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. Ferna´ndez 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 Á Actinorrhizal trees Á Legume trees Á H. A. Leblanc Management practices Á Nitrogen balance Á 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 Martı´nez-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.