Optimising the legume symbiosis in stressful and competitive environments within southern Australia—some contemporary thoughts

Soil Biology & Biochemistry 36 (2004) 1261–1273 www.elsevier.com/locate/soilbio

Optimising the legume symbiosis in stressful and competitive environments within southern Australia—some contemporary thoughts John Howiesona,b,*, Ross Ballardc a

Centre for Rhizobium Studies, Division of Science and Engineering, Murdoch University, Murdoch, WA 6150, Australia b Department of Agriculture Western Australia, Baron Hay Court, South Perth, WA 6151, Australia c South Australian Research and Development Institute, GPO Box 397, Adelaide, SA 5001, Australia

Abstract In the managed agricultural ecosystems of southern Australia, if an edaphic environment is not stressful to root-nodule bacteria (hereafter rhizobia), it is likely to become a competitive environment for nodulation (although not always detrimentally so) soon after the introduction of an inoculated legume. We suggest that stressful environments limit rhizobial communities to less than 100 cells g21 soil at some time during the season. This overview puts forward the hypothesis that in perturbed ecosystems (i.e. those that are intensively managed) such as in the 25 million ha of the southern Australian grain and grazing belts, the rhizobial community is still substantially immature in an evolutionary sense. The rhizobial community is representative of only a few species, primarily those of Mediterranean origin that were accidentally introduced, or have been fostered by legume development programs, or remnants of the populations associated with native legumes. We consider there is little inter-specific competition for substrates because of this relative immaturity, but suggest that intra-specific competition for nodulation is commonplace wherever abiotic stress is absent. We nominate two primary abiotic stresses that are permanently present that have limited rhizobial colonization or legume nodulation for some species in southern Australia and four secondary (temporary) abiotic stresses. We believe that selection of adapted symbioses, or where warranted adapted elite rhizobial strains or legume host genotypes, can overcome these stress factors. We emphasise that where several abiotic stress factors are present they may act synergistically, but that this net effect is still likely to be symbiosis-specific. We acknowledge that genetic transformation in situ is providing new strain variability with which we must contend. We also put forward the suggestion that opportunities exist for the managed introduction of selected genotypes of agricultural legumes that effectively interact with rhizobial communities to achieve optimal N-fixation. In doing so, we give more precise definition to the widely used terms ‘exclusive’, ‘selective’ and ‘promiscuous’ nodulation. q 2004 Published by Elsevier Ltd. Keywords: Abiotic stress; Rhizobia; Nodulation; Competition; N fixation; Acidity; Legumes

1. Introduction Rhizobia is a term used to describe a range of soil bacterial genera including Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium, Allorhizobium and Azorhizobium that are able to enter into symbiosis with plants predominantly of the family Leguminosae (Sprent, 2001; O’Hara et al., 2003). Legumes and rhizobia together fix atmospheric N and because of this feature they are often introduced to managed agricultural ecosystems to improve their organic fertility, N economy or farming system flexibility (Robson, 1990; Reeves and Ewing, 1993; * Corresponding author. E-mail address: [email protected] (J. Howieson). 0038-0717/$ - see front matter q 2004 Published by Elsevier Ltd. doi:10.1016/j.soilbio.2004.04.008

Brockwell et al., 1995; Howieson et al., 2000a). Optimal performance of the N-fixing symbiosis depends upon preselection of both symbiotic partners for adaptation to the target environment, which may in some form present a challenge to rhizobial survival or nodulation (Sessitsch et al., 2002). Ironically, selection and commercialization of elite, well adapted strains of rhizobia can lead to a new management hurdle—the development of large and diverse populations of genetically related rhizobia, which may provide unfavorable competition if they evolve to become ineffective in N fixation. In this paper we provide examples of how different symbioses (combinations of host and rhizobia) and management might be employed to reduce the detrimental effects of abiotic stress, or competitive environments for nodulation that are common in southern Australia.

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2. Stressful environments for nodulation in southern Australia We begin by asserting that: the edaphic environment is considered to be stressful to root nodule bacteria where the proliferation of a rhizobial species is limited to less than 100 cells g21 of bulk soil at some point in time, or where there are adverse effects on the nodulation process. It is considered that legumes will very often respond to inoculation where the rhizobial community is less than 100 cells g21 soil, depending upon soil N status and the rhizobial or legume species (Chatel and Parker, 1973; Thies et al., 1991; Rice and Olsen, 1992; Evans et al., 1993). Hence our arbitrary level has some relevance in the context of the requirement to inoculate agricultural legumes. If we accept this definition of stress in relation to rhizobial populations present in the soil, then there are many Australian environments that are not stressful. Fine textured, neutral and alkaline pH soils in southern Australia commonly contain more than 1000 cells g21 of medic rhizobia (Ballard and Charman, 2000; Brockwell et al., 1991; Brockwell, 2001), clover rhizobia (Coventry et al., 1985; Denton et al., 2000; Ballard et al., 2002) and pea rhizobia (Evans et al., 1993; Ballard et al., 2004), whereas mildly acidic coarse textured soils often contain equally large numbers of lupin and serradella rhizobia (Chatel et al., 1968; Chatel and Parker, 1973). 2.1. The abiotic stresses common in southern Australia The key stress factors in relation to rhizobial survival and legume nodulation in southern Australia have been recently reviewed by Slattery et al. (2001). They consist principally of abiotic factors, some of which can be exacerbated or remediated by agricultural management practices. We observe that the most significant and common abiotic stresses that affect rhizobial proliferation or legume nodulation are permanent features of the environment, whilst the lesser stresses are, more often than not, transient. We term these ‘primary’ and ‘secondary’ stresses respectively, viz.: Primary stresses: † acidity below pH 5.0 or alkalinity above pH 8.5; † clay content below 15%, with cation exchange capacity (CEC) , 10 meq 100 g21. Secondary stresses: † aridity, i.e. low incident rainfall combined with low soil moisture holding capacity; † high soil nitrate concentration in autumn;

