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Nodulation and growth of subterranean clover (Trifolium subterraneum L.) in a drying acid soil
Soil Bid.
Biochem. Vol. ?I. NO. I.pp.l-8, 1989 Britain. All rights rewwd
Copyright 0
F'rin~cd in Great
0038-0717189 53.00 + 0.00 1989 Pergamon Prcsa pk
NODULATION AND GROWTH OF SUBTERRANEAN CLOVER (TRIFOLIUM SUBTERRANEUM L.) IN A DRYING ACID SOIL A. G. DAVEY,A. P. HENDERSONand R. J. SIMPSON Plant and Soil Sciences Section, School of Agriculture and Forestry. The University of Melbourne, Parkville 3052, Australia (Accepted 27 June 1988)
Summary-Subterranean clover (cv. Mt Barker) was grown in undisturbed acid soil (pH 4.3). in cultivated acid soil (PH 4.3) and in time-amended (pH 5.5) soil and after I2 weeks of growth was either watered to field capacity or basally irrigated with the equivalent of half the amount of water transpired by the control plants. Basally-irrigated plants were able to exploit subsoil moisture and maintain adequate plant-water relations whilst the soil at the top of the soil profile dried slowly. Most nodules were confined to the less acidic uppermost and basal regions of the undisturbed acid soil profile and no new nodules were produced on the root system once basal irrigation commenced. The permanent wilting point of the compact, undisturbed acid soil (- I .5 MPa) corresponded with a soil moisture content of 7% w/w and was attained 3 weeks after commencement of basal irrigation. Nitrogen accumulation by the subterranean clover plants grown in this treatment ceased when the top 9 cm of the acid soil profile reached the permanent wilting point. The permanent wilting point of the top 9 cm of the limed and unlimed cultivated soil occurred at a soil moisture content of 5% w/w and was reached 5 weeks after commencement of basal irrigation. Nodules occurred on roots at depth in the limed and unlimed cultivated soil and modulation continued after commencement of basal irrigation compensating for lower nodule numbers on the upper crown roots that were in contact with the drying soil. Nitrogen continued to be accumulated by these plants throughout the experiment. Nodule size varied in accordance with soil cultivation treatment. In particular, larger nodules were produced on the roots of plants grown in undisturbed acid soil whereas smaller, more numerous nodules occurred on the roots of plants grown in lime-amended soil and to a lesser extent on the roots of plants grown in cultivated acid soil. However, total nodule mass was equivalent on plants grown in each of the soil treatments. Continued nodulation at depth on the roots of subterranean clover due to cultivation and incorporation of lime enabled nitrogen fixation to continue when nodules on roots near the soil surface became desiccated and senescent due to the localized moisture deficit.
tNTRODtiCTlON Acidification of soil beneath legume-based pastures is recognized as a widespread problem in south-eastern Australia (Donald and Williams. 1954; Helyar, 1976). For example, a decline in soil pH has occurred at a rate of one pH unit over a SO-yr period in southern New South Wales (Williams, 1980; Bromfield ef al., 1983a). However, high concentrations of hydrogen ions are not distributed uniformly within soil profiles. Often the most extreme soil acidity occurs in the root zone (0-20cm) of the A horizon (Bromtield ef nl., 1983b). In other instances, severe subsoil acidity is also reported (Pinkerton and Simpson, 1986a). Low soil pH may result in toxic concentrations of aluminium, manganese and hydrogen ions as well as deficiencies of calcium, magnesium and molybdenum in agricultural soils (Munns, 1953; Bromfield er ol., 1983a; Coventry er al., 1985a, b). Aluminium toxicity in particular can inhibit root growth and proliferation (Foy, 1984) and has the potential to restrict the ability of plants to fully exploit soil moisture (Bromfield et al., 1983b) and to reduce uptake of nutrients. Root length of species such as lucerne and wheat which are sensitive to acidity can be significantly reduced in very acid soil (Pinkerton and Simpson, 1986a) whilst those of tolerant species such s.0n ?I!,-*
as subterranean
clover may exhibit only slightly restricted root growth when growing under similar conditions (Munns, 1965; Pinkerton and Simpson, 1986b). Nevertheless, Kehoe and Cumow (1963) have reported wilting and premature senescence of subterranean clover growing in acid sand at Marlo in East Gippsland, Victoria. This was considered to be due to restricted development of roots in the acid sand which prevented the plants from utilizing subsoil moisture as the soil profile dried at the end of the growing season. In a drying soil profile, the growth of roots into zones of extreme soil acidity and their distribution relative to nutrients and soil moisture can also have critical effects on plant growth. Soil drying from the top can restrict the availability of nutrients such as phosphorus which are concentrated predominantly in the upper layers of soil profiles (Pinkerton and Simpson, 1986b). Nodulation of legume roots may also be severely restricted in very acid soils (see reviews by Munns, 1978; Dear et al., 1987). Poor productivity of pastures has occasionally been attributed to total nodulation failure in acid soil (Coventry er al., 1985a). Restricted nodulation of legume roots has also been reported (Vincent and Waters, 1954). Even in circumstances
A.
2
G. DAVEY et al.
where roots appear to be adequately nodulated, nodulation patterns can be influenced by localized acidity within a soil profile. Richardson er al. (1988) have shown that up to 86% of root nodules on subterranean clover occurred in the top 4cm of an acid soil profile. Restricted distribution of nodules was associated with the effects of extreme acidity on the growth and distribution of Rhicobium trifofii. In this soil, Rhkobium was essentially confined to the uppermost region of the soil profile which was less acidic and high in organic matter. A larger population of R. trifolii and improved nodulation at depth occurred when lime was incorporated (Richardson and Simpson, 1988). Addition of lime supplies calcium, raises soil pH and reduces aluminium concentrations, all of which also clearly affect the infection of legume roots (Anderson and Moye, 1952; Loneragan and Dowling. 1958; Munns, 1978; Wood and Cooper, 1984). As soils under clover-based pastures dry during periods of short-term drought or toward the end of the growing season, it is likely that nodules restricted to roots near the soil surface by a very acid soil would become desiccated and senescent and may lose their capacity to fix atmospheric nitrogen. This paper outlines experiments in which nodulation, plant growth and nitrogen accumulation was measured on established plants of subterranean clover growing in a drying acid soil profile.
