Consequences of soil acidity and the effect of lime on the nodulation of Trifolium subterraneum L. Growing in an acid soil

Soil Bid. Biwhem. Vol. 20, No. 4, pp. 439-445, Printed in Great Britain. AU rights reserved

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CONSEQUENCES OF SOIL ACIDITY AND THE EFFECT OF LIME ON THE NODULATION OF TRIFOLIUM SUBTERRANEUM L. GROWING IN AN ACID SOIL A. E. RICHARDSON, A. P. HENDERSON, G. S. JAW and R. J. SIMPSON Plant and Soil Sciences Section, School of Agriculture and Forestry, The University of Melbou~e, Parkville 3052, Australia (Accepted 20 Oczober 1987) Summary-Nodulation of subterranean clover roots growing in a very acid soil was influenced by soil pH. Restricted nodulation occurred in regions with very low soil pH and this consequently affected the distribution of nodules on roots throughout the entire soil profile. Nodulation was improved in all profile regions where lime was incorporated into the soil; this resulted in a more uniform distribution of nodules. Surface application of lime influenced nodutation only at the very top of the soil profile. Restrictions to nodulation at low pH were largely compensated for by the plant, either by a shift in the distribution of nodules to less acidic regions of the protiie, or by an increase in the mass of individual nodules. Total nodule mass per plant was therefore generally the same in all pasture treatm~ts despite large differences in the total number of nodules. The major determinants of restricted nodulation of subterranean clover roots in the acid soii appeared to be low numbers of Rhizobium zrifolii or poor coloniaation of the more acidic regions of the soil profile. Inoculation and lime coating of seed did not overcome poor nodulation associated with the very acidic regions within the soil profiie.

INTRODtJmION The formation of nodules on the roots of subterranean clover (Trzfo~ium sabterruneum L.) by ~i~obi~ trifolii is sensitive to low pH and nutrient

imbalances (e.g. Al toxicity and Ca deficiency) associated with acid soils. Increasing soil acidity under permanent pastures throughout much of southern Australia (Bromfield et al., 1983) therefore has important implications for the productivity of subterranean clover growing on these soils. Failure of subterranean clover to nodulate under acidic conditions is possibly due to either (i) poor survival of R. trzfolii or inability of R. trifolii to rapidly multiply under such conditions, or (ii) direct effects on nodule initiation and formation such as the “acid sensitive step” in the infection of roots by rhizobia (Munns, 1968). Multiplication of R. trifolii is restricted in solution culture below pH 5.5, is inhibited by pH < 4.5 and is sensitive to Al concentrations as low as 10~~ (Thornton and Davey, 1983; Wood and Cooper, 1984). In addition, low numbers of R. trzyofii have generally been observed in acid soils (e.g. Jones, 1966; Rice et al., 1977). Low numbers of R. trifolii or the inability to rapidly colonize soil can adversely affect nodulation of subterranean clover growing in an acid soil (Coventry et al., 1985b). Nodulation of roots of subterranean clover can be restricted at pH < 5.0 and completely inhibited at pH c 4.5 (Loneragan and Dowling, 1958; Kim et al., 1985a). Nodulation is further restricted at pH 4.5 in the presence of Al at concentrations as low as 6.4 ~L’M (Kim et al., 1985b). Ca and P to some extent can alleviate restrictions to nodulation associated with low pH and high concentrations of Al (Loneragan and Dowling, 1958; Alva et ai., 1986). Nodulation 439

of roots of legumes can also be affected by other detrimental factors (e.g. micro-nutrient deficiencies and Mn toxicity) commonly associated with very acid soils. These factors may, directly or in association with problems of survival of R. trz~olii in the soil, markedly affect nodulation of subterranean clover roots growing in an acid soil environment. Richardson and Simpson (1988) have shown that localized regions of low soil pH occur in an acid soil profile under a permanent subterranean clover-based pasture. This in turn affected the distribution of the population of R. trifolii, with higher proportions of the total population occurring in more favourable regions of the soil profile. Incorporation of lime into the soil and thus alleviation of low soil pH or Al toxicity, favoured colonization of the soil by R. trzfolii (Richardson and Simpson, 1988; Coventry et al., 1985b). Our objectives were to assess the nodulation of subterranean clover growing in a very acid soil (pH 4.2, 1Om~ CaCl, 1:5, 0-10cm) in relation to the distribution of the population of R. trifolii within an acid soil profile (Richardson and Simpson, 1988) and to study the nodulation of inoculated and uninoculated subterranean clover seeds sown into an acid soil that had been cultivated with or without the inco~oration of lime. MATERIAIS

