated that the N Even stronger negative response to green pruning reserves in roots of 0–5 mm diameter of six legume was observed in Erythrina lanceolata, in which tree species were 60–133 kg [N] ha-1 in 5-year-old nodulation did not recover from complete pruning fallows in a sub-humid area in Coˆte d’Ivoire. A similar twice-a-year and partial pruning every three months amount, 71.5 kg [N] ha-1, was observed in fine roots caused a significant reduction in nodulation and of 0–2 mm diameter of Inga edulis shade trees in an %Ndfa in comparison to unpruned control (Salas organically-grown cacao plantation under humid et al. 2001). In contrast, pruning regime did not affect conditions in Costa Rica (Go´mez Luciano 2008). -1 the %Ndfa in Gliricidia sepium but total N2 fixation in Higher amount, 190 [N] ha , was observed in fine mass terms was reduced by bimonthly partial prunings roots of 0–2 mm in a 7-year-old Acacia polyacantha in comparison to twice-a-year complete pruning fallow in the subhumid zone of Cameroon (Harmand (Nygren et al. 2000). Calliandra calothyrsus, Coda- et al. 2004); whole root system of A. polyacantha riocalyx gyroides (Peoples et al. 1996), Albizia contained 342 kg [N] ha-1. Giller (2001) refers to the lebbeck (L.) Benth., and Leucaena leucocephala range of 26–60 % of legume tree N to be below-

(Kadiata et al. 1997) also actively ﬁxed N2 when ground, under variable ﬁeld and experimental pruned heavily. These data indicate that responses to conditions. 123 Nutr Cycl Agroecosyst

Based on harvestable biomass only, it was estimated competition for soil N with the crop in unpruned shade that G. sepium would fix only 147 kg [N] ha-1 year-1 tree systems and part of the fixed N is recycled to the (Nygren et al. 2000) in the system studied by Dulormne crop in leaf and root litter of the trees (Escalante et al. et al. (2003). However, N2 fixation by G. sepium is the 1984; Nygren and Leblanc 2009; Salas et al. 2001; only N input to this system and N balance taking into Santana and Rosand 1985). Animal diet is enhanced account N export in fodder harvest, N uptake by the by protein-rich tree foliage of the N2-fixing trees in companion grass, and soil N accumulation revealed the fodder production systems, (Blair et al. 1990; Peoples considerably higher estimate (Table 3). Thus, it is et al. 1996). obvious that below-ground N as well as fixed N All N2-fixing plants form the symbiosis for their released by the trees to the environment should be own N supply and release N to the environment only taken into account when estimating the total amount of when they have it in excess (Fig. 1). Nitrogen fixed by

N2 fixation in mass terms. These data are still scarce in the trees may become available for the crop via agroforestry literature. indirect or direct pathways. Here, the indirect pathway refers to the complete microbial N cycle, including mineralisation of organic N in the litter or pruning ? Agronomic importance of N2 fixation residues to NH4 , partial immobilisation by soil by agroforestry trees microbiota and mobilisation because of microbial ? ? turnover, and nitrification of NH4 . While NH4 is State-of-art in agroforestry systems efficiently fixed by cation exchange on soil colloids, - NO3 may be lost from the system via leaching to Our review on the estimates of N2 fixation by several deep soil layers or denitrification, especially if it is tree species used in AFS shows that many of them are available in excess with respect to plant N needs. Soil active N2-fixers (Table 2) and symbiotic N2 fixation N cycle has been widely studied – also in the AFS with may contribute annually tens or hundreds of legume trees (Babbar and Zak 1994, 1995; Dulormne kg [N] ha-1 to the farming system (Table 3). A critical et al. 2003; Hergoualc’h et al. 2007, 2008; Kanmegne review of the original literature in the light of current et al. 2006; Mafongoya et al. 1998)—and a discussion knowledge on the methods for quantifying the N2 of the vast literature on the topic is out of our scope. fixation resulted in 16 estimates on the annual N2 Direct pathway refers here to the transfer of N from fixation per hectare that we considered reliable enough N2-fixing trees to crops either via a common mycelial for Table 3. Obviously, more data are needed at field- network (CMN) of mycorrhizae-forming fungi colon- and farm-level. Even less data are available for evalu- ising both trees and crops (He et al. 2003; Jalonen et al. ating the agronomic importance of N2 fixation in AFS at 2009b) or absorption of tree root exudates by the crop landscape, regional, or global level. Two recent reviews before the complete decomposition cycle (Fustec et al. on the effects of ‘‘fertiliser trees’’ on crop productivity 2010; Jalonen et al. 2009a). The latter pathway (Akinnifesi et al. 2010; Garrity et al. 2010) suggest that requires that either crop plants absorb simple amino inclusion of N2-fixing trees into traditional cropping acids (Lambers et al. 2008;Na¨sholm et al. 2009) from systems may significantly improve crop yields yet the root exudates or the N2-fixing plants exude evaluation of the contribution of the symbiotic N2 inorganic N (Fustec et al. 2010). fixation to the benefits was not evaluated.

