Effects of atmospheric sulphur dioxide on microbial activity in decomposing forest litter

Agriculture, Ecosystems and Environment, 33 ( 1991 ) 263-280

263

Elsevier Science Publishers B.V., Amsterdam

Effects of atmospheric sulphur dioxide on microbial activity in decomposing forest litter P.A. W o o k e y I, P. I n e s o n ~ a n d T.A. M a n s f i e l d 2

IMerlewood Research Station, Institute of Terrestrial Ecology, Grange-over-Sands, Cumbria LA 11 6JU, (Gt. Britain). 2University of Lancaster, Institute of Environmental and Biological Sciences, Bailrigg, Lancaster LA 1 4YQ (Gt. Britain). (Accepted for publication 23 July 1990)

ABSTRACT Wookey, P.A., Ineson, P, and Mansfield, T.A., 1991. Effects of atmospheric sulphur dioxide on microbial activity in decomposing forest litter. Agric. Ecosystems Environ., 33: 263-280. At extremely high concentrations, relative to those occurring in the atmosphere, sulphur dioxide (SO2) has known antimicrobial properties. There is also circumstantial evidence, based on field surveys, to indicate that the occurrence and activities of a number of phylloplane fungi and soil microorganisms are correlated with atmospheric concentrations of SO2 occurring in parts of Europe and North America. The results of these studies need to be corroborated by controlled fumigation experiments applying realistic concentrations of SO2. Unfortunately such experiments have been rare. The suggestion that SO2 may be affecting soil microorganisms merits serious consideration because of the fundamental role of these organisms in maintainingsoil fertility, especially in forests. Events in the forest litter layer are considered to be particularly important because it forms an interface between the atmosphere and the soil system. The research described in this paper involved exposing leaf litter (from a Pinus sylvestris L. stand and a mixed deciduous woodland) to arithmetic mean concentrations of SO2 of ~<0.050/tl 1-i in controlled field-based experiments lasting up to 215 days. Fungal cultures, isolated from the pine litter, were also fumigated with ~<0.053 gl 1- l SO2 in laboratory-based studies. Results showed that arithmetic mean concentrations of SO2 as low as 0.015 gl 1-~ significantly reduced microbial activity (respiration) in both pine and deciduous litter in the open-air fumigation experiment. Results should also be interpreted in relation to the peak SO2 concentrations (often considerably higher than arithmetic means) to which the litter was exposed. Pure cultures of Cladosporium cladosporioides (Fres.) de Vries and Coniothyrium olivaceum Bonord, isolated from the litter, were shown to be sensitive to SO2 concentrations of 40.053 #11- ~in laboratory-based fumigations. It is concluded that the dry deposition of SO2 to forest soils may have important implications for nutrient cycling processes and therefore forest productivity and community structure.

INTRODUCTION D e c o m p o s i t i o n fulfils a f u n d a m e n t a l role in e c o s y s t e m n u t r i e n t cycling p r o c e s s e s ( S w i f t et al., 1 9 7 9 ) b y k e e p i n g p l a n t s s u p p l i e d w i t h e s s e n t i a l n u -

0167-8809/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.

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trients. In forest soils the decomposition of organic plant debris to yield inorganic plant nutrients is performed largely by fungi, bacteria and actinomycetes (Jensen, 1974; Millar, 1974). The work of Miller et al. (1979)clearly indicates that decomposition can become a rate-determining step in the nutrient cycling process, with the potential to alter ecosystem primary productivity. Miller et al. (1979) were aware that any factors capable of disturbing or disrupting nutrient cycling processes might have a detrimental impact. One such factor they identified was pollution. The emission of SO2 to the atmosphere has increased dramatically as a consequence of man's activities, and SO2 is now considered to be a major atmospheric pollutant. Estimated global emissions of SO2 from anthropogenic sources amounted to 180 million tonnes during 1985 (Mrller, 1984). SO2 emissions to the atmosphere are generally associated with regions of industrial and urban activity. Such areas include much of Europe and the north-eastern region of North America. Background concentrations of SO2 have been estimated, on the basis of limited monitoring, to be around 0.00020.0008/tl 1-I for the "continental European" atmosphere (Ryaboshapko, 1983 ) and the potential for man-made emissions to increase the SO2 concentrations substantially is evident from the work of Martin (1980) and EMEP (1984). To provide an example of present conditions in industrialized Europe, Ling and Ashmore ( 1987 ) have characterized the mean annual concentrations of SO2 in central England and central West Germany, 0.005-0.012 #1 1-1 and 0.006-0.017/~1 1-~ being representative values for the two regions, respectively. The suggestion that air pollutants could adversely affect soil biological activity and decomposition processes has resulted in a massive international research effort principally into the effects of wet deposition of acid-forming air pollutants (mainly oxides of sulphur and nitrogen) to soils (see Cowling, 1982 ). In contrast little attention has been given to the possibility that the dry deposition of pollutant gases (such as SO2) could be altering the properties of soils. This represents a major gap in contemporary research because dry deposition of SO2 is quantitatively more significant than wet deposition (in acid precipitation ) over large areas of Europe (Moss, 1978; United Kingdom Review Group on Acid Rain, 1987). Williams et al. (1989) identify dry deposition of SO2 to be the principal deposition mechanism over the majority of England, comprising over 60% of total deposited sulphur in the centre of the country. At very high concentrations, relative to those occurring in the atmosphere, SO2 has antimicrobial properties (Couey and Uota, 1961 ) which have been widely employed in the preservation of fruit and vegetable products (Joslyn and Braverman, 1954). At concentrations characteristic of industrialized regions it is less clear whether or not SO2 can alter the physiology and ecology of microorganisms, although a great deal of empirical and circumstantial evi-