AND METHODS

Nodzdation of subterranean clover growing at the field site

Soil pH, soil exchangeable Al concentrations and other details of the field site have been described by Richardson and Simpson (1988). Six pasture treatments established in February 1984 used in these experiments are summarized in Table 1. All pasture

A. E. RICHARDSON et al.

440

Table 1. Description of pasture treatments established at the field site in February 1984 Description

Treatment

I

Existing subterranean clover-based pasture. Existing pasture (treatment 1) with lime’ applied to the soil surface. Pasture sown with uninoculated subterranean clover seed (cv. Mt Barker; 25 kg ha-‘). Pasture sown as above (treatment 3) with incorporation of lime’ to a depth of 12cm. Pasture sown with lime-coated and inoculatedb seed (cv. Mt Barker clover subterranean 25 kg ha-‘). Pasture sown as above (treatment 5) with incorporation of lime’ to a depth of 12cm.

2 3 4 5

6

‘10 t ha-‘; Calcimo lime, Longford, Victoria; 81% CaCO,. tri/olii, WU95: Nodulaid Group C, Agricultural Laboratories, Scfton.

bRhizobim

treatments were sampled on 6 November 1984 and 3 July 1985 and the existing pasture and inoculated and sown pasture treatments only were sampled on 16 October and early (7th) and late (27th) November 1985. Because nodule dist~bution profiles measured on 16 October 1985 showed similar trends to profiles observed on 7 November 1985, results from 4 of 5 sampling dates only are shown in Fig. 1. At each Exiatinqposture

Inoculated sown pasf~re 6. WIthoutlime

A.

C

Incorporatedlime

0 4

9 13 18

0 4 9 13 18

0 L 9 13 18

0 L 9 13 18

0

40

so Nodule

LO

so

distribution

40

so

(%)

Fig. 1. Percentage nodule distribution on roots of subterranean clover plants at five soil profile depths sampled from the field at four harvest dates during 1984 and 1985. The three pasture treatments shown are: (A) existing subterranean clover-based pasture, (B) inoculated sown pasture and (C) inoculated sown pasture with the incorporation of lime (10 t ha-‘) before sowing. The heavily shaded region (0-4cm) in each profile indicates the percentage of total nodules on the crown root. At each sampling date, bars indicate feast significant difference (P = 0.05) for means of all pasture treatments and all soil profile depths.