The agronomic importance of N2 fixation by the Fate of fixed nitrogen trees depends on the function of the trees in an AFS. Nitrogen in soil and plant residues, including roots, In spite of the low C:N ratio (Mafongoya et al. 1998), after cutting an improved fallow is important for the legume tree mulches seem to have a relatively low succeeding crop (Chikowo et al. 2004; Mercado et al. efficiency as an immediate N source for a crop. Maize 2011; Sta˚hl et al. 2002; 2005). Nitrogen recycling in (Zea mays L.) gained 11 % of its N, ca. 10 kg [N] ha-1, the pruning residues potentially benefits the crop in from pruning residues of Gliricidia sepium and alley cropping and other green manure systems Erythrina poeppigiana in an alley cropping trial under (Haggar et al. 1993; Ladha et al. 1993; Unkovich humid tropical conditions (Haggar et al. 1993). In et al. 2000), while N2 fixation by the trees reduces another experiment, maize used ca. 10 % of the N 123 Nutr Cycl Agroecosyst available in the pruning residues of Leucaena leuco- immediate crop N nutrition (Beer et al. 1998; Haggar cephala (Vanlauwe et al. 1996). This apparently low et al. 1993; Kass et al. 1997). We partially share this efficiency depends on a cascade of factors. First, opinion. However, the role of root litter in the N cycle although amino acids and oligopeptides decompose of AFS has been neglected, although Nye and fast, some proteins decompose slowly or may even be Greenland (1960) proposed more than 50 years ago recalcitrant (Paul and Clark 1996). Thus, not all that dead tree roots may be an important nutrient residual N becomes available for an annual crop source for associated crops because they decompose in during a cropping cycle. close proximity of the absorbing crop roots. Pot Second, the N release is also restricted by the experiments with agroforestry combinations of maize general litter quality: if the mulch C is not easily with Paraserianthes falcataria (L.) I.C. Nielsen available for the decomposer microbes as an energy (Chintu and Zaharah 2003) and cacao with I. edulis source, also N release from the residue slows down. (Ka¨hko¨la¨ et al. 2012) suggest that root litter of the Cellulose and hemicellulose are easily decomposable legume tree may be a more efficient N source for the crop C compounds while lignin is recalcitrant (Paul and than leaf mulch or litter. Further, ca. 300 kg ha-1 year-1 Clark 1996). Further, many legume mulches have high of N fixed by G. sepium was recycled below-ground to content of polyphenols that may be toxic to the the soil and the associated fodder grass Dichanthium decomposers. Nitrogen release rate is significantly aristatum in a cut-and-carry fodder production system, higher from the legume mulches with a high N to where above-ground litter was eliminated by intensive polyphenols ratio than from residues with a low ratio fodder harvesting (Dulormne et al. 2003). (Barrios et al.1997, Ndufa et al. 2009). Following to these factors, 10–30 % of N in legume mulches is Direct N transfer between plants released in a month (Mafongoya et al. 1998) typically followed by an exponential decrease in the N release Reviews of N transfer via CMN (He et al. 2003; Simard rate with time. et al. 2002) and N exudation from legume roots (Fustec Third, soil microbiota are strong competitors for et al. 2010; Wichern et al. 2008)havebeenrecently soil N with the plants; free N in soil solution is published but data on AFS are scarce. Direct N flow absorbed by microbes on average within 24 h after its between plants is bidirectional and its net effect may be release (Jones et al. 2005). Mobility of N in soil almost nil to both components (He et al. 2004;Johansen depends on soil and N type. In a loamy soil, the 24-h and Jensen 1996). Net flow