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Experimental evidence that soil fauna enhance nutrient mineralization and plant nutrient uptake in montane grassland ecosystems
PERGAMON
Soil Biology and Biochemistry 31 (1999) 1007±1014
Experimental evidence that soil fauna enhance nutrient mineralization and plant nutrient uptake in montane grassland ecosystems Richard D. Bardgett *, Kin F. Chan School of Biological Sciences, 3.614 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK Accepted 9 December 1998
Abstract This microcosm study is concerned with understanding those factors which regulate ecosystem processes of nutrient cycling and plant productivity in a montane grassland ecosystem. We examined the eects of dierent groups of soil fauna, namely bacterial-feeding nematodes and Collembola, on nutrient mineralization (N and P) in an acid, organic soil taken from a montane grassland in the Peak District National Park, United Kingdom. We also examined whether faunal in¯uences on nutrient release, a measure of nutrient mineralization, resulted in changes in nutrient uptake and biomass production of an indigenous montane grass species (Nardus stricta (L.)). We found that in the presence of Collembola, and when nematodes and Collembola were combined, N mineralization, nutrient leaching and shoot N contents of N. stricta was signi®cantly increased relative to a defaunated control. We also found that net P mineralization and leaching increased (although not signi®cantly) in the presence of both nematodes and Collembola, resulting in a signi®cant increase in shoot P content of N. stricta. The presence of nematodes alone, which were largely bacterial-feeders, had no eect on the mineralization of N or P, or shoot nutrient content. We suggest that dierences in the eect of faunal treatments on nutrient mineralization are related to the feeding strategies of the added fauna, and to their consequent eect on the size of the soil microbial biomass. The treatments that increased N mineralization and plant nutrient content (N and P) also signi®cantly reduced plant growth (shoot and root). We suggest that high NH4+ ±N concentrations in the soil solution of Collembola treatments inhibited the growth of N. stricta and that the growth of other grassland species may bene®t from this improvement in nutrient availability. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction It is becoming increasingly apparent that soil fauna play a signi®cant role in soil processes through their in¯uence on the quantity, composition and activity of soil micro¯ora. Soil animals may aect microbial communities directly, by feeding on various microorganisms, or indirectly, by mixing and channelling detritus, dispersing microbial propagules and excreting nutrient* Tel.: +44-161-2755959; fax: +44-161-2753938; e-mail: rbardget @fs1.scg.man.ac.uk. Present address: Department of Biological Sciences, Institute of Environmental and Natural Sciences, University of Lancaster, Lancaster LA1 4YQ, UK.
rich wastes (Bardgett and Griths, 1997). Generally, microcosm studies suggest that these soil faunal:microbial interactions result in increased rates of soil nutrient mineralization (Ineson et al., 1982; Bardgett et al., 1998) and an increased uptake of available inorganic nutrients by plants (Ingham et al., 1985). Montane ecosystems in Britain are dominated by soils of high organic matter status and low pH. Primary productivity in these ecosystems is almost entirely dependent on nutrients released by decomposition and, therefore, the activities and interactions of the soil biota (Bardgett and Leemans, 1996). Previous studies have provided evidence that the soil fauna is an important factor regulating soil microbial abundance and community structure, and hence processes
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of decomposition and nutrient release in these harsh ecosystems. A laboratory study by Bardgett et al. (1993a) showed that the feeding activities of an abundant Collembola (Onychiurus procampatus) in¯uenced the growth and activity of a decomposer fungus common to sub-montane grasslands. Likewise, in a study of montane grasslands, Bardgett et al. (1998) showed that both fungal-feeding Collembola and a largely bacterial-feeding nematode community increased the total biomass and activity of micoorganisms in two soils of dierent organic matter content. These biotic interactions, however, were shown to have no eect on net nutrient mineralization (N and P), presumably due to the re-utilization Ð or immobilization Ð of inorganic nutrients by the larger microbial biomass (Visser et al., 1981; Bardgett et al., 1993a; Bardgett et al., 1998). The immobilization of nutrients is likely to be exacerbated by the high C-to-N ratio of these organic soils (Bardgett et al., 1993a). What is less clear, is how these soil biotic interactions in¯uence net nutrient mineralization in the face of competition from plants. Indeed, most previous microcosm experiments on soil biotic interactions in grasslands have been conducted in the absence of plants (e.g. Woods et al., 1982; Bakonyi, 1989; Bardgett et al., 