sampling a single core of soil was taken from each of six replicate plots in each pasture treatment using a modified mechanical ram. Cores of soil were taken directly over subterranean clover plants of similar size and age. Cores of soil were sectioned into five profile regions to 30cm depth on the basis of dist~bution of R. trQ&Iiiand changes in the acidity of the soil profile (Richardson and Simpson, 1988). Highest numbers of R. rr&oIii together with relatively more moderate acidity (pH 4.0-4.2) occurred in the top 4cm of the soil profile, whereas very acidic conditions (pH 3.7-4.0) and generally low numbers of R. trifolii occurred between 4 and 13 cm from the soil surface. More moderate acidity (pH 4.5) was also found below 18 cm, where the soil contained more clay. Subterranean clover roots were carefully washed from each soil profile region and the nodules removed and counted. In the O-4 cm region of the soil profile, crown nodules were designated as being within 1 cm from the main tap root. Total root dry weight and total nodule dry weight was determined for each soil profile region. Modulation of subterranean clover roots in cores of soil removed intact from the field site Cores of soil (30 x 9 cm dia encased in PVC sleeves) were removed intact from the existing pasture (Table 1; treatment 1) at the field site in March 1984 before seasonal rainfall began. After sampling, cores were watered periodically for 4 weeks and germinating seedlings were removed. Four treatments which represented the main treatments in the field trial (Table 1) were then imposed; soil under the existing pasture (treatment 1) was simulated by using undisturbed cores of soil; existing pasture with surface-applied lime (treatment 2) was simulated by undisturbed cores of soil with an equivalence of 10 t ha-’ of lime (Lilydale lime, David Mitchell Estate Ltd, 76% CaCO,) applied to the surface of the soil core. Field treatments in which the soil was cultivated before sowing (treatments 3 and 4) were simulated by removing, mixing and replacing the top 12 cm of soil from each of the soil cores. Where necessary, lime equivalent to 10 t ha-’ was mixed with the soil which was then replaced. The cores of soil were regarded as being similar to the soil profiles in the field (e.g. Richardson and Simpson, 1988) on the basis of their soil pH profiles and dist~bution of R. tr~~o~iiwithin each region of the profile. Surface sterilized and uninoculated seedlings of subterranean clover (cv. Mt Barker) were planted in each soil core and seedlings were later thinned to one plant per core. Plants were grown in a glasshouse in which temperatures ranged between a daily maximum of 25°C and a minimum of 10°C. Soil cores were watered twice weekly to maintain soil moisture at approximately field capacity. Eight replicate cores in each of the 4 treatments were harvested 50, 75, 100, 125 and 150 days after sowing. At harvest, individual cores of soil were sectioned into the 5 profile regions for measurement of root mass, nodule mass and nodule numbers. Soil pH and most probable numbers of R. trl~olii were measured in each region of the soil profile by methods outlined by Richardson and Simpson (1988).

of clover in an acid soil

Nodulation

441

Table 2. Total number of nodules and total nodule mass g-’ root of subterranean clover plants sampled to a depth of 30 cm in the field from existing pasture with or without surface-applied lime and from uninoculated and inoculated sown pasture with or without the incorporation of lime. At each sampling date means not followed by the same letter are significantly different (P < 0.05) Uninoculated sown pasture

Existing pasture Sampling date

Without lime

With lime’

6 Nov 3 July 16 Ckt 7 Nov 27 Nov

1984 1985 1985 1985 1985

171 b 337 b 278 a 428 a 160~

277 b 502 b ND ND ND

6 3 I6 7 27

1984 1985 1985 19X5 1985

214.9 a 172.4 a 214.1 a 129.0 a 31.4 c

216.7a 229.9 a ND ND ND

Without lime

Inoculated sown pasture

With limeb

Without lime

With limeb

Total nodule number (No. g - ’ root dry wt)

249 b 530 b ND ND ND

367 a 781 a ND ND ND

Total nodule mass (mgg-’

Nov July Ott Nov Nov

197.0 a 208.0 a ND ND ND

224 b 485 b 289 a 593 a 349 b

388 a 760a 347 a 626 a 458 a

rool dry wf)

154.8 a 139.3 a ND ND ND

263.8 a 193.7 a 196.0 a 201.9 a 201.7 a

156.2 a ll3.2a 216.2a 205.8 a 104.1 b

‘Surface application of lime (IO t ha-‘). bLime incorporated to l2cm (IO t ha-‘). ND Not determined

Nodulation of subterranean clover roots in specific regions of the soil profile Nodulation of clover roots at different depths in the soil profile was examined by planting uninoculated subterranean clover seedlings into soil collected from 4 specific depths (O-44-9, 9-18 and 18-30 cm) from the existing pasture profile at the field site. Soil was collected before the beginning of the 1984 growing season. After collection, soil from each region of the profile was sieved to remove large pieces of organic matter and was thoroughly mixed. The soil was placed in sterile plastic pots (10 x 9 cm dia) with a sterile clear plastic sleeve which extended the height of the pots to prevent cross-contamination. Five treatments were imposed on soil from each profile region: (i) control; soil from the field without amendment, (ii) soil in which 10.7 g lime kg-’ soil (equivalent to 10 t ha-‘) was incorporated at the time of soil mixing, (iii) limed soil to which 50ml week-’ of a N-free complete nutrient solution was applied, (iv) soil in which 14.7 g of CaSO, (as gypsum) kg-’ soil was incorporated and (v) the CaSO, treatment with additional application of 50ml week-’ of N-free nutrient solution. Where gypsum was applied, the rate of Ca addition to the soil was equivalent to that in the limed treatments. It was envisaged that this would enable distinction between Ca effects and pH effects on nodulation. Where N-free nutrient solutions were applied, small increases in the number and mass of nodules were observed, however, the increases were generally not significant (P < 0.05) and the treatment effects were the same as those where no additional nutrients were applied and are therefore not reported. Uninoculated seedlings of subterranean clover (cv. Mt Barker) were planted in each pot and were later thinned to one plant per pot. Plants were grown in a glasshouse and watered regularly with distilled water to maintain the soil at field capacity. Treatments were randomized. Five replicate plants in each treatment were harvested 50 and 100 days from sowing. Total nodule and crown nodule numbers were recorded at each harvest. However, because differences between

