Nitrogen fixation and growth of non-crop legume species in diverse environments

Perspectives in Plant Ecology, Evolution and Systematics

Vol. 2/2, pp. 149–162 © Urban & Fischer Verlag, 1999 http://www.urbanfischer.de/journals/ppees

Nitrogen fixation and growth of non-crop legume species in diverse environments Janet I. Sprent Department of Biological Sciences, University of Dundee, DD1 4HN, Scotland, UK; e-mail: [email protected]

Abstract Of the 643 legume genera, few have been exploited in agriculture and 40% have not even been evaluated for their ability to nodulate and fix nitrogen. Most of these are in tropical/subtropical regions, with habitats ranging from extremely dry to flooded. Recent work in some of these areas shows that plants can nodulate under conditions previously thought to be disadvantageous. The accepted dogma that nitrogen fixing legumes have a high demand for P is challenged and examples of how legumes can extract P from soils with low available P described. Species tolerant to shading and high soil Al are cited, although the mechanisms of adaptation are not yet clear. Some tropical soils have high nitrate levels and, contrary to perceived wisdom, there are legumes which can nodulate under such conditions. Many tropical tree legumes prefer ammonium to nitrate and are able to fix nitrogen and assimilate ammonium at the same time. In all these cases, there are genotypic differences, both within and among species. Large areas of tropical fresh water, such as the Brazilian Pantanal and the Orinoco floodplain have nodulated legumes predominant in their flora. The ecological potential of these has not been evaluated. One of the sites of nodule evolution is likely to have been in such areas. Modes of infection of legumes by rhizobia vary with taxonomic tribe and may represent evolution for survival in different environments. As more legumes, from more ecosystems are studied, a wider range of adaptations is likely to be found. Work is urgently needed to study these, especially in areas being cleared for agriculture or by logging. Key words: aluminium, legumes, nitrogen fixation, nodules, phosphorus, shading, waterlogging

Introduction The Leguminosae is the third largest family of dicotyledonous plants, consisting of about 643 genera and 18,000 species. It is usually divided into three sub-families, Caesalpinioideae, Mimosoideae and Papilionoideae. Members of the family are found in all parts of the world where angiosperms occur, with one or two exceptions, such as the Falkland Islands (Malvinas) in the South Atlantic Ocean. They show great diversity in growth habit, from small ephemeral herbs to massive long-

lived trees. They show equal diversity in habitat, occurring in all regions from within the Arctic circle to the equator, in mesic, dry, flooded, saline, montane, open, shaded and other environments. Possibly their most widely known characteristic is the ability, not possessed by all, to form nitrogen-fixing symbioses with a group of bacteria collectively known as rhizobia (Sprent & Sprent 1990). The taxonomy of legumes is currently an area of intensive research; many changes 1433-8319/99/2/02-149 $ 12.00/0

150 J. I. Sprent

have been reported in recent literature and far more are expected before the next International Legume Conference in 2001. It is becoming clear that nodulation is a robust taxonomic character, both at the presence/absence level and at the structural/physiological level. However, there are still 40% of legume genera which have never been examined for nodules. All nodulated plants, both legume and non-legume (actinorhizal plants such as Alnus spp, forming nodules with Frankia), in common with members of other families form a separate Rosid clade which may have a predisposition to nodulation which is not always expressed (Soltis et al. 1995). Phylogenetic analysis (Doyle 1994) and evidence from nodule structure/function (Sprent 1994a) suggest that, within the legumes, nodulation has evolved more than once. The enzyme complex, nitrogenase, which has been remarkably well conserved in all N2-fixing microorganisms, is large, slow and expensive to operate (Sprent & Raven 1985). Thus it is generally accepted that even plants which can form N2-fixing root nodules will not do so if they have a plentiful supply of combined nitrogen or if they are short of energy in the form of carbohydrate, or the oxygen required to release that energy. It follows that legume plants must have an internal regulatory system to allow them to adjust N2 fixation to environmental conditions. In the first issue of this Journal, Hartwig (1998) gives an excellent review of ways in which such regulation may occur from the gene to the ecosystem level. However, nearly all the relevant research has been carried out on a few temperate crop and pasture species from the subfamily Papilionoideae, with some recent studies on tropical/subtropical mimosoid genera such as Leucaena and Acacia. Nodulated members of the Caesalpiniodeae have been largely ignored. The aim of this review is to explore the possibility that, if a wider range of legumes is studied, the generally accepted dogmas (e.g. that legumes have a high P requirement because of N2 fixation) will be found to have important exceptions. The first part will consider evidence from physiological/ecological studies on host plants. Effects specifically related to rhizobia or the initial interaction between rhizobia and host cells will not be covered. Subsequently I shall attempt briefly to put these observations into an ecological/evolutionary/ taxonomic framework. It will be clear

from the outset that more questions will be posed than answered and that the amount of knowledge on each topic varies greatly.

