agricultural intensification, soil biodiversity and...

Applied SoU r, co y

ELSEVIER Applied Soil Ecology 6 (1997) 55-76

Agricultural intensification, soil biodiversity and ecosystem function in the tropics: the role of nitrogen-fixing bacteria

J.H.P. Kahindi a, p. Woomer b, T. George c, F.M. de Souza Moreira d, N.K. Karanja a, K.E. Giller e,*

a University ofNairobi, MIRCEN, P.O. Box 30197, Nairobi, Kenya b TSBF, UNESCO-ROSTA, P.O. Box 30592, Nairobi, Kenya

c IRRI-NifFAL Collaborative Program, IRR1, Los Bahos, The Philippines d Departamento de Ciencia do Solo, Escola Superior de Agricultura de Lavaras, CP 37, Lavaras, Minas Gerais, CEP 37200-00, Brazil

e Wye College, University of London, Wye, Ashford, TN25 5AH, UK

Accepted 25 June 1996

Abstract

Among the nitrogen (N2)-fixing bacteria, the rhizobia in symbiosis with legumes are generally the most important in agriculture, although Frankia, cyanobacteria and heterotrophic free-living N~-fixers may fix significant amounts of nitrogen under specific conditions. The taxonomy of N2-fixing bacteria is undergoing substantial revisions due to the advent of molecular methods for phylogenetic analysis, and in certain cases this has proved useful in unravelling ecological relationships among confusing groups. Molecular methods are also proving useful in studies of biodiversity within populations of rhizobial species.

Rhizobia are surprisingly competent free-living bacteria, although few fix nitrogen in the free-living state, and the major factors that determine their population sizes in the absence of legume hosts are environmental stresses (such as soil acidity factors), protozoal grazing and some factors associated with agricultural intensification such as increases in salinity or heavy metal pollution of the soil. Rhizobial populations generally increase in response to the presence of the host legume. Due to the high degree of host-specificity between legume hosts and rhizobial species, loss of a single rhizobial species can result in loss of N2-fixation by that legume, although many legumes can be nodulated by several species of rhizobia. However, as only a single, compatible rhizobial genotype or strain is necessary for establishment of effective N2-fixation (i.e. the basis of the rhizobial inoculant industry), it is questionable whether biodiversity within species is necessary to ensure function, although this may confer resilience in the face of further environmental stresses. © 1997 Elsevier Science B.V.

Keywords: Azorhizobium; Bradyrhizobium; Cyanobacteria; Free-living nitrogen-fixing bacteria; Rhizobium; Legumes; Nitrogen fixation

1. Introduction

1.1. N2-f ixat ion as an e c o s y s t e m f u n c t i o n

The importance of nitrogen (Nz)-fixafion in na- * Corresponding author, ture can best be seen in non-cl imax ecosystems. At

0929-1393/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0929-1393(96)00151-5

56 J.H.P. Kahindi et al. / Applied Soil Ecology 6 (1997) 55-76

early stages of primary succession on freshly weath- ratios required to favour the growth of free-living ering substrata where there is little organic matter in diazotrophs in the soil. A similar situation prevails in the soil, nitrogen is the nutrient most limiting pro- agricultural soils where an accumulation of carbon ductivity and N2-fixing organisms have a major role and nitrogen has been measured in the absence of to play in the accumulation of nitrogen in microbial legumes (Jenkinson, 1977). At later stages of pri- and plant biomass, and in soil organic matter (Fig. mary succession it is postulated that there will be 1). Examples of this are the colonization of bare rock further benefits from N2-fixation in situations where surfaces in tundra by N2-fixing lichens, the abun- nitrogen again becomes limiting, such as in mature dance of legumes and actinorhizal plants such as forests where nitrogen is predominantly bound up in Alnus species in the colonization of outwash gravels the living biomass and in soil organic matter. In from glaciers in mountainous regions. Gorham et al. mature forests there is an abundance of carbon which (1979) suggested that the importance of N2-fixation can act as a substrate for N2-fixation by free-living within a particular ecosystem may be linked to the heterotrophs. In secondary successions, the presence stage of ecosystem development. Phototrophic N 2- of soil organic matter means that the importance of fixation is likely to be critical in early seres where N2-fixation is relatively less than in primary succes- the efficiency of nutrient capture is low. It may also sions. play a crucial role in secondary succession (e.g. after By analogy we can consider agriculture, at least fire) where nitrogen may be lost but other nutrients in production systems where large amounts of nutri- are plentiful. This N2-fixation by symbiotic hetero- ents are not added, as an arrested stage of succes- trophic microorganisms may be important when there sional development in which the rate of N2-fixation is an accumulation of organic matter which is utiliz- is largely dependent on the availability of other able as a carbon source, such as in mature forests nutrients, notably phosphorus. As soil organic matter with an abundance of decaying wood (Giller and reserves, and hence the supply of available nitrogen, Day, 1985). In many mid-successional stages where are reduced, the potential rate of N2-fixation will legumes are absent, appreciable amounts of nitrogen increase as long as other factors are not limiting. A nevertheless accumulate. Under grasslands, there is possible example where rates of N2-fixation are often a gradual accumulation of organic carbon, but strongly limited by water occurs in dry tropical this is normally insufficient to maintain the high C:N climates. Despite the abundance of N2-fixing trees in

l ~Prlmary succession 0

o / \

Successional time

Fig. 1. The relative importance of N2-fixation at different stages of successional development of natural ecosystems (after Gorham et al., 1979).

J.H.P. Kahindi et al. /Applied Soil Ecology 6 (1997) 55-76 57

dry savanna regions such as the Sahel, the main body line arising from two or more ancestries (see Good, of evidence (Giller and Wilson, 1991) would indicate 1974), no longer matters. Macrosymbionts must be that little N2-fixation takes place, accompanied by suitable N2-fixing microsymbionts

to attain a competitive advantage in nitrogen-defi- cient soils and in turn enrich their environment in

2. The diversity of N2-fixing bacteria in soil these organisms. As a result of the coevolution and mutual dependance of N2-fixing associations, a hier-