† salinity; † extremes of temperature. Alkalinity, acidity and low clay content, which are a result of Australia’s ancient geology, are permanent abiotic features in large tracts of southern Australia. Although acidity and low clay content may be remediated at the surface by addition of lime or clay, it is often uneconomic to do so, hence we classify them as the primary and most significant stresses with which legume symbioses must contend. Rhizobia, particularly the fast growing species, are more commonly found in higher number in fine textured, neutral pH soils, than in coarse textured, acid soils (Fred et al., 1932; Jensen, 1943; Hely and Brockwell, 1962; Parker, 1962; Evans et al., 1993) and the protective effect of clay on rhizobia has been well documented (Lahav, 1962; Marshall, 1975; Fuhrmann et al., 1986). The secondary stresses, albeit having substantial effects, are seasonally dependent. We emphasise, however, that the manifestation of these abiotic stresses is often dependent upon the species of rhizobia present and its host. For example, Medicago symbioses are especially sensitive to soil pH below 6 (Munns, 1970), whereas the lupin symbiosis with Bradyrhizobium sp. (Lupinus) will function optimally between pH 4 and 6, but is sensitive to pH above 6.0 (Tang and Robson, 1993). Lupin-nodulating bradyrhizobia are also particularly well suited to survival in soils with clay content below 15% (Chatel and Parker, 1973). Also, it is important to realise that combinations of the above abiotic stresses, whether primary or secondary, are particularly synergistic and it is probably more important to be cognisant of those situations where they are acting together rather than in isolation. For example, the concurrence of acidity, aridity and low clay content are common on approximately 4– 6 million ha of Western Australia and New South Wales, and acting together these abiotic stresses are prejudicial to nodulation of our key agricultural legumes (Robson, 1969; Richardson and Simpson, 1988; Howieson, 1995; Brockwell et al., 1991). This problem of acidity and symbiosis has been so significant in Australian agriculture that it has drawn substantial scientific examination at both the molecular and physiological level (Munns, 1965; Richardson et al., 1988a; Howieson et al., 1993; O’Hara and Glenn, 1994; Glenn et al., 1999; Dilworth et al., 2001; Cheng et al., 2002) as well as at the ecological level (Jensen, 1943; Chatel et al., 1968; Robson and Loneragan, 1970; Howieson and Ewing, 1986; Brockwell et al., 1991; Slattery et al., 1999) over the last 50 years. However, if in this complex of stresses, acidity is replaced by alkalinity (as in large parts of South Australia and N. Victoria), the problem may become not one of rhizobial survival, but of competition for nodulation. Proliferation of the microsymbionts for clover and medic under alkaline conditions has been substantial (Ballard and Charman, 2000; Brockwell, 2001; Denton et al., 2000).

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We discuss intra-specific rhizobial competition for nodulation later in this review, but the point is made that one must consider the effects of combinations of edaphic stress within the context of a specified symbiosis. Soils of low clay content may have concomitant low CEC. This is significant to rhizobial survival because divalent cations are known to ameliorate effects of low pH on cell structural integrity (O’Hara et al., 1989). For example, a soil of pH 5 with 2 meq 100 g21 of Ca is more stressful to rhizobia than a soil of pH 5 that has 20 meq 100 g21 of Ca. Many acid soils in Western Australia have total CECs of less than 10 meq 100 g21, whereas acid soils of the Mediterranean basin, because they are less weathered, have total CECs of . 20 meq 100 g21. This offers some explanation for why many strains collected from acid soils in the Mediterranean basin are not well adapted to acid soils of southern Australia (Howieson, 1995). Alkaline soils of low clay content and low CEC do not appear to present the same challenges to survival of fast-growing species of rhizobia as do acid soils with equivalent clay content. We believe this is because it is the synergistic aspects of multiple abiotic effects that are particularly important. 2.2. Approaches to overcome abiotic stress effects upon rhizobial survival There has been considerable effort expended to select strains of rhizobia tolerant to severe soil conditions in many regions of the world (see reviews by Zahran (1999) and O’Hara et al. (2003)) and in many situations there is no alternative to this approach. However, we believe a fundamental (or perhaps parallel) strategy should be to attempt the selection of well-adapted symbioses for the target environment, before narrowing the research focus to the selection of elite, tolerant rhizobial strains. What do we mean by this? Perhaps a good illustration might be drawn from the 50 years of research to develop annual pasture legume species and rhizobia for the moderately acid, coarse-textured soils of wheat-belt Western Australia and western New South Wales. These regions superficially and broadly suited the cultivation of Medicago (medic) and Trifolium (clover) species, being low and medium rainfall environments (300 – 600 mm annual rainfall) with arable soils that were frequently in crop (Cocks et al., 1980). Medics and clovers had volunteered on neutral and alkaline soils in these vast regions, but had not flourished on the many acid intrusions because the growth and survival of the microsymbionts was reduced by the soil pH(Ca) between 4 and 5.5 (Robson and Loneragan, 1970; Bromfield and Jones, 1980). Whilst first year nodulation was reliably achieved by lime-pelleting the inoculant-coated seed, the regenerating legumes frequently failed to nodulate on the acid soils and subsequently perished because of N-deficiency. The syndrome was termed ‘second year mortality’ (Parker, 1962) and quite rightly ascribed to microbiological problems encountered in