iMATERIALS AND IMETHODS
Cores of an acid soil (9 cm dia x 30 cm depth) were removed intact in PVC sleeves from a subterranean clover-based pasture immediately prior to the commencement of the growing season in May 1986 at “Strathfieldsaye”, via Stratford (38’3’S, 147’16’E) in East Gippsland, Victoria. The duplex soil consisted of an acid loamy sand A horizon with a clay subsoil occurring at 18-20cm depth (principle profile form Dy 4.53; Northcote, 1974). The soil from which the cores were removed had been topdressed annually since 1981 with superphosphate (150 kg ha-‘) which included molybdenum (37.5 g ha-‘) in 1981 and 1984. Three cultivation treatments were imposed on the cores of soil: (i) undisturbed acid soil (control) in which soil pH measured in IO mM CaCl, (1:5 w/v) was pH 4.3; (ii) cultivated acid soil (pH 4.3) in which the top I2 cm of soil was removed, mixed and replaced into the PVC sleeves; and (iii) cultivated soil to which agricultural lime (Calcimo lime, Longford, Victoria, Ca present as 81% CaCO,) was added and mixed at a rate of 5 t ha-’ (based on the surface area of soil in the PVC sleeves). The depth to which lime was incorporated was I2 cm and the pH value of the soil in this region was raised to pH 5.5. The soil pH profile existing beneath undisturbed pasture exhibited relatively moderate acidity in the top few cm (pH 4.5) and wtreme soil acidity (as low as pH 3.8) at depths between 4-l3cm. Below 18cm depth, soil pH increased to levels similar to those occurring at the soil surface. Consequently, levels of exchangeable aluminium in the top 4 cm and below 18 cm down the soil profile ranged between 22 and 32 pg g-i soil and were
lower than levels within the most acid region of the soil profile (44-54pg g-i soil) (Richardson and Simpson, 1988). The level of exchangeable aluminium was decreased substantially after incorporation of lime. Plastic mesh was placed over the base of each PVC sleeve to prevent soil loss and to allow adequate drainage. The soil cores were watered to field capacity over a 6 week period and when necessary, weeds were removed. Soil cores were then sown with seeds of subterranean clover (cv. Mt Barker) inoculated with Rhkobium trifolii (WU95). Seedlings were thinned to one plant per soil core and plants were grown under natural irradiance (June-October) in a glasshouse at the University of Melbourne. Supplementary lighting (incandescent lamps) was used to extend the photoperiod to 14 h. Temperatures were maintained between 25’C (day) and I2C (night). Alkathene plastic beads (ICI Industries, Melbourne) were placed over the soil surface of each core to reduce evaporation. The cores were arranged in a completely randomized design and were watered regularly to field capacity. Flowering commenced on 27 October 1986. After IO weeks of growth, the plants were transferred to a controlled environment plant growth room in which temperatures were maintained at 24’C (day); l8’C (night) and the photoperiod was I4 h. Relative humidity was 70% and the photon flux density (400-700 nm) at plant height was 550 pmol rn-? s-i. After 7 days, half of the soil cores from each of the 3 cultivation treatments were placed in plastic saucers and irrigated from the base only with half the amount of water transpired by the control plants growing in soil cores watered to field capacity. Transpiration by control plants was measured gravimetrically each day. The six treatments (3 cultivation x 2 irrigation treatments) were arranged in a completely randomized design. Five replicate plants per treatment were harvested at weekly intervals for 6 weeks. Plants were harvested 6 h after commencement of the photoperiod. Plants were divided into shoots, roots and nodules. For most purposes, roots and nodules in the top 9cm and remaining 9-30cm regions of the soil profile were of interest. However, a more detailed measurement of root and nodule distribution was also conducted by removing the soil cores from their plastic sleeves and cutting them into regions of O-4, 4-9, 9-13, 13-18 and 18-30 cm from the soil surface. These regions were based on changes in soil pH and the consequent distribution of nodules in the soil profile that had been observed at the field site (Richardson and Simpson, 1988; Richardson et al., 1988). Roots were gently washed from the soil of each profile and all nodules were removed and counted. Roots and nodules from individual profile regions of each replicate were dried separately. Dry weight of plant parts was determined after drying at 70°C for 24 h. Total plant nitrogen content was determined by the Kjeldahl method using Se as the catalyst (Bremner and Mulvaney, 1982). A subsample of soil from each region of the soil core was weighed for gravimetric determination of soil moisture content after drying for 48 h at 80°C. Soil matric potential of the compact undisturbed soil and of the less compact cultivated soil treatments was also measured, using a pressure plate apparatus (Richards, 1965).
Nodulation of clover in a drying acid soil RESULTS
Tim
3
after commencement of sums
(days)
Soil matric potential
The effect of basal irrigation with 50% of the amount of water transpired by control plants was to slowly dry the soil profile from the top. The rate at which soil in the O-9 and 9-30 cm regions of basallyirrigated cores of soil dried to the permanent wilting point (- 1.5 MPa) is presented in Fig. I. The permanent wilting point of the limed and unlimed cultivated soil occurred at a soil moisture content of 5% (w/v) whereas the permanent wilting point of the more compact undisturbed acid soil occurred at a soil moisture content of 7% (w/v) due presumably to the effect of the higher bulk density of the undisturbed soil (data not shown). Within the top 9cm of the drying soil the permanent wilting point was attained after 21 and 30-36 days from the commencement of the moisture deficit for the undisturbed and cultivated soils, respectively (Fig. 1). Nodulation of subterranean clover roots
Numbers of nodules on roots of subterranean clover plants were variable within each treatment. Generally, however, numbers of nodules on plants grown in undisturbed and in cultivated acid soil either declined throughout the experimental period or were less numerous than the number of nodules produced on plants grown in lime-amended soil (data 0-9 cm
E.rjF
-2.0 L
Fig. 1. Soil matric potential in the O-9 cm (closed symbols) and 9-3Ocm (open symbols) regions of the soil profile for
the 3 cultivation treatments after the commencement of basal irrigation. Key: (0, 0) undisturbed acid soil; (m. 0) cultivated acid soil; (A. A) cultivated and limed soil. Bar represents the LSD (P < 0.05).
not shown). Irrespective of the variability in nodule numbers, total nodule mass (dry weight) per plant did not vary significantly and ranged between 40-70, 40-65 and 30-6Omg dry wt per plant for plants grown in undisturbed, cultivated and cultivated and lime-amended soils, respectively (Fig. 2). Basal irrigation did not significantly reduce total nodule dry weight per plant despite drying of the uppermost
Undisturbed acid soil
Cultivated and limed soil
Fig. 2. Dry weight per nodule (a, 0) and total nodule dry weight (a, 0) of well-watered (closed symbols) and basally-irrigated plants (open symbols) growing in (a and b) undisturbed acid soil, (c and d) cultivated acid soil and (e and f) in soil cultivated with the incorporation of lime. Each point is the mean of 5 replicates. Bars represent the LSD (P < 0.05).
