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Australian soils. María A. Pérez-FernándezA,C and Byron B. LamontB. AEcology Area, University of Extremadura, Avenida de Elvas s/n, 06071, Badajoz. Spain.
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Australian Journal of Botany, 2003, 51, 543–553

Nodulation and performance of exotic and native legumes in Australian soils María A. Pérez-FernándezA,C and Byron B. LamontB A

Ecology Area, University of Extremadura, Avenida de Elvas s/n, 06071, Badajoz. Spain. Department of Environmental Biology, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia. C Corresponding author; email: [email protected]

B

Abstract. Six Spanish legumes, Cytisus balansae, C. multiflorus, C. scoparius, C. striatus, Genista hystrix and Retama sphaerocarpa, were able to form effective nodules when grown in six south-western Australian soils. Soils and nodules were collected from beneath natural stands of six native Australian legumes, Jacksonia floribunda, Gompholobium tomentosum, Bossiaea aquifolium, Daviesia horrida, Gastrolobium spinosum and Templetonia retusa. Four combinations of soils and bacterial treatments were used as the soil treatments: sterile soil (S), sterile inoculated soils (SI), non-treated soil (N) and non-treated inoculated soils (NI). Seedlings of the Australian species were inoculated with rhizobia cultured from nodules of the same species, while seedlings of the Spanish species were inoculated with cultures from each of the Australian species. All Australian rhizobia infected all the Spanish species, suggesting a high degree of ‘promiscuity’ among the bacteria and plant species. The results from comparing six Spanish and six Australian species according to their biomass and total nitrogen in the presence (NI) or absence (S) of rhizobia showed that all species benefitted from nodulation (1.02–12.94 times), with R. sphaerocarpa and C. striatus benefiting more than the native species. Inoculation (SI and NI) was just as effective as, or more effective than the non-treated soil (i.e. non-sterile) in inducing nodules. Nodules formed on the Spanish legumes were just as efficient at fixing N2 as were those formed on the Australian legumes. Inoculation was less effective than non-treated soil at increasing biomass but just as effective as the soil at increasing nitrogen content. Promiscuity in the legume–bacteria symbiosis should increase the ability of legumes to spread into new habitats throughout the world. BT03053 MP.erAf.roPméarenzc-Fefornáxnodtiezcand Bn.atBiv.eLlaegmuonetsinAustralia

Introduction Leguminous species are able to establish symbiotic associations with bacteria in the family Rhizobiaceae. This interaction results in the formation of root nodules, in which the bacteria are hosted intracellularly. The advantages to a symbiotic microorganism of living within a plant include access to carbon substrates and micronutrients, and protection from desiccation. In turn, the microorganism fixes atmospheric nitrogen (N2) which is handed over to the plant (ammonium or alanine) for further processing into amino acids and proteins (Long 1996; Waters et al. 1998). Because nitrogen is a limiting nutrient for plant growth in many ecosystems, this symbiosis confers that legumes, providing rhizobia are present, have ability to colonise poor soils. Furthermore, it has been shown that legumes can increase soil N, making it available for adjacent species, and enhance their own growth as well (Pate et al. 1994; Viera-Vargas et al. © CSIRO 2003

1995). Although research on N2 fixation in annual legumes has been intensive, there is a lack of knowledge regarding woody legumes (Allen and Allen, 1981; Streeter, 1994; Unkovich et al. 1997). During the last two decades researchers have become interested in woody legume species because of their role in nutrient cycling ( Wilson and Tilman, 1991; Pate et al. 1993; Chang and Handley 2000), their prospective use in revegetation projects (Dart 1998; Faria et al. 1987; Ndiaye and Ganry 1997) and their capacity to became invaders of new habitats, such as Australian acacias in South Africa (Witkowski 1991a, 1991b; Berger 1993; Högberg and Alexander 1995; Hussey et al. 1997). Despite the usually high legume–bacteria specificity for this symbiosis (Lafay and Burdon 1998; Zahran 2001), it is also known that a high degree of ‘promiscuity’ can be achieved by both plant and microbes (Young and Haukka 1996; Zahran 2001). Thus, the introduction to a given 10.1071/BT03053