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the low pH soil. After considerable effort, acid-adapted and saprophytically-competent strains of rhizobia capable of surviving in these soils were selected (Chatel et al., 1968; Howieson and Ewing, 1986; Watkin et al., 2000) in conjunction with new species of medic-M. polymorpha, M. murex and M. sphaerocarpos (Gillespie and McComb, 1991) which were more symbiotically competent in acid soils (Howieson and Ewing, 1989). Remarkably, the acidtolerant rhizobia and host legumes were together able to establish and maintain symbiosis on many of these stressful soils in the last half of the 20th Century. The need to improve legume reliability and productivity in these same acid, arid regions after the emergence of new biological and economic challenges (Reeves and Ewing, 1993; Howieson et al., 2000a) led to the examination of alternative symbioses from the Mediterranean Basin that had hitherto been largely ignored (Loi et al., 2004). In the first instance this led to the rapid exploitation of Ornithopus spp. (Nutt and Loi, 1999) in these environments, with sowings between 1995 and 2002 over more than 1 million ha. More recently, Biserrula pelecinus (Howieson et al., 1995; Loi et al., 1997) and its specific microsymbiont, which is a Mesorhizobium sp. (Nandasena et al., 2001), have been commercialised for the acid soils containing higher clay content (10 – 25%) in these challenging environments. These symbioses have since proven to be inherently stress tolerant and widely adaptable, without any requirement for intensive selection of elite rhizobial genotypes adapted to the complex of soil acidity, aridity and infertility. The lesson learnt here is that a particular edaphic stress might be almost insurmountable to one symbiosis, but not to another. Only after failing to find ‘naturally’ adapted symbioses for the stressful environment should we embark upon a program to seek elite individuals from less adapted symbioses. 2.3. Nitrate—a secondary stress upon nodulation The effect of nitrate on nodulation is another good example of the species-dependant nature of some abiotic stresses. Nitrate is a transient or seasonal stress in many soils in southern Australia. There is a ‘flush’ of N produced when the autumn break coincides with warm weather that maximises mineralisation of soil N (Simpson, 1962; Unkovich and Pate, 1998). If this mineralisation is not followed quickly by substantial rains that leach N from within the rooting zone of germinating annual legumes, nitrate can cause significant disruption to nodulation in susceptible legume species. Legume symbioses do not react uniformly to nitrate (Harper and Gibson, 1984). It appears likely that some symbioses can delay nodulation in the presence of soil nitrate and then quite adequately proceed to nodulate when the soil reserves of N are diminished. Other symbioses might delay nodulation in the presence of high concentrations of soil N, but because of other factors (such

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Fig. 1. The nodule score* at 7 weeks achieved by two annual pasture legumes growing in soil of pH 4.4 or 6.2 when inoculated with 105 cells ml21 of broth grown inoculant in the presence of nitrate applied in solution (LSD P , 0:05 4.8, unpublished data from S. Carr, J. Howieson and R.Yates, Centre for Rhizobium Studies, Murdoch University, Perth). *nodule score based upon number, size and position of nodules on the root system.

as low populations of rhizobia) cannot achieve satisfactory nodulation when required. The autumn flush of N (as either nitrate or ammonium) can reduce nodulation of annual medics and this can be severe in some species if another stress, such as acidity, is present (Ewing and Robson, 1990). Nodulation failure reported in B. pelecinus grown on acidic sands in the initial evaluation of the species were speculated to result from nitrate inhibition of nodulation, followed by poor survival or colonization of the root system by the biserrula inoculant in acid soils (i.e. the dual abiotic stresses acting synergistically). This hypothesis was examined in sand culture experimental systems. In Fig. 1 we illustrate the differential reaction of the pasture legumes B. pelecinus and Trifolium subterraneum to increasing nitrate concentration at two pH levels. Nitrate was applied in solution twice weekly, at concentrations between 1.2 and 32 mM. Nodulation of B. pelecinus appeared relatively unaffected by N between 1.2 and 9.8 mM nitrate, whereas nodulation of T. subterraneum was strongly enhanced by 6.2 mM nitrate, then depressed in the presence of higher nitrate concentrations. The soil pH did not greatly affect the nodulation response of B. pelecinus to nitrate, whereas there was an interaction between pH and nitrate concentration on nodulation of T. subterraneum. These experiments did not adequately explain the observed nodulation failures of B. pelecinus in situ. In fact, they exposed the resilient nature of the B. pelecinus symbiosis relative to that of T. subterraneum when exposed to the dual abiotic stresses of low pH and high N concentration. 2.4. Other secondary abiotic stresses We included aridity, salinity and extremes of temperature in our list of secondary abiotic stresses because of their well-documented effects on rhizobial proliferation and nodulation (Gibson, 1967; Zahran, 1999; Hungria and Vargas, 2000; Slattery et al., 2001; Peltzer et al., 2002). The gradual salinisation of arable soils in southern Australia and the putative plant-based solutions to that phenomenon (Cocks, 2001) reminds us that we know little of how rhizobial populations will respond to an increasing osmotic potential in soil solutions. In particular, we need to ask