4
A. G. DAMY et al.
regions of soil profiles during the experiment. Within the top 9 cm of the soil profile, individual nodule dry weight was affected by soil treatment. For example, at the fourth harvest, mean individuat nodule dry weights within undisturbed, cultivated and limeamended soils were 0.59, 0.37 and 0.26 mg nodule-‘, respectively, for the well-watered plants and 0.55, 0.35 and 0.25 mg nodule-‘, respectively, for the basally-irrigated plants (Fig. 2). The large nodules which were observed on plants in undisturbed acid soil occurred mostly on the primary root whereas limed and unlimed cultivated soil resulted in more nodules on the lateral roots within the top 9 cm of soil profiles. Smaller nodules were observed on roots in undisturbed acid soil at 9-30 cm depth [Fig. 2(b)] in comparison with those in the uppermost region of that soil profile [Fig. 2(a)]. The size of nodules at depth in the undisturbed acid soil that was drying from the top was also further reduced [Fig. 2(b)]. In contrast, mean dry weight of nodules in both upper (0-9cm) and lower (9-30cm) regions of the soil profile within cultivated [Fig. 2(d)] and lime-amended soil (Fig. 2(f)] did not differ. In undistur~d acid soil, nodule size increased during the first 3 weeks of the experiment. However, the mean size of nodules produced on plants grown in both limed and unfimed cultivated soil either remained relatively unchanged or increased in size to a lesser extent (Fig. 2). Five weeks after imposition of the moisture deficit,
nodulation of roots within O-4.4-9, 9-13, 13-18 and 18-30 cm regions of all soil profiles was analysed. Highest numbers of nodules occurred within the top 4cm of the soil profile on the roots of plants grown in well-watered, lime-amended soil [Fig. 3(a)]. Generally, nodulation of roots of plants grown in wellwatered, undisturbed acid soil was poorer than on plants grown in limed and unlimed cultivated soil [Fig. 3(a)]. More nodules occurred on roots at the base of the well-watered, undisturbed soil cores (18-30 cm). However, total nodule mass in the uppermost region of the undisturbed acid soil profile was equivalent to that in lime-amended soil due to the larger size of crown nodules in undisturbed acid soil [Fig. 3(c)]. Nodulation within basally-irrigated soil profiles was similar to that observed on roots of well-watered plants except that in limed soil nodulation was poorer in the drier top 9cm of the soil profile. Plants grown in limed and unlimed cultivated soil also exhibited significantly higher nodule mass in the more moist 9-13 cm region of the soil profile [Fig. 3(b)]. This was due largely to increasing size rather than number of nodules as the experiment progressed [Figs 2(d), (f) and 3(d)]. Poor root growth occurred at depths below 9cm on plants in the well-watered. u~distur~d acid soil. However, in the top 4 cm. root mass was greater than in most other treatments [Fig. 4(a)]. The extra root growth in the upper regions of undisturbed acid soil
Numberof ncdulcsplant-’ 0
so
loo
150 200 250 0
50
loo
1
1.50 200 2.50
t
a
e
f
(b)
Tota1noduledxyweight(mg plant-’) 0
10
20
30
40
50 60
Well-watered
0
10
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30 40
50 60
f)asaIly-irrigated
Fig. 3 (a and b) Number of nodules per plant and (c and d) total nodule dry weight within the soil profile of well-watered and basally-irrigated plants 5 weeks after commencement of the moisture deficit. Key: (0) undistur~d acid soil; (0) cultivated acid soil; (A) cultivated and limed soil. Each point is the mean of 5 replicates. Bars represent the LSD (P ~0.05).
Nodulation of clover in a drying acid soil Root dry weight (g plant” ) 0
0.5
1.0
1.5
1
0
0.5
1.0
1.5
I
(b) WCll-Watered
Badly-inigatcd
Fig. 4. Root dry weight within the soil profile of (a) well-watered and (b) basally-irrigated plants 5 weeks after commencement of the moisture deficit. Key: (a) undisturbed acid soil; (0) cultivated acid soil; (A) cultivated and limed soil. Each point is the mean of 5 replicates. Bars represent the LSD (P ~0.05). did not occur in the drying soil profile of basally-
irrigated cores (P < 0.05) [Fig. 4(b)]. Root dry weight of plants grown in basally-irrigated soil was generally similar for all treatments although root growth was marginally better at depth in treatments with cultivated soil due presumably to the lower bulk density of the cultivated soil. Total p/am nhogen
and dry matter accumulation
Plants grown in well-watered and basally-irrigated undisturbed acid soil accumulated nitrogen until days 25 and 22 from commencement of the moisture deficit, respectively, after which no further increase in total nitrogen content was measured [Fig. S(a)]. The rate of accumulation of nitrogen by basally-irrigated plants in undisturbed acid soil was less than for well-watered plants and led to a significant difference (P < 0.05) in the total nitrogen content of plants in each treatment at later harvests. In contrast, plants grown in soil cultivated with and without lime showed no significant difference in accumulation of total nitrogen between well-watered and basallyirrigated plants [Fig. 5(c) and (e)]. As well, total plant nitrogen content within these two treatments increased throughout the entire experiment. Despite the difference in total nitrogen content between basallyirrigated and well-watered plants grown in the undisturbed acid soil the accumulation of plant dry matter was less affected by basal irrigation and the small difference in accumulated dry matter was not significant (P c 0.05) [Fig. 5(b), (d) and (f)]. DISCL’SSION
Distribution of nodule and root dry weight in drying acid soil profiles
Localized extreme acidity at depths between 4 and 13 cm in the undisturbed acid soil profile resulted in more prolific nodulation of roots of subterranean clover at the top (-cm) and to a lesser extent the base (IS-30 cm) of that soil profile (Fig. 3). The soil pH of the less acidic regions was still low enough to restrict nodulation, as incorporation of lime (CaCO,) but not gypsum (CaSO,) increased nodulation of roots (Fig. 3; Richardson and Simpson, 1988). However, larger nodules were produced on roots grown in
the very acid undisturbed soil and total nodule mass per plant was therefore not different (Fig. 2). The effectiveness of this mass of nodule tissue is doubtful, however, as specific nitrogenase activity of large nodules is reported to be low in acid soil (Coventry et al., 1985b). Large multi-lobed indeterminate nodules such as occur on clover in very acid soils generally contain high proportions of senescent nodule tissue (Sprent, 1976) which no doubt reduces the rate of nodule specific nitrogenase activity. In the present study, plant-extractable water was depleted more quickly in the upper region of the more compact, undisturbed acid soil (Fig. I) which reached the permanent wilting point at a soil moisture content of 7% as opposed to limed and unlimed cultivated soil which reached the permanent wilting point at a soil moisture content of 5%. Drying topsoil clearly reduced further nodulation of roots in the uppermost region of the lime-amended soil and also to a lesser extent in the cultivated acid soil. Nodulation at the top of the undisturbed acid soil protiles was not affected (Fig. 3) because maximum nodule numbers had been reached prior to commencement of the moisture deficit. Total nodule mass in the undisturbed acid soil was consequently also unaffected by the drying soil. Plants grown in drying, cultivated soils with and without lime, compensated for poorer nodulation at the top of the profile by allowing nodules to increase in size and in number at depths below 9cm (Figs 2 and 3). Soil acidity affected nodulation at depth in the cultivated acid soil. However, the combination of increased nodule size at depth and improved nodulation below 13 cm (Fig. 3) was sufficient to compensate for restricted nodulation in the dry topsoil. Plants grown in limed soil compensated for restricted nodulation in the dry topsoil by increasing nodule numbers at depths between 4-18 cm (Fig. 3) where soil pH had risen from pH 3.8 to between pH 4.3 and 5.5 (Richardson and Simpson, 1988). Nitrogen fixation by these nodules supplemented the nitrogen economy of the plants in the absence of nodule activity elsewhere on the root system allowing continued accumulation of nitrogen by the subterranean clover plants (Fig. 5). It is assumed that burial of Rhkobium inoculum in organic matter from the soil surface during cultivation
