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AUSTRALIAN JOURNAL OF PLANT PHYSIOLOGY Volume 27, 2000 © CSIRO 2000

An international journal of plant function

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Aust. J. Plant Physiol., 2000, 27, 1169–1173

Modification of rhizosphere pH by the symbiotic legume Aspalathus linearis growing in a sandy acidic soil Mmboneni L. Muofhe and Felix D. DakoraA Botany Department, University of Cape Town, Private Bag, Rondebosch 7701, South Africa. A Corresponding author; email: [email protected] Abstract. Aspalathus linearis is a N2-fixing legume used for tea production, and grows in highly acidic soils (pH 3–5.3) of the Cederberg mountains in South Africa. Field and glasshouse studies revealed significantly higher pH in rhizosphere than non-rhizosphere soils. However, when six non-legume species were studied in adjacent fields, there were no differences in pH between rhizosphere and non-rhizosphere soils. The culture of A. linearis plants in sterile Leonard jars similarly showed a marked increase of 2.8 pH units in the nutrient solution bathing the roots of inoculated (nodulated) plants, compared to 1.5 pH units in uninoculated control. The uptake and reduction of NO3– by plants fed 2 mM NO3– also raised the rhizosphere pH by 3.5 units, a value comparable to that of the nodulated plants. The use of titrimetric methods showed that OH– and HCO3– were the components of alkalinity in the nutrient solution bathing roots of A. linearis, and were directly responsible for the increase in rhizosphere pH. These findings suggest that the ability to raise rhizosphere pH is an adaptative feature of this legume symbiosis that overcomes the adverse effects of low pH in enhancing nutrient acquisition and reducing trace element toxicity.

Introduction Aspalathus linearis (Burn. fil.) R. Dahlgr. ssp. linearis is a shrub legume that is the source of a beverage known as ‘Rooibos tea’. It is specifically nodulated by Bradyrhizobium species (Staphorst and Strijdom 1975; Deschodt and Strijdom 1976; Dakora 1998). The plant is grown commercially in nutrient-poor, sandy, acidic soils in the Clanwilliam area of the Cederberg mountains, Western Cape, South Africa (18° 36′ S, 32° 09′ E). Under those conditions, growth of other nodulating legumes and their microsymbionts is limited. Root-nodule bacteria isolated from legumes that are adapted to acidic soils generally have a high chance of being tolerant to low pH, as shown in studies with Medicago spp., Phaseolus bean and lupin (Howieson et al. 1988, 1998; Aarons and Graham 1991). Some indigenous strains of rootnodule bacteria isolated from A. linearis could grow in laboratory medium at pH 3 or 4 (Muofhe and Dakora 1998), a clear indication of their adaptation to the low pH conditions (Muofhe and Dakora 1999). The ability of strains to survive such high acidity and yet nodulate their host plants may be determined by specific genes for acid tolerance (Glenn and Dilworth 1991). Additionally, however, the legume plant must also have mechanisms for overcoming the adverse effects of low pH in order to promote symbiotic establishment and enhance nutrient acquisition (Raven et al. 1990). Plant roots commonly release various chemical molecules as exudate; these may modify the rhizosphere environment and facilitate interactions between the host plant and its microsymbionts. Some low pH-adapted species extrude H+ and organic acids as a © CSIRO 2000