treatments at 50 and 100 days from sowing were similar, only data from the 50 day harvest are presented. Soil pH and exchangeable Al were measured by methods previously outlined by Richardson and Simpson (1988). Numbers of R. trt$ilii were determined by a most probable number plant infection test (Brockwell, 1963) as outlined by Richardson and Simpson (1988). Statistical analysis All data were analysed by analysis of variance and, where variance ratios were significant (P < 0.05). treatment means were compared by least significant difference (P = 0.05). Percentage nodule distribution and percentage crown nodulation were transformed (arcsin) before analysis. RESULTS

At each harvest, subterranean clover plants representative of mean plant size in the pasture were sampled. The uniformity of selection was examined by testing for differences in total dry weight of roots between pasture treatments at each harvest. At all but one harvest (October 1985) total root dry weights per plant were. not significantly different (P c 0.05). In October 1985, the roots of plants growing in the undisturbed existing pasture treatment were 37% smaller (P < 0.05) than those from other pasture treatments. Nodulation of subterranean clover at the field site The numbers of nodules g-’ root (O-30 cm) under the sown pastures were unaffected by inoculating and lime-coating seed (Table 2). However, incorporation of lime at the time of sowing significantly (P < 0.05) increased nodulation of roots (Table 2). Differences between nodulation of roots of plants growing with or without surface-application of lime observed in the field were not significant (P c 0.05) (Table 2). The nodulation of subterranean clover roots under the existing pasture was often similar to that measured under sown pasture where lime had not been incorporated. However, late in the second season poorer

A. E.

442

~XAFUXON

er

nodulation was recorded under the existing (control) pasture. Treatments with lower numbers of nodules g-’ root tended to have larger nodules and as a result, total nodule mass g-l root was not significantly different in any of the pasture treatments (Table 2) at all but one sampling (late November 1985). At this harvest, poor nodulation both in terms of nodule number g-’ root and nodule mass g-’ root was particularly evident in the existing (control) pasture (Table 2). The increase in nodule size associated with growth in unlimed soil ranged between 12 and 262% over all harvests. The largest of these differences in nodule size occurred where there were large differences in the numbers of nodules g-’ root in each pasture treatment (data not shown). Distribution field

of

al.