A. G. DAMYet al.
Cultivatedacid soil 8
; 6
Cultivated and limed soil
The after
cotntncnccment of sums (days)
Fig. 5. Total nitrogen content of the whole plant (0, 0) and dry matter production of shoots (W, 0) and roots (A, A) of well-watered (closed symbols) and basally-irrigated plants (open symbols) growing in (a and b) undisturbed acid soil, (c and d) cultivated acid soil, and (e and f) soil cultivated with the incorporation of lime. Each point is the mean of 5 replicates. Bars represent the LSD (P c 0.05).
combined with leaching of Rhizobium under wellwatered conditions prior to the commencement of basal irrigation may have enabled some continuation of nodulation at depths below I3 cm (pH 4.M.8) in the cultivated acid soil whereas no further nodulation at depth occurred in the undisturbed acid soil. Subterranean clover is considered to be tolerant of soil acidity (Munns, 1978; Pinkerton and Simpson, 1986b) and no large effects of acidity on root growth were observed (Fig. 4). However, root growth was restricted at depth in the undisturbed acid soil but plants in this soil tended to have a greater mass of roots in the surface layers of the soil. In all treatments, root growth near the surface was reduced as the soil dried. Drying of the uppermost region of the soil profile also caused nodule numbers to decline by 90 and 50% in soil cultivated with and without the incorporation of lime, respectively. Thus, senescence and abscission of nodules may have been increased under these conditions but it is also likely that formation of new nodules was inhibited. Worrall and
Roughley (1976) have shown that the number of infection threads was reduced and that nodulation by R. trifofii was consequently inhibited in subterranean clover growing under moisture stress even when the numbers of Rhizobium in the rhizosphere were unaffected by the dry soil conditions. Additionally, some decline in the population of Rhizobhm due to desiccation might be expected (Vincent er al., 1962; Bromfield et al., 1983b). However, it has also been suggested by Pinkerton and Simpson (1986b) that suppression of nodulation and thus nitrogen fixation by subterranean clover in an acid soil may be due to effects of moisture stress on availability of phosphorus in the upper region of the soil profile. Nutrition of subterranean clover in an acid soil
Nitrogen was accumulated by the plants ranean clover grown in limed and unlimed soil throughout the experiment whereas lation of N by plants grown in undisturbed ceased prematurely approx. 20 days after
of subtercultivated accumuacid soil the mois-
7
Nodulation of clover in a drying acid soil ture deficit commenced (Fig. 5). This was associated with attainment of a maximum nodule mass throughout the soil profile and lack of further nodulation of roots either near the soil surface or at depth. Assessment of nodulation at the site from which the soil cores were obtained has also revealed that the roots of subterranean clover harvested from undisturbed acid pasture soil were poorly nodulated late in the growing season (Richardson et al., 1988). Drying of the soil profile further decreased nitrogen fixation (Davey and Simpson, 1989) and N accumulation was slower from 5 to 10 days after the experiment commenced (Fig. 5). The size of nodules in the uppermost region of the drying undisturbed soil did increase. Clearly, however, the symbiotic effectiveness of these nodules was reduced. In other soil treatments where compensatory nodulation of roots occurred at depth, N accumulation by the subterranean clover plants was not significantly inhibited by drying of the upper regions of the soil profile. Continued accumulation of dry matter reflected the ability of the subterranean clover to accumulate N in both well-watered and in drying soil. Nevertheless, yield differences were not significant (P c 0.05) presumably because some growth was still possible in N-deprived plants due to redistribution of N as plants senesced. Premature cessation of nitrogen accumulation by subterranean clover grown in drying undisturbed acid soil is also consistent with data of Engin and Sprent (1973) and DeJong and Phillips (1982) who have suggested that under moisture deficits, decreases in nitrogen fixation are greater than corresponding decreases in shoot dry matter production. In our study, reduced nitrogen fixation was due to a combination of poor nodulation in the most acid region of the soil profile, senescence and decay of large and relatively old nodules found on roots in the extremely acid soil and to desiccation of nodules in the drying soil. Acknonle~~emenrs-Marylyn Williams is thanked for her technical assistance. This work was supported by a grant to R.J.S. from the Wool Research Trust Fund on the recommendation of the Australian Wool Corporation and was carried out whilst A.G.D. was the recipient of an Australian Wool Corporation Postgraduate Scholarship.
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Richardson A. E., Henderson A. P., James G. S. and Simpson R. J. (1988) Consequences of soil acidity and the effect of lime on the nodulation of Triforirrm subterraneum L. growing in an acid soil. Soil Biology % Biochemistry 20, 439-445.
Sprent J. 1. (1976) Water deficits and nitrogen-Axing root nodules. In Warer Deficits and FYant Growth (T. T. Kozlowski, Ed.). Vol. 4. pp. 291-315. Academic Press, New York. Vincent J. M. and Waters L. M. (19.54) The root-nodule bacteria as factors in clover establishment in the red
basaltic soils of the Lismore district, New South Wales. Australian Journal
of Agricultural Research
5, 61-77.
Vincent J. M., Thompson J. A. and Donovan K. 0. (1962) Death of root-nodule bacteria on drying. Ausrrafian Journal of Agricultural Research 13, 258-270.
Williams C. H. (1980) Soii acidification under clover pasture. Australian Journal of Experimental Agricuhure and Animal Husbandry 20, 561-567.
Wood M. and Cooper J. E. (1984) Aluminium toxicity and multiplication of Rhkobium trifolii in a defined growth medium. Soil Bioiogy & Biochhmisrry 16, 571-576. Worrall V. S. and Rouahlev. R. J. (1976) The effect of moisture stress on in&ion of T&&n subterraneum L. by Rhkobium rri/alii Dang. Journal of Experimental Botany 27, 1233-1241.