way of promoting the acquisition of limiting nutrients such as P (Raven et al. 1990; Marschner 1991, 1995). So far, however, there are few reports on plant–microbial systems that raise rhizosphere pH as an adaptive mechanism for enhanced nutrient uptake and the establishment of effective symbiosis in very low pH environments. There is, however, evidence that the pH in parts of the rhizosphere of plants growing in acidic soils can increase through passive H+ influx at the root apices (Marschner 1991, 1995). This study examines changes in pH between rhizosphere and non-rhizosphere soil, and in the nutrient solution bathing roots of A. linearis grown under field and glasshouse conditions. Materials and methods Glasshouse studies of rhizosphere pH in A. linearis Field soil was collected from Clanwilliam into large plastic bags and brought to the laboratory, where it was air-dried, ground, sieved (2 mm), and analysed for pH, soil organic matter and nutrient composition (Muofhe and Dakora 1999) prior to its use in glasshouse studies. Airdried soil was placed in four replicate pots per treatment, watered to field capacity, and planted to seeds of Aspalathus linearis (Burn. fil.) R. Dahlgr. ssp. linearis. A range of nutrient supplements was then provided to the emerging seedlings, arranged in four randomized blocks. The treatments were 0.5 mM NH4NO3 (N), 0.5 mM CaCl2 (Ca) and 0.5 mM K2HPO4/KH2PO4 (P). Additionally, some seedlings were provided with half-strength N-free modified Hoagland’s nutrient solution, or de-ionized distilled water as nutrient-free control (0 mM). At 6 months after planting, the plants were harvested and soil samples taken from the rhizosphere for pH measurements. To measure soil pH, 25 g of the moist rhizosphere soil from each replicate pot was sampled with a spatula, weighed out into a clean 100-mL beaker, and 50 mL of 0.01 M CaCl2 solution added and shaken for 60 min. While stirring, the pH of the soil suspension was measured. 10.1071/PP99198

0310-7841/00/121169

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Glasshouse studies for assessing the direct effects of microbial vs. rhizosphere activity on soil pH elevation Under aerobic conditions, microbial decarboxylation of soil organic anions (e.g. carboxylic acids) in the Krebs cycle can induce a rise in soil pH (Yan et al. 1996). A glasshouse experiment was therefore conducted to determine whether the observed increase in soil pH around the roots of A. linearis stemmed from direct microbial metabolism of soil carbohydrates or from rhizosphere activity. This was done using four replicates of potted Clanwilliam soil with three treatments: (i) plant-free soil watered and covered with aluminium foil to maintain high soil moisture and thus reduce decarboxylation; (ii) plant-free soil watered to field capacity but not covered with aluminium foil, to permit aerobic decarboxylation; and (iii) soil watered to field capacity and planted to A. linearis for determining changes in rhizosphere pH. The treatments were maintained and supplied with de-ionized distilled water three times a week for 4 months. At harvest, the moist soil from each pot was measured for pH. Field studies of rhizosphere pH in A. linearis Field studies were carried out to complement the glasshouse experiments. Rhizosphere soils were collected by using a pickaxe to dig 5–10 cm away from the taproot but around the plant. The root-rich soil (‘rhizosphere soil’) in the zone around the taproot was then shovelled into labelled plastic bags and sealed for transport to the laboratory. Root-free soil (‘non-rhizosphere soil’) was similarly collected from between plant rows and from unplanted outlying ploughed areas surrounding the A. linearis fields. About 0.5 kg soil was collected from the rhizosphere and non-rhizosphere areas of each plant. Fields with plants of differing ages (i.e. 1-, 2-, 3- and 4-year-old plants of A. linearis) were selected for this study to determine the effect of plant age on pH change. In all instances, four replicates of rhizosphere and nonrhizosphere soil samples were collected. The samples were transported to the laboratory in labelled plastic bags, and each moist soil sample sieved (2 mm) within the first 2 d of collection from the field, and used for pH determination Field studies of rhizosphere pH in six non-legume species To test whether other plants growing in the acidic soils of the Clanwilliam area also modify their rhizosphere pH, four replicate soil samples were collected from the rhizosphere of four plants each of six different non-legume species growing in an uncultivated fallow land adjacent to fields of A. linearis. These non-legume species, identified to only the genus level, were Anthospermum sp., Leucospermum sp., Willdenowia sp., Serruria sp., Leucadendron sp. and Nylandtia sp. Twelve non-rhizosphere soils were also collected from the same site as controls for comparison with rhizosphere soils. The standard procedures for soil sample collection and pH analysis, as described above, were used. Comparative study of the effect of inoculation with nodule-forming bacterium and nitrate supply on rhizosphere pH of A. linearis To assess whether the elevation in rhizosphere pH obtained in field and glasshouse studies was symbiosis-related, or induced by NO3– reduction in the roots of A. linearis, an experiment was conducted in Leonard jars containing modified Hoagland’s nutrient solution prepared at pH 6.8 or 4.0 with HCl (unbuffered medium). The treatments were: inoculation with root-nodule bacterial isolate; uninoculated NO3–-free plants; and uninoculated plants fed 2 mM KNO3. The Leonard jars were set up as described by Vincent (1970) and autoclaved. Prior to planting, seeds of A. linearis (obtained from the Rooibos Tea Company, Clanwilliam, South Africa) were surface-sterilized by exposing to 95% ethanol for 2 min, followed by 3 min washing in