Does N2 fixation necessarily impose a high demand for P? In his review, Hartwig (1998) points out that the data indicating that N2 fixation may be limited by low phosphate are inconsistent. It is pertinent to consider why this might be. Legume tissues do not appear to have higher P content than those of other plants. Values for leaves and young shoots reported in the literature tend to fall into two groups, those from agricultural experiments and those from natural or unfertilized field stands (Table 1). Within either of these two groups, legumes and non-legumes overlap. Within nodules, the bacteroids have a high P content, a major component of which is in the form of adenosine nucleotides, needed to fuel the energydemanding nitrogenase reaction (Sprent & Raven 1985). Within the host cell, the bacteria are enclosed by membranes, which also have a high P content. These initial capital costs in a seedling are followed by the costs of additional nodules as plants grow, usually in herbaceous species to maintain nodules at about 5% of plant dry weight. In woody legumes this proportion usually decreases as more nutrients are recycled within the plant. Available data (e.g. Allen et al. 1988) show that the overall P content of nodules, around 0.2% of dry weight (DW), is similar to that of leaves. Since the latter are a much larger fraction of plant DW, the P content of nodules is a relatively small fraction of total plant P. However, what may be important, at least for legumes such as soybean, is that under P stress, dry matter is diverted from root to nodule production (Cassman et al. 1980). In both soybean and lucerne (alfalfa), nodules from plants grown under P deficiency may have a higher concentration of P than those on plants grown with sufficient P and may fix more N2 per unit of P (Drevon & Hartwig 1997). The complex interactions between nodule and root growth under P stress are further considered below. Once capital costs have been incurred, nodule operating costs stem largely from additional respiration, requiring oxygen and reduced carbon. Recent evidence has shown a

Nitrogen fixation and growth of non-crop legume species 151

Table 1. Phosphorus concentration (percent dry weight) considered “adequate” in leaves or young shoots of plants grown in agricultural trials (a) or as observed in the field (b) (n, species number). Type of plant/genus

P content

n

Reference

(a) Data mainly from Australia Trees, mainly fruit spp. Grasses (forage) Grasses (cereal) Legumes (forage) Legumes (grain) Other crop spp.

0.08–0.30 0.18–0.35 0.21–0.60 0.15–0.35 0.20–0.55 0.24–0.40

15 8 5 13 9 3

Reuter & Robinson (1981)

(b) Data from Brazilian cerrado Chamaecrista spp. Stylosanthes spp. Non-legume spp.

0.04–0.19 0.10–0.14 0.03–0.14

17 5 14

Geoghegan & Sprent (1996)

Data from Senegal Legume trees Non-legume trees

0.10–0.22 0.12

7 2

complex relationship between P deficiency in Phaseolus vulgaris and oxygen permeability of nodules (Ribet & Drevon 1995a,b), which could be related to adenylate charge in different cell compartments as found for low pO2 effects on soybean nodules (Kuzma et al. 1999). Further, in soybean, P deficiency may affect carbohydrate transport to nodules (Sa & Israel 1998). However, although these various data do show that P deficiency affects nodule functioning, they do not preclude it having simultaneous effects on other aspects of plant growth and metabolism. In experiments in which P. vulgaris plants were given mineral nitrogen, P deficiency was shown to affect root architecture as a result of both enhanced ethylene production and (possibly) altered sensitivity to ethylene (Borch et al. 1999). Since ethylene is also known to affect nodule development (e.g. Fernandez-Lopez et al. 1998), it is difficult to separate its effects on nodulation and N2 fixation from those on other aspects of plant growth which are concerned with mineral nutrition. Two separate questions need to be answered. First, does N2 fixation per se impose a higher demand for P than other forms of nitrogen acquisition? Second, do legumes vary in their responses to low P supply? Until recently, most studies have been on temperate crop species, normally selected on fertilized soils, which do not encourage a low nutrient economy. Whilst there is a large literature

J.D. Deans, pers. comm.

showing positive responses to P, much of which also shows a positive role for arbuscular mycorrhizas, comparatively few studies have included proper comparisons with plants grown on combined nitrogen. Robson and co-workers in Australia (e.g. Robson 1983) have studied P nutrition in a variety of legumes and have proposed a simple way of checking whether those grown on atmospheric N2 have a genuinely high demand for P. Briefly, they suggest that experiments should examine the interaction between plants grown on fixed and combined N in response to increasing P supply: the same arguments apply to other mineral nutrients. The interaction may be positive, zero or negative. A negative interaction suggests a greater need for P by N2-fixing plants, a zero interaction that plants have the same P requirement for growth on fixed and combined N and a positive one that high P may be inhibitory to N2 fixation. Most people would regard the latter as unlikely. There is, however, evidence that some N2-fixing species, most notably certain Australian acacias, show stunted growth and retarded root development when given levels of P that would be regarded as normal for crop plants (Reuter & Robinson 1981). The possible reasons for this will be discussed later. Sanginga and co-workers have studied N2 fixation by woody plants over many years. In a recent paper (Sanginga et al. 1995) they