seems to be more common ? - diffusion distance of NH4 , amino acids, and NO3 is from a N2-fixing to a non-N2-fixing plant; e.g. from ca. 1.7, 1.5, and 10.2 mm, respectively (Jones et al. soybean to maize (Bethlenfalvay et al. 1991), from 2005). Thus, N mineralisation must occur in the Alnus subcordata C.A. Mey. and Elaeagnus angustifo- rhizosphere to be useful for a plant. Otherwise, it will lia to Prunus avium L. (Roggy et al. 2004), and from be absorbed by microbiota. Based on the 24-h soybean and peanut (Arachis hypogaea L.) to associated diffusion distance and data on fine root densities of weeds (Moyer-Henry et al. 2006). In a pot study, direct cacao in a plantation with Inga edulis shade (Go´mez NtransferfromA. senegal to durum wheat (Triticum Luciano 2008), we estimated that the cacao rhizo- turgidum L.) was enhanced when crop N uptake was ? sphere for NH4 absorption was 45.6 % out of total stimulated by high P availability and competition was soil volume in the 0–2 cm soil depth but only 16.4 % low (Isaac et al. 2012). in the 2–10 cm depth (Nygren and Leblanc, unpub- In several coffee plantations in Burundi with lished). If the soil has a high nitrification rate, the more different legume shade tree species, 6–22 % of N in - mobile NO3 is in better supply for plants but the high coffee leaves was of atmospheric origin (Snoeck et al. mobility makes it also subject to leaching to deeper 2000). In a cut-and-carry fodder production system, soil layers, where it may become unavailable for plant 27–35 % of N in D. aristatum grass (53–68 kg ha-1) roots (Babbar and Zak 1995; Harmand et al. 2007a). was of atmospheric origin. Nitrogen isotopic data The apparently low efficiency of new mulch as a suggested that atmospheric N was directly transferred crop N source has led some authors to argue that from the associated heavily pruned G. sepium (Sierra legume tree mulch is more important for long-term and Nygren 2006). In a cacao plantation with I. edulis build-up of soil organic matter and N reserves than shade, 8–25 % of N in cacao leaves was of 123 Nutr Cycl Agroecosyst atmospheric origin (Nygren and Leblanc 2009). Data further study before we well understand its occurrence on N isotopic relationships also suggest the possibility and importance in different AFS. of direct N transfer from L. leucocephala to understorey weeds in a short-rotation plantation (van Kessel et al. 1994) and from Inga oerstediana Benth. ex Management of N2 fixation Seem. to coffee and non-legume shade tree Liquid- ambar styraciflua L. in organic coffee farms in Possibilities to manage N2 fixation and N recycling are Chiapas, Mexico, (Grossman et al. 2006) although quite limited in AFS with unpruned N2-fixing trees. the authors themselves did not draw this conclusion. According to the data in Table 2, sampling time was Identification of the direct N transfer pathways is one of the factors affecting the %Ndfa. We interpreted difficult under AFS field conditions. Root exudates of this to reflect the seasonality of N2 fixation. If the many legume trees have a remarkably low C:N ratio: climate is seasonal, the trees and crops often follow the 3.3–5.7 in G. sepium (Jalonen et al. 2009b), 4.7–6.8 in same phenological cycle with higher metabolic activ- Robinia pseudoacacia (Uselman et al. 1999), and 5.2 ity and productivity during the same season. Because in I. edulis (Nygren and Leblanc, unpublished data). N2 fixation seems to be connected at least to some Thus, they are potentially good N sources for an extend to the N needs of the canopy (Fig. 1; Nygren associated crop but tree and crop rhizospheres should 1995), it is most active during the time of the highest overlap for the tree root exudates to be useful for the general metabolic activity. Follow-up