0.2% HgCl2 and 10 rinses with sterile de-ionized water (Vincent 1970). About six sterile seeds were sown in each unit, and seedlings thinned out to three per jar after germination. Inoculated plants received a suspension of the bacterial isolate. The strain used in this study, together with other isolates from A. linearis, are being characterized using molecular techniques. Four replicate jars were used for each treatment. Plants were harvested 10 weeks after planting, and nodules counted and weighed. The pH of the nutrient solution bathing the roots of A. linearis was measured for each treatment while stirring thoroughly, as described previously. Determination of the components causing alkalinity in the rhizosphere of A. linearis Titrimetric methods (Vogel 1961) were used to determine total alkalinity and its components in the nutrient solution bathing roots of A. linearis plants grown at pH 4.0 only. Total alkalinity (OH– and HCO3–) was determined by titrating measured volumes of the rootbathed nutrient solution with HCl using bromophenol blue as an indicator. To estimate the amount of OH–, the nutrient solution was heated to 70°C, followed by the addition of 1% BaCl2 to precipitate HCO3–. The remaining OH– was then titrated with HCl using phenolphthalein. Statistical analysis All data were analysed statistically using one-way ANOVA and Statistica software.

Results Rhizosphere pH of glasshouse-grown A. linearis plants Rhizosphere soil pH increased significantly (P < 0.05) in all cases where plants were supplied with nutrient treatments (Fig. 1). Even with the control (0 mM) plants, which received no supplemental mineral nutrients, rhizosphere pH was significantly (P < 0.05) higher than that of the unpotted original soil not used for plant culture. Effects of microbial vs rhizosphere activity on soil pH elevation Microbial activity outside the rhizosphere was assessed for its contribution to soil pH elevation. The pH of rhizosphere 6.2

Rhizosphere soil pH

The pH of the original soil not planted to A. linearis was similarly measured using 25 g of air-dried, 2-mm-sieved material per sample.

M. L. Muofhe and F. D. Dakora

e

6.0 5.8 5.6 5.4

c

5.2 5.0

b

c b

4.8 4.6 4.4 4.2 4.0

a

Original soil

0 mM

N-free

N

Ca

P

Nutrient treatment

Fig. 1. Rhizosphere pH of Aspalathus linearis plants grown in the glasshouse using Clanwilliam soil compared with pH of original bulk soil. As treatments, the plants were provided with distilled, de-ionized water as control (0 mM solution), half-strength Hoagland’s nutrient solution (N-free), 0.5 mM NH4NO3 (N), 0.5 mM CaCl2 (Ca) and 0.5 mM K2HPO4/KH2PO4 (P). Values followed by dissimilar letters differ significantly at P < 0.05. Vertical lines on bars represent S.E. (n = 4).

