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Growth and nitrogen fixation by subterranean clover in response to inoculation, molybdenum application and soil amendment with lime
0038-0717/85$3.00+O.OO
SoilBid Biochem.Vol. 17, No. 6, pp. 191-196,1985
Printed in Great Britain. All rights reserved
Copyright Q 1985 Pergamon Press Ltd
GROWTH AND NITROGEN FIXATION BY SUBTERRANEAN CLOVER IN RESPONSE TO INOCULATION, MOLYBDENUM APPLICATION AND SOIL AMENDMENT WITH LIME D. R. COVENTRY,J. R. HIRTH, T. G. REEVES and V. F. BURNETT Rutherglen Research Institute, Department of Agriculture, Rutherglen, Victoria 3685, Australia (Accepted 30 March 1985)
Summary-Liming an acid soil increased the yield and N content of subterranean clover in both field and glasshouse experiments. Application of MO increased the N concentration of field-grown subterranean clover which corresponded with observed colour and growth differences, but did not change C,H, reduction activity. Herbage MO was not increased by liming, suggesting an absolute deficiency of MO in these acid soils. In the glasshouse liming increased nodulation which increased the amount of N, fixed but the lime had no direct effect on nitrogenase activity as measured by C,H, reduction. In the field both inoculation and lime aunlication increased soil oonulations of R. trifolii. but clover yield was greater with liming alone than with-inoculation alone, indi’caiing the sensitivity of.the host plant to soilacidity.
INTRODUCTION of subterranean clover L.), an annual selfregenerating pasture legume, has been reported for a range of soils and farm systems in south-eastern Australia (Carter et al., 1982; Coventry et al., 1985; Reeves et al., 1984). Although there have been many changes in the management of subterranean clover based pastures, both in permanently grazed (Williams, 1980) and ley systems (Coventry et al., 1985), a feature common to both situations is the association between the decline in production of subterranean clover and a lowering of soil pH (Osborne et al., 1978; Bromfield et al., 1983a,b; Haynes, 1983). Soil acidity, through the direct effect of low pH, Ca deficiency (Munns, 1978) and MO deficiency (Anderson and Spencer, 1950; Dilworth, 1974), can affect nodule function and consequently growth of subterranean clover. We have reported (Coventry et al., 1985) (i) large increases in the nodulation of subterranean clover where soil pH was raised by lime, with the plants growing in unlimed soil having fewer, but much larger nodules and (ii) an increase in R. trzfilii populations to satisfactory levels by sowing inoculated seed. Large increases of clover growth in response to lime were obtained in these experiments. The aim of the field and glasshouse experiments reported here was to determine if soil acidity had a direct effect on nitrogen fixation activity as measured by specific C,H, reduction activity. The decline in production ( Trifolium
subterraneum
MATERIALSAND METHODS Experiment composition
I: Effect of lime and MO on yield, plant and NJ-fixation in the jieid
The C2HZ reduction assay was used for comparison of N,-fixation activity for field grown subterranean clover during two growing seasons. A factorial design
experiment with four replicates was used; the treatments were three rates of lime (0, 0.5, 1.0 t ha-‘; 57% Ca, 5% Mg, neutralizing value 142 as % CaCOA) (13 kg P ha a’, two fertilizer additions and 13 kg P ha-’ plus 62g MO ha-‘). The clover was grown at the Lilliput site (pH 5.2; &lo cm depth, 1: 5 soil : water); details of the establishment, management and site characteristics have been described by Coventry et al. (1985). The soil is low in exchangeable Ca and Mg and high in exchangeable Al and Mn (Coventry et al., 1985). Subterranean clover was carefully transferred from the field, the soil removed and entire plants taken to the laboratory for analysis. The plants were placed in an 885 ml gas-tight jar with a sealed cap and serum stopper for addition and removal of gases and the jars were held at 27°C in a water bath for 25min. The assay was started by injection of C2H, (0.025 atm) and 100 ~1 gas samples were withdrawn periodically for 30 min and analysed by GC. The separation of C2Hz and C,H, was obtained in a 4 mm x 150 cm column of Porapak N run at 100°C with a gas flow of 30 ml min-’ using a Packard Model 437 GC. Ethylene production was linear throughout the assay. The clover plants from each treatment were sampled between 0900 and 1100 h to minimize variation in daily soil temperature, and the time between field sampling and C2Hz injection never exceeded 1.5 h. This restricted the number of samples taken each day and replicate treatment pilots were sampled on successive days. In the 1982 growing season three replicates were analysed at each of 7 harvests and in the 1983 season two replicates were sampled at each of 4 harvests. The clover germinated on 16 March in 1982 and the rainfall was 90.1 mm (cf. 220 mm, 66 yr average) and the mean air temperature 5.6”C (range l-13°C) during the 15 week assay. In 1983 the clover germinated on 30 March and the rainfall was 112 mm (cf. 52 mm, 66yr average) and the mean air temperature 11.5”C (616°C range) for the 5 week assay. 791
192
D. R.
COVENTRY
Plants were also harvested and soil temperature measured every 2 h for 18 h, once in each season, to determine if there were any diurnal patterns of C2H, reduction. Clover herbage dry matter and nodule number were determined from five 0.20m2 quadrats at the fifth harvest in 1982 and the fourth harvest in 1983. The herbage was analysed for N and MO concentration. Nodules collected from the third to fifth harvest in 1982 were also analysed for MO concentration. Experiment 2: Effect of lime and inoculation on yield and numbers of R. trifolii in the field
A factorial experiment was established in April 1982 at the Rutherglen site (pH 5.0; O-10 cm depth, 1: 5 soil:water; Coventry et al., 1985), where soil populations of R. trifolii were known to be very low. The treatments were rates of lime and seed inoculation with R. trifolii (see Table 3). Subterranean clover (cv. Woogenellup) was sown at 10 kg ha-’ with superphosphate supplying 13 kg P ha-‘. Plot size was 1.9 x 10.0 m and there were four replicates. Dry matter production of the establishing clover was determined from one 1.12 x 1.65 m quadrat per plot in October 1983. Soil samples (20 x 3 cm cores, O-8 cm depth composited from four replicates) were taken in June 1983 for estimation of populations of R. trifolii using the plant infection technique (Brockwell, 1963). Experiment 3: Effect of lime on yield, N content and N,-jixation in the glasshouse
Soil for a pot experiment was collected from the Lilliput site to a depth of 10 cm from unlimed plots that had previously grown three consecutive wheat crops. Lime and seed inoculation (see Table 4) were the treatments in a factorial design with six replicates. The lime (1.745 g kg-’ soil, analytical reagent grade CaCO,) was mixed with the soil before filling the pots. Undrained plastic pots (11 cm dia) were filled with 700 g of sieved (6 mm) air-dry soil, watered to field capacity (29x, w/w) and allowed to equilibrate for 10 days. P at 70 pg pot-’ was applied to the soil surface and thoroughly mixed with the soil in the pots prior to sowing. Ten imbibed subterranean clover seeds (cv. Woogenellup) were sown at 1.5 cm depth and a 1 ml dense suspension of R. trifolii was applied in the planting holes to the inoculum treatments. The moisture content of the soil was maintained between 50 and 60% of field capacity for the duration of the experiment. Air temperature was kept below 30°C. Seedlings were thinned to 6 per pot 10 days after emergence. Plants were harvested at 4 weeks and 6 weeks after emergence, the roots were washed and C2H2 reduction of intact plants measured. Nodule number, nodule weight and shoot dry weight were determined and shoot herbage was analysed for N concentration. Plant analyses
Total N was determined by the semi microKjeldah1 method of Bremner (1965) using an electronically controlled digestion block, and automated distillation and titration apparatus. MO was determined
et al.