Biochem. Vol. ?I. NO. I.pp.l-8, 1989 Britain. All rights rewwd
Copyright 0
F'rin~cd in Great
0038-0717189 53.00 + 0.00 1989 Pergamon Prcsa pk
NODULATION AND GROWTH OF SUBTERRANEAN CLOVER (TRIFOLIUM SUBTERRANEUM L.) IN A DRYING ACID SOIL A. G. DAVEY,A. P. HENDERSONand R. J. SIMPSON Plant and Soil Sciences Section, School of Agriculture and Forestry. The University of Melbourne, Parkville 3052, Australia (Accepted 27 June 1988)
Summary-Subterranean clover (cv. Mt Barker) was grown in undisturbed acid soil (pH 4.3). in cultivated acid soil (PH 4.3) and in time-amended (pH 5.5) soil and after I2 weeks of growth was either watered to field capacity or basally irrigated with the equivalent of half the amount of water transpired by the control plants. Basally-irrigated plants were able to exploit subsoil moisture and maintain adequate plant-water relations whilst the soil at the top of the soil profile dried slowly. Most nodules were confined to the less acidic uppermost and basal regions of the undisturbed acid soil profile and no new nodules were produced on the root system once basal irrigation commenced. The permanent wilting point of the compact, undisturbed acid soil (- I .5 MPa) corresponded with a soil moisture content of 7% w/w and was attained 3 weeks after commencement of basal irrigation. Nitrogen accumulation by the subterranean clover plants grown in this treatment ceased when the top 9 cm of the acid soil profile reached the permanent wilting point. The permanent wilting point of the top 9 cm of the limed and unlimed cultivated soil occurred at a soil moisture content of 5% w/w and was reached 5 weeks after commencement of basal irrigation. Nodules occurred on roots at depth in the limed and unlimed cultivated soil and modulation continued after commencement of basal irrigation compensating for lower nodule numbers on the upper crown roots that were in contact with the drying soil. Nitrogen continued to be accumulated by these plants throughout the experiment. Nodule size varied in accordance with soil cultivation treatment. In particular, larger nodules were produced on the roots of plants grown in undisturbed acid soil whereas smaller, more numerous nodules occurred on the roots of plants grown in lime-amended soil and to a lesser extent on the roots of plants grown in cultivated acid soil. However, total nodule mass was equivalent on plants grown in each of the soil treatments. Continued nodulation at depth on the roots of subterranean clover due to cultivation and incorporation of lime enabled nitrogen fixation to continue when nodules on roots near the soil surface became desiccated and senescent due to the localized moisture deficit.
tNTRODtiCTlON Acidification of soil beneath legume-based pastures is recognized as a widespread problem in south-eastern Australia (Donald and Williams. 1954; Helyar, 1976). For example, a decline in soil pH has occurred at a rate of one pH unit over a SO-yr period in southern New South Wales (Williams, 1980; Bromfield ef al., 1983a). However, high concentrations of hydrogen ions are not distributed uniformly within soil profiles. Often the most extreme soil acidity occurs in the root zone (0-20cm) of the A horizon (Bromtield ef nl., 1983b). In other instances, severe subsoil acidity is also reported (Pinkerton and Simpson, 1986a). Low soil pH may result in toxic concentrations of aluminium, manganese and hydrogen ions as well as deficiencies of calcium, magnesium and molybdenum in agricultural soils (Munns, 1953; Bromfield er ol., 1983a; Coventry er al., 1985a, b). Aluminium toxicity in particular can inhibit root growth and proliferation (Foy, 1984) and has the potential to restrict the ability of plants to fully exploit soil moisture (Bromfield et al., 1983b) and to reduce uptake of nutrients. Root length of species such as lucerne and wheat which are sensitive to acidity can be significantly reduced in very acid soil (Pinkerton and Simpson, 1986a) whilst those of tolerant species such s.0n ?I!,-*
as subterranean
clover may exhibit only slightly restricted root growth when growing under similar conditions (Munns, 1965; Pinkerton and Simpson, 1986b). Nevertheless, Kehoe and Cumow (1963) have reported wilting and premature senescence of subterranean clover growing in acid sand at Marlo in East Gippsland, Victoria. This was considered to be due to restricted development of roots in the acid sand which prevented the plants from utilizing subsoil moisture as the soil profile dried at the end of the growing season. In a drying soil profile, the growth of roots into zones of extreme soil acidity and their distribution relative to nutrients and soil moisture can also have critical effects on plant growth. Soil drying from the top can restrict the availability of nutrients such as phosphorus which are concentrated predominantly in the upper layers of soil profiles (Pinkerton and Simpson, 1986b). Nodulation of legume roots may also be severely restricted in very acid soils (see reviews by Munns, 1978; Dear et al., 1987). Poor productivity of pastures has occasionally been attributed to total nodulation failure in acid soil (Coventry er al., 1985a). Restricted nodulation of legume roots has also been reported (Vincent and Waters, 1954). Even in circumstances
A.
2
G. DAVEY et al.
where roots appear to be adequately nodulated, nodulation patterns can be influenced by localized acidity within a soil profile. Richardson er al. (1988) have shown that up to 86% of root nodules on subterranean clover occurred in the top 4cm of an acid soil profile. Restricted distribution of nodules was associated with the effects of extreme acidity on the growth and distribution of Rhicobium trifofii. In this soil, Rhkobium was essentially confined to the uppermost region of the soil profile which was less acidic and high in organic matter. A larger population of R. trifolii and improved nodulation at depth occurred when lime was incorporated (Richardson and Simpson, 1988). Addition of lime supplies calcium, raises soil pH and reduces aluminium concentrations, all of which also clearly affect the infection of legume roots (Anderson and Moye, 1952; Loneragan and Dowling. 1958; Munns, 1978; Wood and Cooper, 1984). As soils under clover-based pastures dry during periods of short-term drought or toward the end of the growing season, it is likely that nodules restricted to roots near the soil surface by a very acid soil would become desiccated and senescent and may lose their capacity to fix atmospheric nitrogen. This paper outlines experiments in which nodulation, plant growth and nitrogen accumulation was measured on established plants of subterranean clover growing in a drying acid soil profile.