EFFECT OF SO2 ON MICROBES IN FOREST LITTER

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dence does suggest that the occurrence and activities of a number of phylloplane and soil microorganisms, and also lichens, are related to ambient concentrations of SO2. Plant pathogenic fungi have received the most attention in this respect (Heagle, 1973; Smith, 1981 ). Studies specifically with soil microorganisms and decomposition processes are exemplified by the work of Lettl (1984), Bewley and Parkinson (1986) and Prescott and Parkinson (1985 ). Much past work has involved examinations of soil properties and processes at various distances along transects away from point sources. Such studies have provided circumstantial evidence that soil processes may be affected by SOz, although they can never conclusively establish that SO2 exposure is the only agent responsible for any observed patterns. Empirical field observations need to be complemented by carefully controlled field- and laboratory-based studies examining the effects of realistic concentrations of SO2 on soil microorganisms and their activities, either in pure culture or naturally occurring in soils. The only experimentally controlled studies in which litter decomposition processes have been examined in relation to realistic SO2 concentrations are those of Dodd and Lauenroth ( 1981 ), Leetham et al. (1983) and Ineson (1983). In all of these examples, decomposition processes, as determined by respirometry (CO2 release) or litter weight loss, were reduced by concentrations of SO2 ranging from 0.035 to 0.249/tl l- I. Unfortunately, the results of these studies provide no information on whether lower concentrations can influence decomposition processes. In the present study it was possible to expose decomposing forest tree litter to controlled concentrations o f SO2 (ranging from an arithmetic mean of 0.005 /tl l- ~ to'a mean of 0.050/A l- 1 ) at an open-air crop fumigation facility at Littlehampton. The experiment provided a unique opportunity to study the impacts of low SOz concentrations on soil microorganisms and their activities. MATERIALS AND METHODS

Open-airfumigation of Scots pine needle litter A full technical description of the open-air fumigation system can be found in McLeod et al., (1985, 1991 ). The system was designed, constructed and maintained by staff of the National Power Technology and Environmental Centre, Leatherhead, and was sited in a 2.6-ha field at the Institute of Horticultural Research, Littlehampton, Sussex (G.R. TQ 045033 ). Although constructed principally to examine interactions between atmospheric SO2 and the growth of winter cereals, the open-air fumigation system also enabled leaf litter decomposition to be studied. Briefly, the system consisted of four field plots, one receiving ambient SO2 exposure (generally less than 0.01/tl 1-~ as

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an annual mean). The remaining three plots contained dispersion pipework to allow enrichment of the atmosphere immediately above them with SO2. Scots pine (Pinus sylvestris L. ) litter was collected from the litter layer of the forest floor in a 32-year-old stand at Gisburn Forest, Lancashire (G.R. SD 750588 ) in October 1985. The litter was air dried after being spread thinly on clean paper in the laboratory and stored at room temperature prior to use in the fumigation experiment. Five g of air-dry litter (4.505 g oven-dry weight) were weighed into each of 72 nylon mesh bags (size 130X 150 mm, 2 mm mesh) which were then closed with polyester thread. On 9 December 1985 three plastic seed trays were placed adjacent to the central sampling area of each plot and filled with vermiculite to approximately 40 m m depth. Eighteen replicate litter bags were placed into each of the four SO2 plots (six bags per tray) and a thin layer of loose P. sy/vestris litter was spread around each bag. The vermiculite moderated the moisture conditions beneath the litter bags by retaining water during dry periods. Small holes were drilled in the bases of the gravel trays to allow excess water to drain. Bags were prevented from blowing off the trays by a wide mesh ( 11 m m ) cotton net over each tray. Trays were left resting on the ground surface during December but were elevated to crop height with adjustable frames as the crop grew. Litter bags were left in the fumigation plots during the growing season of a winter barley (Hordeum vulgate cv. 'Igri' ) crop. They were sampled after 156 INCUBATOR EXTERNAL EXHAUST