Elevation of rhizosphere pH by Aspalathus symbiosis

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soil of A. linearis was significantly (P < 0.05) higher than the pH of covered and uncovered plant-free soils maintained under the same experimental conditions with de-ionized water for 6 months (Fig. 2). However, the pH of the uncovered plant-free soil was also significantly (P < 0.05) greater than the covered plant-free soil (Fig. 2). Rhizosphere pH of field-grown A. linearis plants Except for the 1-year-old plants, where there was no significant difference between rhizosphere and non-rhizosphere soil, the pH of rhizosphere soil was in each case significantly higher than that of non-rhizosphere soil collected from 2-, 3and 4-year-old plants (Fig. 3). The 2-, 3- and 4-year-old plants showed significantly higher rhizosphere pH than 1-year-old or non-rhizosphere soil collected from adjacent uncultivated land (data not shown).

seedlings at that stage of development. Both the uninoculated NO3–-free and the uninoculated 2 mM NO3–-fed plants did not form nodules (Table 1). The increase in pH of the nutrient solution bathing roots of A. linearis was significant (P < 0.05) for plants grown at pH 4, but not at pH 6.8 (Table 1). This rise in pH was, however, lower (P < 0.05) for uninoculated NO3–-free plants compared to nodulated or uninoculated 2 mM NO3–-fed plants (Table 1). In addition, the pH increase was markedly (P < 0.05) higher at pH 4 than at pH 6.8 (Table 1). Measures of alkaline molecules in nutrient solution bathing roots There were large differences in pH elevation of the nutrient solution bathing roots of A. linearis grown in Leonard jars at

Rhizosphere pH of six non-legume species growing in fields adjacent to A. linearis

Non-rhizosphere

Rhizosphere

5.2

Rhizosphere soil pH

Rhizosphere soils collected from six non-legume species were also analysed for changes in pH. Although two species showed significantly (P < 0.05) lower pH for rhizosphere compared to non-rhizosphere soil, the other four species showed similar pH values for both rhizosphere and nonrhizosphere soils (Fig. 4). This indicates that elevation in rhizosphere pH is a special feature of A. linearis symbiosis.

5.4 b

b 5.0

b a

a

4.8 a

a

4.6

a

4.4 4.2

Rhizosphere pH of A. linearis plants inoculated with bacterial isolate or given 2 mM NO3– in Leonard jars Inoculated plants of A. linearis showed effective nodulation when grown at either pH 4 or 6.8. As a result, the number of nodules and the fresh weight of nodules and shoots were similar for plants grown at the two pH levels (Table 1). The extent of nodulation seen in these glasshouse-grown plants was similar to that generally observed in the field for

4.0 Year 1

Year 2

Year 3

Fig. 3. pH of rhizosphere soil of 1-, 2-, 3- and 4-year-old field plants of Aspalathus linearis compared with pH of bulk non-rhizosphere soil collected from the same site. Values followed by different letters for each year differ significantly at P < 0.05. Vertical lines on bars represent S.E. (n = 4).

5.6

4.6

c

5.4

a

4.5

5.2

a

4.8

Soil pH

pH

a

a

4

5

a

4.4

5.0 b

4.6 4.4

Year 4

Plant age

b

4.3 b 4.2

a

4.1

4.2 4.0

4.0

Covered plant-free soil

Uncovered plant-free soil

Rhizosphere soil

Fig. 2. pH of watered plant-free non-rhizosphere soil contained in pots and covered or uncovered with aluminium foil compared with the pH of rhizosphere soil of Aspalathus linearis plants grown in pots and similarly watered for the same length of time. Values followed by dissimilar letters differ significantly at P < 0.05. Vertical lines on bars represent S.E. (n = 4).

1 Non-rhizosphere

2

3

6

Rhizosphere of non-legume species

Fig. 4. A comparison of the pH of rhizosphere soil collected from six native non-legume species with pH of non-rhizosphere bulk soil sampled from the same site. Values followed by dissimilar letters differ significantly at P < 0.05. Vertical lines on bars represent S.E. (n = 4). Plant species: 1, Anthospermum sp.; 2, Leucospermum sp.; 3, Willdenowia sp.; 4, Serruria sp.; 5, Leucadendron sp.; 6, Nylandtia sp.