To group all organisms capable of biological N 2- archy of controls on the biodiversity of NE-fixing fixation into a single category is convenient from the microsymbionts may be assigned. An example is the molecular and physiological perspective but more legume-Rhizobium symbiosis. cumbersome when their ecological roles and biodi- The few N2-fixing species shown to make a real versity are taken into account. The most primitive contribution to tropical cropping systems (Giller and N2-fixing organisms, the anaerobic autotrophs, are Wilson, 1991; Table 1) can be grouped into four among the earliest forms of life on earth and have main categories: the widest distribution as aquatic and soil organisms. 1. The cyanobacteria (blue-green algae), which oc- Furthermore, N2-fixation is widely distributed among cur both as free-living species and in associations the two subdivisions of prokaryotes, including pro- with a variety of plants, most notably Azolla teobacteria, cyanobacteria, green sulphur bacteria and (Giller and Wilson, 1991). methanobacteria. N2-fixation by these free-living or- 2. The actinomycete Frankia species, which form ganisms, in most likelihood, did not emerge until the symbiotic associations with a number of flower- atmospheric nitrogen was readily available as a sub- ing plants. While most of the known host plants strate and prior to the accumulation of atmospheric for Frankia species are temperate plants, Frankia oxygen. Nitrogenase becomes irreversibly damaged species form active N2-fixing symbioses with by exposure to atmospheric levels of oxygen (GiUer some tropical plants such as Casuarina (Giller and Wilson, 1991). We may never know whether and Wilson, 1991; Dreyfus et al., 1987). N2-fixation arose from the evolution of a single, very 3. The free-living diazotrophs, with Klebsiella and primitive bacterial ancestor, or to what extent lateral Azotobacter species as classic examples. This transfer of the N2-fixing genome is responsible for group includes a diverse array of associative N 2- the broad taxonomic distribution of N2-fixation. Re- fixing microorganisms such as Azospirillum and gardless of this, the biodiversity of free-living N 2- Herbaspirillum (Dtibereiner and Pedrosa, 1987). fixers is a ubiquitous phenomenon of several bacte- 4. The rhizobia bacteria, which form symbiotic rela- rial genera which proceeds slowly under the most tionships with legume plants. These contribute the optimal conditions and is regulated by the quantity greatest amounts of biologically fixed nitrogen in and persistence of anaerobic niches and substrate agriculture, and are most intensively studied to availability. Whether or not these organisms actually date. fix nitrogen is related to the availability of 'free' Nitrogenase is an exclusively prokaryotic enzyme, nitrogen in their immediate environment. Complicat- although the largest rates of N2-fixation are found in ing our understanding of biodiversity among the the highly evolved symbioses between bacteria and free-living prokaryotes is our inability to reliably higher plants. Functional N2-fixation thus depends culture them; as a result, many of these organisms on the occurrence of soil bacteria with the capacity remain undiscovered (Ward et al., 1990). to fix nitrogen, and our discussion will thus concen-

The biodiversity of N2-fixing microsymbionts is trate on the biodiversity and ecology of N2-fixing comparatively more readily appreciated and man- bacteria in soil, with particular emphasis on the aged. Symbiosis suggests coevolution of species rhizobia. within discrete centres of origin. Whether the indi-

2.1. Free-living N2-fixing bacteria vidual partner species are monophyletic, arising from single, unrepeatable mutations, or polyphyletic, as In this group are included all those diazotrophs when a new species results from the segregation of a that are 'completely free-living', or in loose associa-


Table 1 A functional classification of the range of NE-fixing bacteria which contribute to agriculture

Type N2-fixing bacteria Symbiont

Heterotrophs Free-living

Anaerobic CIostridium Microaerophilic Frankia, Azospirillum, Aerobic Bradyrhizobium, Azotobacter, Derxia

Root-associated Microaeropbilic AzospiriUum, Herbaspirillum Endophytic Acetobacter Sugar cane (Saccharum spp.) Symbiotic Frankia Casuarina spp., Alnus spp., etc.

Rhizobium, Bradyrhizobium Many legumes Azorhizobium Sesbania rostrata

Autotrophs Free-living

Anaerobic Microaerophilic Rhodospirillum, Bradyrhizobium Aerobic Cyanobacteria

Symbiotic Cyanobacteria Fungi (lichens) Anabaena azollae Azolla slap. Cyanobacteria Cycads Bradyrhizobium Aeschynomene spp.

tion as a result of root (or rhizosphere) and leaf ica. These include maize, sorghum, wheat, rice and (phyllosphere) colonization. Although the data are millet (see Dtibereiner and Pedrosa, 1987). Azospir- incomplete because the ability to fix nitrogen has not illum halopraeferans was found associated with roots always been tested, this group represents 16 families of kallar grass, a salt-tolerant grass grown in saline and over 46 genera of prokaryotes, soils of Pakistan (Reinhold et al., 1987). Several

Non-symbiotic N2-fixing bacteria are phylogeneti- other groups of diazotrophs, notably members of the cally extremely diverse, with representatives occur- genus Azotobacter, have a remarkable ability to fix ring in nine subdivisions of the Eubacteria and in nitrogen aerobically. The two best-known species four subdivisions of the Archaebacteria, whereas Azotobacter chroococcum and Azotobacter symbiotic N2-fixers are found only in three subdivi- vinelandii, have a rather strict requirement for neu- sions of the Eubacteria (Young, 1992). A large hum- tral pH conditions and thus are rare in tropical soils, bet of new genera and species of free-living N2-fix- except in a few near-neutral soils in the humid ing bacteria have been described in the past few tropics (DiSbereiner and Pedrosa, 1987). One species, years and many more non-symbiotic species have Azotobacter paspali, has been found in association been described than there are symbiotic NE-fixers. with the roots of Paspalum notatum (DiSbereiner et New species and genera recently described include al., 1972). Azoarcus (Reinhold-Hurek et al., 1993) Members of Beijerinckia are more tolerant of low

pH, and are hence more common in soils of the 2.1.1. Heterotrophs tropics where acidity prevails. Two species, Beijer-

Perhaps the best known genus of such bacteria is inckia indica and Beijerinckiafluminensis, have been Azospirillum (Tarrand et al., 1978). Strains from shown to associate with the rhizosphere of sugar three species, AzospiriUum brasilense, AzospiriUum cane and other plants (Dtibereiner and Pedrosa, lipoferum and AzospiriUum amazonense, have been 1987). Other genera of free-living diazotrophs of isolated from the rhizosphere of roots of all the importance include Derxia, Herbaspirillum, Pseu- major cereal crops, in both Africa and Latin Amer- domonas, Acetobacter, Klebsiella and Bacillus. For

J.H.P. Kahindi et al. / Applied Soil Ecology 6 (1997) 55-76 59

comprehensive reviews of free-living N2-fixing or- Table 2 ganisms see Eady (1992) and Young (1992). Reported ranges of BNF by different bacterial systems in tropical

soils (after Elkan, 1992) Bacteria with a wide range of ecological adapta- Bacterial system Fixation rates tion are able to fix nitrogen and many of these do not

establish symbioses with plants (Table 1). However, (kg ha- i per year)

many (e.g. Azospirillum, Azotobacter) form close Legume-Rhizobium symbioses 24--584 Azolla- Anabaena symbioses 45-450

associations with the roots of certain plant species, Erankia symbioses 2-362 notably grasses (Boddey and D~ibereiner, 1994), and Free-living and associative bacteria Trace-36 some (e.g. Herbaspirillum, Acetobacter) occur as endophytes within plant shoots (D~bereiner, 1992, 1994).

substantially contribute to the nitrogen requirements 2.1.2. Autotrophs of the rice (Lumpkin and Plucknett, 1982). As the

Free-living autotrophic N2-fixing bacteria include Azolla symbiosis does not strictly occur in soil, it the Cyanobacteria, the photosynthetic bacteria such will not be considered further here. as the purple, non-sulphur bacterium Rhodospirillum and Rhodopseudomonas, and the chemoautotrophs 2.3. Frankia symbiosis such as Thiobacillus.