the question of whether rhizobial species already exposed to the dual stresses of aridity and extreme pH in some environments will cope with the additional stress imposed by salinity.

3. The effects of management on the manifestation of abiotic stress The effects of abiotic stress upon rhizobial proliferation and nodulation may be moderated by management. The following examples serve to illustrate this point: † cultivation has the capacity to disturb protective microsites, so frequent and aggressive cultivation may reduce rhizobial populations (Richardson and Simpson, 1988; Coventry and Hirth, 1992); † herbicide residues have the capacity to damage roots and root hairs and therefore reduce opportunities for nodulation (Koopman et al., 1995); † dry sowing of inoculated legumes can lead to reduced inoculant survival on seed (Roughley, 1988) † the duration of the non-legume phase in rotations can influence rhizobial numbers because rhizobia often multiply to higher numbers in the rhizosphere of their host legume (Coventry et al., 1985); † the type of non-legume in the rotation can affect rhizobial numbers because some species, especially Cruciferae such as Canola, can release bactericidal residues from roots (Riffkin et al., 1996). Other management decisions might ameliorate abiotic stress. For example: † Lime application can reduce acidity at the soil surface to increase rhizobial persistence (Evans et al., 1988; Richardson et al., 1988b; Slattery et al., 2001). † Stubble retention and minimum-tillage can reduce otherwise extreme temperatures at the soil surface (Hungria and Vargas, 2000), as well as increase soil organic matter content. † Clay application to sandy soils can provide microsites for rhizobial survival.

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Hence, through management, we have the opportunity to modify the effects of abiotic stresses on symbiosis. However, in practice, this dimension of optimising the legume symbiosis receives little attention.

4. Competitive environments for nodulation Competition for nodulation is commonplace in parts of southern Australia, as well as worldwide. Indeed for many situations it is the corollary to edaphic stress because: if a particular edaphic environment is not stressful to a species of root-nodule bacteria, and therefore not suppressive of its population development, then a competitive environment for nodulation may emerge soon after the introduction of the inoculated host legume to the soil. There will be competition for nodulation whenever more than a single rhizobial genotype capable of nodulating the legume is resident within the soil (Amarger, 1981; Thies et al., 1991). This may be manifest in the year of establishment of the legume despite seed inoculation (Brockwell et al., 1982; Barran and Bromfield, 1997; Denton et al., 2002), or may develop progressively as inoculant strains become displaced by naturalised rhizobia in seasons subsequent to inoculation (Gibson et al., 1976; Roughley et al., 1976; Streeter, 1994; Evans et al., 2004). Competition for nodulation is most commonly intraspecific, i.e. between strains of a single species of rootnodule bacteria, although there are examples of different rhizobial species able to nodulate a specific legume genotype. Phaseolus vulgaris, for example, may be nodulated by at least five species of root-nodule bacteria (Amarger et al., 1997) and thus in this case inter-specific competition is possible. The resolution of the problem of rhizobial competition for nodulation lies in developing an understanding of the biotic and abiotic factors involved, the dynamics of the soil population, the role of the legume host and the genetic basis of both nodulation and competition for nodulation (Barran and Bromfield, 1997). It is also possible that competition for substrates arises between the many species of rhizobia when they co-exist in communities of soil saprophytes or as rhizosphere inhabitants (Rovira, 1961). However, we believe this form of inter-specific competition (for growth substrates) has little impact on legume performance in southern Australia. As evidence for this, there have been several recent examples where new rhizobial species (e.g. Mesorhizobia for biserrula) have been introduced and are able to proliferate. Further, in experimental plots across southern Australia, we seldom experience problems introducing new species of rhizobia as inocula for other exotic legumes (e.g. Hedysarum and Lotus). This suggests that competition for substrates by other rhizobial species or soil microflora is