152 J. I. Sprent

concluded that in Gliricidia sepium, a species widely used in agroforestry, percentage and amount of N2 fixed were only enhanced at the lowest rate of P applied, probably a reflection of the basic need for P for plant growth. Working with the widely grown Australian legume Acacia mangium, Ribet & Drevon (1996) compared the P requirement of N2-fixing plants with those grown on urea. They found that plants grown on both sources of N had similar external P requirements and also that both used their internal P equally efficiently. A crop species which is widely grown, both in the developed and developing world is Phaseolus vulgaris, a legume which frequently gives nodulation problems and which also forms nodules of varying degrees of effectiveness with a wide range of rhizobia (Michiels et al. 1998). Its uses vary from green vegetables through “navy beans” to dry beans of various colours which, together with a cereal such as rice, form the staple diet in large parts of the developing world. Scientists at the Centre for International Research in Agriculture in the Tropics (CIAT) with its headquarters in Cali, Colombia, have been working on dry beans for many years and are also concerned with low nutrient soils. Because of the long tradition in South America of growing beans on low input farms, CIAT scientists have been able to identify bean germplasm which is adapted to low P soils and have now begun to characterise it (Vadez et al. 1997, and references therein). This general approach of looking at natural populations from low nutrient soils is at last beginning to be more widely adopted. Already it is becoming clear (not surprisingly) that plants, including those which can fix N2, have evolved a variety of methods for obtaining P and other nutrients from soils where they are either in short supply and/or in an unavailable form. As pointed out by Friesen et al. (1997) for tropical pasture systems, a knowledge of such methods would not only benefit farmers on nutrient-poor soils, but would also help to avoid the loss of fertilizer P which occurs with many current farming practices. A good example of the how legumes may be adapted to extract P from soils with low available P is the pigeon pea, Cajanus cajan, a multipurpose grain legume which can be grown as an annual or a short lived perennial. Unlike P. vulgaris, it seldom has nodulation

problems. It has been known for many years to be tolerant of poor soils, but recent studies have shown how it is able to extract P from Fe-P, the dominant form in alfisols (Ae et al. 1990). It does this by producing (p-hydroxybenzyl)tartaric acid (also known as piscidic acid) and its p-O-methyl derivative. These compounds chelate Fe and thus make P available. In addition to the obvious benefit to the pigeon pea, there are two other possible associated benefits. First, by using the P from the Fe complex, other forms of P which may be present could be available to crops such as sorghum, often grown with pigeon pea. Second, residues from pigeon pea are likely to leave mineralisable forms of P in soil. This excellent piece of work shows the importance of considering both the plant and the particular soil to which it is adapted. Some genotypes of groundnut (peanut, Arachis hypogaea) are also able to take up P from an Fe-P complex. In this case P is taken up through the surface of the geocarpic fruit possibly by the hairs on them and also hairs on the pegs (carpophores) (Wissuwa & Ae 1999). Hairs on these structures have previously been thought to assist in water and calcium uptake (Wright & Nageswara Rao 1994). Two widely studied ways of enhancing P extraction from soil are by producing cluster (proteoid) roots and/or mycorrhizas. Cluster roots, although not often reported in legumes are found in many, but not all, species of Lupinus (Skene 1998). Dinkelaker et al. (1989) have shown that P-deficient Lupinus albus roots may exude up to 23% of plant dry weight as citrate, which solubilises soil P. Unusually, especially considering the common signalling systems between rhizobia and plant roots and between arbuscular mycorrhizal fungi and plant roots (Hirsch & Kapulnik 1998), lupin species are essentially non-mycorrhizal (Trinick 1977). Both cluster roots and arbuscular mycorrhizas have been reported in two Australian genera, Daviesia and Viminaria, from the endemic tribe Mirbelieae (see Sprent 1994b, and references therein) and two genera, Aspalathus (Allsopp & Stock 1993) and Cyclopia (A. Spriggs, pers. comm.) endemic to the Fynbos and nearby regions of the Cape province of South Africa. All four of these genera occur in nutrient-poor soils where members of the family Proteaceae are major components of the flora. In the Proteaceae, cluster roots and arbuscular mycorrhizas appear to be alternative mecha-

