- Email: [email protected]
Response of subterranean clover in open-top chambers to chronic exposure of sulphur dioxide
Agriculture, Ecosystems and Environment, 32 (1990) 283-293
283
Elsevier Science Publishers B.V., Amsterdam
Response of subterranean clover in open-top chambers to chronic exposure of sulphur dioxide S.A. Wilson and F. Murray Biological and Environmental Sciences, Murdoch University, Murdoch, Qld., 6150 (Australia) (Accepted for publication 20 March 1990)
ABSTRACT Wilson, S.A. and Murray, F., 1990. Response of subterranean clover in open-top chambers to chronic exposure of sulphur dioxide. Agric. Ecosystems Environ., 32: 283-293. Ten open-top chambers were used to obtain SO2 concentration-response relationships for growth in subterranean clover cv. 'Trikkala', and to study the associated sulphur accumulation. Four-weekold seedlings were exposed to 4, 41, 120, 255 or 515 nl 1-1 of SO2 for 77 days, 4 h day -t. Response variables measured included leaf dry weight, stem weight, runner length, leaf number, average leaf weight, leaf: stem ratio and leaf sulphur concentration. Significant reductions in growth and changes in assimilate distribution occurred at 120 nl 1-1 and higher concentrations;leaf sulphur concentration increased by ~ 70 and 100% at 41 and 120 nl 1-1, respectively.
INTRODUCTION
Subterranean clover ( Trifolium subterraneam L. ) is a winter growing, selfpollinating forage annual which is native to Mediterranean Europe and parts of Asia and Africa. Deliberate use as a pasture legume began early this century in Australia; and in the 1920s in the United States (Knight et al., 1982; Collins and Gladstones, 1984). The value of subterranean clover is in its soil improvement characteristics, its supply of good quality forage and its provision of income from the production of clover seed. In both the United States and Australia the energy crisis and the subsequent high price of mineral nitrogen have added to the importance of subterranean clover, as has the increased competition for land resources amongst commodity crops. In many cases the competition has resulted in pasture crops being grown on thin, marginal soils which are undesirable for other crops; subterranean clover has proved to be an ideal species for these conditions (Knight et al., 1982). More recently, research has been pursuing the potential use of subterranean clover as a nitrogen source for forest plantings (Haines et al., 1978). In addition to supplying nitrogen it is desirable as it provides weed 0167-8809/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
284
S.A. WILSON AND F. MURRAY
control and, as it is low growing and grows during the cool season in Australia, it does not compete with tree seedlings. Underwood and Gladstones (1979) report that subterranean clover has been a key factor in the improvement of soil fertility in Australia and that both crop and livestock production have increased dramatically in the regions in which it has been grown. The net financial benefits are difficult to estimate precisely but Collins and Gladstones (1984) state that its value, in terms of soil nitrogen accumulation alone, may be of the order of $A400 million annually. Sulphur dioxide is an increasingly important pollutant in Australia. SO2 emissions increased from 1.4 million t year -~ in 1976 (Davey, 1980)to 2.2 million t year -~ in 1986 (Murray, 1989). By 1993, SO2 emissions are estimated to be 2.5 million t year- ~ (F. Murray, unpublished data, 1989). However, the economic implications of this trend for Australian agriculture are largely unknown. Agricultural production is affected by air pollution which occurs at significant concentrations over broad regions in developed and developing countries (Wang and Schaap, 1988 ). Because of the obvious great economic importance of subterranean clover and because very little is known about the impact of air pollutants on it, this study was initiated. The objective of the study was to evaluate the concentration-response relationships of subterranean clover exposed to a range of SO2 concentrations and the associated shoot sulphur accumulation. Open-top chambers were used for the study to enable realistic field conditions to be simulated. MATERIALS AND METHODS
Fumigation chambers Ten cylindrical open-top chambers (Heagle et al., 1973 ) were used for fumigation, each 3 m in diameter and 2.4 m tall, consisting of a rigid aluminium frame covered by UV-treated PVC plastic. The upper half of the frame was covered by a single layer of PVC plastic and the lower half was covered by a double thickness of the PVC envelope with the inner layer perforated by holes 25 m m in diameter. Air was drawn by a fan through a dust filter and then forced along a duct, into the chamber through the holes in the lower envelope and then out through the open top. The output of the fan was 1 m 3 s - l enabling ~ 3.5 air changes m i n - 1.
Pollutant dispensing and monitoring Sulphur dioxide was delivered to the inlets of the fumigation chambers after being mixed with dry air and controlled by means of a regulator and series of
RESPONSEOF SUBTERRANEANCLOVERTO SULPHURDIOXIDE
285
TABLE l Treatment characteristics of the SO2 exposure regime in open-top chambers. All concentrations are in nl l - t Treatment
1 2 3 4 5
Fumigated 4-h average conc.
4 41 120 255 515
Standard deviation
4 31 59 104 155
Percentile 10th
90th
2 3 40 111 318
9 79 191 383 677
needle valves. Pollutant monitoring was conducted with a time-sharing system whereby each chamber was sampled for 12 min every 228 min, at approximately canopy height. The concentration of SO2 was determined using a Thermo Electron, Series 43 pulsed fluorescent ambient SO2 analyzer, calibrated using a Thermo Electron, Model 145 calibrator and NBS traceable permeation tubes. Data were recorded with a Starlog portable data logger (Unidata, Australia), with a chart recorder used as a backup. Table 1 displays the characteristics of the fumigation regime with five SO2 treatments, each duplicated. Statistical analysis of the monitoring data, as outlined subsequently, confirmed the validity of the duplicate treatment chamber pairs, at the 0.05 level of probability. Monitored background concentrations at the experimental site of SO2 were < 5 nl l- 1, annual average NOx concentrations were < 6 nl l- ~of which > 90% was NO. Ozone concentrations were believed to be low, as the annual average O3 concentration, measured in the Central Business District of Perth 15 km from the site, was 10 nl l-1 (Public Health Department, 1982).
Cultural practices and growth conditions Seeds of subterranean clover ( T. subterraneum L. cv. 'Trikkala' ) were sown in peat jiffy pots on 30 May 1988, in a glasshouse. Seedlings were inoculated with a commercial inoculum (Nodulaid, Sydney) and later thinned from three to one per pot. Four weeks after sowing they were removed from the glasshouse and 11 plants were randomly assigned to each of l0 chambers. The ground had been pretreated with the herbicide Roundup (Monsanto, active constituent Glyphosate) and mixed with a potting mix (2 sand: 3 sawdust: 3 wood fibre fines: 4 pinebark) and fertilizer containing both macro- and micronutrients. Plants were sprayed once during the fumigation with the insecticide Rogor
286
S.A. WILSON AND E MURRAY
(Chemspray, active constituent Dimethoate) to protect against insect damage. Irrigation took place to supplement natural rainfall, when necessary. Plants were exposed to one of the five SO2 treatments (Table 1 ) for 4 h day -1, 7 days week -1 from 10:00 to 14:00 h for 77 days. The selection of exposure duration and time attempted to simulate atmospheric inversion break-up conditions. The average day length during the period of fumigation was 10 h 47 min and the mean daily total irradiation ( _+SD) was 2827 ( 1088 ) W h m-2. Temperature and relative humidity were measured throughout the exposure period using thermohygrographs in Stevenson screens. The mean ambient daily maximum and minimum temperatures were 19.2 and 9.4 ° C, respectively, and the corresponding relative humidities were 96.0 and 55.8%. Mean temperatures were ~ 1 °C higher inside the chambers than outside and relative humidity was ~ 1.5% lower inside the chambers than outside.