by a modification of the method of Dick and Bingley (1951). Statistical analyses
Factorial analyses of variance were conducted on all data, and where treatment significance (P < 0.05) was attained, the Duncan Multiple Range Test was applied. RESULTS
Experiment 1: Efect of lime and MO on yield, plant composition and N,-jxation in the$eld
Growing conditions during the two seasons varied considerably. During the assay period in 1982 very dry conditions were experienced, whereas in 1983, the conditions were ideal for plant growth. The daily pattern of CIH2 reduction was linear and horizontal in both 1982 and 1983 despite the diurnal fluctuations in soil temperature (e.g. in 1983, 1Ocm soil temperature ranged from 4” to 9°C). The pattern of C,H, reduction of subterranean clover in 1982 is shown in Fig. 1. Nodule activity was significantly greater (P < 0.10) with lime addition at the sampling where C,H, reduction was first detected (9 weeks after germination) and at the samplings 17 and 19 weeks after germination. There were no differences in nodule activity from MO addition. The period of highest C,H, reduction occurred at the time of most rapid plant growth, 12-20 weeks after germination. There were no differences (P = 0.05) in nodule weight and nodule number between treatments and nodule development was restricted by the very dry soil conditions (Fig. 1, Table 1). Acetylene reduction declined after 19 weeks growth, corresponding with the cessation of growth and senescence of the clover. The pattern of C2H, reduction was measured for a shorter period of clover growth in 1983. There were no significant differences (P = 0.10) in C,H, reduction at any sampling time, however when C2H2 reduction was first detected, lime at nil, 0.5 and 1.0 t ha-’ with MO and lime at 1.0 t ha-’ without MO gave higher readings (P = 0.21) than lime at nil and 0.5 t ha-’ without MO. The clover with added MO was a much darker green colour, but this colour difference was not maintained for longer than 8 weeks after germination. The dry matter yield of clover in 1982 increased (P <: 0.05) with lime, but MO did not affect yield although N concentration was increased (Table 1 and 2). MO concentrations increased in both plant tops and nodules with MO fertilization; but the ratio of MO concentration in the nodules to tops declined. This ratio ranged from 13: 1 to 25: 1 in the absence of MO, and from 7: 1 to 11: 1 with added MO. In 1983 there were no significant differences (P = 0.05) in growth (Table l), although MO addition gave larger and darker green plants. The clover stand at this harvest was heavily infested with capeweed (Arctotheca calendula) which gave a very high coefficient of variation to the clover yield data (60%). There was a large increase in the N concentration (P < 0.01) and N content (kg ha-‘) of the clover with MO application (Table 2). Again the MO concentration in the plant tops was much higher with MO application.
Growth and nitrogen fixation of subterranean clover
10
15
20
Time
793
25
(weeks)
Fig. 1. Pattern of C, H, reduction activity and nodule development (0) from germination for field-grown subterranean clover, 1982.Key: No lime (0); 0.5 t ha-’ lime (II); 1.0 t ha-’ (A); solid line, 13 kg P ha-‘, dash line, 13 kg P ha-’ and 62 g MO ha-‘. Vertical bars indicate LSD (10%).
Table 1. Yield and nodulation of field grown subterranean clover (experiment 1) with lime and molybdenum treatments
Lime
MO
(t ha-‘)
(g ha-‘)
0 0 0.S 0.5 1.0
62 0 62 0 62
1.0
0
Nodule number plant-’ August 1982 June 1983
Dry weight (t ha-‘) August 1982 May 1983 1.22a
1.5
1.49 1.18 1.40 1.32 1.27 1.32
1.28a 1.48ab 1.35ab 1S7ab 1.66b
5.5 8.5 9.0 8.9 6.0
23 26 19 20 21 23
Means within a column followed by different letters differ (P < 0.05).
Table 2. N concentration
and content and MO concentration Herbage August 1982
Lime (t ha-‘) 0 0 0.5 0.5
i.0 1.0
MO (g ha-‘) 62 0 62 0 6i 0
(& 4.22 4.05 4.22 4.02 4.23 4.12
N content (kg ha-‘) 51 52 62 54 67 68
of subterranean
clover (experiment 1) Herbage May 1983
Nodules August 1982 _.___-_
~ MO (Pgg-‘) I .30b 0.23a 1.60b 0.25a I .7Oc 0.28a
MO (Icglr’) 13.7 5.6 12.6 3.2 11.5 6.0
(&
N content (kg ha-‘)
MO (Pgg-‘1
3.86b 3.55a 4.02b 3.53a 3.97b 3.85b
58 44 56 47 51 51
0.73b <0.20a 0.75b
Means within a column followed by different letters differ (P i 0.05).
experiment 2: Effect of Iime and inoc~iotio~ on yield and numbers of R. trifolii in the field Large significant differences in dry matter yield were obtained between all treatments in the subterranean clover regenerating in 1983, following a drought-affected establishment year (Table 3). Where clover was not inoculated and no lime was applied, R. trifolii were not detected (< 10 gg’ soil), but with both inoculated clover and limed soil the populations of R. trifoiii increased to detectable numbers (Table 3).
Experiment 3: Effect of lime on yield, N content and N,-fixation in the glasshouse The clover was poorly nodulated in the absence of seed inoculum and lime, and nodulation of the clover without inoculation was increased by lime (Table 4). After 4 weeks there was no effect of lime on nodule numbers in treatments sown with inoculated seed but by 6 weeks the nodulation was greater (P < 0.01) with lime application. The nodules formed on uninoculated plants grown in unlimed soil were larger and
D.