Nitrogen fixation and growth of non-crop legume species 153

nisms for nutrient acquisition, whereas legumes from the same region may have both. The role of arbuscular mycorrhizas in P acquisition generally and in nodulated legumes in particular is usually accepted as being important. However, what is less often appreciated is their cost in carbon terms. In a recent study on dry beans, Nielsen et al. (1998) concluded that maintenance and growth respiration of the fungal component could be a primary limitation to plant growth in low P soils. There is also a P cost in producing fungal hyphae and the extended membrane system at the fungal/plant cell interface. Production and operation of cluster roots and production of compounds such as piscidic acid by pigeon peas clearly also have high carbon costs. Do the benefits justify these costs? This may well depend on the environment to which the plants are adapted, in particular whether their photosynthesis is light-limited. In a study of 14 species of Zimbabwean trees, including both nodulated and non-nodulated legumes, Tuohy et al. (1991) found that leaf photosynthesis was closely related to leaf N. There was no correlation between leaf P and photosynthesis. Some of the species studied, including the nodulated Acacia nigrescens showed a very high light saturation value for photosythesis. Taken overall, these data suggest that if sufficient light is available and leaf N content is high, as in legumes, plants can afford to divert C compounds to the acquisition of P. Since many of the world’s low P soils are in tropical/subtropical areas with high irradiation levels, an examination of native legumes for tolerance of low P is an important goal for ecosystem management and for sustainable agriculture. It may well be, as has been suggested for example by Newman (1997) that P will be a major constraint on agricultural productivity in the next century, but we should not assume that N2-fixing legumes necessarily require more P than other plants.

Are all nodulated legumes shade intolerant? There is plenty of evidence in the literature showing that legumes may be shaded out in mixed plantings. For example in New Zea-

land, introduced tree lupins, Lupinus arboreus, die out when canopies of Pinus radiata close, but when forests are thinned, they regenerate from the seed bank in the soil, nodulate and fix N2 (Sprent & Silvester 1973). Although the total photons received per year may not vary greatly with latitude, the photon flux density is certainly less at lower than at higher latitudes. This may be why the only well documented cases of shading tolerance in legumes that I have been able to find come from the tropics: of 161 species of legume of world economic importance listed by Duke (1981), only 12 were said to have some degree of shade tolerance. One particularly interesting example, not of current economic importance is Dioclea guainensis which grows as a liane in the cloud forests of Venezuela. This species nodulates profusely in dense shade in the surface of the litter layer (Fig. 1). It is also accumulates aluminium (Izaguirre-Mayoral & Flores 1995). It was suggested that shade-tolerant legumes produce large seed, which act as a C and nutrient store during seedling growth; even so, seedlings did not nodulate until the stem was over 10 cm in length. This part of South America, at least, seems to favour shade tolerant nodulated legumes, both in the savanna (Izaguirre-Mayoral et al. 1995) and in forests (Roggy & Prevost 1999). The latter authors distinguished six groups of trees in a large experimental area of French Guiana. Both shade-tolerant and shade-intolerant nodulated species were found in the emergent, upper and lower tree layers and judged by 15N data all were fixing at least some N2. In other areas, such as parts of the Amazon forest, nodulated legumes may be largely confined to gaps (Norris 1969). Many of the legumes whose nodulation status remains unknown are found in tropical forests, for example Haplormosia monophylla, a member of the sub-family Papilionoideae (and therefore likely to be nodulated) from the swampy forests of West Africa. Both these, and others known to be nodulated, such as species of Dalbergia are being heavily exploited for timber and many are on the IUCN world list of threatened trees (Oldfield et al. 1998). Any attempt at sustainable management of tropical forests should take into account their potential for N2 fixation in their natural habitats. It is a sad fact that agroforesters take into account that some trees fix N2, but traditional foresters generally do not.

154 J. I. Sprent

Fig. 1. Nodules of Dioclea guianensis in the surface litter of a deeply shaded part of the cloud forest in Marina State (Venezuela) altitude about 1,750 m a.s.l.

Is combined nitrogen always inhibitory to nodulation and N2 fixation? This is a subject for which there is a voluminous literature. Control may act at the infection, nodule growth, or nodule operation levels. All of these have been well discussed by Hartwig (1998). The possible sources of nitrogen to plants in soil are nitrate, ammonium or simple organic compounds such as amino acids and urea. Because the major form of N in agricultural soils is nitrate, most work has been on this form and the mechanisms by which nitrate controls nodule processes are now partially understood. However, in some tropical soils where legumes grow, there are high concentrations of nitrate in the soil. In some acid soils of West Africa, the normal rapid leaching of nitrate is retarded (Wong et al. 1987). In arid areas such as the Sahel, the soil may contain high concentrations of nitrate, which is not leached because there is insufficient rainfall (Edmunds & Gaye 1997). These areas have a number of native legumes, often annuals, some of which are

important crops for peasant farmers. Two geocarpic species are Vigna (Voandsia) subterranea (Bambara groundnut) and Macrotyloma geocarpum (Kersting’s bean). A recent study in South Africa, using hydroponic culture, Dakora (1998) found that unspecified landraces of these two species nodulated and fixed N2 in the presence of 5 mol m–3 nitrate, much higher than that normally considered to be inhibitory. Although this study was carried out from the perspective of intercropping, it suggests that a search for other nitrate tolerant symbioses from this area may be worthwhile. These would be useful, not only in an ecological context, but also in understanding how nitrate intolerant systems might be modified. The present approach, using nitrate tolerant, supernodulating mutants, has not yet let to increases in N2 fixation (see references in Dakora 1998). In most uncultivated soils, the inorganic form of nitrogen is ammonium. Unlike nitrate, high concentrations of ammonium are generally considered to be toxic to plants, whether or not they can nodulate (Marschner 1995). This toxicity is associated with transport in the xylem of ammonium in excess of that