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M. L. Muofhe and F. D. Dakora

pH 4 or 6.8. The nodulated and uninoculated 2 mM NO3–-fed plants produced significantly (P < 0.05) higher pH levels than uninoculated NO3–-free plants (Table 2). Root release of OH– and HCO3– was responsible for the increase in pH of nutrient solution bathing roots of A. linearis. The concentration of these ions was much higher (P < 0.05) in the rootbathed nutrient solution of nodulated and uninoculated 2 mM NO3–-fed plants compared to the uninoculated NO3–-free control (Table 2). Discussion Both field and glasshouse studies show an increase in soil pH around the rhizosphere of A. linearis. Growing this legume in potted soil increased the pH relative to the original unplanted soil (Fig. 1). These findings were confirmed by field data, which showed a significantly higher pH increase in rhizosphere compared to non-rhizosphere soil of 2-, 3-, and 4-year-old plants, but not of 1-year-old plants (Fig. 3). Although microbial activity outside the rhizosphere also contributed to the rise in soil pH (see covered and uncovered plant-free soil in Fig. 2), possibly from decarboxylation of organic anions (Yan et al. 1996), the largest pH increase was associated with rhizosphere activity of A. linearis (Fig. 2).

Table 1. Nodulation response and changes in pH of root nutrient solution of Aspalathus linearis to NO3– supply and inoculation with root-nodule bacterial isolate at two pH levels The plants were grown in sterile Leonard jars and harvested at 10 weeks of age. Values followed by dissimilar letters in a column are significantly different at P < 0.05

Treatment Inoculated Uninoculated 2 mM NO3– Inoculated Uninoculated 2 mM NO3–

Initial solution pH

pH increase

Nodules plant–1

Nodule FW (mg plant–1)

4.0 4.0 4.0 6.8 6.8 6.8

2.8a 1.5b 3.5a 0.7c 0.6c 0.6c

1.70a 0.00 0.00 1.56a 0.00 0.00

0.92a — — 0.71a — —

Table 2. Effects of inoculation with root-nodule bacterial isolate and NO3– supply on release of OH– and HCO3– by roots of Aspalathus linearis cultured at pH 4 in sterile Leonard jars Values followed by dissimilar letters in a column are significantly different at P < 0.05. Data presented are means ± S.E. (n = 4). The nutrient solution prepared at pH 4 for plant culture initially contained 6.3 ± 0.76 mM total alkalinity, 4.0 ± 0.09 mM OH– and 0.52 ± 0.13 mM HCO3–; these background values were subtracted before estimates of alkalinity and the levels of OH– and HCO3– Level of alkalinity (mM) Treatment pH 4 (uninoc) pH 4 (inoc) pH (NO3–)