794
R. COVENTRY ef al.
Table 3. R. rrijooliipopulations and yield of field-grown subterranean clover (experiment 2) with inoculation (seed inoculated with commerical inoculum, Nodulaid. group C applied in a water slurry) and lime treatments
Lime (t ha -’’ f 0 0 2.5 2.5
Mean R. trifnlii population’ (log no. g-’ soil) June 1983
Treatment
ND 1.41 1.81 2.2 i
- Inoc I- Inoc -bloc +1noc
Dry weight (t ha” ‘) October 1983 2.26a 3.48ab 4sobc 5.87~
ND R. trijb& not detected (< IOg-’ soil). Dry weight means within a column followed by different letters differ (P i 0.05). ‘Absence of letters result of bulking replicates.
heavier (P < O.Ol), but the specific GH, reduction activity of these nodules, measured 4 weeks after ge~ination, was much less than for all other treatments (P < O.Ol), There were no differences in C,H, reduction of nodules on inoculated plants grown in the absence of lime, when compared with the limed treatments (Table 4). The uninoculated clover plants grown without lime were chlorotic, whereas the inoculated plants grown on the same unlimed soil did not develop these symptoms. At both harvests there was no significant difference in the dry matter yield of inoculated and uninoculated clover grown without lime (Table 5). Application of lime increased growth of the clover, with the uninoculated plants producing more dry matter than inoculated plants at the first harvest (P < 0.05); there was a similar, but non-significant, difference at the second harvest (Table 5). The N concentration of the clover increased with both lime addition and inoculation, with lime producing the biggest increase. At the second harvest, the effect of inoculation and lime on N ~ncentration was additive, however there was no such effect with N content, whose level in the presence of lime was unaltered by inoculation treatment.
DISCUSSION
The increase in growth and nitrogen content of subterranean clover obtained in the field with lime and MO, as well as growth responses with inoculation, came from increased N,-fixation. The lime may also have improved plant growth by modifying acid soil properties. In one season lime had a direct effect on the nitrogenase activity, as measured by C,H, reduction, at three of the seven sampling times through the season. Moly~en~ appli~tion resulted in increased N concentration in the herbage in both seasons, and an obvious leaflet colour response in 1983. However no significant increases (P = 0.10) were obtained in nodule C,H, reduction activity with the field-grown clover following MO addition. The MO concentrations in the herbage, without MO application, were low and were not increased by time, suggesting an absolute deficiency of MO in this soil. Liming acid soils usually corrects MO deficiency by increasing the availability of MO already present in the soil (Anderson and Moye, 1952). With the many reports of acidification of Australian soils (Haynes, 1983), as predicted by Donald and Williams (1954), the requirement of pastoral and Icy soils for MO application is now likely to be more extensive. The severe drought and low soil temperatures of 1982 limited both nodulation and N,-fixation in the field (Roughley and Dart, 1970; Gibson, 1971; Sprent, 1972). In 1983, the field conditions were more favourable for nodulation and N,-fixation but again there were no differences between treatments in nodule weight and number. Aluminium and H+ ion toxicities and Ca deficiency are known to restrict nodulation of legumes (Lowther and Loneragan, 1968; Munns, 1978; Carvalho et al., 1982); in the glasshouse experiment Iime application increased nodulation similarly to the field response reported previously with this soil (Coventry ef al., 1985). The subterranean clover grown in the glasshouse (experiment 3) without inoculation and lime produced few nodules and although there was a compen-
Table 4. Nodulation and NI-fixation activity (C,H, reduction) of subterranean clover grown in a glasshouse (excrement 3) with insulation and lime treatments Nodule weight Lime
(gpot ~‘) Treatment 0 0
1.222 1.222
+ +
Inoc lnoc Inoc Inoc
Nodules plant ’ 4 weeks 6 weeks
4 weeks
I .Oa 24.95b 23.92b 26.86b
2.36Ob 0.397a 0.428a 0.33%
7.14a 38.45b 43.86b 53.81c
0%)
Specific nodule activity (nmol C,H, reduced g ’ min ’ ) 4 weeks 232a 1058b 79Sb 948b
Means within a column followed by different letters differ (P i 0.05)
Table 5. Growth, Lime (gmJt--‘) 0
0 1.222 I.222
nitrogen concentration and nitrogen content of subterranean clover grown in a glasshouse (experiment 3) with inoculation and lime treatments Treatment - Inoc + Inoc -1noc +Inoc
Dry weight (gpot-‘) 4 weeks 6 weeks 0.313a 0.318a 0.478b 0.372a
0.613a 0.7 I 8a
1.402~ 1.168b
Herbage N (%) 6 weeks 4 weeks
1.86a 3.04b 3.65c 3.8lc
Means within a column followed by different letters differ (P < 0.05).
1.67a 2.3833 2.441, 2.8cic
N content (mg pot-‘) 6 weeks 4 weeks 5.814a 9.654b 17.532d i4.118c
10.380a 16.331b 33.97Oc 32.394c
Growth
and nitrogen fixation of subterranean clover
satory increase in individual nodule size, the CzHz reduction activity (g-’ tissue) was much less than for the inoculated and limed treatments, suggesting that the volume of bacteroid tissue per nodule was not proportionally increased. The plants with few nodules were chlorotic and typically N-deficient in appearance. With inoculation, the plants were well nodulated; the C2H, reduction activity of the nodules and the herbage N concentration were greater and the plant chlorosis was alleviated. The soils used in experiments 2 and 3 had very low initial populations of R. trifolii and in both experiments either seed inoculation or lime application were required to ensure R. trifolii build-up and adequate nodulation of the clover. The dry matter yield of the inoculated and unlimed plants in experiment 3 was no greater than the uninoculated and unlimed plants indicating that acid soil factors were limiting the growth of the plants on unlimed soil. With addition of lime there was no difference in C,H, reduction activity and nodule size at 4 weeks growth compared with the unlimed and inoculated plants, indicating that the lime effect is not on nodule function. This result differs from reports of direct effects of lime and Ca on the specific nodule activity of subterranean clover (Banath et al., 1966; Munns, 1978). Where lime was applied, nodulation was more rapid, which would account for the increase in N concentration and content of these plants, and for part of the growth difference. The dry matter yield of the clover with inoculation and lime was less at 4 weeks than with the uninoculated plants with lime, but at 6 weeks the yield differences were not as obvious. This contrasted with the field-grown clover (experiment 2) where the yield was greater with inoculation and lime. The reason for the slower initial growth of the limed, inoculated, plants is not obvious, as at 6 weeks these plants had more nodules and higher herbage N concentrations. The large increase in growth of these lime treatment plants at 6 weeks, compared with the inoculated and unlimed treatment plants, may also have resulted from either relief from soil properties limiting plant growth or a direct effect of lime on plant growth, rather than a limitation on N,-fixation (Robson and Loneragan, 1977). A similar result was obtained with the field grown clover in experiment 2, where lime was required to produce the best dry matter yield. Acid soil infertility can be caused by toxicities of Al, Mn and H or deficiencies of Ca, Mg and MO. Many soils in southeastern Australia have become more acid due to shifts in land-use to dryland farming systems based on subterranean clover pastures, and now have high concentrations of both exchangeable Al and Mn (Williams, 1980; Bromfield et al., 1983a). The area of improved pasture affected by acidity is likely to increase in the future (Williams, 1980) and farmers will have to consider corrective measures for restoring pasture productivity. It is therefore important that the various components of the legume-Rhizobium association, and of the nutrition of legume affected by increasing acidity, be identified as this will influence the remedial treatment options. The soils associated with this study, which had previously not required fertilizer MO for subterranean clover productivity, have now been shown to