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environment of either a plant species or a Rhizobium strain might result in effective nodulation (Witkowski 1991b). For legumes, this represents an advantage in the colonisation process. Numerous legumes have invaded Australian and South African landscapes, rapidly displacing native flora. We propose that one of the attributes conferring invasive superiority among some legumes is related to their ability to accept a wide range of rhizobia. Thus, one can ask what is the host range and effectiveness of rhizobia from the Southern Hemisphere on legumes from the Northern Hemisphere. The aims of our project were to determine whether six Spanish leguminous species are able to form effective nodules when grown in selected soils of south-western Australia, and to analyse the efficiency of the nodules from Spanish legume, compared with those on Australian legumes. We hypothesised that legumes introduced from the Iberian Peninsula, Spain, (a) are able to accept bacteria from nodules of Australian legumes as readily as indigenous legumes, and (b) are just as efficient at fixing N2 and enhancing growth as the Australian legumes (Lerouge et al. 1990; Albrecht et al. 1999). These hypotheses led us to identify possible differences in performance between inoculated and non-inoculated indigenous and exotic legume species. This is of special relevance in the case where species are introduced from locations with a predominantly Mediterranean-type climate and poor soils, both characteristics similar to the environment in south-western Australia, where the selected native species occur. In this way, the plant species sharing these climatic requirements can be considered as potentially serious weeds. To test these proposals, we grew six south-western Australian legumes in their own soils ± autoclaved ± inoculated soils, and six Iberian legumes in the ± autoclaved ± inoculated soils of the six host Australian species. Levels of nodulation, biomass production and total N accumulated in the seedlings after 24 weeks of growth were compared. Material and methods Plant material Six Australian and six Spanish (Iberian) legume species were selected according to similarity of growth forms, response to fire, abundance in the region, habitation of representative soils in their regions and ease of cultivation. They are suitable for the revegetation of arable land and areas disturbed by mining and road-building activity. All Spanish species are characterised by relatively good growth on poor soils subject to seasonal drought (Pérez-Fernández et al. 2000). None of the Spanish species, except for C. scoparius, a noxious weed in several parts of the world (Williams 1981; Bossard 1991; Hosking et al. 1998), is yet considered invasive to Australia; however, they could easily be introduced as ornamentals or for land reclamation. The Australian species were Jacksonia floribunda Endl. (treated as synonymous with Jacksonia densiflora Benth., Marchant et al. 1987), Gompholobium tomentosum Labill, Bossiaea aquifolium Benth, Daviesia horrida Preiss ex Meissner, Gastrolobium spinosum Benth and Templetonia retusa (Vent.) R.Br. Seeds were obtained from local seed dealers (Nindethana Seed Service, Albany, WA, and Kimseed Pty