The Cyanobacteria are ubiquitous in tropical soils, Some 279 angiosperm species, mostly trees or occurring in both unicellular (e.g. Aphanothece, shrubs, are known to form symbioses with the N 2- Gloeotrichia) and filamentous forms (e.g. An- fixing actinomycetes of the genus Frankia (Baker abaena, Nostoc). The filamentous, heterocystous and Mullin, 1992). Despite many earlier attempts, forms are predominant in tropical soils but are only Frankia was first isolated in 1978, 90 years after likely to form a large biomass and contribute sub- rhizobia were isolated by Beijerinck (Callaham et al., stantially to agriculture in flooded rice fields (Roger 1978). The difficulty in isolation of Frankia stemmed et al., 1987; Whitton and Roger, 1989). The amounts from its slow growth rate in non-selective media and of nitrogen fixed by cyanobacteria in rice paddies are from the frequent incidence of contaminants during generally moderate (5-25 kg N ha-I per year), but isolation which results from the anatomy of acti- can be improved by management such as fertilization norhizal nodules. Although the plant symbionts be- with phosphorus (Roger and Ladha, 1992). long to a wide range of families, the bacterial sym-

bionts are reasonably homogeneous at the level of 2.2. Cyanobacterial symbioses the genus, even though there exists considerable

heterogeneity between the relatively few strains iso- Cyanobacteria form symbioses with almost every lated in pure culture (Lechevalier and Ruan, 1984).

division of plants and lichens (Giller and Wilson, Recent evidence indicates that a few of these isolates 1991). Although many of these associations play are sufficiently distinct, suggesting that the genus important ecological roles, the only cyanobacterial Frankia will probably be split in future (Lechevalier, symbiosis of importance in tropical cropping systems 1994). is that formed with Azolla. AzoUa is a genus of The biodiversity of Frankia could be assessed aquatic ferns, found free-floating on the water sur- directly in soil. However, such methods are still very face. It forms a symbiotic relationship with the limited. For instance, qualitative bioassays (nodula- cyanobacterium Anabaena azollae whereby Azolla tion tests) do not discriminate between different provides nutrients and a protective leaf cavity for the strains, exclude non-infective strains and neglect the Anabaena, which in turn provides nitrogen for Azolla effects of competition between strains (Akkermans et (Elkan, 1992). Under optimum conditions, this sym- al., 1991). Considering the quantification of propag- biosis results in as much (or more) nitrogen fixed as ule units it is necessary to determine what represents do legume-Rhizobium symbioses (Table 2). If inocu- a nodulation unit (NU). Other difficulties exist dur- lated into a paddy and intercropped with rice, it can ing preparations of serial dilutions because hyphal

60 J.H.P. Kahindi et aL / Applied Soil Ecology 6 (1997) 55-76

fragmentation may inflate or deflate the numbers of from a practical point of view in dealing with rhizo- NUs. Molecular biology methods are also being bial inoculants, this concept has been perpetuated developed to assess biodiversity of Frankia (Akker- although cross-inoculation groups are better regarded mans et al., 1991; Myrold et al., 1994). as a 'series of sieves' than as discrete, neat boxes

(Mytton et al., 1988). Effectiveness groupings within 2.4. Rhizobial biodiversity such cross-inoculation groups were also identified.

A useful concept when considering the nodulation The first root-nodulating NE-fixing bacterium was range of both rhizobia and their legume hosts is that

isolated in 1888 by Beijerinck and named Bacillus of host specifity or promiscuity. An example of a radicicola (Beijerinck, 1888); the name Rhizobium 1 type of rhizobium with restricted host range is Rhi- was coined in 1889 (Jordan, 1984). Initially, all zobium galegae, which appears only to nodulate bacteria isolated from the root nodules of legumes Galega species (Lindstr~Sm, 1989), and that of a were classified as a single genus Rhizobium, even promiscuous strain with a very broad host-range is though it was recognized by Fred et al. (1932) that the fast-growing strain NGR234 (now assigned to the fast-growing rhizobia were in fact more closely Sinorhizobium fredii) (Jarvis et al., 1992) which related to Agrobacterium than they were to the nodulates at least 37 genera of legumes, and the slow-growing rhizobia (see Young, 1994). non-legume tree Parasponia. This tropical tree of

Initial classification of the rhizobia into 'species' the family Ulmaceae is also nodulated by Bradyrhi- was made purely on phenotypic grounds, and largely zobium and is the single known example of a non- based on the ability of the legumes to nodulate legume which can form N2-fixing nodules with rhi- particular legumes, which gave rise to the concept of zobia (Trinick, 1980). 'cross-inoculation' groups. A cross-inoculation group All slow-growing rhizobia were initially grouped can be defined as 'a group of legume host species in the 'cowpea-misceUany', which is still often re- nodulated specifically by one set of rhizobial species, ferred to as the 'predominant tropical type', even and not by any rhizobial species that could induce though little evidence exists to support the view that nodules on legumes not belonging to that cross-inoc- Bradyrhizobium is preponderant in the tropics and ulation group' (Giller and Wilson, 1991). For exam- Rhizobium in temperate regions of the world. Re- ple, Melilotus alba, Medicago sativa and Trigonella cently, phylogenetic analysis on the basis of the species all belong to a cross-inoculation group nodu- sequence of the 16SrRNA gene has become the luted by Rhizobium meliloti (now Sinorhizobium). It standard for classification of bacteria and sufficient was soon recognized that these were not discrete, sequences have become available in the past 5 years non-overlapping groups, and Wilson (1944) pub- to allow this method to be usefully applied to the lished a paper entitled 'Over five-hundred reasons to rhizobia (Young, 1992, 1994; Martinez-Romero, abandon the cross-inoculation group concept' in 1994). This new classification is independent of which, as the title indicated, more than 500 excep- phenotypic traits and yet has confirmed a number of tions to the cross-inoculation groups were listed, taxonomic divisions. The three main genera Azorhi- Even so, because of the attractiveness of this idea zobium, Bradyrhizobium and Rhizobium are clearly

distinguished, as are many of the species that were previously recognized amongst the fast-growing rhizobia (Table 3 and Fig. 2). The new phylogenetic

i A note on the use of terms to apply to bacteria nodulating the classification has also resulted in some surprises. roots of legumes:

I. Rhizobium (singular) and rhizobia (plural) are general terms R h o d o p s e u d o m o n a s pa lus t r i s , a f r ee - l iv ing , p h o t o -

used to describe bacteria capable of forming nodules on the synthetic N2-fixing bacterium was placed in roots (or stems) of legumes. The term (brady)rhizobia can be Bradyrhizobium, although th is d id n o t s e e m so used to refer collectively to slow-growing rhizobia. strange once it was realized that the exciting discov-

2. Azorhizobium, Rhizobium, Sinorhizobium and Bradyrhizobium ely of photosynthetic rhizobium from the stem nod- are genus names. Thus, Rhizobium, Rhizobia, rhizobium, rhizobia, aradyrhizobium, aradyrhizobia, Bradyrhizobia are all ules of Aeschynomene (Dupuy et al., 1992) (which incorrect!! was provisionally placed in a new genus Photorhizo-

J.H.P. Kahindi et al. /Appl ied Soil Ecology 6 (1997) 55-76 61

bium) was also a Bradyrhizobium species. Other lack of clear boundaries in terms of cross-inoculation surprises have been the close relationship between groups of nodulation ability between slow-growing Bradyrhizobium and the soil-borne, cat-scratch bac- rhizobial strains and host legumes led to their being terium Afipia. clumped into a single group termed the 'cowpea