Harvest procedure At harvest, plants were cut at the soil line; leaf number and length of the longest runner were recorded; leaves and stems were separated and then placed in an oven at 70°C and dried to constant weight. Stem and leaf dry weight were later measured. Leaf material was ground to pass a 40 mesh sieve in a Wiley hammer mill prior to total sulphur concentration determination, using the oxygen flask combustion method of Hunt ( 1980 ).
Statistical analysis Prior to statistical analysis, data for all growth variables were transformed to natural logarithms to stabilize variances and because growth is considered exponential (Hunt, 1979). Unfortunately, due to a technical malfunction, plants in one of the duplicate chambers treated with 515 nl 1-1 of SO2 were damaged and the data were discarded. Fumigation monitoring data were statistically analysed using Statview 512 + (Abacus Concepts Inc, 1986 ). Analysis of Variance (ANOVA) was followed by a Fisher's Protected Least Significant Differencetest of the means to indicate duplication of fumigation regimes. As data from duplicate chambers were not significantly different from one another, they were pooled for the descriptive statistics. Regression analysis (Statview 512 + package ) was used to develop concentration-response functions for the response of subterranean clover to SO2. Analyses were conducted on data from individual plants. ANOVA was used to determine treatment effects. Homogeneity of variance was tested using Cochran's C-test. A Duncan's Multiple Range test was used to determine dif-
RESPONSE
OF SUBTERRANEAN
CLOVER
TO SULPHUR
287
DIOXIDE
ferences between response variable means at the 0.05 level of probability. These analyses used the SPSS-X version 3.0 package installed on a U n i x / Microvax system. RESULTS Concentration-response functions were established to depict the effect of SO2 on the growth of subterranean clover cv. 'Trikkala' (Fig. 1 ). The mathe81
10-
,
i
,
,
,
,
,
,
,
,
i
,
o
C
10. 91
3
71
51 ~1.5~
~q 1
.s.I
C
OI
.
,..
.
.. .
.
.
.
,
,
,
,
,
,
,
,
,
10.
8
g ,l.
............ i
~ 6.
~ .54
i ~ .3! 2.
.
o.
-
.
ui
.
0
.
.
.
10o SOt
.
2oo Concentration
300
4o0 (nL/L)
5~
6oo
4. 3.5
~
3 2.5 2.
~1.51 .5 0
1(30 200 300 400 SO2 Conce~ttraUon (nL/L)
500
600
Fig. 1. Concentration-res0onse curves for subterranean clover cv. 'Trikkala' exposed to SO2 in open-top chambers.
288
S.A. WILSON AND F. MURRAY
TABLE 2
Concentration-response functions and correlation coefficients to describe the effects of SO2 on subterranean clover cv. 'Trikkala', in open-top chambers
Response variable
Equation
Correlation coefficient
Probability ( P )
(R) Length ( c m )
y = e ( - 0 . 0 0 4 x + 3.04) y = e ( - 0 . 0 0 8 x + 5.54) y = e ( - 0 . 0 1 2 x + 8.19) y=e(-0.012x+8.28) y = e ( - 0 . 0 1 2 x + 8.95) y=e( -0.004x+2.67 ) y=0.001x+0.33
Leaf number Leaf weight ( m g ) Stem weight ( m g ) Yield ( m g ) Average leaf wt. ( m g ) Sulphur conc. (%)
-0.928 -0.935 -0.901 -0.918 -0.942 -0.874 +0.883
<0.001 < 0.001 <0.00t <0.001 < 0.001 < 0.001 < 0.001
TABLE 3
Arithmetic mean values of response variables and the results of the Duncans Multiple Range Test SO2 concen- Length Leaf Stem (cm) number weight (g)
weight
Total weight
Average leaf Leaf:stem Sulphur weight ratio conc.
(g)
(g)
(mg)
4 41 120 255 515
3.35 a 3.03 a 0.89 b 0.22 ¢ 0.01 c
7.94 a 6.89 a 1.66 b 0.35 c 0.03 ¢
14.10. 12.96" 8.74 b 5.35 c 0.57 a
tration (nl 1 - ' )
19.2" 19.1 ~ 13.7 b 9.0* 3.00
234 a 224" 91 b 39 c 4~
4.50* 3.84 ~ 0.90 b 0.16 c 0.03 a
Leaf
(%) 0.73 a 0.76 a 1.01 b 1.16 b 0.14 ~
0.24 a 0.41 b 0.48 c 0.55 a 0.70*
Means followed by the same superscripts are not significantly different at the 0.05 level of probability.