M. A. Pérez-Fernández and B. B. Lamont

Ltd, Perth,WA). The Iberian leguminous species were Cytisus balansae (Boiss.) Bal, C. multiflorus (L’Hér) Sweet, C. scoparius (L.) Link, C. striatus (Hill) Rothm., Genista hystrix Lange and Retama sphaerocarpa (L.) Boiss. Seeds were collected from plants growing in natural populations located in peneplains and mountain ranges in Salamanca Province (central western Spain), placed in brown envelopes and stored at 21 ± 1°C before the experiments were conducted. All species prefer acidic soils, except the Australian species T. retusa, common in calcareous sands, and the Spanish species R. sphaerocarpa that grows in both acidic and calcareous soils. Bacteria Nodules were obtained from adult plants collected from sites where each of the six Australian species occurred naturally. Nodule collection was carried out in autumn. Roots from three parent plants of each of the Australian species were examined for nodule production and these were collected and transferred to the laboratory. Nodules were excised with a scalpel sterilised in flaming ethanol. Individual nodules were surface-sterilised by immersion in 98% ethanol for 1 min, 4% sodium hypochlorite for 2 min and rinsed with sterile distilled water. Nodules were crushed with a flamed glass rod and the homogenised nodule was streaked onto 1.5% yeast–mannitol agar plates (Vincent 1970). Plates were incubated in darkness at constant temperature of 24°C. Colonies produced after 7–12 days were verified to be rhizobia by inoculation of sterile seedlings of each of the Australian species with isolates from each of three nodules. Pure cultures were obtained after one or several subculturing steps. Seedlings were inoculated at the base of the stem with 2 mL of the appropriate bacterial inoculum. Inocula consisted of a heavy suspension of the log-phase culture on yeast–manitol agar in 20 mL of N-free Jensen solution (Lafay and Burdon 1998), pH 6.8. Experimental design Seeds of the Australian and Spanish species were surface-sterilised by placing them in H2SO4 (95%) for 20 min, followed by 10 consecutives rinses with distilled water (Vincent 1970). Seeds were then placed in boiling water and soaked overnight. After this treatment, they were germinated in Petri dishes on wet filter paper on vermiculite, and placed in a cabinet at a temperature of 15°C for the Australian species and at 22°C for the Spanish ones for 7 days. Each germinated seed was transferred into a black plastic tube (80 mm uppermost diameter, 160 mm long) containing one of six soils in which the Australian species grew naturally. Soils were collected within 50 km of Perth and were representative of the major soil types that occur in south-western Australia (McArthur 1991). Plantings were according to the following four soil treatments: (i) sterile soil, non-inoculated (S); (ii) sterile soil, inoculated (SI); (iii) non-sterile soil, non-inoculated (N); and (iv) non-sterile soil, inoculated (NI). There were six replicates for each combination of treatment, soil and species. Each Australian plant species was sown exclusively in its own soil, whereas the six Spanish species were sown in all six soils. Soil sterilisation was carried out by standard autoclaving (50 min, 124°C). Seedling inoculation was carried out as described above. To prevent soil from external contamination during the growing time, every pot was covered with autoclaved crystal balls of 0.5 mm diameter (Aulabor Industries, S. A. Barcelona, Spain) and the watering was through plastic tubes located in the middle of each pot. Plants were kept in an air-conditioned glasshouse at the Department of Environmental Biology, Curtin University of Technology, Perth, Western Australia, over 6 months. Daily average maximum temperature ± s.d. during the experiment was 24 ± 3°C, and the daily minimum temperature was normally above 20°C. To assist temperature control, the glasshouse was covered with green shadecloth reducing light intensity in the greenhouse to 25% of that outdoors, with a mean photoperiod of 13 h.

Performance of exotic and native legumes in Australia

Data collection and analyses After 6 months, plants were harvested and the roots thoroughly washed with running water. For all harvested plants, shoot and root dry weights (after 48 h at 70°C) were recorded. These values were combined to give total biomass. Root nodules were counted on each plant (as an index of N2-fixing ability). For N analyses, shoots and leaves of plant samples were milled to reduce them to a diameter able to pass through a 1-mm sieve. Root material was not included to ensure that the fixed N2 was utilised by the plant and not retained in the nodules. Total nitrogen contents (2–4 plants per treatment) were determined at the WA Chemistry Centre, Perth, by conventional digestion and titration techniques (Hendry and Grime 1993). Data were analysed by using 3-way ANOVA for overall comparisons between the species and the two treatments. Species within each treatment group were compared by Tukey’s test (significance at P < 0.05). The benefits of nodulation were assessed by comparing biomass or nitrogen content for NI with S by the formula NI/S.

Results Nodule production None of the seedlings produced nodules in any of the sterile non-inoculated soils (S), indicating that the soil sterilisation was successful. Significant differences in nodule production were observed, dependent on species, soil and inoculation treatments. All Australian and Spanish species nodulated in all untreated soils (N), although not all Spanish species produced nodules when grown in soil collected from underneath T. retusa. Invariably, the highest number of nodules for all species was achieved in the non-sterilised inoculated soils (NI) (Fig. 1). Nodule production under the other two treatments (N and SI) was uneven and species-dependent. Inoculation (SI) was just as effective, or more effective than non-inoculated soil (N) in inducing nodules, especially in T. retusa. Among the Spanish species, R. sphaerocarpa proved to be the most promiscuous. The second-most promiscuous species, C. striatus, produced a higher number of nodules than the host native species in five NI soils. The exception was the soil of T. retusa. For the N and SI treatments, nodule production by C. striatus was significantly (P < 0.005) higher than or similar to that of the native species. Both species produced higher numbers of nodules than did the native species in all soils, except B. aquifolium (N) and T. retusa (all treatments). The least promiscuous species was G. hystrix, with little nodule production in the six soils.