iMATERIALS AND IMETHODS
Cores of an acid soil (9 cm dia x 30 cm depth) were removed intact in PVC sleeves from a subterranean clover-based pasture immediately prior to the commencement of the growing season in May 1986 at “Strathfieldsaye”, via Stratford (38’3’S, 147’16’E) in East Gippsland, Victoria. The duplex soil consisted of an acid loamy sand A horizon with a clay subsoil occurring at 18-20cm depth (principle profile form Dy 4.53; Northcote, 1974). The soil from which the cores were removed had been topdressed annually since 1981 with superphosphate (150 kg ha-‘) which included molybdenum (37.5 g ha-‘) in 1981 and 1984. Three cultivation treatments were imposed on the cores of soil: (i) undisturbed acid soil (control) in which soil pH measured in IO mM CaCl, (1:5 w/v) was pH 4.3; (ii) cultivated acid soil (pH 4.3) in which the top I2 cm of soil was removed, mixed and replaced into the PVC sleeves; and (iii) cultivated soil to which agricultural lime (Calcimo lime, Longford, Victoria, Ca present as 81% CaCO,) was added and mixed at a rate of 5 t ha-’ (based on the surface area of soil in the PVC sleeves). The depth to which lime was incorporated was I2 cm and the pH value of the soil in this region was raised to pH 5.5. The soil pH profile existing beneath undisturbed pasture exhibited relatively moderate acidity in the top few cm (pH 4.5) and wtreme soil acidity (as low as pH 3.8) at depths between 4-l3cm. Below 18cm depth, soil pH increased to levels similar to those occurring at the soil surface. Consequently, levels of exchangeable aluminium in the top 4 cm and below 18 cm down the soil profile ranged between 22 and 32 pg g-i soil and were
lower than levels within the most acid region of the soil profile (44-54pg g-i soil) (Richardson and Simpson, 1988). The level of exchangeable aluminium was decreased substantially after incorporation of lime. Plastic mesh was placed over the base of each PVC sleeve to prevent soil loss and to allow adequate drainage. The soil cores were watered to field capacity over a 6 week period and when necessary, weeds were removed. Soil cores were then sown with seeds of subterranean clover (cv. Mt Barker) inoculated with Rhkobium trifolii (WU95). Seedlings were thinned to one plant per soil core and plants were grown under natural irradiance (June-October) in a glasshouse at the University of Melbourne. Supplementary lighting (incandescent lamps) was used to extend the photoperiod to 14 h. Temperatures were maintained between 25’C (day) and I2C (night). Alkathene plastic beads (ICI Industries, Melbourne) were placed over the soil surface of each core to reduce evaporation. The cores were arranged in a completely randomized design and were watered regularly to field capacity. Flowering commenced on 27 October 1986. After IO weeks of growth, the plants were transferred to a controlled environment plant growth room in which temperatures were maintained at 24’C (day); l8’C (night) and the photoperiod was I4 h. Relative humidity was 70% and the photon flux density (400-700 nm) at plant height was 550 pmol rn-? s-i. After 7 days, half of the soil cores from each of the 3 cultivation treatments were placed in plastic saucers and irrigated from the base only with half the amount of water transpired by the control plants growing in soil cores watered to field capacity. Transpiration by control plants was measured gravimetrically each day. The six treatments (3 cultivation x 2 irrigation treatments) were arranged in a completely randomized design. Five replicate plants per treatment were harvested at weekly intervals for 6 weeks. Plants were harvested 6 h after commencement of the photoperiod. Plants were divided into shoots, roots and nodules. For most purposes, roots and nodules in the top 9cm and remaining 9-30cm regions of the soil profile were of interest. However, a more detailed measurement of root and nodule distribution was also conducted by removing the soil cores from their plastic sleeves and cutting them into regions of O-4, 4-9, 9-13, 13-18 and 18-30 cm from the soil surface. These regions were based on changes in soil pH and the consequent distribution of nodules in the soil profile that had been observed at the field site (Richardson and Simpson, 1988; Richardson et al., 1988). Roots were gently washed from the soil of each profile and all nodules were removed and counted. Roots and nodules from individual profile regions of each replicate were dried separately. Dry weight of plant parts was determined after drying at 70°C for 24 h. Total plant nitrogen content was determined by the Kjeldahl method using Se as the catalyst (Bremner and Mulvaney, 1982). A subsample of soil from each region of the soil core was weighed for gravimetric determination of soil moisture content after drying for 48 h at 80°C. Soil matric potential of the compact undisturbed soil and of the less compact cultivated soil treatments was also measured, using a pressure plate apparatus (Richards, 1965).
Nodulation of clover in a drying acid soil RESULTS
Tim
3
after commencement of sums
(days)
Soil matric potential
The effect of basal irrigation with 50% of the amount of water transpired by control plants was to slowly dry the soil profile from the top. The rate at which soil in the O-9 and 9-30 cm regions of basallyirrigated cores of soil dried to the permanent wilting point (- 1.5 MPa) is presented in Fig. I. The permanent wilting point of the limed and unlimed cultivated soil occurred at a soil moisture content of 5% (w/v) whereas the permanent wilting point of the more compact undisturbed acid soil occurred at a soil moisture content of 7% (w/v) due presumably to the effect of the higher bulk density of the undisturbed soil (data not shown). Within the top 9cm of the drying soil the permanent wilting point was attained after 21 and 30-36 days from the commencement of the moisture deficit for the undisturbed and cultivated soils, respectively (Fig. 1). Nodulation of subterranean clover roots
Numbers of nodules on roots of subterranean clover plants were variable within each treatment. Generally, however, numbers of nodules on plants grown in undisturbed and in cultivated acid soil either declined throughout the experimental period or were less numerous than the number of nodules produced on plants grown in lime-amended soil (data 0-9 cm
E.rjF
-2.0 L
Fig. 1. Soil matric potential in the O-9 cm (closed symbols) and 9-3Ocm (open symbols) regions of the soil profile for
the 3 cultivation treatments after the commencement of basal irrigation. Key: (0, 0) undisturbed acid soil; (m. 0) cultivated acid soil; (A. A) cultivated and limed soil. Bar represents the LSD (P < 0.05).
not shown). Irrespective of the variability in nodule numbers, total nodule mass (dry weight) per plant did not vary significantly and ranged between 40-70, 40-65 and 30-6Omg dry wt per plant for plants grown in undisturbed, cultivated and cultivated and lime-amended soils, respectively (Fig. 2). Basal irrigation did not significantly reduce total nodule dry weight per plant despite drying of the uppermost
Undisturbed acid soil
Cultivated and limed soil
Fig. 2. Dry weight per nodule (a, 0) and total nodule dry weight (a, 0) of well-watered (closed symbols) and basally-irrigated plants (open symbols) growing in (a and b) undisturbed acid soil, (c and d) cultivated acid soil and (e and f) in soil cultivated with the incorporation of lime. Each point is the mean of 5 replicates. Bars represent the LSD (P < 0.05).