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Fig. 1. Laboratory-based apparatus for the determination of microbial respiration rates or exposing fungal cultures to controlled SO2 concentrations.

EFFECT OF SO2 ON MICROBES IN FOREST LITTER

267

and 215 days of exposure and stored at 0-5°C until analyses could be performed. Bags sampled after 156 days (six replicate bags from each plot) were analysed for CO2 release (microbial respiration), chemistry of leachates (Ineson and Wookey, 1988 ) and needle calcium, magnesium, potassium and nitrogen contents. The bags sampled after 215 days were similarly analysed, but additional microbiological investigations were performed (described later) and needle analyses were excluded. Microbial respiration (at 15 °C) was determined by infrared gas analysis (Jarvis and Sandford, 1985 ) after the litters had been brought to field capacity with distilled water. The release of CO2 from the litter was monitored by an ADC model 225 Mk3 plant physiology infra-red gas analyser (IRGA) (The Analytical Development Co. Ltd., Pindar Road, Hoddesdon, Hertfordshire) operated in differential mode and in the absence of SO2 (see Fig. 1 ). Subsequently the litter was oven dried to constant weight at 80°C. All results were expressed on an oven-dry weight basis and results were analysed statistically by analysis of variance (ANOVA) and Tukey's comparison of means test. Where necessary, data were transformed arithmetically prior to performing the tests.

Open-airfumigation of mixed deciduous leaf litter On I 1 November 1986 mixed deciduous leaf litter was collected randomly from the forest floor of Meathop Wood in Cumbria. Meathop Wood is a mixed deciduous coppice-with-standards woodland, located on the northern edge of Morecambe Bay (G.R. SD 436795 ). The tree species comprise Quercus petraea Mattuschka (Liebl.) (sessile oak), Fraxinus excelsiorL. (common ash ), Betula pubescens Ehrh. (downy birch ), Betula pendula Roth (silver birch) and Acer pseudoplatanus (sycamore), with an understorey of Corylus avellana L. (hazel). The leaf litter was collected from the surface soil layer and mainly consisted of intact litter which had fallen that autumn and was relatively fresh. On 26 November 1986 72 litter bags were prepared as described for Scots pine litter above, except that field-weight litter (rather than air dried) was used to fill the bags. The moisture content of the deciduous litter was 80.5%. Once constructed and sealed, litter bags were stored at 0-5 °C prior to use. On 1 December 1986 18 replicate bags were placed into each treatment plot using the same procedures as those for the Scots pine experiment described above. Nine litter bags were collected from each of the experimental plots on each of the two sampling dates, 169 and 204 days from the start of the experiment. Similar analyses and statistical tests were performed as are described for the Scots pine experiment but no mycological investigations were performed with the deciduous litter.

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The laboratory-based respirometry/fumigation system This system was used in two different ways: ( l ) as an apparatus for determining litter respiration rates using Perspex "microcosm" chambers (microcosms modified from the design of Anderson and Ineson, 1982 ) to contain litters (see Fig. l ); respiration was only measured when the SO2 supply was switched off; (2) for fumigating pure cultures of fungi, when the chambers shown in Fig. 2 were attached in place of the microcosm chambers indicated in Fig. 1. The air flow through the system was maintained by a diaphragm p u m p situated subsequent to all analytical stages. Incoming air was passed through activated charcoal to remove ambient SO2, and this "SO2-free" air then flowed into a large sealed plastic bin which served to smooth out any fluctuations in the CO2 concentration. After mixing, the air was humidified and split into two airstreams, each one entering a separate manifold. At this stage SO2-enriched air from a compressed gas cylinder (BOC Special Gases, Deer Park Road, London SW 19 3UF) containing 10 #1 l- l SO2 could be added, for pure culture fumigations only, to one of the manifolds through a needle valve and flow meter. This SO2 was immediately diluted on mixing with the air in the manifold to give concentrations of less than 0.100/A l150 mm C I omp L..