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not sufficiently intense to limit the development of rhizobial communities. Our ability to readily introduce new rhizobial species to southern Australia is also indicative that the current rhizobial flora has the capacity to develop to a new level of equilibrium. Prior to European settlement of Australia there is little doubt that a mature rhizobial population existed in our soils, in association with the diverse suite of native legumes. Little is known of the qualitative aspects of that population, although recent studies (Lawrie, 1983; Barnet and Catt, 1991; Marsudi et al., 1999; Yates et al., 2004a) suggest it was probably more diverse than previously described (Lange, 1961). A surprising finding was that many of the rhizobia associated with Australia’s native legumes are fast growing (Yates et al., 2004a). However, we speculate that the rhizobial communities associated with the native legumes became suppressed, both qualitatively and quantitatively, as their hosts progressively disappeared and new plant species which were incapable of nodulating were grown after European settlement (Lange and Parker, 1960). The incursion of Mediterranean legumes, with their specific inoculant root-nodule bacteria, and the cultivation of them in near-monoculture conditions over approximately 95% of arable southern Australia, has caused a massive perturbation to the pre-existing rhizobial equilibrium. Despite this change in rhizobial ecology, the introduced rhizobial strains only represent a very narrow range of species and genotypes, because only a few non-indigenous legume species have become prominent in southern Australian agriculture. The few rhizobial species that have recently proliferated in southern Australia include Rhizobium leguminosarum, Sinorhizobium spp. and Bradyrhizobium sp. (Lupinus). These species are primarily those that have been fostered by the limited number of legume genera that were introduced either accidentally (e.g. Medicago, Trifolium) or by institutional legume development programs (e.g. Lupinus, Ornithopus, Vicia and Pisum). Where the edaphic environment has been favourable to these hosts and their rhizobia, large populations of the microsymbiont have quickly developed. They often exceed 1000 and occasionally 1 £ 106 cells g21 of bulk soil (Chatel and Parker, 1973; Evans et al., 1993). For example, in Fig. 2 the composite data from several recent surveys shows that in soils where medics are often grown, nearly 70% contain more than 1000 Sinorhizobium spp. g21. Similarly, for soils collected from regions where clovers are grown, about 70% contain more than 1000 R. leguminosarum bv. trifolii g21. It should be noted that the more acidic, stressful soils of Western Australia comprise only a small number of these samples. For these soils, qualitatively diverse populations of Bradyrhizobium spp. are generally present (A. McInnes, unpub. PhD thesis, University of Western Australia, 2000). Where rhizobial communities exceed 1000 cells g21 of soil they are considered to present a formidable barrier to the introduction of new inoculant strains (Thies et al., 1991). There are few well documented examples, perhaps with

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Fig. 2. Class distribution of southern Australian agricultural soils based upon their population size of Sinorhizobium spp. (480 soils, left) and Rhizobium leguminosarum bv. trifolii (236 soils, right) as assessed by the MPN soil dilution method (Brockwell, 1963). Data sourced from Brockwell et al., 1982; Jones and Curnow, 1986; Bowman et al., 1998; Brockwell et al., 1991; Unkovich and Pate, 1998; Riffkin et al., 1999; Slattery et al., 1999; Ballard and Charman, 2000; Denton et al., 2000; Brockwell, 2001; Ballard et al., 2002; Ballard et al., 2003; unpub. data from R.A. Ballard and N. Charman, South Australian Research and Development Institute, Adelaide.

the exception of WU95 (Gibson et al., 1976), where the rhizobial strain in an Australian pasture legume inoculant has been able to overcome these competition barriers for more than several years after the inoculant strain is introduced to the soil. For pulse legumes which are resown annually, the inoculant strain can be given a positional and numerical advantage at sowing and hence increase its chances of nodule occupation. 4.1. Evidence for intra-specific diversity With the aid of molecular typing (Richardson et al., 1995; Thies et al., 2001) communities of naturalised rhizobia are being increasingly well-described (Demezas et al., 1995; Hebb et al., 1998; Ballard et al., 2004; Evans et al., 2004). We are now left with little doubt that the range of strains we find nodulating Mediterranean legumes in southern Australia is far greater than the number of strains ever released as inoculants. This diversity of strains does not appear to correspond with experimental genotypes that have escaped from introduction and selection experiments. Further, as far as we have been able to ascertain, very few of the introduced legumes in agriculture are able to nodulate with the bacteria commonly associated with our native legumes (Lange, 1961; Yates et al., 2004a). Exceptions include Chamaecytisus proliferus, Lupinus cosentinii and Ononis natrix (Yates et al., 2004a). As evidence for this diversification, Fig. 3 illustrates the different nodule occupants collected from T. purpureum 6 years after the introduction of several elite strains to the field as inocula near Northam, Western Australia. Very few of the banding patterns from strains occupying the nodules of T. purpureum appear to correspond closely with the original inoculant strains. However, approximately half the nodule occupants (after 6 years in the soil) were more closely related to WSM409 and WSM1328 (at the top of the dendrogram), than to WU95, TA1 or CC2483 g (at the base of the dendrogram) based upon analyses of the shared bands. Where isolates share common bands produced with the ERIC primer (which is a chromosomal directed primer) it suggests a close relationship between them and it is possible