of annual bio- crop. mass and N2 fixation dynamics of Erythrina lanceo- Formation of an effective CMN between trees and lata used as living support for vanilla (Vanilla crops in an AFS requires that both share compatible planifolia Andrews) under a seasonal climate in Costa strains of mycorrhizal fungi: anastomoses, functional Rica indicated that main litterfall occurred during the connections that allow material transfer between dry season (Berninger and Salas 2003) when no N2 mycelia of two fungi, have been observed only fixation was detected (Salas et al. 2001). Litterfall was between fungi of the same population yet they may close to nil during time of the most active foliage differ genetically (Croll et al. 2009). Glomus etunic- growth and N2 fixation. Thus, although the timing of atum Becker & Gerdemann and Gigaspora albida N2 fixation and N recycling in unpruned multistrata Schenck & Perez used for inoculating Calliandra AFS has not received much attention, we may expect calothyrsus spread their mycelia also to associated the N2 fixation to occur at the time of the highest maize and common bean (Phaseolus vulgaris L.) productivity of both trees and crops, thus, reducing (Ingleby et al. 2007). Millet (Pennisetum americanum competition between trees and crops. (L.) Leeke) was effectively colonised by inocula from An interesting case is the ‘‘reverse phenology’’ of Acacia nilotica (L.) Willd. ex Delile, A. tortilis Faidherbia albida in the African drylands (Garrity (Forssk.) Hayne, and Prosopis juliflora (Sw.) DC. et al. 2010): the tree is leafy during the dry season. The (Diagne et al. 2001). Inga edulis positively responded low water consumption during the cropping season to AMF originating from roots of cacao in a cross- and the use of deep-water reserves out of cropping inoculation trial indicating the potential of formation season reduce competition for water with the associ- of a CMN between these species (Iglesias et al. 2011). ated crops (Roupsard et al. 1999). However, N2 In a pot study, N was transferred from G. sepium to fixation is effective only 2–3 months during a year D. aristatum both via the CMN and root exudates when foliage growth begins (Roupsard 1997). Because (Jalonen et al. 2009b). Under field conditions, both N2 fixation occurs out of the cropping season F. albida species were colonised by Rhizosphagus intraradices adds N for crop via litterfall at the beginning of (ex. Glomus intraradices) and grass colonisation was cropping season. Green pruning may also reverse more abundant between the tree rows than in an phenology; Central American cattle ranchers prune adjacent pure grass plot (Jalonen et al. 2012). G. sepium at the beginning of dry season so that the It seems obvious that the potential for direct N trees remain leafy and provide dry season fodder transfer from legume trees to crops in AFS exists and (Hernańdez and Benavides 1994). positive evidence on this interaction has been found in Green pruning is generally timed according to the a few cases. However, the phenomenon requires crop phenology. Costa Rican coffee farmers 123 Nutr Cycl Agroecosyst traditionally prune shade trees leaving only a few decomposing tree mulches (‘‘high-quality’’ and ‘‘low- branches (ca. 5–10 % of foliage) to promote coffee quality’’ mulch) for enhancing the synchrony between flowering at the end of the drier season and make a the crop N demand and N release (Mafongoya et al. complete pruning for promoting the ripening of berries 1998; Nair et al. 1999;Palm1995; van Noordwijk about half year later. The highest coffee N demand et al. 1996). These authors, however, ignored the occurs 6–17 weeks after blossoming when the grow- effect of nodule turnover on trees’ N uptake. We argue ing berries may consume 95 % of newly assimilated N that temporarily non-N2-fixing trees enforce the root (DaMatta et al. 2007). The popular shade tree safety-net (van Noordwijk et al. 1996) by at least E. poeppigiana remains unnodulated about 10 weeks partially reabsorbing N leaching from the mulch following a complete pruning (Nygren and Ramı´rez application. Further, the nodule and root turnover 1995) but it retains reduced nodulation after a partial following the pruning may form a ‘‘high-quality pruning (Chesney and Nygren 2002). Similar pattern mulch’’ that releases N at the beginning of the has been observed in G. sepium but its renodulation cropping cycle. The trees renodulate and fix N2 – rate is higher after a pruning (Nygren and Cruz 1998). unless too heavily pruned – during the crop flowering Thus, in coffee AFS we may envision partial and seed production, thus, reducing competition with synchrony between coffee N needs and N2 fixation and the crop. N recycling from the shade trees. The green mulch Although at least partial synchrony between N2 from the pruning to promote blossoming may release fixation, N recycling, and crop N use is possible in N for active coffee foliage growth that occurs at the AFS with both perennial and annual crops, it must be time of coffee flowering (DaMatta et al. 2007) but noted that precision agriculture is not possible in green trees fix only limited amount of N2 for their own manure systems. Thus, the main benefit of N2-fixing foliage regrowth, thus, potentially competing for soil trees in AFS is probably the long-term accumulation N and reabsorbing N released from the pruning of soil N reserves (Haggar et al. 1993) although below- residues. Nitrogen released from nodule (Escalante ground N recycling and direct N transfer between et al. 1984; Nygren and Ramı´rez 1995) and fine root plants probably provide N for more immediate crop N turnover (Chesney and Nygren 2002) forms an needs than foliage litter or mulch applied on soil additional N source, which may be more efficient surface (Chintu and Zaharah 2003; Dulormne et al. than above-ground mulch (Chintu and Zaharah 2003; 2003; Jalonen et al. 2009b;Ka¨hko¨la¨ et al. 2012). The Ka¨hko¨la¨ et al. 2012). During the high coffee N need long-term accumulation of organic matter and nutri- for growing berries (DaMatta et al. 2007), the shade ents to soil is essential in improved fallow systems, in trees probably fix actively N2, which reduces compe- which cropping relies on the residual N after the fallow tition between the trees and the crop. The coffee N enhanced with N2-fixing trees (Chikowo et al. 2004; needs are low during the second annual pruning for Mercado et al. 2011; Sta˚hl et al. 2002, 2005). Few promoting berry ripening (DaMatta et al. 2007), and attempts have been made for studying the fate of the the residual N probably contributes to regrowth of the high N release to soil after clearing the fallow for trees themselves, accumulation of soil organic N, or N cropping. Nitrate leaching rates two year after clearing - losses through NO3 leaching. 7-year-old Acacia polyacantha, Eucalyptus camaldul- In alley cropping, hedgerow trees are pruned before ensis Dehnh., and Senna siamea fallows were similar, -1 sowing of the annual crop and, if the crop requires and ca. 10–15 kg [NO3–N] year (Oliver et al. 2000). trees tolerate, in a later phase of cropping season (Akinnifesi et al. 2010; Haggar et al. 1993; Kang et al. 1981; Vanlauwe et al. 1996). The pruning often refers Crop N needs and tree N2 fixation to complete defoliation. Thus, we may expect that a complete nodule turnover follows (Nygren and Cruz Perennial crops coffee, cacao, and tea (Camellia 1998; Nygren and Ramı´rez 1995) and trees do not fix sinensis (L.) Kuntze) cover globally ca. 21.6 Mha of