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EFFECT OF SO2 ON MICROBES IN FOREST LITTER

269

Air from the manifolds was drawn either into microcosm chambers or pure culture chambers (Fig. 2 ). Twelve microcosm chambers (and one reference chamber) were connected if the system was being used for respirometry. In contrast, only six pure culture chambers were used for fumigation experiments; three receiving "SOz-free" air and three receiving SO2-enriched air. All chambers were housed within an incubator and air flow rates through each of the chambers were adjustable. The air leaving each chamber was drawn into an ADC Model WA-161-Mk3/12 12-channel gas handling unit which provided a balanced flow through all of the chambers and supplied the selected airstreams from particular chambers to the IRGA if microbial respiration was to be measured. A Meloy Model SA 185-2 flame photometric detection sulphur analyser (Meloy Laboratories Inc., Instruments and Systems Division, 6715 Electronic Drive, Springfield, VA 22151, U.S.A. ) was used to measure concentrations of SO2. Sulphur hexafluoride (SF 6) in air (BOC Special Gases, op. cit. ), at a concentration of 0.085/tl 1- l , was used as a calibration source for the analyser.

Fumigation experiments with fungal pure cultures isolated from Scots pine litter Pure cultures of a Trichoderma sp., Cladosporium cladosporioides (Fres.) de Vries and Coniothyrium olivaceum Bonord, isolated from Scots pine litter from the open-air fumigation experiment (Wookey, 1988 ), were exposed to "SO2-free" ( ~<0.003/~1 1- ~ SO2) or SO2-enriched air within the laboratorybased fumigation system (Fig. 1 ). Each species was studied in a separate fumigation experiment to allow greater replication and to eliminate the possibility of cross-contamination. The fungal type to be exposed to SO2 was grown on 2% malt extract agar (MA) until a substantial radial growth of the colony was achieved. Discs (8 mm diameter) were then cut from the colony edges using a sterile cork borer and 24 of these were placed, colony surface uppermost, on fresh 2% MA in 90-mm-diameter polystyrene Petri dishes. The 24 Petri dishes containing the fungal discs were then used to fill six specially designed Petri plate chambers of the type shown in Fig. 2. Four Petri dishes, inverted with the lids removed, were placed into each chamber using a unidirectional laminar downflow cabinet to minimize contamination. The six fumigation chambers were then connected to the laboratory-based SO2 exposure system (Fig. 1, and described above), three chambers receiving "SO2-free" and the other three SO2-enriched airstreams. Thus, with four plates in each chamber, there were 12 replicates for each treatment. Air flow through each chamber was 1 1m i n - 1giving just over one air change every 2 min. SO2 concentrations were monitored at four positions within the

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chambers (Fig. 2, positions marked with an "X") immediately above the agar surfaces. Fumigations were performed at approximately 15°C (see Fig. 5 for exact temperatures) and were continued for a period sufficient to allow radial colony growth, or colony area, to be determined accurately. Colony margins were marked on the base of each Petri dish and the dishes were then photocopied. Paper "colonies" were then cut from the photocopies and these were passed through a planimeter to determine colony area. ANOVA was used to test for treatment effects. RESULTS

Open-air fumigation of Scots pine needle litter Table 1 gives a summary of the SO2, NO, NO2 and 03 concentrations at the field site during the course of the experiment. Microbial respiration rates (CO2 release from the litter), expressed in relation to SO2 exposure, indicated that microbial activity decreased in association with greater concentrations of SO2 (Fig. 3 ). For the first sampling, after 156 days of experimental exposure, the low S O 2 treatment (0.016 gl 1-1) showed a 9.8% reduction in microbial respiration ( P < 0.05 ) compared with that in the ambient SO2 plot (0.008 #11- ~). The results obtained after 215 clays of experimental exposure confirmed those of the first sampling although a more marked difference was observed TABLE 1 Characterization of SO2, NO, NO2 and 03 exposure of Scots pine needle litter by open-air fumigation. Values are arithmetic means of hourly mean concentrations (/tl 1- l ) with standard deviation in parentheses Treatment plot

Duration ofexposure

156 days

215 days

Mean

Standard deviation

Maximum hourly value

Mean

Standard deviation

Maximum hourly value

Ambient SO2 Low SO2 Medium SO2 High SO2

0.008 0,016 0.031 0.050

(0,009) (0,015) (0,021) (0.033)

0.071 0.140 0.143 0.174

0.007 0.014 0.030 0.048

(0.008) (0.014) (0.021) (0.033)

0.071 0.140 0.143 0.174

Ambient O3 Ambient NO Ambient NO2

0.018 0.002 0.015

(0.012) (0.007) (0.012)

0.074 0.108 0,068

0.020 0.001 0.012

(0.015) (0.006) (0.011 )

0.112 0.108 0.068

Details of data collection and examples of typical frequency distributions of hourly mean values are given by McLeod et al. ( 1991 ).