Total

OH–

HCO3–

3.70 ± 1.30a 9.70 ± 0.08b 8.70 ± 0.27b

3.6 ± 1.30a 5.6 ± 0.01b 5.3 ± 0.01b

1.48 ± 0.35a 7.48 ± 0.01b 6.28 ± 1.50b

In the field study, six different native species (identified only to the genus level) were also evaluated to determine whether the increase in rhizosphere pH was widespread among plants growing in the acidic soils that supported the cultivation of A. linearis. The results obtained were inconsistent with those for the legume. A species of Anthospermum and Leucospermum showed decreased pH in the rhizosphere relative non-rhizosphere soil (Fig. 4), a mechanism commonly used by species adapted to acidic soils for promoting nutrient uptake (Marschner 1991, 1995). The pH of soils from the rhizosphere of the remaining four species (namely Willdenowia sp., Serruria sp., Leucadendron sp. and Nylandtia sp.) were, however, not significantly different from the pH of bulk non-rhizosphere soil collected from the same site in the field (Fig. 4). The findings with these six species suggested that the elevation in rhizosphere pH is a special feature of A. linearis. The tea legume was then tested in another study to determine whether the pH increase was symbiosis-related. This was done by comparing the pH increase from NO3– reduction by the plant with the effect of inoculation with bacterial isolate. As shown in Table 1, inoculated plants at pH 4 increased the pH of their root nutrient solution from pH 4 to 6.8. This increase was similar in magnitude to that of 2 mM NO3–-fed plants, which raised their pH from 4 to 7.5. The uninoculated NO3–-free treatment was significantly (P < 0.05) lower in pH elevation (Table 1). There was, however, little change in pH with treatments when plants were grown at pH 6.8 (Table 1). The components of pH increase were determined from the use of titrimetric analysis. The data showed that OH– and HCO3– were the two alkaline ions affecting pH change in the root nutrient solution when plants were grown at pH 4. Both bacterial inoculation of A. linearis and NO3– assimilation by this legume triggered the extrusion of similar levels of OH– and HCO3– into the root nutrient solution (Table 2). As a result, the total alkalinity was also similar for the two treatments (Table 2). However, compared to uninoculated A. linearis plants, the levels of OH– and HCO3– and total alkalinity from inoculated and NO3–-fed plants were significantly (P < 0.05) higher in the nutrient solution bathing the root system (Table 2). Root excretion of HCO3– through NO3– uptake and OH– extrusion as a result of NO3– reduction by nitrate reductase are known features of NO3– nutrition in field plants, and may therefore account for the presence of these anions in the root medium of NO3–-fed A. linearis plants. However, the detection of the same anions as products of root exudation by nodulated A. linearis is inconsistent with the popular view of symbiotic legumes. In fact, rhizosphere acidification is normal among N2-fixing legume and non-legume species growing in root media at neutral pH (Jarvis and Hatch 1985; Lui et al. 1989; Marschner 1995). It is, however, interesting to note that a recent study in tropical Asia also isolated a symbiotic strain of soybean root-

Elevation of rhizosphere pH by Aspalathus symbiosis

nodule bacteria (strain BjGJ3a), presumably from an acidic soil, that grew at pH 3.5 (Abe et al. 2000). Strain BjGJ3a could also raise the pH of its unbuffered culture medium from pH 3.5 to 7.5, an increase similar in magnitude to that of the Aspalathus-infecting root-nodule bacterium isolated from Clanwilliam soil (Table 1). In addition to other enzymes that were expressed in acidic conditions, strain BjGJ3a could maintain its acid phosphatase activity even at pH 3.5; this was unlike other strains, which lost that activity on culturing at only pH 5 (Abe et al. 2000). These findings of pH increase with A. linearis and soybean isolates would suggest the presence of natural populations of root-nodule bacteria in the soils of Clanwilliam and Java able to elevate pH, either on their own, or in the rhizosphere with the legume host. Although it makes ecological sense for symbioses such as those of A. linearis and soybean strain BjGJ3a, which have evolved in acidic environments, to produce substances that neutralize the low pH effects, the mechanisms by which they do so are still not understood. We do know, however, that in addition to the excretion of OH– and HCO3– through NO3– assimilation (Touraine et al. 1992; Imsande and Touraine 1994; Marschner 1995) and decarboxylation of organic acids (Imsande and Touraine 1994; Yan et al. 1996), field plants employ other mechanisms to effect an increase in rhizosphere pH. These include the release of OH– from nitrogenase activity in root nodules (Kennedy 1988) and passive H+ influx at root apices (Marschner 1991, 1995). Although the mechanism used by A. linearis is still unclear, the observed root exudation of HCO3– in this study (Table 2) suggests decarboxylation of organic acids, which led to the pH increase in the rhizosphere. This is consistent with a report that showed increased translocation of malate to roots and its subsequent decarboxylation, which simultaneously stimulated HCO3– excretion and pH increase in the rooting zone of soybean plants (Touraine et al. 1992). Acknowledgments This study was supported with funds from the National Research Foundation, Pretoria, and the University Research Committee of the University of Cape Town. References Aarons SR, Graham PH (1991) Response of Rhizobium leguminosarum bv. phaseoli to acidity. Plant and Soil 134, 145–151. Abe M, Fukuda S, Nuntagij A, Prana T, Uchiumi T, Suzuki A, Higashi S, Prijambada ID, Widianto D (2000) Acid-tolerant and fast-growing symbiotic bacteria isolated from soybean nodule in tropical Asia. In ‘Nitrogen fixation: from molecules to crop productivity’. (Eds FO Pedrosa, M Hungria, MG Yates and WE Newton) p. 503. (Kluwer Academic Publishers: Dordrecht)