4
A. G. DAMY et al.
regions of soil profiles during the experiment. Within the top 9 cm of the soil profile, individual nodule dry weight was affected by soil treatment. For example, at the fourth harvest, mean individuat nodule dry weights within undisturbed, cultivated and limeamended soils were 0.59, 0.37 and 0.26 mg nodule-‘, respectively, for the well-watered plants and 0.55, 0.35 and 0.25 mg nodule-‘, respectively, for the basally-irrigated plants (Fig. 2). The large nodules which were observed on plants in undisturbed acid soil occurred mostly on the primary root whereas limed and unlimed cultivated soil resulted in more nodules on the lateral roots within the top 9 cm of soil profiles. Smaller nodules were observed on roots in undisturbed acid soil at 9-30 cm depth [Fig. 2(b)] in comparison with those in the uppermost region of that soil profile [Fig. 2(a)]. The size of nodules at depth in the undisturbed acid soil that was drying from the top was also further reduced [Fig. 2(b)]. In contrast, mean dry weight of nodules in both upper (0-9cm) and lower (9-30cm) regions of the soil profile within cultivated [Fig. 2(d)] and lime-amended soil (Fig. 2(f)] did not differ. In undistur~d acid soil, nodule size increased during the first 3 weeks of the experiment. However, the mean size of nodules produced on plants grown in both limed and unfimed cultivated soil either remained relatively unchanged or increased in size to a lesser extent (Fig. 2). Five weeks after imposition of the moisture deficit,
nodulation of roots within O-4.4-9, 9-13, 13-18 and 18-30 cm regions of all soil profiles was analysed. Highest numbers of nodules occurred within the top 4cm of the soil profile on the roots of plants grown in well-watered, lime-amended soil [Fig. 3(a)]. Generally, nodulation of roots of plants grown in wellwatered, undisturbed acid soil was poorer than on plants grown in limed and unlimed cultivated soil [Fig. 3(a)]. More nodules occurred on roots at the base of the well-watered, undisturbed soil cores (18-30 cm). However, total nodule mass in the uppermost region of the undisturbed acid soil profile was equivalent to that in lime-amended soil due to the larger size of crown nodules in undisturbed acid soil [Fig. 3(c)]. Nodulation within basally-irrigated soil profiles was similar to that observed on roots of well-watered plants except that in limed soil nodulation was poorer in the drier top 9cm of the soil profile. Plants grown in limed and unlimed cultivated soil also exhibited significantly higher nodule mass in the more moist 9-13 cm region of the soil profile [Fig. 3(b)]. This was due largely to increasing size rather than number of nodules as the experiment progressed [Figs 2(d), (f) and 3(d)]. Poor root growth occurred at depths below 9cm on plants in the well-watered. u~distur~d acid soil. However, in the top 4 cm. root mass was greater than in most other treatments [Fig. 4(a)]. The extra root growth in the upper regions of undisturbed acid soil
Numberof ncdulcsplant-’ 0
so
loo
150 200 250 0
50
loo
1
1.50 200 2.50
t
a
e
f
(b)
Tota1noduledxyweight(mg plant-’) 0
10
20
30
40
50 60
Well-watered
0
10
20
30 40
50 60
f)asaIly-irrigated
Fig. 3 (a and b) Number of nodules per plant and (c and d) total nodule dry weight within the soil profile of well-watered and basally-irrigated plants 5 weeks after commencement of the moisture deficit. Key: (0) undistur~d acid soil; (0) cultivated acid soil; (A) cultivated and limed soil. Each point is the mean of 5 replicates. Bars represent the LSD (P ~0.05).
Nodulation of clover in a drying acid soil Root dry weight (g plant” ) 0
0.5
1.0
1.5
1
0
0.5
1.0
1.5
I
(b) WCll-Watered
Badly-inigatcd
Fig. 4. Root dry weight within the soil profile of (a) well-watered and (b) basally-irrigated plants 5 weeks after commencement of the moisture deficit. Key: (a) undisturbed acid soil; (0) cultivated acid soil; (A) cultivated and limed soil. Each point is the mean of 5 replicates. Bars represent the LSD (P ~0.05). did not occur in the drying soil profile of basally-
irrigated cores (P < 0.05) [Fig. 4(b)]. Root dry weight of plants grown in basally-irrigated soil was generally similar for all treatments although root growth was marginally better at depth in treatments with cultivated soil due presumably to the lower bulk density of the cultivated soil. Total p/am nhogen
and dry matter accumulation
Plants grown in well-watered and basally-irrigated undisturbed acid soil accumulated nitrogen until days 25 and 22 from commencement of the moisture deficit, respectively, after which no further increase in total nitrogen content was measured [Fig. S(a)]. The rate of accumulation of nitrogen by basally-irrigated plants in undisturbed acid soil was less than for well-watered plants and led to a significant difference (P < 0.05) in the total nitrogen content of plants in each treatment at later harvests. In contrast, plants grown in soil cultivated with and without lime showed no significant difference in accumulation of total nitrogen between well-watered and basallyirrigated plants [Fig. 5(c) and (e)]. As well, total plant nitrogen content within these two treatments increased throughout the entire experiment. Despite the difference in total nitrogen content between basallyirrigated and well-watered plants grown in the undisturbed acid soil the accumulation of plant dry matter was less affected by basal irrigation and the small difference in accumulated dry matter was not significant (P c 0.05) [Fig. 5(b), (d) and (f)]. DISCL’SSION
Distribution of nodule and root dry weight in drying acid soil profiles
Localized extreme acidity at depths between 4 and 13 cm in the undisturbed acid soil profile resulted in more prolific nodulation of roots of subterranean clover at the top (-cm) and to a lesser extent the base (IS-30 cm) of that soil profile (Fig. 3). The soil pH of the less acidic regions was still low enough to restrict nodulation, as incorporation of lime (CaCO,) but not gypsum (CaSO,) increased nodulation of roots (Fig. 3; Richardson and Simpson, 1988). However, larger nodules were produced on roots grown in
the very acid undisturbed soil and total nodule mass per plant was therefore not different (Fig. 2). The effectiveness of this mass of nodule tissue is doubtful, however, as specific nitrogenase activity of large nodules is reported to be low in acid soil (Coventry et al., 1985b). Large multi-lobed indeterminate nodules such as occur on clover in very acid soils generally contain high proportions of senescent nodule tissue (Sprent, 1976) which no doubt reduces the rate of nodule specific nitrogenase activity. In the present study, plant-extractable water was depleted more quickly in the upper region of the more compact, undisturbed acid soil (Fig. I) which reached the permanent wilting point at a soil moisture content of 7% as opposed to limed and unlimed cultivated soil which reached the permanent wilting point at a soil moisture content of 5%. Drying topsoil clearly reduced further nodulation of roots in the uppermost region of the lime-amended soil and also to a lesser extent in the cultivated acid soil. Nodulation at the top of the undisturbed acid soil protiles was not affected (Fig. 3) because maximum nodule numbers had been reached prior to commencement of the moisture deficit. Total nodule mass in the undisturbed acid soil was consequently also unaffected by the drying soil. Plants grown in drying, cultivated soils with and without lime, compensated for poorer nodulation at the top of the profile by allowing nodules to increase in size and in number at depths below 9cm (Figs 2 and 3). Soil acidity affected nodulation at depth in the cultivated acid soil. However, the combination of increased nodule size at depth and improved nodulation below 13 cm (Fig. 3) was sufficient to compensate for restricted nodulation in the dry topsoil. Plants grown in limed soil compensated for restricted nodulation in the dry topsoil by increasing nodule numbers at depths between 4-18 cm (Fig. 3) where soil pH had risen from pH 3.8 to between pH 4.3 and 5.5 (Richardson and Simpson, 1988). Nitrogen fixation by these nodules supplemented the nitrogen economy of the plants in the absence of nodule activity elsewhere on the root system allowing continued accumulation of nitrogen by the subterranean clover plants (Fig. 5). It is assumed that burial of Rhkobium inoculum in organic matter from the soil surface during cultivation