EFFECT O F SO2 ON MICROBES IN FOREST LITTER

271

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Fig. 3. Microbial respiration rates of Scots pine needle litter exposed to SO2 by open-air fumigation. Mean respiration values (n = 6) for litter from each treatment are plotted against mean SO2 concentration and joined by lines. Differences in respiration rates between the two sampling dates were not tested statistically. Statistical comparisons refer to differences between the fumigation plots only: means with the same letters are not significantly different from each other (P> 0.05).

between litter from the ambient SO2 plot and that from the low S O 2 treatment plot: an increase of the SOz concentration from a mean of 0.007 to 0.014/~1 1-1 was associated with an 18.4% reduction in microbial respiration ( P < 0.001 ). Further increments of the SO2 concentration were not associated with such a marked reduction in microbial respiration. The timescales involved in this experiment were not of sufficient length to enable weight loss of litter bag contents to be used as a reliable measure of decomposition rates.

Open-airfumigation of mixed deciduous leaf litter Table 2 shows the concentrations of SO2, NO, NO2 and 03 recorded at the field plots during the experiment. After 169 days, microbial respiration rates showed a general reduction with increasing concentrations of SO2, although statistical significance was only attained between litter exposed to ambient air (0.006/111-' SO2) and that exposed to 0.040/tl 1-' SO2 (Fig. 4). Thirtyfive days later microbial respiration was similarly reduced in association with increasing concentrations of SO2 but statistical significance was achieved between the ambient plot (0.005/A 1-1 ) and all of the SO2 treatment plots (P<0.05).

Owing to the length of the fumigation period, and also the problems of comparing initial fresh litter weights with final oven-dry weights, we consider

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TABLE 2 Characterization of SO2, NO, NO2 and 03 exposure of mixed deciduous leaf litter by open-air fumigation. Values are arithmetic means of hourly mean concentrations (/tl l- ~) with standard deviation in parentheses Treatment plot

Duration of exposure 169 days

204 days

Mean

Standard deviation

Maximum hourly value

Mean

Standard deviation

Maximum hourly value

Ambient SOz Low SO2 Medium SO2 High SO2

0.006 0.016 0.030 0.040

(0.007) (0.015) (0.026) (0.033)

0.062 0.232 0.252 0.171

0.005 0.015 0.029 0.040

(0.007) (0.014) (0.026) (0.033)

0.062 0.232 0.252 0.172

Ambient O3 Ambient NO Ambient NO 2

0.018 0.004 0.011

(0.014) (0.011 ) (0.008)

0.093 0.101 0.056

0.020 0.003 0.011

(0.015) (0.010) (0.008)

0.093 0.101 0.056

Details of data collection and examples of typical frequency distributions of hourly mean values are given by McLeod et al. ( 1991 ).

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0101 0102 0103 0104' 0105 Mean SO2 concentration (pI I "1) Fig. 4. Microbial respiration rates of mixed deciduous leaf litter exposed to SO~ by open-air fumigation. Mean respiration values (n = 9) for litter from each treatment plot are plotted against mean SO2 concentration and joined by lines. Differences in respiration rates between the two sampling dates were not tested statistically. Statistical comparisons refer to differences between the fumigation plots only: means with the same letters are not significantly different from each other (P> 0.05).

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273

TABLE 3 Open-air fumigation of deciduous litter. Oven-dry weights of litter bag contents after 169 and 204 days of open-air fumigation, expressed as a percentage of the initial estimated oven-dry weights at the start of fumigation. Values in parentheses are the standard error of the mean ( n = 8 or 9 for each treatment plot for each sampling date) Treatment plot

Duration ofexposure 169 days

Ambient SO2 Low SO2 Medium SO2 High SO2

204 days

Mean % of initial weight

Standard error

Mean % of initial weight

Standard error

95.4 a 89.3 a 86.6 a 89.2 ~

( 3.88 ) (3.12) ( 1.35 ) ( 3.42 )

99. I a 92.0 ab 81.6 b 84.0 b

( 2.90 ) (3.23) (3.16 ) (2.64)

Statistical comparisons refer to differences between the fumigation plots only; differences between the two sampling dates were not tested. Means with the same superscripts are not significantly different from each other ( P > 0.05 ).

microbial respiration a more appropriate and reliable measure of microbial activity than rates of litter weight loss. It is interesting to note, however, that after 204 days of open-air fumigation, rates of litter weight loss were highest in plots receiving the highest concentrations ( > 0.029/d l - l ) of SO2 (Table 3 ). These results seem to contradict the respirometry data since they suggest that decomposition was more rapid at higher concentrations of SO2. The implications are considered further in the Discussion section.