N2 at the beginning of the cropping cycle. Nitrogen agricultural land (Table 4). Perennial crops are often recycled in pruning residues is a strong input to the soil grown in AFS with N2-ﬁxing shade trees (Beer et al. when the crop is relatively small. Many authors 1998; Kass et al. 1997) although global estimates are recommend a combination of rapidly and slowly missing for other perennials than cacao; 7.8 Mha of 123 Nutr Cycl Agroecosyst

- cacao is cultivated in AFS (Zomer et al. 2009), which This results in NO3 leaching loss equivalent to N is 89 % out of the global cacao cultivation area of 8.73 needed for producing 1–4 Mg of green coffee (Babbar Mha (Table 4). Central American, Caribbean, and and Zak 1995; Harmand et al. 2007a, b) and increased

Andean coffee is mostly shaded while full-sun culti- emissions of nitrous oxide (N2O) (Hergoualc’h et al. vation prevails in large Brazilian plantations. 2008), which is a greenhouse gas with 206 times

We calculated the average yield of the perennial higher atmospheric forcing potential than CO2. crops based on the 2009 cultivation area and produc- Including N2-fixing trees to highly fertilised coffee tion data (FAOStat 2011) and estimated the N export farms increases N2O emissions but the emissions peak in crop harvest using the estimates on N needed for after fertilisation in both shaded and unshaded systems producing 1 Mg of harvest according to Bertsch (Hergoualc’h et al. 2008). -1 (2003): 30, 36, and 57 kg Mg for green coffee, Perhaps the best known practice for combining N2- cacao beans, and tea leaves, respectively. The average fixing trees with annual crops is alley cropping yields of all these three crops were relatively modest (Akinnifesi et al. 2008; Kang et al. 1981) that was and N export in the harvest was, consequently, quite intensively studied in the 1980s and 1990s but later low (Table 4). In addition to the N export in the abandoned. However, it showed some promise in harvest, part of N is used for producing permanent certain humid and subhumid areas (Akinnifesi et al. biomass. We compiled data on N accumulation to 2008; Kass et al. 1997) and an improved version is permanent biomass of coffee, cacao, non-legume now widely adopted by small-scale farmers in South- shade trees Cordia alliodora (Ruiz & Pav.) Oken ern Africa for maize cropping (Akinnifesi et al. 2008, and Eucalyptus deglupta Blume, and legume shade 2010). The main improvement was achieved by tree Erythrina poeppigiana in Table 5. Combining modifying the spatial arrangement of the trees for these few data with statistics in Table 4, we can reducing interference competition between trees and roughly estimate that on average ca. maize. Gliricidia sepium has been the most successful 40–50 kg [N] ha-1 year-1 is exported in the average tree species in these systems (Akinnifesi et al. 2008), harvest or immobilised in permanent biomass in which have been adopted by over 120,000 Malawian unshaded cacao and coffee plantations and ca. farmers (Garrity et al. 2010). 60–80 kg [N] ha-1 year-1 in shaded plantations. Palm (1995) estimated that 40 kg of N is needed for Table 3 shows that many trees commonly used in 1 Mg of maize yield. Gliricidia sepium may recycle AFS may fix enough N for fulfilling the current or even 70–126 kg ha-1 year-1 of N fixed from atmosphere increasing N needs of perennial crops. As green (estimated from alley cropping data in Ladha et al. pruning seems to enhance N2 fixation both proportion- 1993 and Rowe et al. 1999), which is sufficient for ally (Table 2) and absolutely (Table 3), it is a recom- harvesting ca. 1.7–3.1 Mg ha-1 year-1 of maize. mended practice for shade trees over perennial crops. Akinnifesi et al. (2010) report even higher sustained Green pruning of legume trees Erythrina fusca, maize yields with G. sepium without fertiliser in E. poeppigiana,andI. edulis under typical management Malawi (around 4 Mg ha-1 year-1, depending on the potentially recycles 80, 70–115, and 100 kg ha-1 year vs. ca. 1.5 Mg ha-1 year-1 in pure maize with year-1, respectively, of N fixed from atmosphere to low N fertilisation). This may imply that also the tree the soil (Leblanc et al. 2007), which is enough for root safety-net, which retains nutrients in the system compensating N accumulation to permanent crop out of the cropping season (van Noordwijk et al. 1996), -1 biomass and harvest loss in1.5–3 Mg ha yield. is important for these systems. Further, N2 fixation is Intensively managed large-scale perennial crop probably underestimated because the estimates of plantations may be over-fertilised; e.g. the typical Ladha et al. (1993) and Rowe et al. (1999) are based on coffee fertilisation rate in Costa Rica ranging from 150 tree prunings only. Dulormne et al. (2003) found that (Harmand et al. 2007a) to 350 kg [N] ha-1 year-1 160 kg ha-1 year-1 of N fixed by G. sepium accumu-