to speculate these frequently isolated nodule occupants have a common predecessor—WSM409 or WSM1328. However, this would need to be verified by a more comprehensive assessment of chromosomal and symbiotic DNA. Complementary work examining 17 soils mainly from south-eastern Australia has similarly shown that the many different strains of rhizobia which nodulate balansa clover (Trifolium michelianum) exhibit little resemblance to the strains used in commercial clover inoculants (I.R. Sitepu, R.A. Ballard and P.J. Murphy, unpub. data). McInnes (unpub. PhD thesis, University of Western Australia, 2000) was able to detect up to 25% nodule occupancy by commercial inoculant strains for serradella after several years in the soil at one site in the eastern wheatbelt of WA, however, the inoculant strains were found to occupy nodules in very low frequency at other sites. In contrast, perhaps because of soil acidity restricting community development, Evans et al. (2004) was able to demonstrate the dominance of Sinorhizobium meliloti strain WSM922 in uninoculated plots of M. sativa 3 years after sowing. One must then ask the question that if we have managed rhizobial introduction to southern Australia well (by releasing elite genotypes of a few species) how has this rather large intra-specific diversity developed? We are left with options that include genetic re-arrangement in situ, mutation, and the possibility that large numbers of genotypes arrived accidentally in forage transported by the early arrivals to our shores. 4.2. Development of genetic diversity Genetic diversification in rhizobial populations in situ was given significant prominence and understanding through the work of Dr Clive Ronson and colleagues in New Zealand (Sullivan et al., 1995, 2002; Sullivan and Ronson, 1998). They demonstrated that the suite of Lotus nodulating organisms in their experimental fields increased in genetic diversity over a 7 – year period through transfer of a 500 kb ‘symbiosis island’ to resident soil bacteria. In Australia, too, there is now strong evidence of rhizobial diversification through exchange of mobile genetic

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Fig. 3. Banding patterns of nodule occupants (uninoc. 1–19) of T. purpureum 6 years after the introduction to soil of a range of commercial inoculants— CC2483g, TA1, WU95, WSM409 and WSM1328 (see Howieson et al. (2000b) for trial details). The ERIC primer was used in a REP-PCR reaction (Versalovic et al., 1991) with modifications as described by Yates et al. (2004b). The banding patterns were analysed and grouped by means of the Unweighted Pair-Group Method using Arithmetic Average (UPGMA) cluster analysis dendrogram (matched by Rf calibration with tolerance 100) performed by the Phoretix 1D software (Nonlinear Dynamics LTD).

elements. Five years after the introduction of an unique Mesorhizobium sp. as inoculant for the monospecific legume B. pelecinus, Nandasena et al. (2004) have described the emergence of a low proportion (3.5%) of genetically distinct nodule occupants. Their work reveals that these nodule occupants have arisen through transfer of symbiotic DNA from the experimental inoculant strain WSM1271 to resident soil bacteria of, as yet, unknown origin. These new nodulating organisms appear to be very poor at N-fixation with B. pelecinus, hence the challenge exists to understand the mechanisms of this genetic exchange and, if possible, to control it. Fast growing species of rhizobia typically carry up to 40% of their genome as relatively stable plasmids (Honeycutt et al., 1993). If the transfer of nodulating ability is conferred through plasmid exchange then there is the possibility of regulating this event through manipulation of the genes required for plasmid

transfer and integration. The transfer genes Tra1, Msi173 and Msi174 are thought to regulate symbiosis island gene transfer in M. loti (Sullivan et al., 2002) and these might be initial targets to stabilize the commercial inoculants for B. pelecinus. 4.3. Other ways to manage competition for nodulation Before addressing this issue, it is relevant to acknowledge that the development of rhizobial communities does not always produce a competitive environment for nodulation that is detrimental to N-fixation. For example, as previously indicated, there is substantial competition for nodulation of Ornithopus spp. by strains of Bradyrhizobium sp. (Lupinus) on the acid sands of Western Australia, yet most of the competitors are highly effective microsymbionts (A. McInnes, unpub. PhD thesis, University of Western

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Australia, 2000). Similarly, the strains of R. leguminosarum bv. viciae that nodulate field pea grown on the alkaline soils of South Australia are generally effective microsymbionts (Ballard et al., 2004). These ‘effective’ scenarios need not be managed, aside from ensuring that new legume cultivars released into agriculture are compatible with the dominant rhizobial genotypes in the communities. Unfortunately, the same cannot be said for the communities of Sinorhizobium spp. and R. leguminosarum bv. trifolii which reside in many Australian soils. The alkaline and neutral pH soils of South Australia and western Victoria contain a myriad of strain genotypes of S. meliloti and R. l. bv. trifolii that substantially compromise N-fixation by some species of annual medic and clover, respectively (Slattery et al., 1999; Ballard and Charman, 2000; Denton et al., 2000, 2002). Sessitsch et al. (2002) have listed a range of approaches available for managing competition. We do not wish to repeat that information, but to concentrate instead upon Australian work investigating the capacity of current and prospective pasture legumes to establish effective symbioses with the naturalised rhizobia in the soils on which they are likely to be grown. This is a complex problem in Australia because many (at least 12) different species of Trifolium and Medicago are already used in agriculture or are being developed by national pasture breeding and evaluation programs. These species interact with the rhizobia in the soils of southern Australia in different ways. Some are nodulated and establish mostly effective (although not always optimal) symbioses; some nodulate, but form a high frequency of ineffective symbioses; others do not form nodules with the naturalised rhizobia (Table 1). We are eager to encourage the use of lucerne (Medicago sativa) and other legumes that behave (symbiotically) in a similar manner to generally form effective symbioses. Conversely, we believe the use of legume species such as M. rigiduloides (previously used as a source of cold tolerance in medic breeding programs) that are prone to form ineffective symbioses with soil rhizobia, and for which highly competitive inoculants would need to be selected, should be discouraged. There are also those novel species, such as Trigonella caelesyriaca, that are nodulated by Sinorhizobium spp. but fail to nodulate with the genotypes dominant in Australian soils (Table 1). Such species may provide us with the opportunity to introduce new and effective rhizobial strains in the absence of intense competition for nodulation that is usually provided by the resident soil rhizobia. This aspect is discussed under the heading of ‘exclusive’ nodulation in Section 4.3.2. It is also pertinent, in this section, to remind the reader that competition for nodulation may be largely avoided if new legume species with unique inoculant requirements are introduced to our agricultural systems. Legumes nodulated by Mesorhizobium spp., such as B. pelecinus and Hedysarum spinosissimum (Kishinevsky et al., 2003) fall into this category.