795
require MO fertilization. It has also been established that soil populations of R. trtfolii are very low following cropping in a clover-ley rotation, which frequently results in poor nodulation of subterranean clover. The R. trtyolii populations can be quickly re-established to adequate levels by either seed inoculation or lime application when the clover is being re-established. However Bromfield et al. (1983b) have warned that nodulation problems may develop in permanent annual pastures as a result of relatively small further increases in soil acidity. Soil acidity also is having an effect on the growth of subterranean clover separate from effects on the N,-fixing association which has been overcome by liming the soil. It is likely that surface-applied lime will be used extensively on ley-rotation acid soils where crop yields are decreased because of the decreased soil pH. However liming soils for renovation of permanent pastures often is not feasible as the existing pasture has to be disrupted for incorporating the lime and then re-established at considerable cost. There is a need for the development of management systems on permanently grazed pastures which reduce the activity of the toxic soil components, and maintain soil biological activity. Acknowledgements-We wish to acknowledge the assistance of Mr E. G. Baker and Sons, Lilliput and Messrs W. R. and R. W. Francis, Rutherlgen, on whose properties the experiments were conducted. The Rhizobium analyses were done by Mr H. R. Jones, Department of Agriculture, Bendigo and some chemical analyses by staff of the State Chemistry Laboratory, Melbourne. The assistance of G. R. Morrison, S. A. Shaw, D. K. Jarvis, J. M. Roberts and B. C. Allen is also acknowledged. This work was/ supported by a grant from the Wool Research Trust Fund on the recommendation of the Australian Wool Corporation.
REFERENCES
Anderson A. J. (1956) Molybdenum as a fertilizer. AdL1ance.s in Agronomy 8, 163-202. Anderson A. J. and Moye D. V. (1952) Lime and molybdenum in clover development on acid soils. Australian Journal of Agricultural Research 3, 95-l 10. Anderson A. J. and Spencer D. (1950) Molybdenum in nitrogen metabolism of legumes and non-legumes. Australian Journal of Scientific Research, Series B 3,414430. Banath C. L., Greenwood E. A. N. and Loneragan J. F. (1966) Effect of calcium deficiency on symbiotic nitrogen fixation. Plant Physiology 42, 760-763. Bremner J. M. (1965) Total nitrogen. In Methods of Soil Analysis Part 2, (C. A. Black, Ed.), pp. 1149-1178. American Society of Agronomy, Madison. Brockwell J. (1963) Accuracy of a plant-infection technique for counting populations of Rhizobium trifolii. Applied Microbiology 11, 377-383. Bromfield S. M., Cumming R. W., David D. J. and Williams C. H. (1983a) Change in soil pH, manganese and aluminium under subterranean clover pasture. Australian Journal of Experimental Agriculture and Animal Husbandry 23, 181-191. Bromfield S. M., Cumming R. W., David D. J. and Williams C. H. (1983b) The assessment of available manganese and aluminium status in acid soils under subterranean clover pastures of various ages. Australian Journal of Experimental Agriculture and Animal Husbandry 23, 192-200. Carter E. D., Wolfe E. C. and Francis C. M. (1982)
796
D. R. COVENTRY ef al.
Problems of maintaining pastures in the cereal-livestock areas of Southern Australia. In Proceedings of the Second Australian Agronomy Conference, Wagga Wagga, (M. J. T. Norman, Ed.), pp. 68-87. The Australian Society of Agronomy, Victoria. Carvalho M. M. de, Edwards D. G., Asher C. J. and Andrew C. S. (1982) Effects of aiuminium on n~ulation of two Stylosanthes sp. grown in nutrient solution. Plant and Soil 64, 141-152. Coventrv D. R.. Hirth J. R.. Reeves T. G. and Jones H. R. (198.5) Development of p&pulations of Rhizobium trifblii and nodulation of subte~~~n clover following the cropping phase in crop-pasture rotations in southeastern Australia. Soil Biology & Biochemistry 17, 17-22. Dick A. T. and Bingley J. B. (195 I) Further observations on the determination of molybdenum in plant and animal tissue. Australian Journal of Experimental Biology and Medical Science 29, 459462. Dilworth M. J. (1974) Dinitrogen fixation. Annual Review of Plant Physiology 25, 81-114. Donald C. M. and Wjlliams C. H. (1954) Fertility and pr~uctivity of a podzolic soil as inlluenced by subterranean clover (Trt,foIium ~bterr~e~ L.) and superphosphate. Austrahan Journal of Agricultural Research 5, 664-687. Gibson A. H. (1971) Factors in the physical and biological enviro~~t affecting nodulation and nitrogen fixation by legumes. Plant and Soif Special Volume 1971,139-152. Haynes R. J. (1983) Soil acidification induced by leguminous crops. Grass and Forage Science 38, l-l I.
Lowther W. L. and Loneragan J. F. (1968) Calcium and nodulation in subterranean clover (Trifolium subterraneum L.). Plant Physiology 43, 1362-1366. Munns D. N. (1978) Soil acidity and nodulation. In h4inerul Nutrition of Legumes in Tropical and Subtropical Soils (C. S. Andrew and E. J. Kamprath, Eds), pp. 247-263, C.S.I.R.O., Australia. Osborne G. J., Wright W. A. and Sykes J. (1978) Increasing soil acidity threatens farming systems. Agrieuburai Gazette. New South Wales 89. 21. Reeves’ T. G., Haines P. J. ‘and Coventry D. R. (1984) Growth of wheat and subte~ane~ clover on soil artificially compacted at various depths. Plant and Soil 80, 135-138. Robson A. D. and Loneragan J. F. (1977) Responses of pasture plants to soil chemical factors other than nitrogen and phosphorus, with particular emphasis on the legume symbiosis. In Plant Relations in Pastures (J. R. Wilson, Ed.). C.S.I.R.O., Australia. Roughley R. J. and Dart P. J. (1970) Growth of TrijXium s~terr~e~ L. selected for sparse and abundant nodulation as affected by root temperature and Rhizobium strain. Journal of Experiments Botany 21, 776-786. Sprent J. I. (1972) The effects of water stress on nitrogenfixing root nodules. IV EKects on whole plants. Neut Phytologist 71, 603-611. Williams C. H. (1980) Soil acidification under clover pasture. ~us~ra~ia~ Journal of Experimental Agri~lture and Animal husbandry 20, 56 I-567.