I

t.’2b

oo

nodules on the roots of plants in the

The largest proportion of nodules on roots of subterranean clover were always observed in the uppermost region of the soil profile (Fig. I). For instance, under the existing (control) pasture 86% of the nodules were observed in the 04 cm region of the profile in November 1984. This was also evident at all sampling dates during 1985, except in late November. At this sampling date, late spring rainfall which followed a dry period (Richardson and Simpson, 1988), enabled root and nodule development deep in the soil profile ( > 18 cm) and reduced the proportion of nodules at the top of the profile to 69% of the total (O-30 cm). Nodulation was apparently restricted in the most acidic regions (4-18 cm) of the soil profile, as high proportions of nodules only formed above and below this region (Fig. 1). Significantly (P < 0.05) larger proportions of nodules were found in the 4-9 cm region of soil profiles where lime had been incorporated (Fig. 1). Improved nodulation in this region was partly attributable to significantly (P c 0.05) improved root growth (e.g. at the 3 July 1985 sampling, 20.7 and 15.3% of the total root mass occurred in the 4-9 cm region of the soil profile in sown and inoculated pasture treatments with or without incorporated lime respectively). However, nodule number g-’ root was also significantly (P < 0.05) improved in the 4-9 cm region of soil profiles where lime had been incorporated (e.g. 578 and 103 nodules g-’ root in the 4-9 cm region of the sown and inoculated pasture treatments with and without incorporated lime, respectively, on 3 July 1985). Plants growing in soil with incorporated lime did not form large numbers of nodules at depths below 18 cm as was observed in both of the unlimed soil profiles or where there was only a surface application of lime. The distribution of nodules observed within soil profiles in pastures sown with uninoculated seed (data not shown) were similar to those reported (Fig. 1) in pastures sown with inoculated seed. Nodulation of subterranean clover roots in cores of soi! removed intact from the field site The distribution of nodules within soil profiles of each pasture treatment were similar to those observed

Days after

sawing

Fig. 2. Total nodule number g-’ root (a) and mean nodule mass (bl for subterranean clover slants crown in cores of I

,

1

soil removed intact from the field so as to simulate pasture treatments in the field. Treatments are: (i) existing subterranean clover-based pasture (a), (ii) existing pasture with surface applied lime (0) (simulated by cores of soil removed intact from the field site), (iii) sawn pasture (m) and (iv) sown pasture with the incorporation of lime (0) (simulated by cultivating soil in cores to 12 cm depth). At each harvest, bars indicate least significant difference. (P = 0.05) for the means of the four pasture treatments.

in equivalent treatments in the field (Fig. 1) and are consequently not shown. Under well-watered conditions in the glasshouse, relatively high proportions of nodules were also observed in the less acidic regions below 18 cm in both the control treatment and the cultivated treatment without lime. This was similar to observations in the field when late spring rain fell during 1985. Nodulation of roots growing in cores of soil from the field was also significantly improved (P < 0.05) where lime had been incorporated [Fig. 2(a)]. Application of lime to the surface of the existing soil profile also improved (P c 0.05) nodulation of roots, but only until 75 days after sowing [Fig. 2(a)]. Nodules formed on roots growing in soil with incorporated lime were always significantly (P < 0.05) smaller than nodules from plants growing in cultivated soil without the incorporation of lime [Fig. 2(b)]. Nodulation of subterranean clover roots growing in specific regions of the soil pro$le Potential restriction of nodulation of subterranean clover roots at specific depths in the soil profile was further examined by planting sterile uninoculated seedlings into soil collected from specific regions of the soil profile. Numbers of R. trifofii at the time of soil collection (March 1984) were highest in the 0-4cm region of the profile and were lower at depth (Table 3). Application of lime to each profile region raised soil pH and correspondingly reduced

Nodulation of clover in an acid soil

443

concentrations of exchangeable Al. Conversely, the application of gypsum at an equivalent amount of Ca, caused a slight decrease in soil pH and increased concentrations of exchangeable Al (Table 3). Most nodules were observed in those regions of the soil profile where the soil pH was more favourable (i.e. greater than at least pH 4.17). At lower soil pH, nodulation was improved if relatively high initial numbers of R. trifolii occurred (e.g. w cm region, Table 3). In all cases where nodulation was improved there were increased proportions of crown nodules and larger total numbers of nodules per plant. Application of lime improved nodulation in soil from all regions of the soil profile. Incorporation of gypsum however, decreased nodulation relative to the control treatment (Table 3).