Nitrogen fixation and growth of non-crop legume species 155

which can be assimilated by roots. However, ammonium is generally considered to be less inhibitory to N2 fixation than nitrate (Hartwig 1998), although nitrogenase activity may be inhibited indirectly following both ammonium and nitrate assimilation, by feedback regulation involving glutamine in the phloem (see references in Dakora 1998 and Hartwig 1998). However, work in our laboratory on some genotypes of the N2-fixing legume trees

Table 2. Effects of combined N on nodulation and nitrogen fixation by some legume trees. (a) Mimosa caesalpiniaefolia. Plants were grown in a greenhouse, inoculated with an effective rhizobial strain and either without added N or given 10 mg N per week as either calcium nitrate or ammonium sulphate. Plants were grown in a glasshouse and harvested at 76 d. Nodules formed in the presence of ammonium appeared normal, but those of nitrated treated plants were grossly distorted. Nitrate significantly reduced number of nodules, but ammonium had no effect. Ammonium-treated plants had significantly more N than those of the other treatments (data from Goi et al. 1997). N source

Number of nodules/plant

N/plant (mg)

N2 NO3 NH4

38 2 41

8.55 13.70 49.64

(b) Gliricidia sepium. Plants were grown for 12 weeks in a greenhouse, inoculated with an effective rhizobial strain and with one of two spp. of Glomus. The mycorrhizal species had no effect on nitrogen fixation and data are averaged over the two Glomus species. Plants were given 3 mol m–3 N as potassium nitrate, ammonium sulphate or urea enriched with 5% excess 15N and nitrogen fixation estimated by the 15N dilution technique. Although ammonium-treated plants had more N than those of the other treatments, differences were not statistically significant. Nitrate significantly reduced the proportion of plant N derived from fixation, whereas ammonium had no effect. Total plant N includes than derived from the seed (data from Twum-Ampofo 1995). N source

Total N/plant (mg)

N derived from fixation (mg)

N2 NO3 NH4 Urea

83.4 84.8 91.9 91.3

74.0 62.5 72.6 71.2

including Acacia auriculiformis (Goi 1993), Mimosa caesalpinifolia (Goi 1993; Goi et al. 1997), both sub-family Mimosoideae, and Gliricidia sepium (Papilionoideae, Twum-Ampofo 1995) has shown that they can both nodulate and fix N2 in the presence of ammonium (Table 2). There is considerable genotypic variation in the response, as Sun et al. (1992) have shown for A. auriculiformis. Uptake of organic nitrogen is usually associated with mycorrhizal roots, especially ectotrophic forms (Alexander 1989). It has been suggested that in legumes ectotrophic mycorrhizas and nodulation are alternative strategies, not occurring in the same species, but this is now known not to be the case (Alexander 1989; Sprent 1994b). Apart from urea, to which nodulation and N2 fixation seem fairly resistant (e.g. Hine & Sprent 1988), the effects of soil organic nitrogen on N2 fixation by legumes have not been studied. If compounds such as glutamine can be absorbed by and translocated out of roots, they might inhibit nodulation and N2 fixation, by contributing to the internal feedback control system discussed by Hartwig (1998). On the other hand, if taken up by ectomycorrhizal fungi, they might be divorced from this. Recent evidence suggest that some tropical forests (including those known to contain nodulated legumes, have excess N, whereas most temperate forests are N-limited (Martinelli et al. 1992, 1999). How N2 fixation is controlled in these tropical forests is completely unknown.

How does waterlogging affect nitrogen fixation by legumes? The effects of waterlogging are generally considered to result from anoxia, and within limits, to be reversible (Hartwig 1998). For some crop plants, N2 fixation appears to be more sensitive than assimilation of nitrate (Bacanamwo & Purcell 1999). However, there are very wide differences in adaptation of legume nodules to waterlogging. Within the temperate genus Lotus, major differences were found between L. corniculatus which is terrestrial and L. uliginosus which is much more tolerant of flooding. Seed of the former could not germinate under water, whereas those of the latter germinated equally well under both flooded and non-flooded condi-