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with greater biomass production in the soils with bacteria (SI, N and NI) and lower in their absence. In terms of biomass production in the presence (NI) and absence (S) of bacteria, the species that benefited most from the presence of nodules (NI v. S) were C. striatus (4.34×) and R. sphaerocarpa (3.18×) (Table 1). In the NI soils, these two species, respectively, produced 4.34× and 3.18× more biomass than they did in the S soils. Species that benefited least were C. balansae and C. scoparius, which produced only 1.7× more biomass in the NI soil than in the S soil. The Australian species were intermediate, producing 2.03× more biomass in the NI soil than in the S soil. Nitrogen content Nitrogen accumulation varied significantly between species and within species grown in sterile and non-sterile soils. The Spanish and the Australian species showed the same response patterns, with a clear trend to increase total N content in plants grown in the presence of bacteria (SI, N and NI) and a reduction in their absence (S). Inoculation (SI) was just as effective as non-sterile soil (N) in increasing N content, although there was a strong species effect. Nitrogen content was highest in the NI treatment (Fig. 3). In terms of N accumulation, the species that benefited most from the presence of nodules were R. sphaerocarpa (3.78×) and C. striatus (2.41×) (Table 1). In the NI soils, these two species, respectively, produced 3.78× and 2.41× more biomass than they did in the S soils. Similarly, the species that benefited least were G. hystrix (1.09×) and C. scoparius (1.13×). In the NI soils, the Australian species accumulated on average 1.23× more N than in the S soils. Nitrogen content was significantly positively correlated with nodule production for all species in each soil, except for T. retusa (Fig. 4). In four soils (J. floribunda, G. tomentosum, B. aquifolium and D. horrida) nodules on the Spanish legumes were as efficient at fixing N2 as the Australians. They were more efficient than the Australians in the G. spinosum soil and there was no pattern in the T. retusa soil. Results of a 3-way ANOVA comparing the effects of soil treatment and bacterial inoculation on nodulation, biomass production and nitrogen accumulation by the six Spanish legumes grown in six Australian soils, and the six Australian legumes grown in their own soils are given in Appendix.

Biomass production All seedlings grew in all soils and under all treatments (Fig. 2). Biomass varied significantly between species as well as within species grown in non-sterile and sterile soils. Inoculation (SI) was less effective than non-sterile soil (N) at increasing biomass, except for C. striatus, which was able to produce at least as much biomass in the N soils as in the SI soils (Fig. 2). The Spanish and the Australian species showed the same response patterns in regard to the soil treatments,

Discussion Soil sterilisation (S) prevented nodule formation in all species grown in any of the Australian soils, indicating that the sterilisation procedure was successful and that subsequent nodulation in the SI treatment was only due to the presence of the inoculated bacteria. Non-sterile soil yielded as many nodules per plant as the inoculated treatment, except for T. retusa soil. This indicates that the natural rhizobia in

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75 60

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Fig. 1. Number of root nodules (mean ± s.e.) produced by each plant species in each soil under the following four treatments: N, non-treated soil; NI, non-treated inoculated soil; SI, sterilised, inoculated soil. Sterilised soil (S) was omitted as no plants produced nodules. Species names on the graphs refer to the Australian plant species under which the soil was collected from. The Australian species native to that soil is given first in the sequence (solid bar) followed by the six Spanish species (open bars). Different letters on top of the columns indicate significant differences between species per treatment, based on Tukey’s test. Jfl, J. floribunda; Gto, G. tomentosum; Baq, B. aquifolium; Dho, D. horrida; Gsp, G. spinosum; Tre, T. retusa; Cba, C. balansae; Cmu, C. multiflorus; Csc, C. scoparius; Cst, C. striatus; Ghy, G. hystrix; Rsp, R. sphaerocarpa. Note different scales.