The genus Sinorhizobium was originally proposed miscellany'. for the fast-growing rhizobia isolated from the roots The only 'species' recognized separately was ini- of soyabean (Rhizobium fredii), but this proposal tially Rhizobium japonicum, which became was rejected as 16SrRNA gene sequences indicated Bradyrhizobium japonicum on the description of the that Rhizobium fredii was in fact closely related to genus Bradyrhizobium (Jordan, 1982). The grounds Rhizobium meliloti ((Jarvis et al., 1992) Young, for recognition of Bradyrhizobiumjaponicum were 1992). Further research which has identified two questionable as this 'species' clearly never repre- further closely related species has also led to the sented a clearly defined cross-inoculation grouping suggestion that Rhizobium meliloti be assigned to with many Bradyrhizobium japonicum forming ef- the genus Sinorhizobium, which would also contain fective nodules on cowpea and many cowpea strains Rhizobium fredii and the new species from Senegal forming effective nodules on soyabean. Distinct Sinorhizobium teranga and Sinorhizobium saheli (De groups within Bradyrhizobium japonicum have long Lajudie et al., 1992, 1994). The slow-growing been distinguished on both genetic and phenotypic bradyrhizobia have received much less attention. The grounds (Young, 1992), which eventually led to the

Table 3 The classification of rhizobia (for further details see Young (1992, 1994)

Genus and species Host plants Reference

Azorhizobiurn

A. caulinodans Sesbania rostratu Dreyfus et al. (1988)

Bradyrhizobium

B. elkanii Glycine, Vigna Kuykendall et al. (1992) B. japonicum Glycine Jordan (1982) Bradyrhizobium sp. (Arachis) Arachis Jordan (1984) Bradyrhizobium sp. ( Cajanus) Cajanus

Rhizobium

R. ciceri ~ Cicer Nour et al. (1994) R. etli Phaseolus Segovia et al. (1993) R. galegae Galega Lindstri~m (1989) R. huakuii * Astragalus Chen et al. ( 1991) R. leguminosarum Jordan (1984)

biovar phaseoli Phaseolus by. viciae Vicia, Pisum by. trifolii Trifolium

R. loti * Lotus Jarvis et al. (1982) R. tropici Leucaena, Phaseolus Martinez-Romero et al. (1991) R. tianshanense * Giyaine Chen et al. (1995)

Sinorhizobium

S. fredii Glycine (and many others) S cholla and Elkan (1984) S. meliloti Melilotus Jordan (1984) S. saheli Sesbania, Acacia De Lajudie et al. (1994) S. teranga Sesbania, Acacia

Note added in proof: Since this article was written in 1995 the genus Mesorhizobium has been proposed to include the species marked with an asterisk.


Agrobacterium Rhizobium loti _tumefaciens

• \ R. hualcuii / P~Uo~mum L L - R c i ~ / R. sp. OK55

abortus N \ ~ ~ /Sinorhizobiura meliloti X \ \ / / s. freaii

\ \ \ / . / ~ s. ~li

Rhizobium e t h ' ~ . _ "R. sp. FL27

/ N R. sp. '¢ \ --R. sp. Or191

I I IX \ N Blastobacter / / ] \ ~ --d~itri~n, Azorhizobium / BTAil \ ~ _ _ caulinodans Rhodopseudomonas B. radyr, hizobium

Bradyrhizobium I ap°nwum elkanii

Fig. 2. A phylogenic tree showing propable relationships between rhizobia and related bacteria (redrawn from Martinez-Romero, 1994, with additions)• Distances not to scale.

description of a separate species Bradyrhizobium urgent attention to the taxonomy of the bradyrhizo- elkanii (Kuykendall et al., 1992; Anonymous, 1993). bia is apparent. Bradyrhizobium elkanii comprises strains which be- longed to DNA homology group II of soyabean 2.5. Order from chaos? rhizobia, which has marked similarities to other cowpea bradyrhizobia (Hollis et al., 1981; Fuhrmann, In ecological terms, the new revisions of rhizobial 1993). taxonomy start finally to help us make some sense of

The major research activity on Bradyrhizobium what have long been confusing groups. The case of has concentrated on inoculant strains and strains Phaseolus vulgaris is one interesting example of indigenous to the soils of the USA and Brazil this, where originally all isolates were lumped into (Rumanjek et al., 1993) because of the importance of Rhizobium phaseoli. Although isolates from nodules the soyabean inoculant industry there. This undoubt- of Phaseolus vulgaris were invariably fast-growing, edly represents another gross imbalance compared to it was recognized that this 'species' encompassed a the distribution of the tropical food and forage wide range of colony characteristics in culture and of legumes such as Arachis, Calapogonium, Cajanus, ecological adaptation, for instance to acidity. Centrosema, Desmodium, Pueraria and Vigna Successive revisions resulted initially in Rhizo- species which all nodulate with bradyrhizobia. If we bium phaseoli being placed in Rhizobium legumi- further consider the other wild species and trees nosarum bv. phaseoli, as strains isolated from which nodulate with bradyrhizobia, the need for Phaseolus vulgaris grown in UK soils were found to


be closely related to strains isolated from pea 3. Ecology of N2-fixing bacteria ( Rhizobium leguminosarum) and clover ( Rhizobium trifolii). As the ability to nodulate different host plants could be transferred between strains simply by 3.1. The saprophytic existence of rhizobia in soil

replacing the symbiotic plasmid (pSym), these three rhizobial 'species' were grouped together into ~t sin- An important consideration for optimizing nitro- gle species Rhizobium leguminosarum with three gen fixation in the legume-rhizobial symbiosis is the biovars (Jordan, 1984). Whilst this classification was response of the microsymbiont and the nodule to a perhaps appropriate for the range of strains initially dynamic soil environment. In the absence of the studied, research in Mexico revealed further hetero- host, for instance, rhizobia are just ordinary soil geneity among the rhizobia from Phaseolus vulgaris bacteria, with any genetic or ecological advantage nodules in America, which led to the description of realized only in the presence of a host root (Keyser two further species: Rhizobium tropici (Martinez- et al., 1992). As free-living members of the soil Romero et al., 1991) and Rhizobium etli (Segovia et microflora, rhizobia are subject to prevailing al., 1993). Rhizobium tropici and Rhizobium etli 2 physicochemical and biological conditions, and are represent rhizobia which can nodulate Leucaena leu- forced to compete with other organisms for limited cocephala and many other legumes, whereas Rhizo- resources. When free-living, rhizobia are in their bium leguminosarum bv. phaseoli are more host- saprophytic phase. When presented with the root of a specific with nodulation ability restricted to Phaseo- receptive host, rhizobia gain an advantage because lus vulgaris of the legumes on which they have been they possess the ability to infect and gain entry into tested. Strains belonging to Rhizobium tropici are the root (infective phase) and eventually cooperate in also intrinsically more tolerant of acidity than many the formation of a functioning root nodule (symbio- rhizobia (Graham et al., 1994) and in fact all of the tic phase) (Keyser et al., 1992). The ability of rhizo- acid-tolerant rhizobial strains for Phaseolus fall into bia to persist in the absence of their legume hosts is this species, crucial to the successful establishment and persis-