SoilBid Biochem.Vol. 17, No. 6, pp. 191-196,1985
Printed in Great Britain. All rights reserved
Copyright Q 1985 Pergamon Press Ltd
GROWTH AND NITROGEN FIXATION BY SUBTERRANEAN CLOVER IN RESPONSE TO INOCULATION, MOLYBDENUM APPLICATION AND SOIL AMENDMENT WITH LIME D. R. COVENTRY,J. R. HIRTH, T. G. REEVES and V. F. BURNETT Rutherglen Research Institute, Department of Agriculture, Rutherglen, Victoria 3685, Australia (Accepted 30 March 1985)
Summary-Liming an acid soil increased the yield and N content of subterranean clover in both field and glasshouse experiments. Application of MO increased the N concentration of field-grown subterranean clover which corresponded with observed colour and growth differences, but did not change C,H, reduction activity. Herbage MO was not increased by liming, suggesting an absolute deficiency of MO in these acid soils. In the glasshouse liming increased nodulation which increased the amount of N, fixed but the lime had no direct effect on nitrogenase activity as measured by C,H, reduction. In the field both inoculation and lime aunlication increased soil oonulations of R. trifolii. but clover yield was greater with liming alone than with-inoculation alone, indi’caiing the sensitivity of.the host plant to soilacidity.
INTRODUCTION of subterranean clover L.), an annual selfregenerating pasture legume, has been reported for a range of soils and farm systems in south-eastern Australia (Carter et al., 1982; Coventry et al., 1985; Reeves et al., 1984). Although there have been many changes in the management of subterranean clover based pastures, both in permanently grazed (Williams, 1980) and ley systems (Coventry et al., 1985), a feature common to both situations is the association between the decline in production of subterranean clover and a lowering of soil pH (Osborne et al., 1978; Bromfield et al., 1983a,b; Haynes, 1983). Soil acidity, through the direct effect of low pH, Ca deficiency (Munns, 1978) and MO deficiency (Anderson and Spencer, 1950; Dilworth, 1974), can affect nodule function and consequently growth of subterranean clover. We have reported (Coventry et al., 1985) (i) large increases in the nodulation of subterranean clover where soil pH was raised by lime, with the plants growing in unlimed soil having fewer, but much larger nodules and (ii) an increase in R. trzfilii populations to satisfactory levels by sowing inoculated seed. Large increases of clover growth in response to lime were obtained in these experiments. The aim of the field and glasshouse experiments reported here was to determine if soil acidity had a direct effect on nitrogen fixation activity as measured by specific C,H, reduction activity. The decline in production ( Trifolium
subterraneum
MATERIALSAND METHODS Experiment composition
I: Effect of lime and MO on yield, plant and NJ-fixation in the jieid
The C2HZ reduction assay was used for comparison of N,-fixation activity for field grown subterranean clover during two growing seasons. A factorial design
experiment with four replicates was used; the treatments were three rates of lime (0, 0.5, 1.0 t ha-‘; 57% Ca, 5% Mg, neutralizing value 142 as % CaCOA) (13 kg P ha a’, two fertilizer additions and 13 kg P ha-’ plus 62g MO ha-‘). The clover was grown at the Lilliput site (pH 5.2; &lo cm depth, 1: 5 soil : water); details of the establishment, management and site characteristics have been described by Coventry et al. (1985). The soil is low in exchangeable Ca and Mg and high in exchangeable Al and Mn (Coventry et al., 1985). Subterranean clover was carefully transferred from the field, the soil removed and entire plants taken to the laboratory for analysis. The plants were placed in an 885 ml gas-tight jar with a sealed cap and serum stopper for addition and removal of gases and the jars were held at 27°C in a water bath for 25min. The assay was started by injection of C2H, (0.025 atm) and 100 ~1 gas samples were withdrawn periodically for 30 min and analysed by GC. The separation of C2Hz and C,H, was obtained in a 4 mm x 150 cm column of Porapak N run at 100°C with a gas flow of 30 ml min-’ using a Packard Model 437 GC. Ethylene production was linear throughout the assay. The clover plants from each treatment were sampled between 0900 and 1100 h to minimize variation in daily soil temperature, and the time between field sampling and C2Hz injection never exceeded 1.5 h. This restricted the number of samples taken each day and replicate treatment pilots were sampled on successive days. In the 1982 growing season three replicates were analysed at each of 7 harvests and in the 1983 season two replicates were sampled at each of 4 harvests. The clover germinated on 16 March in 1982 and the rainfall was 90.1 mm (cf. 220 mm, 66 yr average) and the mean air temperature 5.6”C (range l-13°C) during the 15 week assay. In 1983 the clover germinated on 30 March and the rainfall was 112 mm (cf. 52 mm, 66yr average) and the mean air temperature 11.5”C (616°C range) for the 5 week assay. 791
192
D. R.
COVENTRY
Plants were also harvested and soil temperature measured every 2 h for 18 h, once in each season, to determine if there were any diurnal patterns of C2H, reduction. Clover herbage dry matter and nodule number were determined from five 0.20m2 quadrats at the fifth harvest in 1982 and the fourth harvest in 1983. The herbage was analysed for N and MO concentration. Nodules collected from the third to fifth harvest in 1982 were also analysed for MO concentration. Experiment 2: Effect of lime and inoculation on yield and numbers of R. trifolii in the field
A factorial experiment was established in April 1982 at the Rutherglen site (pH 5.0; O-10 cm depth, 1: 5 soil:water; Coventry et al., 1985), where soil populations of R. trifolii were known to be very low. The treatments were rates of lime and seed inoculation with R. trifolii (see Table 3). Subterranean clover (cv. Woogenellup) was sown at 10 kg ha-’ with superphosphate supplying 13 kg P ha-‘. Plot size was 1.9 x 10.0 m and there were four replicates. Dry matter production of the establishing clover was determined from one 1.12 x 1.65 m quadrat per plot in October 1983. Soil samples (20 x 3 cm cores, O-8 cm depth composited from four replicates) were taken in June 1983 for estimation of populations of R. trifolii using the plant infection technique (Brockwell, 1963). Experiment 3: Effect of lime on yield, N content and N,-jixation in the glasshouse
Soil for a pot experiment was collected from the Lilliput site to a depth of 10 cm from unlimed plots that had previously grown three consecutive wheat crops. Lime and seed inoculation (see Table 4) were the treatments in a factorial design with six replicates. The lime (1.745 g kg-’ soil, analytical reagent grade CaCO,) was mixed with the soil before filling the pots. Undrained plastic pots (11 cm dia) were filled with 700 g of sieved (6 mm) air-dry soil, watered to field capacity (29x, w/w) and allowed to equilibrate for 10 days. P at 70 pg pot-’ was applied to the soil surface and thoroughly mixed with the soil in the pots prior to sowing. Ten imbibed subterranean clover seeds (cv. Woogenellup) were sown at 1.5 cm depth and a 1 ml dense suspension of R. trifolii was applied in the planting holes to the inoculum treatments. The moisture content of the soil was maintained between 50 and 60% of field capacity for the duration of the experiment. Air temperature was kept below 30°C. Seedlings were thinned to 6 per pot 10 days after emergence. Plants were harvested at 4 weeks and 6 weeks after emergence, the roots were washed and C2H2 reduction of intact plants measured. Nodule number, nodule weight and shoot dry weight were determined and shoot herbage was analysed for N concentration. Plant analyses
Total N was determined by the semi microKjeldah1 method of Bremner (1965) using an electronically controlled digestion block, and automated distillation and titration apparatus. MO was determined
et al.