Performance of exotic and native legumes in Australia

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Fig. 2. Total biomass (mean ± s.e.) produced by each plant species in each soil under four treatments. Abbreviations and Australian species names are as in Fig. 1. Different letters on top of the columns indicate significant differences between species per treatment, based on Tukey’s test. Note different scales.

2.62 (2) 1.95 (7) 1.25 (7) 2.75 (3) 1.40 (7) 2.23 (1) 2.03 (4)

1.50 (4) 1.19 (4) 1.19 (3) 1.07 (5) 1.07 (6) 1.37 (4) 1.23 (4)

J. floribunda G. tomentosum B. aquifolium D. horrida G. spinosum T. retusa Overall mean

Australian species

1.02 (6) 1.19 (5) 1.03 (7) 1.24 (3) 1.13 (4) 1.33 (5) 1.17 (5)

1.98 (7) 2.66 (5) 1.31 (5) 1.62 (7) 1.44 (6) 1.19 (7) 1.70 (6)

C. balansae

2.00 (6) 2.18 (6) 1.45 (4) 1.91 (5) 1.47 (5) 1.20 (6) 1.70 (7) 1.17 (5) 1.17 (6) 1.08 (5) 1.05 (6) 1.08 (5) 1.23 (6) 1.13 (6)

Total nitrogen 1.59 (3) 1.50 (3) 1.12 (4) 1.22 (4) 1.30 (2) 1.69 (1) 1.40 (3)

02.75 (2) 01.81 (1) 05.81 (1) 01.29 (2) 01.20 (3) 01.58 (3) 02.41 (2)

02.21 (3) 04.21 (2) 12.71 (1) 02.99 (2) 02.32 (1) 01.58 (3) 04.34 (1)

Spanish species C. scoparius C. striatus

Biomass 2.12 (5) 2.67 (4) 2.38 (3) 2.42 (4) 1.81 (3) 1.32 (5) 2.12 (3)

C. multiflorus

— — 1.06 (6) — — 1.12 (7) 1.09 (7)

2.16 (4) 2.71 (3) 1.29 (6) 1.71 (6) 2.31 (2) 1.49 (4) 1.95 (5)

G. hystrix

03.15 (1) 01.62 (2) 01.93 (2) 12.94 (1) 01.38 (1) 01.64 (2) 03.78 (1)

03.54 (1) 04.79 (1) 02.83 (2) 04.56 (1) 01.62 (4) 01.68 (2) 03.18 (2)

R. sphaerocarpa

Comparison of total biomass production and nitrogen accumulation in plants grown in non-sterile, inoculated (NI) and sterile (S) soils Results are given as NI/S. Values in parentheses indicate the species rankings (1 = highest)

J. floribunda G. tomentosum B. aquifolium D. horrida G. spinosum T. retusa Overall mean

Soil source

Table 1.

548 Australian Journal of Botany M. A. Pérez-Fernández and B. B. Lamont

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Fig. 3. Total N content (mean ± s.e.) accumulated by each plant species in each soil under four treatments. Abbreviations and Australian species names are as in Fig. 1. Different letters on top of the columns indicate significant differences between species per treatment, based on Tukey’s test. Note different scales.

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Total nitrogen (mg)

500

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Fig. 4. Relationship between the number of root nodules formed by plants grown in sterile (S) and non-sterile, inoculated (NI) soils and total shoot nitrogen content of plants. Best-fit equations and coefficients of determination (r2) are given for each soil. Each point is the mean of six replicates. Solid symbols represent Australian species; open symbols represent Spanish species.