Pure culture fumigations Laboratory-based fumigation experiments with the fungal pure cultures (Figs. 5 (a), (b) and (c)) indicated that radial colony growth of the Trichoderma sp. was unaffected by 62 h of exposure to SO2-enriched air. In contrast, the radial growth of both C. cladosporioides and C. olivaceum was significantly reduced by exposure to SO2-enriched air. The exact concentrations of SO2 used differed in each fumigation e x l ~ m e n t , as did the length of time for which the cultures were exposed to SO2 (see Fig. 5 ): C. olivaceum and C. cladosporioides grew considerably more slowly than the Trichoderma sp. and they therefore had to be exposed for much longer to enable accurate determinations of SO2 effects. For this reason broad comparisons of response between species are of limited applicability.

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P.A. W O O K E Y ET AL.

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Fig. 5. Pure culture fumigations. (a) Mean colony area ( n = 12) for the Trichoderma sp. after 62 h exposure to 0.002 gl 1-~ (control) or 0.035-0.050 #11- t SO2 (SO2). Temperatures during incubation were 14.5-15.5°C. (b) Mean colony radius ( n = 12) for Cladosporium cladosporioides after 12 days exposure to 0.001-0.002/A 1- t (control) or 0.015-0.035 #11-1802 ( 802 ). Temperatures during incubation were 15.2-15.6°C. (c) Mean colony area ( n = 12) for Coniothyriurn olivaceum after 10 days exposure to 0.001-0.003 gl 1- ~ (control) or 0.034-0.053 #1 l - ~ SO2 (SO2). Temperatures during incubation were 15.5-16.3°C. pH values are for the agar surface after fumigation. Vertical bars show_+ 1 SE (**P< O.O1, ***P< 0.001 ). DISCUSSION

These experiments indicated that environmentally realistic concentrations of SO2 were inhibitory to the metabolism of litter decomposers. Microbial respiration (CO2 release) was used as an index of rates of carbon mineralization (Reiners, 1968) and significant reductions in CO2 release from decomposing Scots pine and mixed deciduous leaf litter were observed during controlled open-air fumigations (Figs. 3 and 4). These effects were evident at arithmetic mean concentrations of SO2, (0.014-0.016 #1 1-l ) known to occur over large areas of Europe. It must be stressed, however, that peak SO2 concentrations, often considerably higher than the arithmetic means (see Tables 1 and 2), may have contributed significantly to the observed effects. Carbon dioxide efflux has been used by other workers to assess the impact of SO2 on microbial activity. Many studies have been of the empirical "field survey" type and they are of interest because they have often revealed that microbial respiration is reduced at more polluted sites. Examples of such an approach include the work of Langkramer and Lettl (1982, cited by Lettl,