156 J. I. Sprent

tions (James & Sprent 1999). Nodules formed had extensive production of aerenchyma; a feature widely found in nodules growing in very moist soils, see for example, Pankhurst & Sprent (1975) for soybean. This aerenchyma was continuous with that on roots and stems, allowing oxygen diffusion from the air to the outer parts of the nodule (James & Crawford 1998). One of the best known aquatic legumes is the mimosoid Neptunia oleracea and closely related species. Nodules are found in the axils of adventitious roots on the floating stems and are formed by a “crack” infection pathway, first described in Arachis hypogaea (groundnut, peanut) (James et al. 1992b; Subba Rao et al. 1995). Plants are well adapted to fix N2 in static flooded soils and nitrogenase can be inhibited by over-vigorous aeration (James et al. 1992a). Interestingly, when grown terrestrially, N. oleracea can form root hairs and have a “standard” root hair infection pathway (James et al. 1994). There have been isolated reports in the literature that a single species of legume may have more than one infection pathway (e.g. Dart 1977), but only recently has it become apparent that this may

be an adaptation to environmental conditions. In Lotus uliginosus, some infections under flooded conditions were observed to occur through the bases of swollen epidermal cells rather than root hairs (James & Sprent 1999), a route which may be used by terrestrial legumes such as Mimosa scabrella which do not normally produce root hairs (de Faria et al. 1988). One of the largest areas of freshwater in the world which is still relatively undisturbed is the Pantanal region of Brazil, covering an area greater than the size of France. The nitrogen content of its waters is generally low, and nodulated legumes feature prominently in its flora. Two shrubby genera found there are Discolobium and Aeschynomene, members of the papilionoid tribe Aeschynomeneae whose nodules are associated with lateral or adventitious roots, and are characterised by “crack” infection and internal tissue containing few or no uninfected cells (Sprent et al. 1989). Nodules on Pantanal species, which may be formed on roots or stems conform generally to this pattern with certain exceptions in detail (Loureiro et al. 1994, 1995). In particular, Discolobium pulchellum re-

Fig. 2. Typical vegetation on the banks of the Orinoco river, near the branch of the Mapire river in Venezuela, during the wet season. Almost all of the trees are nodulated legumes, including Campsiandra laurifolia (see Barrios & Herrera 1993). Height of trees above water level approximately 30 m.

Nitrogen fixation and growth of non-crop legume species 157

quires immersion in water for maintenance of healthy stem nodules: in both stem and root nodules, the infected tissue is penetrated by branches of the nodule vascular system, a highly unusual situation. This species appears to be adapted to continuously flooded conditions whereas Aeschynomene fluminense occurs more in regions which are seasonally flooded (when nodules may be 2 m below water); its stem nodules, like other stem nodulated species such as Sesbania rostrata (Parsons et al. 1993) require high humidity but not total immersion. Another major area where seasonal flooding occurs is in the Orinoco region of Venezuela. A tributary of this river, the Mapire has been well studied. For large distances its banks (and that of the parent Orinoco, Fig. 2) are dominated by nodulated legume trees from all sub-families (pers. observ.). The caesalpinioid species Campsiandra laurifolia has perennial nodules, which show some nitrogenase activity throughout the year (Barrios & Herrera 1993). The infection pathway for this species has not been studied, but, like other perennial nodules whose infected tissue may senesce during adverse conditions it may maintain a healthy meristem from which new N2-fixing tissue may be formed. Many other species, such as Mimosa pellita and Vigna longifolia, native to areas which are prone to flooding have nodule anatomy which permits continued N2 fixation albeit not always at maximum rates (e.g. Viminaria juncea in Western Australia, Walker et al. 1983). For a detailed discussion of possible physiological mechanisms operating in nodules of wetland legumes, see the review by Loureiro et al. (1998). In global terms, a significant fraction of legume N2 fixation may occur in wetlands, areas which are centres of great plant and animal biodiversity. There is an urgent need to study the ecological role of flooding tolerant legumes in the maintenance of these fragile ecosystems.

Other constraints Because of the paucity of evidence from noncrop legumes, this section will cover only some of those other factors listed by Hartwig (1998) as affecting N2 fixation. For over twenty years, a small group of scientists (e.g. Anon 1979; Duke 1981) has been noting legumes of actual or potential economic importance which have properties such as acid tolerance, which could be exploited. An analysis of the species listed by Duke (1981) for example shows that over half have been shown to tolerate soils pH values ranging from below 6 and to above 7.5 (Table 3). The majority of legumes studied show considerable acid tolerance. Far fewer show tolerance to high pH although some, such as Leucaena leucocephala will grow above pH 8. However, to my knowledge, there has been no targetted search for legumes in general which can grow in high pH soils. As Hartwig (1998) points out, extreme soil pH levels are accompanied by a suite of problems with mineral availability including toxic heavy metals. One problem associated with many acid soils is aluminium toxicity. However, recent studies of legumes growing in soils containing naturally high levels of aluminium show that they can nodulate and in some cases even accumulate aluminium (Izaguirre-Mayoral & Flores 1995; Geoghegan & Sprent 1996, although unpublished EDAX examination of some of the material used in the latter showed some of the Al to be in an inorganic form embedded in the leaf). In the low P, high Al cerrado area of Brazil (Goedert 1983), there are numerous species of the caesalpinioid genus Chamaecrista, together with mimosoids Mimosa and Calliandra and papilionoid Stylosanthes. All nodulate in the field and 15N data suggest that they fix N2 there (Sprent et al. 1996). Clearly, the accepted wisdom that aluminium and N2 fixation in legumes are incompatible needs to be re-examined critically, taking into account the