by a modification of the method of Dick and Bingley (1951). Statistical analyses
Factorial analyses of variance were conducted on all data, and where treatment significance (P < 0.05) was attained, the Duncan Multiple Range Test was applied. RESULTS
Experiment 1: Efect of lime and MO on yield, plant composition and N,-jxation in the$eld
Growing conditions during the two seasons varied considerably. During the assay period in 1982 very dry conditions were experienced, whereas in 1983, the conditions were ideal for plant growth. The daily pattern of CIH2 reduction was linear and horizontal in both 1982 and 1983 despite the diurnal fluctuations in soil temperature (e.g. in 1983, 1Ocm soil temperature ranged from 4” to 9°C). The pattern of C,H, reduction of subterranean clover in 1982 is shown in Fig. 1. Nodule activity was significantly greater (P < 0.10) with lime addition at the sampling where C,H, reduction was first detected (9 weeks after germination) and at the samplings 17 and 19 weeks after germination. There were no differences in nodule activity from MO addition. The period of highest C,H, reduction occurred at the time of most rapid plant growth, 12-20 weeks after germination. There were no differences (P = 0.05) in nodule weight and nodule number between treatments and nodule development was restricted by the very dry soil conditions (Fig. 1, Table 1). Acetylene reduction declined after 19 weeks growth, corresponding with the cessation of growth and senescence of the clover. The pattern of C2H, reduction was measured for a shorter period of clover growth in 1983. There were no significant differences (P = 0.10) in C,H, reduction at any sampling time, however when C2H2 reduction was first detected, lime at nil, 0.5 and 1.0 t ha-’ with MO and lime at 1.0 t ha-’ without MO gave higher readings (P = 0.21) than lime at nil and 0.5 t ha-’ without MO. The clover with added MO was a much darker green colour, but this colour difference was not maintained for longer than 8 weeks after germination. The dry matter yield of clover in 1982 increased (P <: 0.05) with lime, but MO did not affect yield although N concentration was increased (Table 1 and 2). MO concentrations increased in both plant tops and nodules with MO fertilization; but the ratio of MO concentration in the nodules to tops declined. This ratio ranged from 13: 1 to 25: 1 in the absence of MO, and from 7: 1 to 11: 1 with added MO. In 1983 there were no significant differences (P = 0.05) in growth (Table l), although MO addition gave larger and darker green plants. The clover stand at this harvest was heavily infested with capeweed (Arctotheca calendula) which gave a very high coefficient of variation to the clover yield data (60%). There was a large increase in the N concentration (P < 0.01) and N content (kg ha-‘) of the clover with MO application (Table 2). Again the MO concentration in the plant tops was much higher with MO application.
Growth and nitrogen fixation of subterranean clover
10
15
20
Time
793
25
(weeks)
Fig. 1. Pattern of C, H, reduction activity and nodule development (0) from germination for field-grown subterranean clover, 1982.Key: No lime (0); 0.5 t ha-’ lime (II); 1.0 t ha-’ (A); solid line, 13 kg P ha-‘, dash line, 13 kg P ha-’ and 62 g MO ha-‘. Vertical bars indicate LSD (10%).
Table 1. Yield and nodulation of field grown subterranean clover (experiment 1) with lime and molybdenum treatments
Lime
MO
(t ha-‘)
(g ha-‘)
0 0 0.S 0.5 1.0
62 0 62 0 62
1.0
0
Nodule number plant-’ August 1982 June 1983
Dry weight (t ha-‘) August 1982 May 1983 1.22a
1.5
1.49 1.18 1.40 1.32 1.27 1.32
1.28a 1.48ab 1.35ab 1S7ab 1.66b
5.5 8.5 9.0 8.9 6.0
23 26 19 20 21 23
Means within a column followed by different letters differ (P < 0.05).
Table 2. N concentration
and content and MO concentration Herbage August 1982
Lime (t ha-‘) 0 0 0.5 0.5
i.0 1.0
MO (g ha-‘) 62 0 62 0 6i 0
(& 4.22 4.05 4.22 4.02 4.23 4.12
N content (kg ha-‘) 51 52 62 54 67 68
of subterranean
clover (experiment 1) Herbage May 1983
Nodules August 1982 _.___-_
~ MO (Pgg-‘) I .30b 0.23a 1.60b 0.25a I .7Oc 0.28a
MO (Icglr’) 13.7 5.6 12.6 3.2 11.5 6.0
(&
N content (kg ha-‘)
MO (Pgg-‘1
3.86b 3.55a 4.02b 3.53a 3.97b 3.85b
58 44 56 47 51 51
0.73b <0.20a 0.75b
Means within a column followed by different letters differ (P i 0.05).
experiment 2: Effect of Iime and inoc~iotio~ on yield and numbers of R. trifolii in the field Large significant differences in dry matter yield were obtained between all treatments in the subterranean clover regenerating in 1983, following a drought-affected establishment year (Table 3). Where clover was not inoculated and no lime was applied, R. trifolii were not detected (< 10 gg’ soil), but with both inoculated clover and limed soil the populations of R. trifoiii increased to detectable numbers (Table 3).
Experiment 3: Effect of lime on yield, N content and N,-fixation in the glasshouse The clover was poorly nodulated in the absence of seed inoculum and lime, and nodulation of the clover without inoculation was increased by lime (Table 4). After 4 weeks there was no effect of lime on nodule numbers in treatments sown with inoculated seed but by 6 weeks the nodulation was greater (P < 0.01) with lime application. The nodules formed on uninoculated plants grown in unlimed soil were larger and
D.