Table 1 Differences in the nodulation and N fixation capacity of three pasture legume genera when inoculated with extracts from Australian agricultural soils Legume

Medicago sativa (lucerne) Medicago littoralis (strand medic) Medicago polymorpha (burr medic) Medicago rigiduloides Trigonella balansae Trigonella caelesyriaca

Survey soils with rhizobial populations which formed nodules (%)

Mean effectiveness of rhizobia forming nodules (% of an effective inoculation treatment)

85

80

.95

53

.95

27

90

,5

88 0

65 0

Trifolium subteranneum .90 (subterranean clover) Trifolium michelianum .80 (balansa clover) Trifolium glanduliferum 30a (gland clover)

47 52 41

Data sourced from nine experiments (using a minimum of 24 soils) conducted at SARDI (South Australian Research and Development Institute) by N. Charman and R.A. Ballard. a Only soil rhizobial populations which formed nodules on at least 4 out of 5 plants of T. glanduliferum have been included in this value. Many soil populations produced only infrequent nodules on this clover species.

4.3.1. Selection of hosts that form effective symbioses For the legume species that are widely grown and frequently nodulated, but often form sub-optimal symbioses (e.g. M. littoralis, M. polymorpha and T. michelianum) we have examined a wider range of plant genotypes for symbiotic variation. Our most extensive efforts here have been with M. polymorpha (222 accessions) but the approach has delivered little success (Charman and Ballard, 2004). Indeed, symbiotic capacity within M. polymorpha appears remarkably conserved. An alternative approach, which we have employed to improve the symbiotic capacity of M. littoralis, has been to hybridise it with a genotype of M. truncatula selected for its broad symbiotic capacity with most test soils (promiscuous nodulation, see 4.3.2). We are encouraged because a number of fourth generation progeny that have M. littoralis characteristics (leaf markers and pods) also have high symbiotic capacity. Others (Howieson et al., 2000c) have cautioned that hybrids of M. littoralis and M. truncatula may have altered symbiotic capacity relative to their parents but note that the effects may be unpredictable. We are also mindful that plant breeding approaches focussed only upon symbiotic characteristics have historically been unsuccessful (Herridge et al., 2001). Hence our current efforts are closely linked to the Australian medic breeding program where progeny selections are being backcrossed to adapted cultivars.

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4.3.2. Genetic control for effective symbiosis—selection or exclusion of partners? This work also leads us to ask which genetic mechanisms confer effective symbiosis, since understanding them may allow us to increase N fixation in competitive environments. Vincent and Waters (1953) and later Robinson (1969) published a series of articles reporting observations of the ability of several clover species to select a preferred nodule occupant when strains of lesser N fixing ability were present. These observations and those more recently (Sadowski and Graham, 1998) seem incongruous amongst the many contemporary published reports of ineffective nodulation and attendant constraints to N-fixation in competitive environments (Bottomley, 1992; Denton et al., 2002, 2003; Brockwell et al., 1995; Brockwell, 2001). In attempting to resolve this apparent contradiction and to unravel the mechanisms involved in ‘selective nodulation’, we suggest grouping examples into those where: (a) effective microsymbionts are preferentially selected by the host from a pool of variably effective strains within a species that are all capable of nodulating the host. This might be termed selective nodulation as described by Robinson (1969). The second group is where (b) a proportion of microsymbionts within a pool of strains from a species normally able to nodulate the host are unable to do so. We might term this exclusive nodulation as described for Woogenellup subterranean clover in relation to strain TA1 (Gibson, 1968) and Afghanistan pea (Davis et al., 1988). For a third group of legumes (c) there is no apparent selection or exclusion from within the pool of strains but the resultant symbiosis that occurs with a diverse range of strain genotypes is nearly always effective. We term this promiscuous nodulation as described for many tropical legumes (Singleton et al., 1992; Date, 2000; Mpepereki et al., 2000) and Ornithopus spp. in southern Australia (A. McInnes, unpub. PhD thesis, University of Western Australia, 2000). In both selective and exclusive nodulation, it is almost certain the phenomenon is controlled within the set of complex biochemical communications between the legume and microsymbiont which precede nodulation (Perret et al., 2000). The latter situation has been well examined and documented with respect to the exclusive nodulation of Pisum sativum cv. Afghanistan pea carrying the gene Sym-2 by geographically isolated strains of R. l leguminosarum bv viceae carrying the nodX symbiotic gene that acetylates nod-factor (Davis et al., 1988; Geurts et al., 1997). The situation with T. caelesyriaca described in the previous section is a local example of a species that could be classed as capable of exclusive nodulation. T. glanduliferum, a relatively new species to Australian agriculture (Loi et al., 2004), is also infrequently infected by the naturalised clover