Table 3. Percentage (out of a total of 161) legumes of economic importance tolerant for different ranges of soil pH (data from Duke 1981). Tolerance level

pH range

Species (%)

Tolerant of both acid and alkaline soils Acid intolerant Alkali tolerant Requiring more-or-less neutral soil

< 6.0– >7.5 < 7.5 > 7.1 6.0–7.5

60 37 1 2

158 J. I. Sprent

exact form of aluminium available to the plant and, if taken up, how it is dealt with by the plant. An essential element which is needed in greater amounts by N2-fixing that combined-N assimilating plants is molybdenum, a constituent of nitrogenase. Crop legumes grown in acid soils may suffer from molybdenum deficiency (see Sprent & Sprent 1990, p. 118), but it is not known whether legumes native to acid soils are more tolerant to low availability of molybdenum. It is known that arbuscular mycorrhias can take up zinc and copper (Marschner 1995), but can they also take up molybdenum? Do cluster roots have a role to play in some soils? Drought and salinity are two conditions limiting plant growth in much of the world. Effects of drought on N2 fixation by crop plants have been quite widely studied in recent years and genotypes with drought resistance properties are being actively selected (e.g. Pimental et al. 1990). A wider study of legumes shows that they have evolved many of the ”standard” mechanisms for drought tolerance or avoidance (ephemeral species) although none is known to have either CAM or C4 metabolism. Thus many examples of geophytic legumes are known, such as Tylosema esculentum (marama bean, not nodulated) and Sphenostylis stenocarpa (african yam bean, nodulated), both of which make large, edible underground storage structures (Anon 1979). Ability to maintain a positive carbon balance under dry conditions may be achieved by production of phyllodes in many acacias (e.g. A. harpophylla, which can open its stomata at a water potential of -5.5 MPa, Tunstall & Connor 1975), or by the shedding of some or all of the leaflets in other acacias (pers. observ.) and shedding of all leaves in many genera, with a reliance on photosynthetic stems (Nilsen et al. 1993). There are various more extreme adaptations to dry conditions which have not been studied. Argentina has an area with a number of native xerophytic legumes, none of which has been examined for nodulation. These include the small papilionoid tree Ramorinoa girolae which has spine-tipped branches and leaves reduced to small brown scales. The extent of N2 fixation by non-agricultural species in dry soils is largely unknown, partly because of the lack of good methods for estimating it (Sprent & Sprent 1990). The genus Prosopis has 44 species, a number of which are drought and/or salt toler-

ant (Anon 1979). Prosopis tamarugo grows on the salt flats of the Atacama desert in Chile – the only tree which can survive there. Although it can nodulate and fix N2, how significant this is in the field remains obscure. Australian research has been aimed at finding native species which can nodulate and fix N2 under saline conditions and some success has been achieved, most notably with Acacia ampliceps (Marcar et al. 1991; Zou et al. 1995). This species nodulated and fixed N2 when treated with 100 mol m–3 NaCl as well as it did with none. One of the adaptations which may have allowed it to do this was a high level of succulence in its phyllodes.

General considerations As discussed earlier, most of the information available about legume responses to environmental stress has come from agriculturally important species. Further, until recently, the aim of the research was mainly to provide practical recommendations for farmers. It is also true that only recently have grain legumes been bred or selected on other than optimum conditions of fertilizer and water supply, usually including combined nitrogen. Selection will then have tended either to be neutral towards N2 fixation and use of nutrients such as P, or even favouring those genotypes with high requirements. Ecologists, on the other hand have been considering how plants cope with nutrient limitation for many years, both at an individual plant and at a community level (e.g. Chapin 1980). Chapin et al. (1986) make the distinction between crop species, which respond more slowly to increases in nutrients at the low end of the scale, but continue their response over a much wider range of nutrient concentrations, and plants adapted to low nutrients which respond rapidly at the low end of the scale, but cease to respond much earlier. Grubb (1998) suggested that plants have three types of strategy which they can adopt to cope with shortages of resources, low flexibility, switching and gearing down. However the responses of plants to stress are categorised, I believe that most will be found to contain nodulated legumes. Species of the genus Acacia alone shows enormous versatility. Growth rates vary greatly, as does their ability to respond to added nutrients. This variation reflects, not