794
R. COVENTRY ef al.
Table 3. R. rrijooliipopulations and yield of field-grown subterranean clover (experiment 2) with inoculation (seed inoculated with commerical inoculum, Nodulaid. group C applied in a water slurry) and lime treatments
Lime (t ha -’’ f 0 0 2.5 2.5
Mean R. trifnlii population’ (log no. g-’ soil) June 1983
Treatment
ND 1.41 1.81 2.2 i
- Inoc I- Inoc -bloc +1noc
Dry weight (t ha” ‘) October 1983 2.26a 3.48ab 4sobc 5.87~
ND R. trijb& not detected (< IOg-’ soil). Dry weight means within a column followed by different letters differ (P i 0.05). ‘Absence of letters result of bulking replicates.
heavier (P < O.Ol), but the specific GH, reduction activity of these nodules, measured 4 weeks after ge~ination, was much less than for all other treatments (P < O.Ol), There were no differences in C,H, reduction of nodules on inoculated plants grown in the absence of lime, when compared with the limed treatments (Table 4). The uninoculated clover plants grown without lime were chlorotic, whereas the inoculated plants grown on the same unlimed soil did not develop these symptoms. At both harvests there was no significant difference in the dry matter yield of inoculated and uninoculated clover grown without lime (Table 5). Application of lime increased growth of the clover, with the uninoculated plants producing more dry matter than inoculated plants at the first harvest (P < 0.05); there was a similar, but non-significant, difference at the second harvest (Table 5). The N concentration of the clover increased with both lime addition and inoculation, with lime producing the biggest increase. At the second harvest, the effect of inoculation and lime on N ~ncentration was additive, however there was no such effect with N content, whose level in the presence of lime was unaltered by inoculation treatment.
DISCUSSION
The increase in growth and nitrogen content of subterranean clover obtained in the field with lime and MO, as well as growth responses with inoculation, came from increased N,-fixation. The lime may also have improved plant growth by modifying acid soil properties. In one season lime had a direct effect on the nitrogenase activity, as measured by C,H, reduction, at three of the seven sampling times through the season. Moly~en~ appli~tion resulted in increased N concentration in the herbage in both seasons, and an obvious leaflet colour response in 1983. However no significant increases (P = 0.10) were obtained in nodule C,H, reduction activity with the field-grown clover following MO addition. The MO concentrations in the herbage, without MO application, were low and were not increased by time, suggesting an absolute deficiency of MO in this soil. Liming acid soils usually corrects MO deficiency by increasing the availability of MO already present in the soil (Anderson and Moye, 1952). With the many reports of acidification of Australian soils (Haynes, 1983), as predicted by Donald and Williams (1954), the requirement of pastoral and Icy soils for MO application is now likely to be more extensive. The severe drought and low soil temperatures of 1982 limited both nodulation and N,-fixation in the field (Roughley and Dart, 1970; Gibson, 1971; Sprent, 1972). In 1983, the field conditions were more favourable for nodulation and N,-fixation but again there were no differences between treatments in nodule weight and number. Aluminium and H+ ion toxicities and Ca deficiency are known to restrict nodulation of legumes (Lowther and Loneragan, 1968; Munns, 1978; Carvalho et al., 1982); in the glasshouse experiment Iime application increased nodulation similarly to the field response reported previously with this soil (Coventry ef al., 1985). The subterranean clover grown in the glasshouse (experiment 3) without inoculation and lime produced few nodules and although there was a compen-
Table 4. Nodulation and NI-fixation activity (C,H, reduction) of subterranean clover grown in a glasshouse (excrement 3) with insulation and lime treatments Nodule weight Lime
(gpot ~‘) Treatment 0 0
1.222 1.222
+ +
Inoc lnoc Inoc Inoc
Nodules plant ’ 4 weeks 6 weeks
4 weeks
I .Oa 24.95b 23.92b 26.86b
2.36Ob 0.397a 0.428a 0.33%
7.14a 38.45b 43.86b 53.81c
0%)
Specific nodule activity (nmol C,H, reduced g ’ min ’ ) 4 weeks 232a 1058b 79Sb 948b
Means within a column followed by different letters differ (P i 0.05)
Table 5. Growth, Lime (gmJt--‘) 0
0 1.222 I.222
nitrogen concentration and nitrogen content of subterranean clover grown in a glasshouse (experiment 3) with inoculation and lime treatments Treatment - Inoc + Inoc -1noc +Inoc
Dry weight (gpot-‘) 4 weeks 6 weeks 0.313a 0.318a 0.478b 0.372a
0.613a 0.7 I 8a
1.402~ 1.168b
Herbage N (%) 6 weeks 4 weeks
1.86a 3.04b 3.65c 3.8lc
Means within a column followed by different letters differ (P < 0.05).
1.67a 2.3833 2.441, 2.8cic
N content (mg pot-‘) 6 weeks 4 weeks 5.814a 9.654b 17.532d i4.118c
10.380a 16.331b 33.97Oc 32.394c
Growth
and nitrogen fixation of subterranean clover
satory increase in individual nodule size, the CzHz reduction activity (g-’ tissue) was much less than for the inoculated and limed treatments, suggesting that the volume of bacteroid tissue per nodule was not proportionally increased. The plants with few nodules were chlorotic and typically N-deficient in appearance. With inoculation, the plants were well nodulated; the C2H, reduction activity of the nodules and the herbage N concentration were greater and the plant chlorosis was alleviated. The soils used in experiments 2 and 3 had very low initial populations of R. trifolii and in both experiments either seed inoculation or lime application were required to ensure R. trifolii build-up and adequate nodulation of the clover. The dry matter yield of the inoculated and unlimed plants in experiment 3 was no greater than the uninoculated and unlimed plants indicating that acid soil factors were limiting the growth of the plants on unlimed soil. With addition of lime there was no difference in C,H, reduction activity and nodule size at 4 weeks growth compared with the unlimed and inoculated plants, indicating that the lime effect is not on nodule function. This result differs from reports of direct effects of lime and Ca on the specific nodule activity of subterranean clover (Banath et al., 1966; Munns, 1978). Where lime was applied, nodulation was more rapid, which would account for the increase in N concentration and content of these plants, and for part of the growth difference. The dry matter yield of the clover with inoculation and lime was less at 4 weeks than with the uninoculated plants with lime, but at 6 weeks the yield differences were not as obvious. This contrasted with the field-grown clover (experiment 2) where the yield was greater with inoculation and lime. The reason for the slower initial growth of the limed, inoculated, plants is not obvious, as at 6 weeks these plants had more nodules and higher herbage N concentrations. The large increase in growth of these lime treatment plants at 6 weeks, compared with the inoculated and unlimed treatment plants, may also have resulted from either relief from soil properties limiting plant growth or a direct effect of lime on plant growth, rather than a limitation on N,-fixation (Robson and Loneragan, 1977). A similar result was obtained with the field grown clover in experiment 2, where lime was required to produce the best dry matter yield. Acid soil infertility can be caused by toxicities of Al, Mn and H or deficiencies of Ca, Mg and MO. Many soils in southeastern Australia have become more acid due to shifts in land-use to dryland farming systems based on subterranean clover pastures, and now have high concentrations of both exchangeable Al and Mn (Williams, 1980; Bromfield et al., 1983a). The area of improved pasture affected by acidity is likely to increase in the future (Williams, 1980) and farmers will have to consider corrective measures for restoring pasture productivity. It is therefore important that the various components of the legume-Rhizobium association, and of the nutrition of legume affected by increasing acidity, be identified as this will influence the remedial treatment options. The soils associated with this study, which had previously not required fertilizer MO for subterranean clover productivity, have now been shown to
795
require MO fertilization. It has also been established that soil populations of R. trtfolii are very low following cropping in a clover-ley rotation, which frequently results in poor nodulation of subterranean clover. The R. trtyolii populations can be quickly re-established to adequate levels by either seed inoculation or lime application when the clover is being re-established. However Bromfield et al. (1983b) have warned that nodulation problems may develop in permanent annual pastures as a result of relatively small further increases in soil acidity. Soil acidity also is having an effect on the growth of subterranean clover separate from effects on the N,-fixing association which has been overcome by liming the soil. It is likely that surface-applied lime will be used extensively on ley-rotation acid soils where crop yields are decreased because of the decreased soil pH. However liming soils for renovation of permanent pastures often is not feasible as the existing pasture has to be disrupted for incorporating the lime and then re-established at considerable cost. There is a need for the development of management systems on permanently grazed pastures which reduce the activity of the toxic soil components, and maintain soil biological activity. Acknowledgements-We wish to acknowledge the assistance of Mr E. G. Baker and Sons, Lilliput and Messrs W. R. and R. W. Francis, Rutherlgen, on whose properties the experiments were conducted. The Rhizobium analyses were done by Mr H. R. Jones, Department of Agriculture, Bendigo and some chemical analyses by staff of the State Chemistry Laboratory, Melbourne. The assistance of G. R. Morrison, S. A. Shaw, D. K. Jarvis, J. M. Roberts and B. C. Allen is also acknowledged. This work was/ supported by a grant from the Wool Research Trust Fund on the recommendation of the Australian Wool Corporation.