A. G. DAMYet al.
Cultivatedacid soil 8
; 6
Cultivated and limed soil
The after
cotntncnccment of sums (days)
Fig. 5. Total nitrogen content of the whole plant (0, 0) and dry matter production of shoots (W, 0) and roots (A, A) of well-watered (closed symbols) and basally-irrigated plants (open symbols) growing in (a and b) undisturbed acid soil, (c and d) cultivated acid soil, and (e and f) soil cultivated with the incorporation of lime. Each point is the mean of 5 replicates. Bars represent the LSD (P c 0.05).
combined with leaching of Rhizobium under wellwatered conditions prior to the commencement of basal irrigation may have enabled some continuation of nodulation at depths below I3 cm (pH 4.M.8) in the cultivated acid soil whereas no further nodulation at depth occurred in the undisturbed acid soil. Subterranean clover is considered to be tolerant of soil acidity (Munns, 1978; Pinkerton and Simpson, 1986b) and no large effects of acidity on root growth were observed (Fig. 4). However, root growth was restricted at depth in the undisturbed acid soil but plants in this soil tended to have a greater mass of roots in the surface layers of the soil. In all treatments, root growth near the surface was reduced as the soil dried. Drying of the uppermost region of the soil profile also caused nodule numbers to decline by 90 and 50% in soil cultivated with and without the incorporation of lime, respectively. Thus, senescence and abscission of nodules may have been increased under these conditions but it is also likely that formation of new nodules was inhibited. Worrall and
Roughley (1976) have shown that the number of infection threads was reduced and that nodulation by R. trifofii was consequently inhibited in subterranean clover growing under moisture stress even when the numbers of Rhizobium in the rhizosphere were unaffected by the dry soil conditions. Additionally, some decline in the population of Rhizobhm due to desiccation might be expected (Vincent er al., 1962; Bromfield et al., 1983b). However, it has also been suggested by Pinkerton and Simpson (1986b) that suppression of nodulation and thus nitrogen fixation by subterranean clover in an acid soil may be due to effects of moisture stress on availability of phosphorus in the upper region of the soil profile. Nutrition of subterranean clover in an acid soil
Nitrogen was accumulated by the plants ranean clover grown in limed and unlimed soil throughout the experiment whereas lation of N by plants grown in undisturbed ceased prematurely approx. 20 days after
of subtercultivated accumuacid soil the mois-
7
Nodulation of clover in a drying acid soil ture deficit commenced (Fig. 5). This was associated with attainment of a maximum nodule mass throughout the soil profile and lack of further nodulation of roots either near the soil surface or at depth. Assessment of nodulation at the site from which the soil cores were obtained has also revealed that the roots of subterranean clover harvested from undisturbed acid pasture soil were poorly nodulated late in the growing season (Richardson et al., 1988). Drying of the soil profile further decreased nitrogen fixation (Davey and Simpson, 1989) and N accumulation was slower from 5 to 10 days after the experiment commenced (Fig. 5). The size of nodules in the uppermost region of the drying undisturbed soil did increase. Clearly, however, the symbiotic effectiveness of these nodules was reduced. In other soil treatments where compensatory nodulation of roots occurred at depth, N accumulation by the subterranean clover plants was not significantly inhibited by drying of the upper regions of the soil profile. Continued accumulation of dry matter reflected the ability of the subterranean clover to accumulate N in both well-watered and in drying soil. Nevertheless, yield differences were not significant (P c 0.05) presumably because some growth was still possible in N-deprived plants due to redistribution of N as plants senesced. Premature cessation of nitrogen accumulation by subterranean clover grown in drying undisturbed acid soil is also consistent with data of Engin and Sprent (1973) and DeJong and Phillips (1982) who have suggested that under moisture deficits, decreases in nitrogen fixation are greater than corresponding decreases in shoot dry matter production. In our study, reduced nitrogen fixation was due to a combination of poor nodulation in the most acid region of the soil profile, senescence and decay of large and relatively old nodules found on roots in the extremely acid soil and to desiccation of nodules in the drying soil. Acknonle~~emenrs-Marylyn Williams is thanked for her technical assistance. This work was supported by a grant to R.J.S. from the Wool Research Trust Fund on the recommendation of the Australian Wool Corporation and was carried out whilst A.G.D. was the recipient of an Australian Wool Corporation Postgraduate Scholarship.
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Anderson A. J. and Moye D. V. (1952) Lime and molybdenum in clover development on acid soils. Australian Journal of Agricultural Research 3, 95 I IO. Bremner J. M. and Mulvaney C. S. (1982) Nitrogen-total. In Methods of Soil Analysis, Part 2, Chemical and Microbiological Properties (A. L. Page, R. H. Miller and D. R. Keeney. Eds). pp. 595624. Soil Science Society of America, Madison. Bromtield 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 181-191.
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Dear B. S., Cregan P. D. and Hochman Z. (1987) Factors restricting the growth of subterranean clover in New South Wales and their implications for further research. In Temperate Pastures: Their Production, Use and Management (J. L. Wheeler, C. J. Pearson and G. E. Robards, Eds), pp. 55-57. Australian Wool Corporation-CSIRO, Melbourne. DeJong T. M. and Phillips D. A. (1982) Water stress effects on nitrogen assimilation and growth of Trtfolium subterraneum L. using dinitrogen or ammonium nitrate. Plant Physiology 69, 4 16-420. Donald C. M. and Williams C. H. (1954) Fertility and productivity of a podzolic soil as influenced by subterranean clover (rrrifolium subterraneum L.) and superphosphate. Australian Journal of Agricultural Research 5, 664-687.
Engin M. and Sprent J. I. (1973) Effects of water stress on growth and nitrogen-fixing activity of Tri/olium repens. New Phytologist 72, I 17-126. Foy C. D. (1984) Physiological effects of hydrogen, aluminium, and manganese toxicities in acid soil. In Soil Acidity and Liming (F. Adams, Ed.), pp. 57-97. American Society of Agronomy. Madison. Helyar K. R. (1976) Nitrogen cycling and soil acidification. Journal of the Australian 42, 217-221.
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