these soils were highly infective, as has been demonstrated previously for Australian isolates (Lafay and Burdon 1998). Higher nodulation levels in plants under the N than SI treatments indicates that either the N soils were an extra source of infective rhizobia, or that autoclaving had some other inhibitory effect on root growth or eliminated other micoorganisms that are required in the nodulation process (Albrecht et al. 1999). The conclusion that nodulation was limited by a supply of suitable rhizobia was supported by their presence at highest levels in the NI treatment. While the extra nodules resulting from inoculation enhanced N accumulation, overall carbon fixation (biomass) was significantly enhanced in only one soil. The exceptions were R. sphaerocarpa and C. striatus, whose nodulation, biomass and nitrogen content were greatly increased by inoculation. Both biomass and N accumulation were enhanced in non-autoclaved soils. How different sources of rhizobia could cause different effects on biomass independent of N uptake is worthy of further investigation. If the ultimate expression of nodule effectiveness is the extent of biomass increase, then the soil appears to be a better source of suitable rhizobia than nodules obtained from species growing in that soil. Rhizobial isolates from six Australian legumes were able to re-infect seedlings of the

Australian host species and infect seedlings of six legumes from the Iberian Peninsula. The most infective isolates (those inducing the highest number of nodules per species) were those cultured from B. aquifolium and G. tomentosum, whereas the least infective was that cultured from T. retusa (except on R. sphaerocarpa). With respect to the six Spanish species, R. sphaerocarpa and C. striatus had the greatest capacity to accept rhizobia, although the six species were able to produce nodules in the presence of all Australian rhizobia (Fig. 1). The reverse of this pattern has been reported in seedlings of Australian species that can nodulate in the presence of Iberian rhizobial strains (Rodríguez-Echeverría 2002). This supports our hypothesis that both the native Australian rhizobial strains and the exotic Spanish legumes have a high degree of symbiotic promiscuity (Ulrich and Irmtraut 2000), while the Spanish strains and the Australian legumes have poor promiscuity. We predicted that indigenous rhizobia would be as effective on exotic species as on the native ones. We tested this at three levels: biomass production of the most heavily infected plants (NI) v. sterile plants (S), shoot nitrogen content, and nitrogen content relative to nodule production. Overall, three Spanish species (R. sphaerocarpa, C. striatus and C. multiflorus) outperformed the Australians on the

Performance of exotic and native legumes in Australia

basis of relative biomass and nitrogen accumulation (Table 1). R. sphaerocarpa and C. striatus in particular benefited greatly from inoculation or the presence of soil rhizobia (Fig. 2). The presence of red pigment (haemoglobin) in nodules of all species and treatments confirmed that they were capable of fixing N2 (Batzli et al. 1992). Nevertheless, two species (C. scoparius and C. balansae) showed little increase in nitrogen content in two or three Australian soils compared with the uninfected plants. For the Spanish species in five of the Australian soils there was a strong relationship between number of nodules and nitrogen content (Fig. 4). The relationship was much more erratic in the calcareous T. retusa soil, partly because nodulation was so poor. Nitrogen content per nodule was just as high as the Australian legumes in five soils and higher in the G. spinosum soil. While there may be wide variation about the mean, we conclude that the Spanish species benefit by at least as much as the Australian species by the presence of Australian-sourced rhizobia. Having evolved in soils with a much higher N content than those of the Australian species it is surprising that the Spanish legumes performed so well (Austin et al. 1988; Bellingham et al. 2001). In the absence of nodules, the Spanish species sometimes grew as well as the Australians; when fully nodulated (N and NI), they sometimes grew better. It is possible that autoclaving released extra nitrogen from the soil (Schmidt et al. 1997); extra nitrogen in the sterile soils might explain survival of seedlings growing in these soils in the absence of nodules. Thus, selection for tolerance of low nitrogen availability does not seem to have operated at the level of the N2-fixing process. Our study revealed high levels of symbiont promiscuity and effectiveness among Australian rhizobia, indicating a high capacity for exotic legumes to overcome nitrogen impoverishment in Australian soils. In particular, R. sphaerocarpa and C. striatus accepted more rhizobia and benefited more from their presence than Australian species in their own soil. This suggests that they could become invasive should they ever be introduced into Australia, as has already occurred with Cytisus scoparius (Hosking et al. 1998), also included in our study. It is worth noting that C. scoparius performed the worst of all species in our study, indicating that extra vigilance could be required over the quarantining of these species in Australia and possibly elsewhere (Bossard 1991; Williams 1981). Acknowledgments This work was supported by grants from Ministerio de Ciencia y Tecnología (Spain), Project no. REN20010749/GLO, Programa Propio de la Universidad de Extremadura (Spain), Ayudas para estancias cortas en el Extranjero and infrastructure support scheme at Curtin University. We thank Dr Dave Allen at the WA Chemistry