Problems of the instability of Phaseolus rhizobia tence of legumes in field and pasture systems (see have often been observed with strains being notori- Fig. 3). Relationships between selected environmen- ously unstable in symbiotic properties. This instabil- tal variables and the abundance of free-living rhizo- ity can be attributed to genetic rearrangements which bia, hosts and the symbionts have been examined in commonly occur in the plasmids of strains which detail (Lawson et ai., 1987; Woomer et al., 1988; belong to Rhizobium etli and Rhizobium legumi- Yousef et al., 1987). Fundamental differences in the nosarum bv. phaseoli. Thus the new species of adaptation to prevalent stress conditions exist be- Phaseolus rhizobia distinguish groups which have tween rhizobia at the genera, species and strain distinct ecological properties from what was earlier a levels. confusing mixture of types (Graham et al., 1982). Woomer (1990) found that Rhizobium species Unfortunately, the story does not end there, as many and Bradyrhizobium japonicum were intolerant of more 'species' of rhizobia occur amongst strains that the stress conditions associated with highly weath- have been isolated from Phaseolus nodules, indicat- ered soils of low pH, and were moderately adapted ing that further clarification of the taxonomy of to semi arid conditions. Of the Rhizobium species, Phaseolus rhizobia is necessary. This attests to the those associated with Leucaena leucocephala were permissive nature of Phaseolus as a host for rhizobia best suited to the highly weathered and the semiarid (Segovia et al., 1993; Giller et al., 1994). soils. Bradyrhizobium japonicum was well suited to

the soils representative of the humid tropics and intolerant to high temperatures and desiccation. This difference in the adaptation of the two genera also

2 Rhizobium etli was originally thought to be host-specific to Phaseolus vulgaris in its nodulation, but later research (Hernan- occurs among indigenous populations in a wide range dez-Lucas et al., 1995) demonstrated that it is also able to of soil conditions on the Island of Maui (Woomer e t

nodulate many legumes, al., 1988), and in the symbiotic relationships with


~ - : ' . . . . 'co pe rs is t ;oil

Environmental stresses limit

nodule function

Ineffeclive symbiosis

. . . . . . !, ~ ;

Environmental stresses limit nodule number

Fig. 3. The critical ecological stages of the legume symbiosis (after Woomer and Bohlool, 1989).

Lupinus species (Miller and Pepper, 1988) and bv. viciae to be more acid sensitive than bv. trifolii, Prosopis glandulosa (Jenkins et al., 1987) in desert but less so than Sinorhizobium meliloti. Halliday and soils of North America. The different abilities of Somasegaran (1983) identified TAL 1145 as a highly these two genera to colonize soils may be an adapta- effective isolate on Leucaena that is persistent in tion to the preferred habitats of their respective host acid soils, compared to other strains of the same legumes (Norris, 1965). species.

Of all the fast-growing rhizobia, Rhizobium trop- 3.2. Acid stress ici appears to be the species most tolerant of soil

acidity (Graham et al., 1994; see above). It has been Rhizobial strains differ in their tolerance to soil suggested that the ability of the slow-growing

acidity. For example, Graham and Parker (1964) bradyrhizobia to produce an alkaline reaction in cul- demonstrated that Sinorhizobium meliloti was the ture may be related to their ability to colonize acid most sensitive of several species of Rhizobium to the soils rich in aluminium (Karanja and Wood, 1988). effects of low pH in broth culture. Similar results However, the ability to produce alkali is a function were obtained when the population sizes of indige- of the nutrient-rich culture media and there is no nous Sinorhizobium meliloti in soils of various pH in evidence that bradyrhizobia can reduce tha acidity of Canada were examined (Rice et al., 1977). Richard- their surrounding environment in the soil. In a study son and Simpson (1988) reported that bv. trifolii of Phaseolus vulgaris rhizobia in two Kenyan soils associated with Trifolium subterraneum L. is intoler- of contrasting pH, the population of rhizobia in an ant of low pH soils (pH 4.3). Rice et al. (1977) acid soil was actually more diverse than the rhizobial recovered higher populations of bv. trifolii than population in a soil of moderate pH (Anyango et al., Sinorhizobium meliloti in low pH soils of Canada. 1995). The population in the acid soil, however, was Other authors have concluded that Rhizobium legu- dominated by Rhizobium tropici, whereas the popu- minosarum bv. trifolii is less sensitive to low pH lation in the higher pH soil was dominated by spe- effects than Sinorhizobium meliloti (see Lowendorf, cific rhizobia similar to Rhizobium etli. 1980). Liming the soil resulted in increased popula- Liming the soil resulted in increased population tion density of indigenous bv. trifolii in pastures that density of indigenous Rhizobium leguminosarum bv. were both planted and not planted with the legume trifolii in pastures that were both planted and not host (Richardson and Simpson, 1988). Fred and Dav- planted with the legume host (Richardson and Simp- enport (1918) identified Rhizobium leguminosarum son, 1988). The effect liming is likely to have on


biodiversity of rhizobia is unclear. Under severe acid Graham and Rosas, 1979; Cassman et al., 1980; stress, the number of rhizobial strains (and species) Jacobsen, 1984; Israel, 1987). Thus, when legume is likely to be reduced, but if the stress is alleviated growth is limited, in this case by decreased phospho- completely this may allow individual, highly compet- rus availability, host enrichment of introduced elite itive strains to become dominant. It is likely that strains or highly competitive indigenous strains would legume hosts that are adapted to soil acidity would also be limited, thereby influencing their biodiver- enrich rhizobia tolerant to acid soil conditions, sity.

3.3. Effect o f phosphorus 3.4. Soil aeration

Survival and effectiveness of rhizobial popula- Soil aeration status could be a determinant of tions are dependent on levels of soil nutrients. Phos- rhizobial biodiversity. Soil aeration does not undergo phorus is an important nutrient in this regard. Rhizo- drastic changes in upland agricultural soils, but in bia differ markedly in their external phosphorus re- most of the rice lowlands aerobic-to-anaerobic soil quirements and in their phosphorus storage and uti- transition is a feature of the production system. Soil lization capacities (Cassman et al., 1981; Smart et flooding could decrease rhizobial diversity and num- al., 1984; Beck and Munns, 1985). Further, the bers, but strains differ in survival ability (Boonkerd differences among rhizobia in their ability to use and Weaver, 1982), and often sufficient numbers of phosphorus are expressed variably at different phos- rhizobia thrive through flooding (Weaver et al., phorus levels (K.G. Cassman, unpublished data; Sin- 1987). Ladha et al. (1989) reported survival in high gleton et al., 1985, 1992), but phosphorus nutrition is numbers of rhizobia of the aquatic legume Sesbania important in interpreting the N2-fixing capacity of rostrata in flooded rice rhizosphere. rhizobia (Singleton et al., 1985). The effects at- tributable to rhizobia are pronounced when phospho- 3.5. Other environmental factors ms supply is low, a condition synonymous with the acid infertile uplands in the tropics. Singleton et al. There is evidence that at least some of the pesti- (1985, 1992) reported discrimination among rhizo- cides used in agriculture can have adverse effects on bial strains in soybean response to phosphorus. Un- the survival of rhizobia or on nodulation of legumes der field conditions, Singleton et al. (1992) found (Roberts, 1992). Particular attention must be paid if that strains equally effective at moderate phosphorus legumes are to be inoculated with rhizobia by seed fertilization differed substantially when phosphorus coating when agrochemicals are also applied to the supply was high (600 kg P ha -1) in a highly phos- seed surface (Graham et al., 1980). Pollution of phorus-fixing Hawaiian ultisol, agricultural soils caused by the addition of heavy