DISCUSSION

We have examined the effects of soil acidity on the survival and distribution of R. trifolii (see Richardson and Simpson, 1988) and on the nodulation of clover roots in a non-uniform acid soil profile. Nodulation of roots (Fig. 1) and numbers of R. trifolii in the soil (Richardson and Simpson, 1988) were markedly influenced by soil pH, with the largest numbers of R. trifolii and most nodules g-’ roots occurring in those regions of the soil profile where soil was less acidic. These regions were more favourable for either saprophytic existence or rapid colonization of the soil by the bacterium (Richardson and Simpson, 1988). Rapid colonization and high numbers of Rhizobium have been shown to be necessary for early infection and successful nodulation for a variety of legumes (e.g. Vincent and Waters, 1954; Purchase and Nutman, 1957; Robson and Loneragan, 1970). In our experiments, restricted nodulation of clover roots was most apparent when low soil pH occurred in combination with low numbers of R. trifofii (e.g. at 4-18 cm depth). Larger initial numbers of R. trifoiii in the 04cm region of the soil profile improved nodulation of clover roots in this region. This region however, had a more moderate soil pH, lower concentrations of exchangeable Al and higher organic C content and was clearly conducive to rapid colonization by the bacterium (Richardson and Simpson, 1988). Nodulation of subterranean clover roots was improved by the incorporation of lime into the soil. This enhanced colonization of the soil by R. trifolii (Richardson and Simpson, 1988) and consequently favoured nodulation. It is also possible that higher soil pH may have directly improved nodulation by eliminating the potential influence of an acid-sensitive step in the nodulation process (Munns, 1968). However, from the observation that restricted nodulation of roots was most apparent when low soil pH occurred in combination with low numbers of R. trifolii (e.g. at 4-18 cm depth; Table 3) it did appear that relatively low numbers of R. trifofii or poor colonization of the more acidic regions of this soil, were more likely to be the major determinants of nodulation of subterranean clover roots. The net effect of localized low soil pH within the soil profile in our experiments was therefore to alter

444

A. E. RICHARDSON et al.

the distribution of nodules on the roots of subterranean clover. Nodulation was largely confined to roots in the upper region of the soil profile in all pasture treatments except where lime had been incorporated. Roots growing in regions of the soil profiles that received lime, consistently had more nodules and consequently subterranean clover plants in limed treatments had more nodules g-i root over the whole root system. Application of gypsum did not increase nodulation of roots, indicating that the improved nodulation was primarily due to alteration of soil pH rather than addition of Ca. Improved nodulation due to the incorporation of lime was maintained throughout the entire period of plant growth, whereas surface application of lime only increased early nodulation of plants. Where lime was applied to the soil surface, the number of nodules g-l root (0-30cm) declined as roots developed deeper into the soil where surface lime had not affected soil pH (Fig. 2a). Thus, if lime was not incorporated into the soil, low soil pH at depth restricted nodulation when roots grew out of the limed region of the soil profile. It is presumed that the restriction of large proportions of nodules to the top few cm of an acid soil profile may result in nodules being subjected to wider extremes of soil temperature (Quinlivan, 1961) and localized moisture deficits as surface layers of soil dry during dry periods or at the end of a growing season (Pinkerton and Simpson, 1986). Both of these influences can adversely affect nodule activity (e.g. Gibson, 1969; Sprent, 1976). Despite marked differences in the distribution of nodules on roots, restrictions to nodulation associated with low soil pH were largely compensated by the plant. Firstly, larger proportions of the nodule population were found in less acidic regions of the soil profile. Large proportions of the nodules below 18 cm were only observed in unlimed soil and in soil with a surface application of lime. Incorporation of lime, but not surface-application of lime, which alleviated low soil pH at depth resulted in improved nodulation of roots throughout the soil profile and these plants consequently did not form large numbers of nodules below 18 cm. Secondly, despite significant differences in the number of nodules gg’ root in the various pasture treatments, total nodule mass g-’ root was generally the same (Table 2). A relationship of this nature has previously been described for subterranean clover (Nutman, 1967). However, it has been reported that larger nodules associated with restricted nodulation in an acidic soil have lower specific acetylene reduction activities (Coventry et al., 1985a). Rhizobia introduced to the soil through inoculating and lime-coating seed had no effect on nodulation of clover roots probably because relatively high numbers of R. trifofii were already present in the top few centrimetres of the soil (Richardson and Simpson, 1988). Similarly, this treatment did nothing to alleviate the restrictions to nodulation observed in the more acidic regions at depth where soil acidity clearly resulted in relatively low numbers of R. trifolii and poor colonization of the soil (Richardson and Simpson, 1988). Possibly the only effect of inoculating seed was to increase the proportion of the inoculum strain of R. trifolii in the nodules formed at