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Manuscript received 14 April 2003, accepted 18 August 2003

http://www.publish.csiro.au/journals/ajb

0.0001 0.0001 0.0100 0.0018 0.2284 0.2593 0.9208 0.0001 0.0001 0.0001 0.1038 0.0001 0.0024 0.014 0.0001 0.0001 0.0001 0.0001 0.0711 0.0008 0.0036 0.0001 0.0049 0.0001 0.0554 0.2304 0.0020 0.0115

5 1 1 5 5 1 5

5 1 1 5 5 1 5

5 1 1 5 5 1 5

0.0001 0.0001 0.8823 0.0001 0.1069 0.7404 0.0547

0.0001 0.0001 0.7832 0.0001 0.0005 0.3633 0.0001

0.0001 0.0001 0.0001 0.0001 0.0015 0.0528 0.0442

0.0001 0.0001 0.2736 0.0001 0.1066 0.1759 0.1054

P-value No. of nodules Total biomass

5 1 1 5 5 1 5

d.f.

0.0001 0.0029 0.0175 0.0596 0.0216 0.3493

0.0081 0.0079 0.0041 0.9303 0.0192 0.0057

0.0652 0.0417 0.0095 0.5725 0.0229 0.0081

0.0314 0.0050 0.0353 0.1494 0.0123 0.0508

N content Gastrolobium spinosum Species Soil (± autoclaved) Inoculum Species × soil Species × inoculum Soil × inoculum Species × soil × inoculum Templetonia retusa Species Soil (± autoclaved) Inoculum Species × soil Species × inoculum Soil × inoculum Species × soil × inoculum Australian species Species Soil (± autoclaved) Inoculum Species × soil Species × inoculum Soil × inoculum Species × soil × inoculum 5 1 1 5 5 1 5

5 1 1 5 5 1 5

5 1 1 5 5 1 5

d.f.

0.0412 0.0327 0.0006 0.6716 0.0963 0.2284 0.5263

0.0001 0.0113 0.0001 0.7652 0.0001 0.0996 0.6295

0.0001 0.0001 0.0001 0.0001 0.0171 0.0522 0.0794

No. of nodules

0.0001 0.0126 0.0792 0.0185 0.2333 0.1738 0.0269

0.0001 0.5091 0.3017 0.7648 0.0443 0.2068 0.0001

0.0001 0.0011 0.4390 0.0008 0.1206 0.0691 0.0104

P-value Total biomass

0.0022 0.0035 0.0476 0.6446 0.0327 0.1330

0.0051 0.0005 0.0092 0.0173 0.0095 0.5556

0.0027 0.0001 0.0038 0.0040 0.0259 0.0923

N content

Results of a 3-way ANOVA comparing the effects of soil treatment (autoclaving) and bacterial inoculation on nodulation, biomass production and nitrogen (N) accumulation by six Spanish legumes grown in six Australian soils, and six Australian legumes grown in their own soils n = 6 per species per treatment for nodules and biomass; n = 1 for N content

Jacksonia floribunda Species Soil (± autoclaved) Inoculum Species × soil Species × inoculum Soil × inoculum Species × soil × inoculum Gompholobium tomentosum Species Soil (± autoclaved) Inoculum Species × soil Species × inoculum Soil × inoculum Species × soil × inoculum Bossiaea aquifolium Species Soil (± autoclaved) Inoculum Species × soil Species × inoculum Soil × inoculum Species × soil × inoculum Daviesia horrida Species Soil (± autoclaved) Inoculum Species × soil Species × inoculum Soil × inoculum Species × soil × inoculum

Appendix.

Performance of exotic and native legumes in Australia Australian Journal of Botany 553