matical functions and the correlation coefficients of these curves are listed in Table 2. After preliminary analyses, a first degree polynomial exponential model was considered the most appropriate model for the data. ANOVA was performed on the data to characterize statistically significant individual treatment effects to supplement the clearly defined trends in the data established by regression analysis. Regression analyses of the data indicated a highly significant (P<0.001) negative linear relationship between SO2 concentration and all of the response parameters. The exception was leaf sulphur concentration, which displayed a highly significant ( P < 0.001 ) positive relationship. Analysis of variance revealed that 41 nl 1- ~ SO2 had no statistically significant effect on the growth parameters analysed. However, the leaf sulphur concentration was increased by 70% (Table 3 ). Exposure to 120 nl 1- x of SO2, or higher, significantly reduced all growth parameters and further increased leaf sulphur concentration (see Table 3 ). The runner length was least affected, being reduced by 30%. The average individual leaf weight and leaf number were reduced by 38 and 61%, respec-
RESPONSE OF SUBTERRANEAN CLOVER TO SULPHUR DIOXIDE
289
tively; this resulted in a 73% decrease in the total leaf weight per plant. Stem weight decreased more substantially, showing an 80% reduction. The leaf to stem ratio increased from ~ 0.7 to ~ 1.0 as a result of the SO2 induced changes in leaf and stem weight. The significant increase in the ratio is the result of a greater decrease in stem weight in comparison with the decrease in leaf weight. In all response variables, except leaf: stem ratio, 255 and 515 nl 1-i of SO2 had a significantly greater effect than 120 nl 1-1. DISCUSSION
The results indicate that under these experimental conditions the concentration at which a significant growth reduction in subterranean clover cv. 'Trikkala' occurs is 120 nl 1- ~SO2 for a 4-h period; a concentration frequently occurring in some parts of Australia. Near an industrial area at Kalgoorlie (Western Australia) maximum 3-h average concentrations for each month in 1987 and 1988 ranged between 150 and 700 nl 1-1, the mean monthly maximum 3-h average concentrations for 1987 and 1988 were 380 and 315 nl 1-~, respectively (EPA, 1989 ). Clearly, SO2 can have quite dramatic effects on the growth of subterranean clover. As subterranean dover is a widely grown fodder crop, changes in shoot biomass are particularly important. The changes in biomass were the result of changes to both leaf and stem dry weight; stem dry weight, however, was reduced by SO2 to a greater extent than leaf dry weight. Many studies have reported that atmospheric pollution can affect the distribution ofphotosynthate in plants. Predominantly a change in the root:shoot ratio occurs, reflecting a greater proportion of assimilate being allocated to the shoots, mainly to the leaves, at the expense of the roots. The difference in allocation has been measured as changes to the root: shoot ratio on a dry weight basis (Ashenden, 1979; Freer-Smith, 1985; Murray, 1985; Pande and Mansfield, 1985; Gould and Mansfield, 1988 ) or as a reduced carbohydrate movement out of leaves or into roots using radiotracer studies (Noyes, 1980; Jones and Mansfield, 1982; McLaughlin and McConathy, 1983; Gould and Mansfield, 1988 ). Changes in assimilate distribution may be an adaptive mechanism within the plant to maintain the relative growth rate in conditions of reduced carbohydrate availability (Darrall, 1989 ). However, with increasing concentrations of pollutants plants become unable to maintain growth, as a result of injury to the physiological processes and enhanced rates of respiration for repair and detoxification processes (Murray, 1985; Darrall, 1989 ). In the current experiment a change in resource partitioning occurred, represented by changes to the leaf: stem ratio. The control plants and those exposed to 41 nl l -I SO2 had a leaf:stem ratio of ~0.7. Plants exposed to 120 and 255 nl l- ~SO2 displayed significant growth reductions and the leaf: stem
290
S.A. WILSON AND F. MURRAY
ratio became ~ 1.0. This could be explained by a greater proportion of assimilates being retained by the leaves at the expense of the stem, as an adaptive mechanism to maximize photosynthetic potential. Other studies have reported that the retention of assimilate by leaves is also at the expense of the roots (Ashenden, 1979; Jones and Mansfield, 1982; Okano et al., 1984; FreerSmith, 1985; Gould and Mansfield, 1988 ). Assimilate distribution changes observed in plants exposed to atmospheric pollutants can be attributed to one or more of the following: a malfunctioning of the phloem loading processes; reduced gaseous exchange, therefore a reduction in photosynthetic carbon fixation; or greater demand for assimilate at the source (Cooley and Manning, 1987; Griffith and Campbell, 1987). Active nitrogen fixing nodules are strong sinks for photo-assimilates (Hardy and Havelka, 1976); therefore, SO2 induced changes in assimilate distribution in subterranean clover could markedly reduce its nitrogen fixing capacity, and hence, have a significant impact on agriculture. Very little is known about the effects of SO2 on nitrogen fixation in legumes, however, most studies undertaken to date report its inhibitory effects. Sheridan ( 1979 ) fumigated soya bean with SO2 for 14 days continuously and found a 25, 60 and 75% reduction in nitrogen fixation at 50, 100 and 1000 nl 1- ~exposure, respectively. Griffith and Campbell ( 1987 ) exposed soya bean plants for 5 days, 4 h day -~ to 18/tmol SO2 m -3 (433 nl 1-1 ) and found no effect on specific nodule activity (SNA); however, exposure to 36/~mol SO2 m -3 (866 nl 1-~ ) significantly reduced (SNA) by 59%. Tingey et al. (1980) found that exposure of lucerne throughout its growing season to ~ 160 and 215 nl 1-~ SO2 significantly reduced nitrogen fixation but 21 and 65 nl 1had no effect. Prasad and Rao ( 1982 ) suggest that leguminous plants are more sensitive to SO2 than grasses. Perhaps the impact of SO2-induced assimilate distribution changes on nitrogen fixation contributes to the sensitivity. Associated with the growth reductions in subterranean clover was a substantial increase in leaf sulphur concentration. Sulphate uptake by roots is the main source of sulphur for the synthesis of sulphur-containing compounds but plants are able to use atmospheric SO2 absorbed through the leaves (Cowling and Lockyer, 1976). Runeckles (1974) and Fowler and Cape (1982) suggest that plant response is closely related to pollutant absorbed, rather than just concentration or duration of exposure. The results of this experiment indicate that leaf sulphur concentration provides a useful estimate of net SO2 absorbed, as a very close relationship (R = 0.88 ) was displayed between sulphur concentration of subterranean clover leaves and ambient SO2 concentration. This direct relationship has been found in other plant species (Pratt et al., 1983; Lauenroth et al., 1985; Bytnerowicz et al., 1987; Adaros et al., 1988). Gilbert and Robson (1984) define the critical sulphur concentration (that required for 90% of the maximum yield) for 'Trikkala' to be 0.20%; the con-
RESPONSE OF SUBTERRANEAN CLOVER TO SULPHUR DIOXIDE
291
trol plants in this experiment had a leaf sulphur concentration of 0.24%, and 41 nl l - 1 SO2 increased this to 0.41%. The 70% increase in sulphur concentration, however, did not correspond with a significant reduction in growth. Significant growth reductions occurred at SO2 concentrations of 120 nl l-1 or higher, where the leaf sulphur concentration was increased by at least 100%. This indicates a possible threshold response to increased foliar sulphur concentration as plant sulphur status moves through the adequate range to luxury and then into toxicity. CONCLUSION Subterranean clover showed quite dramatic reductions in above-ground plant dry weight in response to SO2 at concentrations that occur in Australia. Reductions occurred in both stem and leaf dry weight, but to differing extents, reflecting changes in assimilate distribution in favour of leaf tissue. Associated with the decrease in growth was an increase in leaf sulphur concentration. Clearly, these effects could have a significant impact on agriculture due to the reduction in fodder production and the possible reduction in nitrogen fixation and the associated losses in soil quality. ACKNOWLEDGEMENTS Support was provided under the National Energy Research D e v e l o p m e n t and D e m o n s t r a t i o n Programme, which is administered by the Australian Department o f Primary Industries and Energy. Gratitude is expressed to Fergus O ' H a r a for provision of expert technical assistance.