(Hergoualc’h et al. 2007) would be enough for lated in soil. Direct N transfer to a non-N2-ﬁxing crop compensating the N export in the harvest of may also occur (Sierra and Nygren 2006). As far as we 4–11 Mg ha-1 of green coffee yet the average yield know, the studies by Dulormne et al. (2003) and in Costa Rica is 0.93 Mg ha-1 (calculated from Bernhard-Reversat (1996) are the only ones that cropping area and production data in FAOStat 2011). account for the N ﬁxed from atmosphere in the soil 123 Nutr Cycl Agroecosyst

Table 4 Global cultivation area and production of perennial crops in 2009 (FAOStat 2011), and an estimate of annual N export in the harvest of these crops Commodity and Total area (ha) Total production (Mg) Yield (Mg ha-1) N export (kg ha-1 year-1) Total N export area (Mg year-1)

Cacao beans Sub-Saharan Africa 5,935,274 2,639,788 0.445 16.0 95,032 Tropical Americas 1,585,728 548,360 0.346 12.4 19,741 South and East Asia 1,068,476 837,766 0.784 28.2 30,160 Worlda 8,733,093 4,082,270 0.467 16.8 146,962 Green coffee Sub-Saharan Africa 2,060,527 1,006,318 0.488 14.7 30,190 Tropical Americas 5,474,941 4,807,644 0.878 26.3 144,229 South and East Asia 2,250,612 2,468,351 1.097 32.9 74,051 Worlda 9,841,317 8,342,636 0.848 25.4 250,279 Tea leaves Sub-Saharan Africa 283,161 530,992 1.875 106.9 30,267 Tropical Americas 45,539 85,477 1.877 107.0 4,872 South and East Asia 2,680,869 3,326,333 1.241 70.7 189,601 Worlda 3,014,909 3,950,047 1.310 74.7 225,153 a Includes areas of low production not listed separately and associated crop in addition to the trees. These kinds still poorly understood functions in the tripartite of studies would be most welcome for different AFS symbiosis. They also form CMN between trees’ and but the main constraint is the lack of sufﬁcient time crops’ symbionts, which create a direct N transfer series for constructing the system N balance over years. pathway.

Quantification of N2 fixation under field conditions is a challenging yet necessary task as controlled- Concluding remarks environment studies with seedlings provide little information on functioning of mature trees in an

Symbiotic N2 fixation in AFS should not be studied as AFS. Nitrogen isotopic analyses or detailed, long-term an isolated process because even the ‘‘N2-fixing whole system N balances may provide the most component’’ in an AFS is a tripartite symbiotic system reliable estimates on the N2 fixation by trees. The based on a plant capable of forming symbiosis with general average of %Ndfa for 19 tree species used in both N2-fixing bacteria and mycorrhizae-forming AFS was 59, with a higher proportion in juvenile and fungi. The bacterial partners of the symbiosis are pruned trees and lower in unpruned trees. High highly diverse both taxonomically and functionally. variability was observed in drylands while N2 fixation So far, 98 rhizobial species forming N2-fixing symbi- was active in most of the AFS in the humid and sub- oses with legumes have been identified with most of humid areas. In mass terms, N2-fixation may annually the diversity probably still remaining unrecognised. add from tens to hundreds of kilograms of N per Actinorrhizal symbioses are much less studied than hectare to an AFS. Few data were available on fixed N legume systems and they form an unexplored resource in system components other than the trees. The reports for AFS. Plants rely more on the N2-fixing symbiosis published so far indicate that estimates on annual N2 when the plant N needs exceed soil N supply. Thus, fixation based on trees only may hide considerable soil N supply reduced in AFS by the crop N use and N direct transfer of fixed N from trees to crops and an export in harvest may enhance the N2 fixation above important rhizodeposition of tree N to the soil. It levels observed in natural ecosystems. Mycorrhizae seems that symbiotic N2 fixation is indeed an under- enhance P nutrition but they also appear to have other estimated resource in AFS. 123 Nutr Cycl Agroecosyst