Clearly, these rhizobial characteristics influence metal contaminated sewage sludges has been shown their ability to survive and establish in soils and to completely suppress N2-fixation in white clover thereby their biodiversity. Leung and Bottomley (Trifolium repens) due to the toxicity of heavy met- (1987) demonstrated the superior ability of bv. trials to Rhizobium (Giller et al., 1989). folii strain S-6, compared to others, to persist in Growth and survival of rhizobia (and free-living phosphorus-depleted media, which indicates that not N2-fixing bacteria) will be influenced by competition only are there differences in saprophytic competence and antagonism from other organisms. Some mi- between rhizobial species, but also between strains croorganisms, including fungi and other bacteria, and within species. Low phosphorus supply greatly will compete for nutrients, while direct antagonistic diminishes the likelihood of a response to rhizobia effects include production of toxic bacteriocins, lysis by reducing legume growth (Cassman et al., 1981; by bacteriophages, predation by protozoa or para- Crasswell et al., 1987). Further, nodules are strong sitism by Bdellovibrio (Roughley, 1985). Whilst such sinks for phosphorus. Many authors have reported phenomena can be demonstrated in laboratory media, effects of phosphorus on nodulation and nodule func- the extent of their importance in soil is unknown. tion in many legumes (Gates and Muller, 1979; Grazing of rhizobia in soil by protozoa has been


shown to reduce the populations of rhizobia in soil by Sylvester-Bradley et al. (1983, 1991) in which (Danso et al., 1975), and susceptibility of strains to rhizobia were screened in cores of unsterile soil from bacteriophages may result in their poor survival the Colombian savannahs. These soils were acidic (Barnet, 1980). oxisols with high aluminium saturation which con-

Heijnen et al. (1987) observed an increase in the rained dense populations of (brady)rhizobia compati- persistence of bv. trifolii resulting from the addition ble with the tropical pasture legumes for which of 10% bentonite clay to soil. Bushby and Marshall rhizobia were screened. Thus in a single screening (1977) noted a similar effect with montmorillonite, test the rhizobia were evaluated not only for their In rhizosphere studies, Moawad and Bohlool (1984) effectiveness in N2-fixation, but also in competitive found that TAL 1145 was extremely competitive for ability to establish nodules despite the resident in- nodulation against other introduced and indigenous digenous population of rhizobia and in adaptation to strains in both a Mollisol and an Oxisol. Sanginga et the hostile soil environment (see Giller and Wilson, al. (1988) found 310 and 580 Leucaena rhizobia g - l 1 9 9 1 ) .

soil in a secondary forest and grassland, respectively, 3.6. The influence of host symbionts and suggested that strain IRc 1045 was highly competitive and somewhat persistent in a sandy Alfisol The establishment and functioning of an effective in Ibadan, Nigeria. Ishizawa (1953)identified Rhizo- symbiosis is dependent on genetic determinants in bium leguminosarum bv. viciae as similar to bv. both the plant and the bacterium. Some 45 genes trifolii in high-temperature tolerance, and less toler- across eight legume species have now been identi- ant then Rhizobium meliloti. Overall, populations of fled as affecting nodulation and N2-fixation (Vance Rhizobium and Bradyrhizobium species have been et al., 1988). The establishment of rhizobia in the shown to vary in their tolerance to major environ- symbiotic state results from a competition for nodule mental factors, and subsequently the screening for sites among strains in the nodules of legumes grown tolerant strains has been pursued (Keyser et al., in soils that contain indigenous populations of other 1992). Keyser and Munns (1979) found that cowpea rhizobia (Ham, 1980; Brockwell et al., 1988). Previ- rhizobia, previously identified as acid- and alu- ous inoculation and continued cropping of a legume minium-tolerant, formed more nodules in two acid confer a formidable advantage in numbers and envi- soils than did sensitive strains. Acid-tolerant strains ronmental adaptation to the indigenous population in of Sinorhizobium meliloti helped establish large ar- competition with introduced strains (May and eas of Medicago polymorpha on acid soils in Aus- Bohlool, 1983). Recent experiments from standard- tralia (Howieson and Ewing, 1986). In the tropical ized field inoculation trials at 29 sites showed that Africa region, for instance, most biological nitrogen 59% of the variation in inoculation response could fixation work has concentrated on the screening of be accounted for by numbers of indigenous rhizobia rhizobial strains for their tolerance to various envi- (Thies et al., 1991). However, in the absence of ronmental stresses such as temperature (Gitonga et indigenous rhizobia, the pattern of competition be- al., 1989), pH and aluminium (Karanja and Wood, tween strains for nodulation of soybean was found to 1988). be a stable, detectable characteristic, independent of

In contrast, Bottomley (1991) suggested that the rhizosphere population size, nitrogen fertilizer appli- influences of specific abiotic factors such as organic cations, temperature, or soil type (Abaidoo et al., matter or clay content, pH base saturation, etc., 1990). which were shown by various researchers to influ- Rhizobial biodiversity could also be affected by ence saprophytic rhizobia, are much less important the nutritional status of the legume host. Legume than their interactive effects. This could be true in growth is restricted when conditions are un- that, in view of all the efforts that have been directed favourable for an effective symbiosis (Bohlool et al., to identifying highly effective and environmentally 1992; George et al., 1992), and inoculation with compatible strains, not much impact has been superior N2-fixing rhizobia frequently fail to provide recorded in terms of increased legume production. A a yield response due to limiting growth factors other different, simple but elegant approach was pursued than nitrogen (Singleton et al., 1992).


4. Managing bacterial biodiversity bia into soils facilitates future nodulation by highly effective microsymbionts (Chatel et al., 1968).

Populations of Rhizobium leguminosarum bv. tri- 4.1. Controlled biodiversity through rhizobial intro- folii in soils from pasture areas where clover did not duction occur ranged from 1.78 to 3.76 log10 g-1 soil, and

these populations became enriched 30-1600-fold in The peristence of Bradyrhizobium japonicum in- nearby areas where legumes were present (Woomer