REFERENCES Adaros, G., Weigel, H.J. and Jager, H.J., 1988. Effects of sulphur dioxide and acid rain alone or in combination on growth and yield of broad bean plants. New Phytol., 108: 67-74. Ashenden, T.W., 1979. Effects of SO2 and NO2 pollution on transpiration in Phaseolus vulgaris L. Environ. Pollut., 18: 45-50. Bytnerowicz, A., Olszyk, D.M., Kats, G., Dawson, P.J., Wolf, J. and Thompson, C.R., 1987. Effects of SO2 on physiology, elemental content and injury development of winter wheat. Agric. Ecosystems Environ., 20: 37-47. Collins, W.J. and Gladstones, J.S., 1984. Breeding to improve subterranean clover in Australia. In: R.F. Barnes, P.R. Ball, R.W. Brougham, G.C. Marten and D.J. Minson (Editors), Forest Legumes for Energy-Efficient Animal Production. Proceedings of a Trilateral Workshop, Palmerston North New Zealand, April 30-May 4, U.S. Dept. of Agriculture, Agricultural Research Service, St. Paul, MN, pp. 308-315. Cooley, D.R. and Manning, W.J., 1987. The impact of ozone on assimilate partitioning in plants: A Review. Environ. Pollut., 47:95-113. Cowling, D.W. and Lockyer, D.R., 1976. Growth of perennial ryegrass (Lolium perenne L. ) exposed to a low concentration of sulphur dioxide. J. Exp. Bot., 27:411-417.
292
S.A. WILSON AND F. MURRAY
Darrall, N.M., 1989. The effects of air pollutants on physiological processes in plants. Plant Cell Environ., 12: 1-30. Davey, T.R.A., 1980. Anthropogenic sulfur balance for Australia, 1976. In: J.R. Freney and A.J. Nicolson (Editors), Sulfur in Australia. Australian Academy of Science, Canberra, pp. 7587. EPA, 1989. Air Quality Measurements 1987 and 1988 Perth, Kwinana, Kalgoorlie and Port Hedland, Technical Series No. 29. Environmental Protection Authority, Perth, Western Australia, pp. 8-9. Fowler, D. and Cape, J.N., 1982. Air pollutants in agriculture and horticulture. In: M.H. Unsworth and D.P. Ormrod (Editors), Effects of Gaseous Air Pollution in Agriculture and Horticulture. Butterworths Scientific, London, pp. 3-26. Freer-Smith, P.H., 1985. The influence of SO2 and NO2 on the growth development and gas exchange ofBetulapendula Roth. New Phytol., 99:417-a30. Gilbert, M.A. and Robson, A.D., 1984. Sulfur nutrition of temperate pasture species. II. A comparison of subterranean clover cultivars, medics and grasses. Aust. J. Agric. Res., 35: 389398. Gould, R.P. and Mansfield, T.A., 1988. Effects of sulphur dioxide and nitrogen dioxide on growth and translocation in winter wheat. J. Exp. Bot., 39: 389-399. Griffith, S.M. and Campbell, W.F., 1987. Effects of sulfur dioxide on nitrogen fixation, carbon partitioning and yield components in snapbean. J. Environ. Qual., 16: 77-80. Haines, S.G., Haines, L.W. and White, G., 1978. Leguminous plants increase sycamore growth in northern Alabama. Soil Sci. Soc. Am. J., 42: 130-132. Hardy, R.W.F. and Havelka, U.D., 1976. Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. In: P.S. Nutman (Editor), Symbiotic Nitrogen Fixation in Plants. Cambridge University Press, Cambridge, pp. 421-439. Heagle, A.S, Body, D.E. and Heck, W.W., 1973. An open top field chamber to assess the impact of air pollution on plants. J. Environ. Qual., 2: 365-368. Hunt, J., 1980. Determination of total sulphur in small amounts of plant material. Analyst, 105: 83-85. Hunt, R., 1979. Plant growth analysis: The rationale behind the use of the fitted mathematical function. Ann. Bot., 43: 245-249. Jones, T. and Mansfield, T.A., 1982. Studies on dry matter partitioning and distribution of ~4Clabelled assimilates in plants ofPhleum pratense exposed to SO2 pollution. Environ. Poilut., 28:199-207. Knight, W.E., Hagedorn, C., Watson, V.H. and Friesner, D.L., 1982. Subterranean clover in the United States. Adv. Agron., 35: 43-48. Lauenroth, W.J., Detling, J.K., Milchunas, D.G. and Dodd, J.L., 1985. Impact of SO2 exposure on the response ofAgropyron smithii to defoliation. J. Range Manag., 38:16-20. McLaughlin, S.B. and McConathy, R.K., 1983. Effects of SO2 and 03 on allocation of ~4C-labelled photosynthate in Phaseolus vulgaris. Plant. Physiol., 73: 630-635. Murray, F., 1985. Changes in growth and quality characteristics of lucerne (Medicago sativa L.) in response to sulphur dioxide exposure under field conditions. J. Exp. Bot., 36: 449457. Murray, F., 1989. Acid rain and acid gases in Australia. Arch. Environ. Contam. Toxicol., 18: 131-136. Noyes, R.D., 1980. The comparative effects of sulphur dioxide on photosynthesis and translocation in bean. Phys. Plant Pathol., 16: 73-9. Okano, K., Ito, O., Takeba, G., Shimiza, I. and Totsuka, T., 1984. Alteration of 13C-assimilate partitioning in plants ofPhaseolus vulgaris exposed to ozone. New Phytol., 97:155-63. Pande, P.C. and Mansfield, T.A., 1985. Responses of spring barley to SO2 and NO2 pollution. Environ. Pollut. (Ser. A), 38: 87-97.
RESPONSEOF SUBTERRANEANCLOVERTO SULPHURDIOXIDE
293
Prasad, B.J. and Rao, D.N., 1982. Relative sensitivity of a leguminous and a cereal crop to sulphur dioxide pollution. Environ. Pollut. (Ser. A), 29: 57-70. Pratt, G.C., Kromroy, K.W. and Krupa, S.V., 1983. Effects of ozone and sulphur dioxide on injury and foliar concentrations of sulphur and chlorophyll in soybean Glycine max. Environ. Pollut. (Ser. A), 32: 91-99. Public Health Department, 1982. Annual Report for the Year Ended 31 December 1982. Public Health Department, Perth, Western Australia. Runeckles, V.C., 1974. Dosage of air pollutants and damage to vegetation. Environ. Cons., l" 305-308. Sheridan, R.P., 1979. Effects of airborne particulates on nitrogen fixation in legumes and algae. Phytopathology, 69:1011 - 1018. Tingey, D.T., Neely, G.E. and Gumpertz, M.L., 1980. The effects of low concentrations of sulfur dioxide on the growth, quality and nitrogen fixation of alfalfa. In: E.M. Preston and D.W. O'Guinn (Editors), The Bioenvironmental lmpact of a Coal-Fired Power Plant. Fifth interim report, Colstrip, Montana, EPA, Corvallis, OR, EPA-600/3-80-052, pp. 279-291. Underwood, E.J. and Gladstones, J.S., 1979. Subterranean clover and other legumes. In: G.H. Burvill (Editor), Agriculture in Western Australia 1829-1979, University of Western Australia Press, Perth, pp. 139-156. Wang, D. and Schaap, W., 1988. Air pollution impacts on plants - current research challenges. ISI Atlas of Science - Animal and Plant Sci., l: 33-39~
283
Elsevier Science Publishers B.V., Amsterdam
Response of subterranean clover in open-top chambers to chronic exposure of sulphur dioxide S.A. Wilson and F. Murray Biological and Environmental Sciences, Murdoch University, Murdoch, Qld., 6150 (Australia) (Accepted for publication 20 March 1990)
ABSTRACT Wilson, S.A. and Murray, F., 1990. Response of subterranean clover in open-top chambers to chronic exposure of sulphur dioxide. Agric. Ecosystems Environ., 32: 283-293. Ten open-top chambers were used to obtain SO2 concentration-response relationships for growth in subterranean clover cv. 'Trikkala', and to study the associated sulphur accumulation. Four-weekold seedlings were exposed to 4, 41, 120, 255 or 515 nl 1-1 of SO2 for 77 days, 4 h day -t. Response variables measured included leaf dry weight, stem weight, runner length, leaf number, average leaf weight, leaf: stem ratio and leaf sulphur concentration. Significant reductions in growth and changes in assimilate distribution occurred at 120 nl 1-1 and higher concentrations;leaf sulphur concentration increased by ~ 70 and 100% at 41 and 120 nl 1-1, respectively.