EFFECT OF SO2 ON MICROBES IN FOREST LITTER

275

1984) in the Slavkov Forest of Czechoslovakia, and that of Bewley and Parkinson (1986) and Prescott and Parkinson (1985 ) around the West Whitecourt sour gas-processing plant in Alberta, Canada. Both of these studies revealed that microbial respiration was lowest at the "most polluted" sites, or nearest to the gas-processing plant. Unfortunately, SO2 concentration data were not published and it is therefore difficult to relate the results to other forest areas. There have been only few controlled field- or laboratory-based studies which have used SO2 concentrations comparable with those occurring in ambient air, and the most relevant studies to compare with the present one are those of Dodd and Lauenroth ( 1981 ), Ineson (1983) and Leetham et al. (1983). The work of Dodd and Lauenroth ( 1981 ) and Leetham et al. (1983) formed part of a wider project examining the potential impacts of SO2 pollution in the Great Plains region of North America. Controlled open-air fumigation was used in a mixed grass prairie ecosystem, and the decomposition of western wheatgrass (Agropyron smithii) was studied in four test plots having seasonal geometric mean concentrations of SO2 ranging from < 0.010 to 0.076 /zl 1-1. Results revealed that concentrations as low as 0.046/A 1- ~significantly reduced rates of litter weight loss. Ineson ( 1983 ) found that experimentally controlled fumigation of decomposing Pinus nigra litter with a mean of 0.035 /zl 1-1 SO2 over 82 days resulted in a 33% reduction in microbial respiration. During the present study it became apparent that rates of CO2 efflux from litter provided an accurate and reliable measure of microbial activity. In contrast, the use of litter weight loss to infer rates of microbial activity was subject to a number of limitations, due both to the length of the fumigation period and also to the "resource quality" of the litter (Swift et al., 1979 ). In the open-air fumigation of P. sylvestris needles, the duration of the experiment was insufficient to enable weight loss to be used to measure decomposition rates. The experiment with the mixed deciduous leaf litter highlighted the significance of factors other than microbial activity: the more rapid weight loss of litter exposed to the higher concentrations of SO2 (Table 3 ) was probably the outcome of more rapid chemical "weathering" of the litter; this occurring as a result of the dry deposition of SO2 with the subsequent formation of sulphate and acidity under moist conditions (Ineson and Wookey, 1988 ). Such an effect was noted by Abrahamsen et al. (1980) for decomposing Pinus contorta needles receiving artificial acid rain of pH 3 (acidified with sulphuric acid). Ineson ( 1983 ) has made similar observations on decomposing petioles of Pteridium aquilinum. Rates of litter weight loss are therefore not simply a reflection of microbial activity alone, and this may be particularly true in studies assessing the biological impacts of acidifying pollutants. None the less, the studies of Dodd and Lauenroth ( 1981 ) and Leetham et al. ( 1983 ), in which rates of litter weight loss were decreased by exposure to SO2, suggest that chemical weathering of the litter was less significant under their experimental conditions than reductions in microbial activity in response to SO2.

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Data obtained in the present study broadly corroborate the findings of a number of other research projects, both of the field survey type and also of the controlled fumigation type. The present study differs from previous studies, however, because arithmetic mean concentrations of SO2 of 0.014-0.016 #11-1 were demonstrated to have a significant impact on microbial activity, these concentrations being considerably lower than those reported by other authors. Unfortunately the biological significance of peak hourly SO2 concentrations (Tables 1 and 2 ) cannot be assessed from the data, although the potential impacts of such peaks should not be overlooked. It should also be stressed that these data cannot be directly related to conditions within a forest ecosystem. Some of the more important issues are considered below. The open-air fumigation environment, an arable field, obviously contrasted markedly in microclimate with a forest floor. Mycological studies, however, revealed that components of the fungal flora colonizing the Scots pine needles (Wookey, 1988 ) were consistent with those to be expected at a forest floor (Millar, 1974). A more serious limitation concerns the concentrations of SO2 that occur beneath forest canopies, since there are few published records of monitoring data from beneath or within such canopies (Dasch, 1987 ). It is therefore not clear whether the concentrations used in this study were representative of the forest floor environment. The ecological significance of the results will become clearer once pollution climates within forests are better characterized. The ambient plot at Littlehampton experienced a mean concentration of 0.005-0.008 #11-1 SOe during the fumigation studies (Tables 1 and 2) and such concentrations may be higher than those background concentrations thought to characterize "clean" air (Ryaboshapko, 1983 ). The experiments therefore provided no data on the dose-response relationship below 0.005 #1 1-1 SOa. It is well known that saprophytic fungi are of overriding importance in the decomposition of conifer litter (Millar, 1974). The observation that exposure to SOa altered rates of microbial respiration in both Scots pine and mixed deciduous litter therefore justified some laboratory-based fumigation experiments with pure cultures of decomposer fungi. The three species studied were isolated from Scots pine litter and are representative of important and widespread groups of saprophytes. Indeed Abrahamsen et al. ( 1980 ) isolated Trichoderrna harzianum, a Coniothyrium species and Cladosporium macrocarpum from Pinus contorta litter and found them to be active cellulosedecomposing fungi. The exposure of pure cultures to SO2 suggested that radial growth of the Trichoderma sp. was unaffected by the SO: (Fig. 5 ( a ) ) . It is possible that the Trichoderma sp. tested was indeed tolerant of atmospheric concentrations of SO> It must be stressed, however, that the agar surface on which the colonies were growing was different, both physicochemically and biologically, compared with the pine litter substrate from which the fungus was isolated. A