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rhizobia resident in many Australian soils (exclusive nodulation). However, T. glanduliferum readily nodulates with the commercial inoculant strain for annual clovers (WSM409) and so this legume could be used strategically to establish WSM409 in the field. Mpepereki et al. (2000) have recorded the capacity of locally derived soyabean genotypes to nodulate effectively when sown in southern Africa without inoculation. In contrast, soyabean cultivars from the developed world sown in the same experiments failed to nodulate effectively with the indigenous rhizobial communities. We are currently unsure if the reaction of the African genotypes represents selective or exclusive nodulation phenomena, or whether it is an example of the more frequently described ‘promiscuous nodulation’ character. From the studies in our laboratories, we have been aware of the conundrum of releasing into the agricultural environment of southern Australia effective inocula for perennial clovers that are ineffective on annual clovers (Yates et al., 2003; Howieson et al., 2004). In the target farming systems productivity is still currently very much reliant upon N-fixation from the annual clover T. subterraneum and we do not wish to compromise this valuable source of fixed N. However, recent information from studies with perennial clovers in Uruguay indicates that the South American perennial clover T. polymorphum can select its preferred effective microsymbiont from a pool of variably effective strains introduced to its rhizosphere as inocula for the Mediterranean clovers T. purpureum and T. vesiculosum (Yates et al., 2004b). Similarly, in later experiments, T. purpureum has exhibited the capacity to select an effective microsymbiont from soil communities containing infective, yet highly ineffective, rhizobial strains that nodulate T. polymorphum, i.e. selective nodulation (R.J. Yates, Centre for Rhizobium Studies, Murdoch University, Perth, unpub. data). The challenge, therefore, is one of determining under which circumstances the communication between legume and microsymbiont results in selective, exclusive or promiscuous nodulation behaviour and exploiting these attributes to maximize N fixation.

5. Optimising the symbiosis Where does this lead us in the context of optimising the legume symbiosis in stressful and competitive environments within Australia? In our view it infers that we have the opportunity to manage the stressful situations through the deliberate selection and introduction of adapted legume and rhizobial genotypes. It may not always be possible to achieve this aim, or to influence production, because some environments are very extreme. As well, whilst we may be able to select appropriate rhizobia for these environments, we may not always be able to introduce their host legume because of intrinsic agronomic shortcomings. However, the stressful environments often represent a vacuum for

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rhizobial species and this should be seen as an opportunity, not a threat. For less-stressful soils, where the abiotic stresses are not present or synergistic, new rhizobial strains may be introduced. However, this can only be achieved readily in the absence of strong competition for nodulation within rhizobial communities. For these competitive situations we may have the opportunity to manage competition, but nearly always this will require some manipulation of the legume host or of the microsymbiont. Conventional (and lessconventional) approaches to manage competition from the perspective of the microsymbiont have been reviewed by Sessitsch et al. (2002). In the current review, we highlight the use of legumes that are able to select effective strains of rhizobia from mixed populations, to exclude ineffective microsymbionts, or simply to form an effective symbiosis with a wide range of rhizobial genotypes (i.e. promiscuous nodulation). However, we must be cognisant that identification of legume lines with improved symbiotic capacity is usually only the first step in a complex process to improve net N fixation from legumes. Further development will often be needed to correct other agronomic shortcomings in material selected for enhanced N fixation, so it is critical that these efforts are closely aligned with breeding and evaluation programs. Even if this can be accomplished, the adoption of new legumes into agriculture is strongly influenced by farming profitability and in some cases the sociology of the farming community (Sessitsch et al., 2002). It would be futile to embark on a legume or rhizobial selection program for symbiotic improvement to overcome stressful environments without first giving consideration to these factors. Finally, we acknowledge that within the rhizobial communities there is genetic flux. Transfer of symbiotic capacity within and between these communities is a reality with which rhizobiologists must contend if we are to continue maximising N fixation from legumes. Acknowledgements The authors wish to thank Mr Ron Yates, Dr Steven Carr and Mr Nigel Charman for data included in the manuscript, other colleagues at the Centre for Rhizobium Studies, Murdoch University and the South Australian Research and Development Institute (SARDI), as well as the Grains Research and Development Corporation (GRDC) for funding to the National Rhizobium Program (NRP) whose collaborators have contributed to the research covered in this manuscript. References Amarger, N., 1981. Competition for nodule formation between effective and ineffective strains of Rhizobium meliloti. Soil Biology & Biochemistry 13, 475–480.

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