INTRODUCTION
Subterranean clover ( Trifolium subterraneam L. ) is a winter growing, selfpollinating forage annual which is native to Mediterranean Europe and parts of Asia and Africa. Deliberate use as a pasture legume began early this century in Australia; and in the 1920s in the United States (Knight et al., 1982; Collins and Gladstones, 1984). The value of subterranean clover is in its soil improvement characteristics, its supply of good quality forage and its provision of income from the production of clover seed. In both the United States and Australia the energy crisis and the subsequent high price of mineral nitrogen have added to the importance of subterranean clover, as has the increased competition for land resources amongst commodity crops. In many cases the competition has resulted in pasture crops being grown on thin, marginal soils which are undesirable for other crops; subterranean clover has proved to be an ideal species for these conditions (Knight et al., 1982). More recently, research has been pursuing the potential use of subterranean clover as a nitrogen source for forest plantings (Haines et al., 1978). In addition to supplying nitrogen it is desirable as it provides weed 0167-8809/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
284
S.A. WILSON AND F. MURRAY
control and, as it is low growing and grows during the cool season in Australia, it does not compete with tree seedlings. Underwood and Gladstones (1979) report that subterranean clover has been a key factor in the improvement of soil fertility in Australia and that both crop and livestock production have increased dramatically in the regions in which it has been grown. The net financial benefits are difficult to estimate precisely but Collins and Gladstones (1984) state that its value, in terms of soil nitrogen accumulation alone, may be of the order of $A400 million annually. Sulphur dioxide is an increasingly important pollutant in Australia. SO2 emissions increased from 1.4 million t year -~ in 1976 (Davey, 1980)to 2.2 million t year -~ in 1986 (Murray, 1989). By 1993, SO2 emissions are estimated to be 2.5 million t year- ~ (F. Murray, unpublished data, 1989). However, the economic implications of this trend for Australian agriculture are largely unknown. Agricultural production is affected by air pollution which occurs at significant concentrations over broad regions in developed and developing countries (Wang and Schaap, 1988 ). Because of the obvious great economic importance of subterranean clover and because very little is known about the impact of air pollutants on it, this study was initiated. The objective of the study was to evaluate the concentration-response relationships of subterranean clover exposed to a range of SO2 concentrations and the associated shoot sulphur accumulation. Open-top chambers were used for the study to enable realistic field conditions to be simulated. MATERIALS AND METHODS
Fumigation chambers Ten cylindrical open-top chambers (Heagle et al., 1973 ) were used for fumigation, each 3 m in diameter and 2.4 m tall, consisting of a rigid aluminium frame covered by UV-treated PVC plastic. The upper half of the frame was covered by a single layer of PVC plastic and the lower half was covered by a double thickness of the PVC envelope with the inner layer perforated by holes 25 m m in diameter. Air was drawn by a fan through a dust filter and then forced along a duct, into the chamber through the holes in the lower envelope and then out through the open top. The output of the fan was 1 m 3 s - l enabling ~ 3.5 air changes m i n - 1.
Pollutant dispensing and monitoring Sulphur dioxide was delivered to the inlets of the fumigation chambers after being mixed with dry air and controlled by means of a regulator and series of
RESPONSEOF SUBTERRANEANCLOVERTO SULPHURDIOXIDE
285
TABLE l Treatment characteristics of the SO2 exposure regime in open-top chambers. All concentrations are in nl l - t Treatment
1 2 3 4 5
Fumigated 4-h average conc.
4 41 120 255 515
Standard deviation
4 31 59 104 155
Percentile 10th
90th
2 3 40 111 318
9 79 191 383 677
needle valves. Pollutant monitoring was conducted with a time-sharing system whereby each chamber was sampled for 12 min every 228 min, at approximately canopy height. The concentration of SO2 was determined using a Thermo Electron, Series 43 pulsed fluorescent ambient SO2 analyzer, calibrated using a Thermo Electron, Model 145 calibrator and NBS traceable permeation tubes. Data were recorded with a Starlog portable data logger (Unidata, Australia), with a chart recorder used as a backup. Table 1 displays the characteristics of the fumigation regime with five SO2 treatments, each duplicated. Statistical analysis of the monitoring data, as outlined subsequently, confirmed the validity of the duplicate treatment chamber pairs, at the 0.05 level of probability. Monitored background concentrations at the experimental site of SO2 were < 5 nl l- 1, annual average NOx concentrations were < 6 nl l- ~of which > 90% was NO. Ozone concentrations were believed to be low, as the annual average O3 concentration, measured in the Central Business District of Perth 15 km from the site, was 10 nl l-1 (Public Health Department, 1982).
Cultural practices and growth conditions Seeds of subterranean clover ( T. subterraneum L. cv. 'Trikkala' ) were sown in peat jiffy pots on 30 May 1988, in a glasshouse. Seedlings were inoculated with a commercial inoculum (Nodulaid, Sydney) and later thinned from three to one per pot. Four weeks after sowing they were removed from the glasshouse and 11 plants were randomly assigned to each of l0 chambers. The ground had been pretreated with the herbicide Roundup (Monsanto, active constituent Glyphosate) and mixed with a potting mix (2 sand: 3 sawdust: 3 wood fibre fines: 4 pinebark) and fertilizer containing both macro- and micronutrients. Plants were sprayed once during the fumigation with the insecticide Rogor
286
S.A. WILSON AND E MURRAY
(Chemspray, active constituent Dimethoate) to protect against insect damage. Irrigation took place to supplement natural rainfall, when necessary. Plants were exposed to one of the five SO2 treatments (Table 1 ) for 4 h day -1, 7 days week -1 from 10:00 to 14:00 h for 77 days. The selection of exposure duration and time attempted to simulate atmospheric inversion break-up conditions. The average day length during the period of fumigation was 10 h 47 min and the mean daily total irradiation ( _+SD) was 2827 ( 1088 ) W h m-2. Temperature and relative humidity were measured throughout the exposure period using thermohygrographs in Stevenson screens. The mean ambient daily maximum and minimum temperatures were 19.2 and 9.4 ° C, respectively, and the corresponding relative humidities were 96.0 and 55.8%. Mean temperatures were ~ 1 °C higher inside the chambers than outside and relative humidity was ~ 1.5% lower inside the chambers than outside.