REFERENCES
Anderson A. J. (1956) Molybdenum as a fertilizer. AdL1ance.s in Agronomy 8, 163-202. Anderson A. J. and Moye D. V. (1952) Lime and molybdenum in clover development on acid soils. Australian Journal of Agricultural Research 3, 95-l 10. Anderson A. J. and Spencer D. (1950) Molybdenum in nitrogen metabolism of legumes and non-legumes. Australian Journal of Scientific Research, Series B 3,414430. Banath C. L., Greenwood E. A. N. and Loneragan J. F. (1966) Effect of calcium deficiency on symbiotic nitrogen fixation. Plant Physiology 42, 760-763. Bremner J. M. (1965) Total nitrogen. In Methods of Soil Analysis Part 2, (C. A. Black, Ed.), pp. 1149-1178. American Society of Agronomy, Madison. Brockwell J. (1963) Accuracy of a plant-infection technique for counting populations of Rhizobium trifolii. Applied Microbiology 11, 377-383. Bromfield S. M., Cumming R. W., David D. J. and Williams C. H. (1983a) Change in soil pH, manganese and aluminium under subterranean clover pasture. Australian Journal of Experimental Agriculture and Animal Husbandry 23, 181-191. Bromfield S. M., Cumming R. W., David D. J. and Williams C. H. (1983b) The assessment of available manganese and aluminium status in acid soils under subterranean clover pastures of various ages. Australian Journal of Experimental Agriculture and Animal Husbandry 23, 192-200. Carter E. D., Wolfe E. C. and Francis C. M. (1982)
796
D. R. COVENTRY ef al.
Problems of maintaining pastures in the cereal-livestock areas of Southern Australia. In Proceedings of the Second Australian Agronomy Conference, Wagga Wagga, (M. J. T. Norman, Ed.), pp. 68-87. The Australian Society of Agronomy, Victoria. Carvalho M. M. de, Edwards D. G., Asher C. J. and Andrew C. S. (1982) Effects of aiuminium on n~ulation of two Stylosanthes sp. grown in nutrient solution. Plant and Soil 64, 141-152. Coventrv D. R.. Hirth J. R.. Reeves T. G. and Jones H. R. (198.5) Development of p&pulations of Rhizobium trifblii and nodulation of subte~~~n clover following the cropping phase in crop-pasture rotations in southeastern Australia. Soil Biology & Biochemistry 17, 17-22. Dick A. T. and Bingley J. B. (195 I) Further observations on the determination of molybdenum in plant and animal tissue. Australian Journal of Experimental Biology and Medical Science 29, 459462. Dilworth M. J. (1974) Dinitrogen fixation. Annual Review of Plant Physiology 25, 81-114. Donald C. M. and Wjlliams C. H. (1954) Fertility and pr~uctivity of a podzolic soil as inlluenced by subterranean clover (Trt,foIium ~bterr~e~ L.) and superphosphate. Austrahan Journal of Agricultural Research 5, 664-687. Gibson A. H. (1971) Factors in the physical and biological enviro~~t affecting nodulation and nitrogen fixation by legumes. Plant and Soif Special Volume 1971,139-152. Haynes R. J. (1983) Soil acidification induced by leguminous crops. Grass and Forage Science 38, l-l I.
Lowther W. L. and Loneragan J. F. (1968) Calcium and nodulation in subterranean clover (Trifolium subterraneum L.). Plant Physiology 43, 1362-1366. Munns D. N. (1978) Soil acidity and nodulation. In h4inerul Nutrition of Legumes in Tropical and Subtropical Soils (C. S. Andrew and E. J. Kamprath, Eds), pp. 247-263, C.S.I.R.O., Australia. Osborne G. J., Wright W. A. and Sykes J. (1978) Increasing soil acidity threatens farming systems. Agrieuburai Gazette. New South Wales 89. 21. Reeves’ T. G., Haines P. J. ‘and Coventry D. R. (1984) Growth of wheat and subte~ane~ clover on soil artificially compacted at various depths. Plant and Soil 80, 135-138. Robson A. D. and Loneragan J. F. (1977) Responses of pasture plants to soil chemical factors other than nitrogen and phosphorus, with particular emphasis on the legume symbiosis. In Plant Relations in Pastures (J. R. Wilson, Ed.). C.S.I.R.O., Australia. Roughley R. J. and Dart P. J. (1970) Growth of TrijXium s~terr~e~ L. selected for sparse and abundant nodulation as affected by root temperature and Rhizobium strain. Journal of Experiments Botany 21, 776-786. Sprent J. I. (1972) The effects of water stress on nitrogenfixing root nodules. IV EKects on whole plants. Neut Phytologist 71, 603-611. Williams C. H. (1980) Soil acidification under clover pasture. ~us~ra~ia~ Journal of Experimental Agri~lture and Animal husbandry 20, 56 I-567.