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Dakora FD (1998) Nodulation specificity of Aspalathus linearis subsp. linearis, a shrub tea legume indigenous to the Western Cape. In ‘Biological nitrogen fixation for the 21st century’. (Eds C Elmerich, A Kondorosi and WE Newton) pp. 671–672. (Kluwer Academic Publishers: Dordrecht) Deschodt CC, Strijdom BW (1976) Effective nodulation of Aspalathus linearis ssp. linearis by rhizobia from other Aspalathus species. Phytophylactica 8, 103–104. Glenn AR, Dilworth MJ (1991) The biology and biochemistry of nitrogen fixation: a look forward. In ‘Biology and biochemistry of nitrogen fixation’. (Eds AR Glenn and MJ Dilworth) pp. 1–8. (Elsevier: Amsterdam) Howieson JG, Ewing MA, D’Antuono MF (1988) Selection for acid tolerance in Rhizobium meliloti. Plant and Soil 105, 179–188. Howieson JG, Fillery IRP, Legocki AB, Sikorski MM, Stepkowski T, Minchin FR, Dilworth MJ (1998) Nodulation, nitrogen fixation and nitrogen balance. In ‘Lupins as crop plants: biology, production and utilization’. (Eds JS Gladstones, CA Atkins and J Hamblin) pp. 149–179. (CAB International: London) Imsande J, Touraine B (1994) Nitrogen demand and the regulation of nitrate uptake. Plant Physiology 105, 3–7. Jarvis SC, Hatch DJ (1985) Rate of hydrogen ion efflux by nodulating legumes grown in flowing solution culture with continuous pH monitoring and adjustment. Annals of Botany 55, 41–51. Kennedy IR (1988) The molecular basis of symbiotic nitrogen fixation. In ‘Microbiology in action’. (Eds WG Murrell and IR Kennedy) pp. 143–155. (Research Studies Press: London) Lui WC, Lund LJ, Page AL (1989) Acidity produced by leguminous plants through symbiotic dinitrogen fixation. Journal of Environmental Quality 18, 529–534. Marschner H (1991) Mechanisms of adaptation of plants to acid soils. Plant and Soil 134, 1–20. Marschner, H (1995) ‘Mineral nutrition of higher plants.’ Second edition. (Academic Press: London) Muofhe ML, Dakora FD (1998) Bradyrhizobium species isolated from indigenous legumes of the Western Cape exhibit high tolerance of low pH. In ‘Biological nitrogen fixation for the 21st century’. (Eds C Elmerich, A Kondorosi and WE Newton) p. 519. (Kluwer Academic Publishers: Dordrecht) Muofhe ML, Dakora FD (1999) Nitrogen nutrition in nodulated field plants of the shrub tea legume Aspalathus linearis assessed using 15 N natural abundance. Plant and Soil 209, 181–186. Raven JA, Franco AA, de Jesus EL, Jacob-Neto J (1990) H+ extrusion and organic-acid synthesis in N2-fixing symbioses involving vascular plants. New Phytologist 114, 369–389. Staphorst JL, Strijdom BW (1975) Specificity in the Rhizobium symbiosis of Aspalathus linearis (Burn. fil.) R. Dahlgr. ssp. linearis. Phytophylactica 7, 95–96. Touraine B, Muller B, Grignon C (1992) Effect of phloem-translocated malate on NO3– uptake by roots of intact soybean plants. Plant Physiology 93, 1118–1123. Vincent, JM (1970) ‘A manual for the practical study of root-nodule bacteria.’ (Blackwell Scientific Publications: Oxford) Vogel, AI (1961) ‘Quantitative inorganic analyses. Third edition.’ (Longmans, Green and Co.: New York) Yan F, Schubert S, Mengel K (1996) Soil pH increase due to biological decarboxylation of organic anions. Soil Biology and Biochemistry 28, 617–624. Manuscript received 3 December 1999, accepted 5 September 2000

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