Harvest procedure At harvest, plants were cut at the soil line; leaf number and length of the longest runner were recorded; leaves and stems were separated and then placed in an oven at 70°C and dried to constant weight. Stem and leaf dry weight were later measured. Leaf material was ground to pass a 40 mesh sieve in a Wiley hammer mill prior to total sulphur concentration determination, using the oxygen flask combustion method of Hunt ( 1980 ).
Statistical analysis Prior to statistical analysis, data for all growth variables were transformed to natural logarithms to stabilize variances and because growth is considered exponential (Hunt, 1979). Unfortunately, due to a technical malfunction, plants in one of the duplicate chambers treated with 515 nl 1-1 of SO2 were damaged and the data were discarded. Fumigation monitoring data were statistically analysed using Statview 512 + (Abacus Concepts Inc, 1986 ). Analysis of Variance (ANOVA) was followed by a Fisher's Protected Least Significant Differencetest of the means to indicate duplication of fumigation regimes. As data from duplicate chambers were not significantly different from one another, they were pooled for the descriptive statistics. Regression analysis (Statview 512 + package ) was used to develop concentration-response functions for the response of subterranean clover to SO2. Analyses were conducted on data from individual plants. ANOVA was used to determine treatment effects. Homogeneity of variance was tested using Cochran's C-test. A Duncan's Multiple Range test was used to determine dif-
RESPONSE
OF SUBTERRANEAN
CLOVER
TO SULPHUR
287
DIOXIDE
ferences between response variable means at the 0.05 level of probability. These analyses used the SPSS-X version 3.0 package installed on a U n i x / Microvax system. RESULTS Concentration-response functions were established to depict the effect of SO2 on the growth of subterranean clover cv. 'Trikkala' (Fig. 1 ). The mathe81
10-
,
i
,
,
,
,
,
,
,
,
i
,
o
C
10. 91
3
71
51 ~1.5~
~q 1
.s.I
C
OI
.
,..
.
.. .
.
.
.
,
,
,
,
,
,
,
,
,
10.
8
g ,l.
............ i
~ 6.
~ .54
i ~ .3! 2.
.
o.
-
.
ui
.
0
.
.
.
10o SOt
.
2oo Concentration
300
4o0 (nL/L)
5~
6oo
4. 3.5
~
3 2.5 2.
~1.51 .5 0
1(30 200 300 400 SO2 Conce~ttraUon (nL/L)
500
600
Fig. 1. Concentration-res0onse curves for subterranean clover cv. 'Trikkala' exposed to SO2 in open-top chambers.
288
S.A. WILSON AND F. MURRAY
TABLE 2
Concentration-response functions and correlation coefficients to describe the effects of SO2 on subterranean clover cv. 'Trikkala', in open-top chambers
Response variable
Equation
Correlation coefficient
Probability ( P )
(R) Length ( c m )
y = e ( - 0 . 0 0 4 x + 3.04) y = e ( - 0 . 0 0 8 x + 5.54) y = e ( - 0 . 0 1 2 x + 8.19) y=e(-0.012x+8.28) y = e ( - 0 . 0 1 2 x + 8.95) y=e( -0.004x+2.67 ) y=0.001x+0.33
Leaf number Leaf weight ( m g ) Stem weight ( m g ) Yield ( m g ) Average leaf wt. ( m g ) Sulphur conc. (%)
-0.928 -0.935 -0.901 -0.918 -0.942 -0.874 +0.883
<0.001 < 0.001 <0.00t <0.001 < 0.001 < 0.001 < 0.001
TABLE 3
Arithmetic mean values of response variables and the results of the Duncans Multiple Range Test SO2 concen- Length Leaf Stem (cm) number weight (g)
weight
Total weight
Average leaf Leaf:stem Sulphur weight ratio conc.
(g)
(g)
(mg)
4 41 120 255 515
3.35 a 3.03 a 0.89 b 0.22 ¢ 0.01 c
7.94 a 6.89 a 1.66 b 0.35 c 0.03 ¢
14.10. 12.96" 8.74 b 5.35 c 0.57 a
tration (nl 1 - ' )
19.2" 19.1 ~ 13.7 b 9.0* 3.00
234 a 224" 91 b 39 c 4~
4.50* 3.84 ~ 0.90 b 0.16 c 0.03 a
Leaf
(%) 0.73 a 0.76 a 1.01 b 1.16 b 0.14 ~
0.24 a 0.41 b 0.48 c 0.55 a 0.70*
Means followed by the same superscripts are not significantly different at the 0.05 level of probability.