Table 5 Nitrogen Species Management N accumulation References accumulation to the (kg ha-1 year-1) permanent woody biomass in coffee (Coffea arabica L.) Coffea arabica Shaded 11 Fassbender (1987) and cacao (Theobroma cacao L.) plantations Cordia alliodora Free growth 25 System 36 Coffea arabica Shaded 11 Fassbender (1987) Erythrina poeppigiana Pruned 11 System 22 Coffea arabica Unshaded 15 Harmand et al. (2007a, b) Coffea arabica Shaded 13 Harmand et al. (2007a, b) Eucalyptus deglupta Free growth 7 System 20 Theobroma cacao Shaded 9 Fassbender et al. 1988 Cordia alliodora Free growth 28 System 37 Theobroma cacao Shaded 9 Fassbender et al. 1988 Erythrina poeppigiana Free growth 30 System 39

Variation in the N2 fixation by trees in AFS was observed in improved fallows and intensive tree depends on both intrinsic factors of the trees and their fodder production systems. Thus, we may envision symbionts and the environment. Tree species selec- significant contribution of N2 fixation to the production and in some cases inoculation with compatible tion of N-rich mulch for soil improvement in the bacteria are the main options for managing the improved fallows and to the high yields of N-rich intrinsic factors. Species and provenance selection is browse for domestic animals in protein banks and also the only available response to the challenges other tree fodder systems. caused by the macro environment, especially in In order to manage the symbiotic N2 fixation by dryland AFS, while several options exist for managing trees in AFS, basic research is needed on the the microenvironment such as proper spacing of trees functioning of the tripartite tree-rhizobia-mycorrhizae and crops. Green pruning that is practiced for reducing symbiosis; on the tree N rhizodeposition to soil via crop shading and enhancing nutrient cycling, seems to exudates and root turnover; on the direct N transfer rejuvenate the trees and maintain high levels of N2 from trees to crops; and if the trees really enhance P fixation. However, green pruning may temporarily supply by excretion of extracellular phosphatases. impede N2 fixation by simultaneously reducing both Applied research is needed on tree and symbiont the C flow to the roots and microsymbionts and tree N selection, especially for drylands, and on the effects of requirements. This may reduce the synchrony of N2 AFS management on N2 fixation. fixation with crop N needs. The N2-fixing trees mostly improve the crop N supply by long-term build-up of Acknowledgments An early version of this review was soil N reserves. presented in the 2nd World Congress of Agroforestry (Nairobi, August 2009). We thank Dr Anne-Marie Domenach We may envision a role for N2-fixing trees in for inspiring discussions and comments on a draft of this review. different kinds of AFS. In small-scale, low-input The contribution of PN was funded by the Academy of Finland (grant 129166). perennial cropping systems, N2-fixing trees may provide enough N for sustained or increasing yields. In large-scale, highly fertilised perennial cropping References systems, they contribute to the reduction in the use of industrial fertilisers. Recent developments of legume Acosta-DurańC,Martıńez-Romero E (2002) Diversity of rhizobia trees mixed with annual crops show much promise for from nodules of the leguminous tree Gliricidia sepium,anatural low-input farming. The highest N2 fixation activity host of Rhizobium tropici. Arch Microbiol 178:161–164 123 Nutr Cycl Agroecosyst

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