the top of the soil profile (Roughley et al., 1976; Brockwell et al., 1982). By contrast, inoculation of clover seed sown into very acid cropping soils has been shown to improve markedly the nodulation of the roots of subterranean clover plants (Coventry et al., 1985b). However, in these instances the R. trifolii inoculum was introduced into soil which, after cultivation and a period in which the host plant was absent, contained few R. trifolii. Acknowledgements-Our

work was supported by a grant from the Wool Research Trust Fund on the recommendation of the Australian Wool Corporation, by the Estate of the late Sir A. E. Rowden White and bv Research Development Grants from the University of &Ielbourne. A. E. Richardson was a recipient of a Commonwealth Postgraduate Research Award. We thank J. Brockwell for his critical evaluation of the manuscript. REFERENCES

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Brockwell J. (1963) Accuracy of a plant-infection technique for counting populations of Rhizobium rrifolii. Applied Microbiology 11, 377-383.

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Bromfield S. M., Cumming R. W., David D. J. and Williams C. H. (1983) Change in soil pH, manganese and aluminium under subterranean clover pasture. Ausrralian Journal of Experimental Husbandry 23, 181-191.

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Coventry D. R., Hirth J. R., Reeves T. G. and Jones H. R. (1985b) Development of populations of Rhizobium rrifolii and nodulation of subterranean clover following the cropping phase in crop-pasture rotations in Southeastern Australia. Soil Biology & Biochemistry 17, 17-22. Gibson A. H. (1969) Physical environment and symbiotic nitrogen fixation. VII. Effect of fluctuating root temperature on nitrogen fixation. Ausfralian Journal of Biological Sciences 22, 8399846.

Jones D. G. (1966) The contribution of white clover to a mixed upland sward. II. Factors affecting the density and effectiveness of Rhizobium lrijolii. Planf and Soil 24, 25@260.

Kim Moo-Key, Edwards D. G., Date R. A. and Asher C. J. (1985a) Effects of pH on nodulation and growth of subterranean clover cultivars. In Proceedings XV International Grasslands Congress, Kyoto, Japan, pp. 543-544. Science Council of Japan & Japanese Society Grassland Science, Tochigi-ken, Japan. Kim Moo-Key, Asher C. J., Edwards D. G. and Date R. A. (1985b) Aluminium toxicity: effects on growth and nodulation of subterranean clover. In Proceedings XV International Grasslands Congress, Kyoto, Japan, pp. 501-503. Science Council of Japan & Japanese Society Grassland Science, Tochigi-ken, Japan. Loneragan J. F. and Dowling E. J. (1958) The interaction of calcium and hydrogen ions in the nodulation of

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and distribution of Rhizobium trifolii under n subterranean clover-based pasture growing in an acid soil. Soil Biology & Biochemisky 20, 431-438. Robson A. D. and Loneranan J. F. (1970) Nodulation and growth of Medicago*truncatula‘on &id soils. II. Colonization of acid soils by Rhizobium meliloti. Australian Journal of Agricultural Research 21, 435-445. Roughley R. J., Blowes W. M. and Herridge D. F. (1976) Nodulation of Trifalium subterraneum by introduced rhizobia in competition with naturalized strains. Soil Biology & Biochemistry 8, 403-407. Sprent J. I. (1976) Nitrogen fixation by legumes subjected to water and light stresses. In Symbiotic Nitrogen Fixation in Pfants (P. S. Nutman, Ed.), pp. 405-420. Cambridge University Press, London. Thornton F. C. and Davey C. B. (1983) Acid tolerance of Rhizobium trifolii in culture media. Soil Science Society of America Journal 47, 49650 1. Vincent J. M. and Waters L. M. (1954) The root nodule bacteria as factors in clover establishment in the red basaltic soils of the Lismore district, New South Wales. II. Survival and success of inocula in laboratory trials. Australian Journal of Agrirultural Research S, 61-76. Wood M. and Cooper J. E. (1984) Aluminium toxicity and multiplication of Rhizobium trifolii in a defined growth medium. Soil Biology & Biochemistry 16, 571-576.