matical functions and the correlation coefficients of these curves are listed in Table 2. After preliminary analyses, a first degree polynomial exponential model was considered the most appropriate model for the data. ANOVA was performed on the data to characterize statistically significant individual treatment effects to supplement the clearly defined trends in the data established by regression analysis. Regression analyses of the data indicated a highly significant (P<0.001) negative linear relationship between SO2 concentration and all of the response parameters. The exception was leaf sulphur concentration, which displayed a highly significant ( P < 0.001 ) positive relationship. Analysis of variance revealed that 41 nl 1- ~ SO2 had no statistically significant effect on the growth parameters analysed. However, the leaf sulphur concentration was increased by 70% (Table 3 ). Exposure to 120 nl 1- x of SO2, or higher, significantly reduced all growth parameters and further increased leaf sulphur concentration (see Table 3 ). The runner length was least affected, being reduced by 30%. The average individual leaf weight and leaf number were reduced by 38 and 61%, respec-
RESPONSE OF SUBTERRANEAN CLOVER TO SULPHUR DIOXIDE
289
tively; this resulted in a 73% decrease in the total leaf weight per plant. Stem weight decreased more substantially, showing an 80% reduction. The leaf to stem ratio increased from ~ 0.7 to ~ 1.0 as a result of the SO2 induced changes in leaf and stem weight. The significant increase in the ratio is the result of a greater decrease in stem weight in comparison with the decrease in leaf weight. In all response variables, except leaf: stem ratio, 255 and 515 nl 1-i of SO2 had a significantly greater effect than 120 nl 1-1. DISCUSSION
The results indicate that under these experimental conditions the concentration at which a significant growth reduction in subterranean clover cv. 'Trikkala' occurs is 120 nl 1- ~SO2 for a 4-h period; a concentration frequently occurring in some parts of Australia. Near an industrial area at Kalgoorlie (Western Australia) maximum 3-h average concentrations for each month in 1987 and 1988 ranged between 150 and 700 nl 1-1, the mean monthly maximum 3-h average concentrations for 1987 and 1988 were 380 and 315 nl 1-~, respectively (EPA, 1989 ). Clearly, SO2 can have quite dramatic effects on the growth of subterranean clover. As subterranean dover is a widely grown fodder crop, changes in shoot biomass are particularly important. The changes in biomass were the result of changes to both leaf and stem dry weight; stem dry weight, however, was reduced by SO2 to a greater extent than leaf dry weight. Many studies have reported that atmospheric pollution can affect the distribution ofphotosynthate in plants. Predominantly a change in the root:shoot ratio occurs, reflecting a greater proportion of assimilate being allocated to the shoots, mainly to the leaves, at the expense of the roots. The difference in allocation has been measured as changes to the root: shoot ratio on a dry weight basis (Ashenden, 1979; Freer-Smith, 1985; Murray, 1985; Pande and Mansfield, 1985; Gould and Mansfield, 1988 ) or as a reduced carbohydrate movement out of leaves or into roots using radiotracer studies (Noyes, 1980; Jones and Mansfield, 1982; McLaughlin and McConathy, 1983; Gould and Mansfield, 1988 ). Changes in assimilate distribution may be an adaptive mechanism within the plant to maintain the relative growth rate in conditions of reduced carbohydrate availability (Darrall, 1989 ). However, with increasing concentrations of pollutants plants become unable to maintain growth, as a result of injury to the physiological processes and enhanced rates of respiration for repair and detoxification processes (Murray, 1985; Darrall, 1989 ). In the current experiment a change in resource partitioning occurred, represented by changes to the leaf: stem ratio. The control plants and those exposed to 41 nl l -I SO2 had a leaf:stem ratio of ~0.7. Plants exposed to 120 and 255 nl l- ~SO2 displayed significant growth reductions and the leaf: stem
290
S.A. WILSON AND F. MURRAY
ratio became ~ 1.0. This could be explained by a greater proportion of assimilates being retained by the leaves at the expense of the stem, as an adaptive mechanism to maximize photosynthetic potential. Other studies have reported that the retention of assimilate by leaves is also at the expense of the roots (Ashenden, 1979; Jones and Mansfield, 1982; Okano et al., 1984; FreerSmith, 1985; Gould and Mansfield, 1988 ). Assimilate distribution changes observed in plants exposed to atmospheric pollutants can be attributed to one or more of the following: a malfunctioning of the phloem loading processes; reduced gaseous exchange, therefore a reduction in photosynthetic carbon fixation; or greater demand for assimilate at the source (Cooley and Manning, 1987; Griffith and Campbell, 1987). Active nitrogen fixing nodules are strong sinks for photo-assimilates (Hardy and Havelka, 1976); therefore, SO2 induced changes in assimilate distribution in subterranean clover could markedly reduce its nitrogen fixing capacity, and hence, have a significant impact on agriculture. Very little is known about the effects of SO2 on nitrogen fixation in legumes, however, most studies undertaken to date report its inhibitory effects. Sheridan ( 1979 ) fumigated soya bean with SO2 for 14 days continuously and found a 25, 60 and 75% reduction in nitrogen fixation at 50, 100 and 1000 nl 1- ~exposure, respectively. Griffith and Campbell ( 1987 ) exposed soya bean plants for 5 days, 4 h day -~ to 18/tmol SO2 m -3 (433 nl 1-1 ) and found no effect on specific nodule activity (SNA); however, exposure to 36/~mol SO2 m -3 (866 nl 1-~ ) significantly reduced (SNA) by 59%. Tingey et al. (1980) found that exposure of lucerne throughout its growing season to ~ 160 and 215 nl 1-~ SO2 significantly reduced nitrogen fixation but 21 and 65 nl 1had no effect. Prasad and Rao ( 1982 ) suggest that leguminous plants are more sensitive to SO2 than grasses. Perhaps the impact of SO2-induced assimilate distribution changes on nitrogen fixation contributes to the sensitivity. Associated with the growth reductions in subterranean clover was a substantial increase in leaf sulphur concentration. Sulphate uptake by roots is the main source of sulphur for the synthesis of sulphur-containing compounds but plants are able to use atmospheric SO2 absorbed through the leaves (Cowling and Lockyer, 1976). Runeckles (1974) and Fowler and Cape (1982) suggest that plant response is closely related to pollutant absorbed, rather than just concentration or duration of exposure. The results of this experiment indicate that leaf sulphur concentration provides a useful estimate of net SO2 absorbed, as a very close relationship (R = 0.88 ) was displayed between sulphur concentration of subterranean clover leaves and ambient SO2 concentration. This direct relationship has been found in other plant species (Pratt et al., 1983; Lauenroth et al., 1985; Bytnerowicz et al., 1987; Adaros et al., 1988). Gilbert and Robson (1984) define the critical sulphur concentration (that required for 90% of the maximum yield) for 'Trikkala' to be 0.20%; the con-
RESPONSE OF SUBTERRANEAN CLOVER TO SULPHUR DIOXIDE
291
trol plants in this experiment had a leaf sulphur concentration of 0.24%, and 41 nl l - 1 SO2 increased this to 0.41%. The 70% increase in sulphur concentration, however, did not correspond with a significant reduction in growth. Significant growth reductions occurred at SO2 concentrations of 120 nl l-1 or higher, where the leaf sulphur concentration was increased by at least 100%. This indicates a possible threshold response to increased foliar sulphur concentration as plant sulphur status moves through the adequate range to luxury and then into toxicity. CONCLUSION Subterranean clover showed quite dramatic reductions in above-ground plant dry weight in response to SO2 at concentrations that occur in Australia. Reductions occurred in both stem and leaf dry weight, but to differing extents, reflecting changes in assimilate distribution in favour of leaf tissue. Associated with the decrease in growth was an increase in leaf sulphur concentration. Clearly, these effects could have a significant impact on agriculture due to the reduction in fodder production and the possible reduction in nitrogen fixation and the associated losses in soil quality. ACKNOWLEDGEMENTS Support was provided under the National Energy Research D e v e l o p m e n t and D e m o n s t r a t i o n Programme, which is administered by the Australian Department o f Primary Industries and Energy. Gratitude is expressed to Fergus O ' H a r a for provision of expert technical assistance.