troduced into soils was the topic of many previous et al., 1990). The importance of legume hosts has investigations (Bohlool and Schmidt, 1973; Vidor also been demonstrated by Lawson et al. (1987) for and Miller, 1980; Crozat et al., 1982; Dunigan et al., Rhizobium leguminosarum bv. trifolii in Australian 1984; Ellis et al., 1984; Corman et al., 1987). A pastures and Yousef et al. (1987) for Bradyrhizo- rapid decline in population size is reported for bium species associated with peanut in drylands in Bradyrhizobium japonicum following release into Iraq. soils containing high populations of indigenous Bradyrhizobium japonicum (Ellis et al., 1984; Cor- 4.2. Dispersal of rhizobia in soils man et al., 1987). Consequently, their research was unable to measure the effects of severe stress condi- Rhizobia do not form resting structures, which tions because of the lower limit (103 cells g - t soil) facilitate wind dispersion, but may be physically of the fluorescent antibody technique (Crozat et al., protected by close association of microcolonies with 1987). None of these studies has followed population clay minerals. Rhizobia do not colonize aquatic envi- kinetics in the range of 101-102 applied rhizobia ronments, but are readily mobilized by rain-splatter (Singleton and Tavares, 1986). Some researchers and surface runoff and erosion, as well as being (Crozat et al., 1982; Corman et al., 1987) have tolerant of salt water. Azorhizobium caulinodans, modelled the persistence kinetics of Bradyrhizobium which resides saprophytically in the soil but infects japonicum introduced into soils. Crozat et al. (1982) and forms nodules on the stems of Sesbania ros- mathematically described the persistence of trata, must be able to bridge the space between the Bradyrhizobium japonicum with a logistic function, two (Adebayo et al., 1989). Similarly, an animal that In this 5 year experiment, the bacteria reached a carries legume seeds by adhesion to the exterior of stable population size in soils after 900 days, and the its body is also likely to transport soils from which level of persistence was independent of the quantity that legume was grown, on its feet and body. A of rhizobia released. Consequently, the rate of de- unique feature in the movement of the microsym- cline but not the ultimate outcome of the release was biont is its chemotropic attraction to host roots and determined by the population density of the release, its movement along them (Woomer, 1990). The nat- Populations released at less than stable densities ural dispersal mechanisms are sufficient to allow slowly increased with time. Only two soils were used legumes to steadily exploit new habitats accompa- in this study and no mathematical analysis of the nied by their microsymbiont, and vice versa. But coefficients obtained with different soils was possi- some wind and water dispersal mechanisms clearly ble. Corman et al. (1987) had difficulty in applying place either partner beyond one another's company. the logistical model of Crozat et al. (1982) to the Rhizobia transmitted long distances by dust storms persistence of Bradyrhizobium japonicum in three may often settle into new floristic zones. Canavalia French soils; rather, the Gompertz equation was maritima forms large, buoyant seeds that are trans- selected to describe the kinetics of persistence. Again, ported by ocean currents and waves to new beaches the stable population density was independent of the where these vines stabilize primary sand dunes. Seeds levels applied. While the N2-flxation resulting from adhering to animals along lengthy migration routes the inoculation of legumes with rhizobial strains is that include many river crossings are likely' to be- not always in direct proportion to the saprophytic come deposited onto soils uninhabited by their co- abilities of those strains (Robert and Schmidt, 1983), evolutionary microsymbiont. When this separation the successful introduction of highly effective rhizo- occurs, the fate of the organism and the biodiversity

68 J.H.P. Kahindi et al. /Appl ied Soil Ecology 6 (1997) 55-76

of the symbiotic community becomes regulated by prolonged disturbances due to agricultural intensifi- the degree of symbiotic specificity, cation, populations of rhizobia will re-establish very

The rate of movement of rhizobia in soils has slowly in most cases unless they are deliberately received limited attention in the past, primarily be- reintroduced. cause this rate is sufficiently slow to allow uninocu- lated host legumes not to be nodulated by adjacent 4.3. Availability of suitable macrosymbionts treatments (Frazier and Fred, 1921). The importance of downward movement of applied rhizobia to infec- The Leguminosae is the third largest family of tion sites prior to or during germination has been plants, consisting of over 500 genera and 11 000 recognized (Brockwell and Whaley, 1979; Madsen species, with a natural distribution on all continents and Alexander, 1982; McDermott and Graham, except Antartica (see Good, 1974). Many of the 1989). Hamdi (1971) examined the role of particle Leguminosae enter into N2-fixing symbiosis with size and water passage on the vertical passage of bacteria belong to the genera Azorhizobium, Rhizobium leguminosarum bv. trifolii through soils. Bradyrh&obium, Rhizobium and Sinorhizobium Woomer (1990) observed that different sets of envi- (Sprent and Sprent, 1990). Differences in specificity ronmental parameters influence the survival and dis- between the two symbionts for nodulation and N 2- persal of rhizobia. The horizontal movement of rhi- fixation exist for different relationships. In some zobia in soils has received less attention. Using cases, legumes belonging to different subfamilies Rhizobium leguminosarum bv. trifolii, Hamdi (1971) (Caesalpinioideae, Papillionoidae and Mimosoideae) demonstrated that movement of rhizobia in sterile are able to enter into symbiosis with a single rhizo- soils is slowed by increasing water tension and re- bium, and several genera and species of rhizobia are quires that water-filled pore spaces remain continu- able to effectively nodulate a single host (Dreyfus et ous. Sorby and Bergman (1983) used behavioral al., 1987). In other cases, symbiotic partners are mutants to demonstrate that active chemotaxis in- more restricted, as when a single genus (Trifolium) is creases the rate of horizontal movement of Sinorhi- restricted to a subspecies of Rhizobium legumi- zobium meliloti in sterile soils. The rate of move- nosarum (bv. trifolii). While rhizobia may not be ment in soils at high moisture tensions varies among obligate symbionts and in some cases can persist for other genera of bacteria (Wong and Griffin, 1975). long periods as saprophytic organisms, rbizobia tend These authors demonstrated the restrictive influence to be enriched in the presence of a compatible host of soil bacteria. The role of earthworms in the in- (Section 3.1). crease of soil microbial populations and presumably the movement of water and bacteria in soils was 4.4. Unknowing human dispersal of rhizobia? demonstrated by Parle (1963). Increases in the microbial densities of bacteria and actinomycetes, but Humans have transported legumes, and to a far not for fungi, were noted when earthworm intestines lesser extent other BNF macrosymbionts, from one were compared to the bulk soil. Woomer (1990) area of the world to another for many centuries. noted that the populations of rhizobia introduced at Phaseolus lunatus, assigned to the South American the rate of 450 000 g - J soil that survived within the centre of diversity, was discovered in 6000-5000 release area were 43-359 for Bradyrizobiumjapon- BC at excavations in Peru and in Mexico dating icum and 215-6094 for Leucaena rhizobia 2 years from 500-300 BC. During the 16th century, following release. The number of rhizobia recovered Spaniards carried this bean across to the Philippines 4 m from the release area containing these rhizobia from where it spread to tropical Asia (Duke, 1981). ranged from 1 to 5 cells g i soil. Less dispersal Glycine max and Vigna mungo, grain legumes be- occurred in upslopc and upwind directions, particu- lieved to be of Asian origin, are unknown in the wild larly in drier environments. This is consistent with but have since become widely distributed throughout the findings of Hamdi (1971), who noted little or no the world (Table 4). More recently, leguminous trees movement of rhizobia in soils at high moisture ten- have become widely distributed. Leucaena leuco- sions. Thus, if rhizobia are lost from a soil due to cephala, Calliandra calothrysus and Gliricidia


sepium are three widely planted tree species within 5. The b iod ive rs i ty of N2-fixing o rgan i sms u n d e r

agroforestry systems which originate from tropical a g r i c u l t u r a l in tens i f ica t ion America and have specialized rhizobial requirements. Presumably the reason for the success of We have summarized the various types of N2-fix- these legumes in countries where they have been ing organisms, their niches and ecological regulation. introduced must rely either on the host being suffi- Now, we address the disturbances resulting from ciently promiscuous to nodulate with rhizobia in- land management and how these disturbances impact digenous to the soils in the new countries (as is upon those niches and the biodiversity of N2-fixing l ikely with Vigna unguiculata, for example), or on organisms. Conversion of natural ecosystems neces- the introduction of compatible rhizobia along with sitates the removal of native vegetation and soil the seed of the legumes. In the case of Phaseolus disturbance. The level of disturbance may vary with vulgaris, introduced to Afr ica by the Portuguese in different conversion practices; for purposes of sim- the 16th century, compatible rhizobia were demon- plicity we will address a conversion process where strated to be present in African soils with no history all above-ground natural vegetation is felled and of cultivation (Gil ler et al., 1994), although the removed, regrowth of propagules is subsequently possibil i ty that rhizobia had also been dispersed with controlled by weeding, waterlogged areas are drained, the crop could not be discounted, the entire soil surface is tilled and monocrops planted.