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major limitation of any fumigation experiments in which heterotrophic organisms are grown on artificial media is that much of the deposited SO2 may be rapidly detoxified by complexation reactions with the sugars contained in the medium (Ingram, 1948; Joslyn and Braverman, 1954). This also applies to the fumigation experiments with C. cladosporioides and C. olivaceum. The exposure of pure cultures of C. cladosporioides and C. olivaceum to SO2 (Figs. 5 (b) and (c), respectively ) did, none the less, result in significant reductions in radial growth of both species, even though they were grown on a sugar-containing medium. Both C. cladosporioides and C. olivaceum were fumigated for considerably longer than the Trichoderma sp. and this may also, to some extent, explain why these species appeared more sensitive to SO2. Comparisons between species are therefore of limited value but the important point to note is t h a t radial growth of two species was clearly demonstrated to be sensitive to low concentrations of SO2. The results of these pure culture fumigations agree with observations made by Magan and McLeod ( 1988 ) who have shown that in vitro exposure of certain leaf yeasts and filamentous phylloplane fungi to between 0.050 and 0.200/211-1 for 24 h reduced growth as measured by colony size (leaf yeasts), percentage spore germination or germ-tube extension (filamentous fungi ). Figure 5 shows the pH of the agar surfaces (measured by antimony electrode) after each fumigation experiment and it is clear that SO2 acidified the agar. Sulphur dioxide may exert its toxicity through the action of its solution products - sulphurous acid ( H 2 5 0 3 ) , bisulphite ( H S O 3 - ) and sulphite (SO32- ) - or it may be toxic by its ability, once oxidized to sulphate (SO42- ), to generate protons (acidify). In the present study it was unclear whether acidity or direct SO2 toxicity was responsible for the results obtained, both in the open-air fumigations and the pure culture fumigations. The difficulties of distinguishing effects of acidity from effects of aqueous SO2 have been discussed by Lettl (1984). In the open air fumigation experiments it is also possible that SO2 exerted an effect by altering the base status (principally the Ca and Mg content) of the litter since decomposition rate is related to base status (see Williams and Gray, 1974). Indeed, both Scots pine litter and deciduous litter were markedly depleted of bases after exposure to SO2; this is thought to result from the oxidation of SO2 to sulphate at the litter surfaces during moist conditions with the subsequent leaching of base cations (Ca 2÷, Mg 2+ and K ÷ ) in response to the leaching of mobile sulphate anions (Ineson and Wookey, 1988 ). If litter samples had been removed more frequently from the field site it may have been possible to gain a clearer insight into the interrelationship between SO2, litter pH, litter base status and microbial activity. Such studies would also have been of value in determining whether microbial activity was similarly affected by SO2 at other stages in the process of decomposition.

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CONCLUSIONS Studies using the open-air fumigation system at Littlehampton revealed that mean concentrations of SO2 as low as 0.015 Ftl 1- ~ significantly reduced the rate of CO2 efflux (a measure of the activity of decomposer organisms) from decomposing Scots pine litter and mixed deciduous leaf litter. Such SO2 concentrations occur over large regions of industrialized Europe at monitoring sites in the open, although it is less clear whether these concentrations commonly occur beneath forest canopies. The full implications of the results can only be assessed when more data become available concerning the pollution regimes within forests. Three fungal pure cultures, isolated from the Scots pine litter, were exposed to ~<0.053/zl 1-1 SO2 in vitro, and these studies revealed that the radial growth of Cladosporiurn cladosporioides and Coniothyrium olivaceum was sensitive to SO2 whereas a Trichoderma sp. seemed to be unaffected. The data obtained during the course of this study have indicated that the dry deposition of SO2 to forest litter layers may result in alterations in the metabolic activities of some decomposer organisms. If decomposition processes are impaired in forest ecosystems the ultimate result may be a reduction in primary productivity, with long-term implications for ecosystem structure and functioning. The data are also of indirect relevance to agricultural systems since they add weight to the hypothesis that the occurrence and activities of certain crop pathogenic fungi may also be affected by SO2. ACKNOWLEDGEMENTS We would like to thank the Natural Environment Research Council for funding this research. P.I. would also like to acknowledge the support of the Department of Environment. The field-based experiments were conducted at the National Power Technology and Environmental Centre open-air fumigation system at Littlehampton, and we are particularly grateful to Dr. A.R. McLeod for enabling us to work at the site. We are also indepted to Dr. Juliet Frankland and Doreen Howard of Merlewood Research Station for their advice on mycology and statistical analyses respectively. Peter Williams and Philip Smith (Institute of Environmental and Biological Sciences, Lancaster University) constructed and helped to design the chambers for exposing fungal cultures to SO2.

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