REFERENCES Adaros, G., Weigel, H.J. and Jager, H.J., 1988. Effects of sulphur dioxide and acid rain alone or in combination on growth and yield of broad bean plants. New Phytol., 108: 67-74. Ashenden, T.W., 1979. Effects of SO2 and NO2 pollution on transpiration in Phaseolus vulgaris L. Environ. Pollut., 18: 45-50. Bytnerowicz, A., Olszyk, D.M., Kats, G., Dawson, P.J., Wolf, J. and Thompson, C.R., 1987. Effects of SO2 on physiology, elemental content and injury development of winter wheat. Agric. Ecosystems Environ., 20: 37-47. Collins, W.J. and Gladstones, J.S., 1984. Breeding to improve subterranean clover in Australia. In: R.F. Barnes, P.R. Ball, R.W. Brougham, G.C. Marten and D.J. Minson (Editors), Forest Legumes for Energy-Efficient Animal Production. Proceedings of a Trilateral Workshop, Palmerston North New Zealand, April 30-May 4, U.S. Dept. of Agriculture, Agricultural Research Service, St. Paul, MN, pp. 308-315. Cooley, D.R. and Manning, W.J., 1987. The impact of ozone on assimilate partitioning in plants: A Review. Environ. Pollut., 47:95-113. Cowling, D.W. and Lockyer, D.R., 1976. Growth of perennial ryegrass (Lolium perenne L. ) exposed to a low concentration of sulphur dioxide. J. Exp. Bot., 27:411-417.
292
S.A. WILSON AND F. MURRAY
Darrall, N.M., 1989. The effects of air pollutants on physiological processes in plants. Plant Cell Environ., 12: 1-30. Davey, T.R.A., 1980. Anthropogenic sulfur balance for Australia, 1976. In: J.R. Freney and A.J. Nicolson (Editors), Sulfur in Australia. Australian Academy of Science, Canberra, pp. 7587. EPA, 1989. Air Quality Measurements 1987 and 1988 Perth, Kwinana, Kalgoorlie and Port Hedland, Technical Series No. 29. Environmental Protection Authority, Perth, Western Australia, pp. 8-9. Fowler, D. and Cape, J.N., 1982. Air pollutants in agriculture and horticulture. In: M.H. Unsworth and D.P. Ormrod (Editors), Effects of Gaseous Air Pollution in Agriculture and Horticulture. Butterworths Scientific, London, pp. 3-26. Freer-Smith, P.H., 1985. The influence of SO2 and NO2 on the growth development and gas exchange ofBetulapendula Roth. New Phytol., 99:417-a30. Gilbert, M.A. and Robson, A.D., 1984. Sulfur nutrition of temperate pasture species. II. A comparison of subterranean clover cultivars, medics and grasses. Aust. J. Agric. Res., 35: 389398. Gould, R.P. and Mansfield, T.A., 1988. Effects of sulphur dioxide and nitrogen dioxide on growth and translocation in winter wheat. J. Exp. Bot., 39: 389-399. Griffith, S.M. and Campbell, W.F., 1987. Effects of sulfur dioxide on nitrogen fixation, carbon partitioning and yield components in snapbean. J. Environ. Qual., 16: 77-80. Haines, S.G., Haines, L.W. and White, G., 1978. Leguminous plants increase sycamore growth in northern Alabama. Soil Sci. Soc. Am. J., 42: 130-132. Hardy, R.W.F. and Havelka, U.D., 1976. Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. In: P.S. Nutman (Editor), Symbiotic Nitrogen Fixation in Plants. Cambridge University Press, Cambridge, pp. 421-439. Heagle, A.S, Body, D.E. and Heck, W.W., 1973. An open top field chamber to assess the impact of air pollution on plants. J. Environ. Qual., 2: 365-368. Hunt, J., 1980. Determination of total sulphur in small amounts of plant material. Analyst, 105: 83-85. Hunt, R., 1979. Plant growth analysis: The rationale behind the use of the fitted mathematical function. Ann. Bot., 43: 245-249. Jones, T. and Mansfield, T.A., 1982. Studies on dry matter partitioning and distribution of ~4Clabelled assimilates in plants ofPhleum pratense exposed to SO2 pollution. Environ. Poilut., 28:199-207. Knight, W.E., Hagedorn, C., Watson, V.H. and Friesner, D.L., 1982. Subterranean clover in the United States. Adv. Agron., 35: 43-48. Lauenroth, W.J., Detling, J.K., Milchunas, D.G. and Dodd, J.L., 1985. Impact of SO2 exposure on the response ofAgropyron smithii to defoliation. J. Range Manag., 38:16-20. McLaughlin, S.B. and McConathy, R.K., 1983. Effects of SO2 and 03 on allocation of ~4C-labelled photosynthate in Phaseolus vulgaris. Plant. Physiol., 73: 630-635. Murray, F., 1985. Changes in growth and quality characteristics of lucerne (Medicago sativa L.) in response to sulphur dioxide exposure under field conditions. J. Exp. Bot., 36: 449457. Murray, F., 1989. Acid rain and acid gases in Australia. Arch. Environ. Contam. Toxicol., 18: 131-136. Noyes, R.D., 1980. The comparative effects of sulphur dioxide on photosynthesis and translocation in bean. Phys. Plant Pathol., 16: 73-9. Okano, K., Ito, O., Takeba, G., Shimiza, I. and Totsuka, T., 1984. Alteration of 13C-assimilate partitioning in plants ofPhaseolus vulgaris exposed to ozone. New Phytol., 97:155-63. Pande, P.C. and Mansfield, T.A., 1985. Responses of spring barley to SO2 and NO2 pollution. Environ. Pollut. (Ser. A), 38: 87-97.
RESPONSEOF SUBTERRANEANCLOVERTO SULPHURDIOXIDE
293
Prasad, B.J. and Rao, D.N., 1982. Relative sensitivity of a leguminous and a cereal crop to sulphur dioxide pollution. Environ. Pollut. (Ser. A), 29: 57-70. Pratt, G.C., Kromroy, K.W. and Krupa, S.V., 1983. Effects of ozone and sulphur dioxide on injury and foliar concentrations of sulphur and chlorophyll in soybean Glycine max. Environ. Pollut. (Ser. A), 32: 91-99. Public Health Department, 1982. Annual Report for the Year Ended 31 December 1982. Public Health Department, Perth, Western Australia. Runeckles, V.C., 1974. Dosage of air pollutants and damage to vegetation. Environ. Cons., l" 305-308. Sheridan, R.P., 1979. Effects of airborne particulates on nitrogen fixation in legumes and algae. Phytopathology, 69:1011 - 1018. Tingey, D.T., Neely, G.E. and Gumpertz, M.L., 1980. The effects of low concentrations of sulfur dioxide on the growth, quality and nitrogen fixation of alfalfa. In: E.M. Preston and D.W. O'Guinn (Editors), The Bioenvironmental lmpact of a Coal-Fired Power Plant. Fifth interim report, Colstrip, Montana, EPA, Corvallis, OR, EPA-600/3-80-052, pp. 279-291. Underwood, E.J. and Gladstones, J.S., 1979. Subterranean clover and other legumes. In: G.H. Burvill (Editor), Agriculture in Western Australia 1829-1979, University of Western Australia Press, Perth, pp. 139-156. Wang, D. and Schaap, W., 1988. Air pollution impacts on plants - current research challenges. ISI Atlas of Science - Animal and Plant Sci., l: 33-39~