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only factors classically recognised in growth analysis, such as root:shoot ratio, leaf area ratio, but also root architecture and symbiotic relations. Acacia albida (also known as Faidherbia albida) is well known as a drought tolerant species, which often shows reverse phenology, producing its leaves in the dry season and shedding them in the wet season. It can do this, because of its ability to produce a very long tap root immediately following germination, to reach the water table. Gitonga (1994) grew seedlings of this species, uninoculated, inoculated with rhizobia, with no added N, or with combined N in the form of ammonium or nitrate. All produced tap roots which reached one metre in length in six weeks (in Scotland!), but which varied in diameter, dry weight and degree of branching. In a nutrient-poor soil, production of such a tap root imposes a severe drain on seed resources, so, not surprisingly, plants responded to added N by increasing dry weight, earlier production of additional leaves and earlier nodulation. Other acacias may nodulate using seed reserves alone. Flexibility in leaf morphology and anatomy has been mentioned earlier with respect to drought response. Additionally it may be a response to shade. All phyllodinous acacias produce pinnate leaves following germination, which often occurs in shaded conditions, and then change to phyllode production on emergence through the canopy or earlier when growing in the open. Another feature of many phyllodinous acacias is the ability to form, not only root nodules, but also both arbuscular and ecto-mycorrhizas (Sprent 1994b). To do this, they can utilize microbes from soils not only in their native Australia, but also Brazil and Africa. This may underlie the success of these plants when introduced into other countries where they grow very fast and can be exploited for purposes such as wood production, reclamation of mine spoils and land stabilisation (Franco & de Faria 1997). It may also be why they are invading and destroying vast areas of the fragile fynbos area of south Africa (Holmes & Cowling 1997). However, it may also be why some acacias, when grown on soils with higher nutrient levels than those to which they are adapted, show toxicity symptoms, as shown for A. baileyana, A. retinoides, A. decurrens and A. iteaphylla (Reuter & Robinson 1981) for phosphorus. Whether this is a direct effect or due to ion imbalance (see Marschner 1995) is not known.

Since legumes first evolved, probably in the late cretaceous, they have radiated into almost all corners of the world. Although it is now generally agreed that nodulation evolved more than once (Doyle 1994; Sprent 1994a), we have no idea when this occurred. The variations in nodule infection processes and in nodule structure, both of which are closely related to taxonomic position, suggest that selection pressures favoured particular processes in particular environments. Although there is very little evidence beyond the genus/species level of co-evolution of legumes and their rhizobial symbionts (Sprent 1994a,b), it is noteworthy that some stem nodules are formed with photosynthetic rhizobia, which may represent one of the ancestral forms (Sprent 1994a). Stem nodules only occur in flooded regions, and are always characterised by infections via junctions of adventitious roots. Apart from Sesbania rostrata, which is in a closely related tribe, all those so far described are from the tribe Aeschynomeneae. Nodules of this type probably evolved in wet conditions. The classical mode of infection, via root hairs, could have evolved in drier situations. Acacias, which are highly successful in dry areas, have indeterminate nodules with apical meristems which can resume growth after periods of stress. They also have a phellogen which can produce corky cells in the dry season and aerating tissue when the soil is moist (Sprent 1988). There are probably many other adaptations yet to be discovered, especially bearing in mind the large number of legumes whose nodulation status is still unknown. Many of the latter are in parts of the world subject to great environmental pressures, so it is vital that their potential is evaluated and managed. There are large areas where legumes do not occur naturally in large numbers, for example the dipterocarp forests of Asia, and others where most of the legumes are nonnodulated members of the Caesalpiniodeae. In still other areas, nodulated and non-nodulated legumes occur side by side. In some tropical rain forests nodulated (and apparently N2-fixing) legumes are dominant even when the whole ecosystem is not nitrogen limited (Martinelli et al. 1992, 1999). Understanding the occurrence and role of legumes in these different plant communities is a major challenge.

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Conclusion I can do no better than to quote Corner (1976): “Leguminosae are one of the great lines of dicotyledonous evolution. They far exceed the idea of an order or family, except within the narrow confines of nomenclature. The neap-tide of modern botany never uncovers its riches.” I hope I may have helped to turn the tide!

Acknowledgments I should like to thank, first my colleagues and students of the last 25 years who have weaned me away from temperate crop legumes and escorted me to so many fascinating places; second, various funding agencies, in particular EU, NERC, ODA (now DFID), British Council and the Leverhulme Trust; third, those who have allowed me to quote unpublished data (Douglas Deans, ITE Edinburgh; Amy Spriggs, University of Capetown) or work in press (Luis Martinelli, Sao Paulo; Fanchon Prevost, French Guyana); fourth, two referees for very helpful suggestions; finally Walter Herrmann for introducing me to the apt quotation from Corner (1976).

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Received 4 June 1999 Revised version accepted 6 October 1999