Table 4 Selected legume hosts, their microsymbionts, center of diversity a and current distribution

Legume host Microsymbiont Center of diversity Distribution

Acacia mearnsii Bradyrhizobium sp. Australia Australia, East Africa Acacia nilotica Bradyrhizobium sp. Africa Africa, India Acacia senegal Sinorhizobium teranga Africa Africa Arachis hypogaea Bradyrhizobium sp. South America Worldwide Astragalus cicer Rhizobium haukuii Eurosiberia Southern Europe Cicer arietinum Rhizobium ciceri Central Asia Middle East, India, South America,

Europe Desmodium intortum Bradyrhizobium sp. South America Tropics and subtropics Glycine max Bradyrhizobium japonicum, China USA, Brazil, Argentina, China, Japan,

Sinorhizobium fredii Russia, Zambia Lablab purpureus Bradyrhizobium sp. Africa India, SE Asia, US Lens culinaris Rhizobium leguminosarum bv. viciae Near East Mediterrean, subtropics and warm

temperate regions Leucaena leucocephala Rhizobium tropici Middle America SE Asia, Mexico, Hawaii Medicago sativa Sinorhizobium meliloti Near East Asia, Europe, Americas, Australia Phaseolus vulgaris Rhizobium leguminosarum by. phaseoli, Middle America Americas, East Africa, Europe

Rhizobium tropici, R. etti Pisum sativum Rhizobium leguminosarum bv. viciae Near East Temperate and upland tropics Sesbania cannabina Sinorhizobium saheli Africa Africa, USA Sesbania rostrata Sinorhizobium saheli, Africa Africa

Sinorhizobium teranga, Azorhizobium caulinodans

Sesbania sesban Sinorhizobium teranga Africa Pantropical Stylosanthes humilis Bradyrhizobium sp. South America Pantropical Trifolium repens Rhizobium leguminosarum bv. trifolii Near East Temperate and highland tropics Vicia faba Rhizobium leguminosarum bv. viciae Central Asia Americas, East Africa, India,

SE Asia, China Vigna mungo Bradyrhizobium sp. Hindustan India, USA, West Indies Vigna r a d i a t a Bradyrhizobium sp. Indochina China, East Africa, SE Asia,

West Indies, USA Vigna unguiculata Bradyrhizobium sp. Africa Tropics, subtropics, warm temperate

a Centers of diversity after Duke (1981) and distribution from Duke (1981) and personal observations of the authors.


Naturally occurring organisms are by necessity Some forms of land intensification improve the acclimatized to the periodic stresses in their environ- biodiversity of N~-fixing organisms, particularly in ments, but land conversion may result in new intensi- improved, inoculated pastures and mixed fanning ties and types of stresses beyond past acclimatiza- systems (Fig. 4). For example, introduction of rhizo- tion. Following land conversion and tillage, there is a bia into soils is frequently the result of inoculation short-lived increase in soil microbial activity result- use, dependent, in large part, on the saprophytic ing from organic inputs (Jenkinson, 1977); however, competence of the strains contained within the inocu- the persistence and function of N2-fixing autotrophs lant. The persistence of the relatively ineffective will, with time, depend on the changes in the abun- soybean rhizobia cluster USDA 123 (group of strains dance of free water surfaces and anaerobic niches. USDA 123, 127 and 129) in the soils of mid-western Photosynthetic bacteria capable of N2-fixation that USA is a case in point where an indigenous strain is rely upon standing water surfaces will become re- dominant to the extent of excluding other inoculant duced in number as soils are tilled and drained, strains (Moawad and Bohlool, 1984; Schmidt et al., Microsymbionts whose populations in the soil are 1986). However, subsequent naturalization of inocu- dependant upon specialized associations with natural lants into soil microbial communities is not in- vegetation will become directly affected as host in- evitable because rhizobial strain selection procedures fection sites and rhizospheres are no longer avail- have now focused on the infective and symbiotic able, while more promiscuously associated mi- capabilities of isolates rather than on their sapro- crosymbionts may find opportunity for association phytic capabilities. with the introduced crops (Thies et al., 1995). If the introduced crop species are N2-fixing, and effective microsymbionts either never occurred in the previous 6. Future trends natural ecosystem or failed to persist during land conversion, then one land management option is to It can be argued that the limitations of both the introduce the desired microsymbiont through inocu- past and the current rhizobial taxonomy are largely lation. From this senario, we envisage some naturally due to the limited range of rhizobial isolates that occurring, N2-fixing organisms being removed or have been studied. These have concentrated on suppressed during land conversion, and others being species of largely herbaceous species of the subfam- introduced during subsequent land management, ily Papilionoideae from which isolates have been

~iiii~i~i!i!!!i!ili!il]!i!!!!i]iiiii]ii~i!7:~ ~,..

• '.!ii~iii~ii~i!!iiiiiiiiiiiiiiiiiili::~iii~: :~!~ii: "..~ii!~i~ii!iiiiiiiiiiiiiiii!iiiiiiiii~

~iiiiiiiiiiiiiii~i!iii!ili!i!~,il ~. ~'iiiiii~i,~iiiiiiiiiii!iiiiii!~i ~~ ~ 1 ~

Fig. 4. Niches and diversity of N2-fixing organisms in tropical ecosystems.

J.H.P. Kahindi et al. /Applied Soil Ecology 6 (1997) 55-76 71

made. Since the 1984 edition of Bergey's Manual of internal cycling of nutrients and low loss rates, the Systematic Bacteriology in which four species were input of nitrogen from free-living diazotrophs may in described (Jordan, 1984), nine additional species have fact be sufficient to meet the requirements for main- been named (Table 3). Most of these new species tenance of productivity. Whether agricultural intensi- were isolated from legumes belonging to the subfam- fication is always directly linked to reductions in ily Papilionoideae. Whether this can be taken to biodiversity of rhizobial species, and strains within indicate the Papilionoideae as a particular source of those species, remains to be established, but future rhizobial diversity, or whether this is simply due to benefits of the legume-rhizobium symbiosis, whether the lack of research on rhizobia from nodules of for food, fodder, soil fertility or forestry, will depend legumes which belong to the other two subfamilies, on the expolitation of natural biodiversity of both the the Mimosoideae and the Caesalpinioideae, remains legume hosts and the bacterial strains present in the to be established. Jordan (1984) pointed out that soils of the tropics. Only when we better understand rhizobia classification would require gradual modifi- the factors regulating this biodiversity will we be cation as the large reservoir of tropical leguminous able to conserve it for the future and exploit it fully species are examined. Recent surveys of the nodula- in tropical agriculture. tion ability of tropical legume species from the Ama- zon and Atlantic Brazilian forests have resulted in the isolation of many new strains from nodules of

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