1993a; Bardgett et al., 1998) where nutrient availability will be regulated largely by microbial mineralization±immobilization dynamics. In the ®eld, plant roots may compete eectively with soil microorganism for inorganic nutrients that have been excreted by the microbial-feeding fauna (Ingham et al., 1985). Therefore, the question remains, does faunal grazing on microorganisms in organic, montane grassland soils translate into (a) increased nutrient mineralization and, therefore, (b) increased plant nutrient uptake and growth of indigenous grassland plants? An additional question is whether dierent groups of soil fauna have a greater in¯uence on nutrient mineralization when combined, or when added to microcosms independently. It has been hypothesized that most mineralization of nutrients in soil is facilitated by the action of the most ecient microbial grazers, the microfauna, and that microarthropods only contribute signi®cantly to these processes when the activities of microfauna are inhibited in some way (Vreeken-Buijs et al., 1997). To test these hypotheses, we selected the fungal-feeding Collembola O. procampatus, which makes-up some 95% of total numbers of Collembola in these montane soils (Bardgett et al., 1993a), and a mixed community of nematodes, dominated by bacterial-feeders, taken from the ®eld site (Bardgett et al., 1998). The nematode community that develops in these microcosm systems has been characterized by Bardgett et al. (1998). The microcosm study focuses on N and P release and their uptake by Nardus stricta (L.), a ubiquitous grass species of sheep-grazed mon-
tane grasslands in the United Kingdom (Rodwell, 1992). 2. Methods and materials 2.1. Sites and soils In June 1997 soil was collected from the surface horizon (to 10 cm depth) of a grassland site in the Peak District National Park, roughly 500 m altitude and 24 km east of Manchester, United Kingdom. The site is a Nardus stricta dominated grassland with a humic stagnopodzol soil (Hafren series; pH 4.4, organic matter 47%, C-to-N ratio 40). Soil was passed through a 6 mm sieve and stored at 48C until use. In order to eliminate the fauna, the sieved soil from both sites was subjected to air drying, followed by freezing at ÿ808C for 24 h, and then further air drying for 24 h (Bardgett et al., 1998). Subsequent samples taken from the defaunated soil revealed that the procedure had completely eliminated the multicellular fauna (nematodes, microarthropods and larger fauna). Sheep dung collected from the same grassland site, was defaunated by freezing for 24 h at ÿ808C. 2.2. Microcosms The experiments were carried out in microcosms, as described by Anderson and Ineson (1982). Microcosms were prepared by weighing 40 g (dry mass) of defaunated soil into opaque plastic containers (3.5 cm dia., 10 cm height). To all microcosm chambers, 0.6 g dry mass defaunated sheep dung, collected from the ®eld site, was added to the surface of the soil. Our previous studies have shown that the addition of sheep dung to these soils is essential to ensure rapid colonisation and reproduction of added fauna (Bardgett et al., 1998). Thereafter, microcosms were reinoculated with micro¯ora by adding 10 ml of a soil suspension made from the appropriate soil type; the suspension was made by mixing 1 kg of sieved soil in 2 l of sterile distilled water which was then centrifuged at 2500 rpm for 5 min to obtain a supernatant that was ®ltered through a 10 mm sieve. Soils were adjusted to moisture contents of 60% (w/w) (equivalent to a water potential of ÿ5 kPa) by adding sterile distilled water. Microcosms were then incubated at 158C (average summer 0±5 cm soil depth temperature; Bardgett and Leemans, 1996) for 10 d to allow establishment of micro¯ora. Pots were then planted with ®ve seedlings of N. stricta which had been germinated from surface sterilized seeds obtained from Emorsgate Seeds, King's Lynn, UK. Soil fauna used in the experiment were collected as described by Bardgett et al. (1998). Brie¯y, nematodes
R.D. Bardgett, K.F. Chan / Soil Biology and Biochemistry 31 (1999) 1007±1014
were extracted from samples of bulked soil using Whitehead and Hemming (1965) trays to give an inoculum of 1200 per microcosm which was then applied in 2 ml of distilled water. The nematode community that develops in these montane soil microcosms has been shown to be dominated by bacterial-feeding nematodes (rhabditids, plectids and cephalobids), however, a few plant-feeding nematodes and fungal-feeding aphelenchids also persisted (Bardgett et al., 1998). For Collembola, the species Onychiurus procampatus was used, since it is a known fungal-feeder, and is present in large numbers in upland soils (Bardgett et al., 1993a,b,c, 1998). Brie¯y, specimens of O. procampatus were collected by gentle heat extraction from large soil samples taken from each of the sites, and were stored in Petri dishes with a base of moist plaster of Paris and fed on brewers' yeast extract. Seventeen O. procampatus individuals of sizes 2±3 mm were added to each of the Collembola treatments. Numbers of soil animals added were based on mean ®eld densities recorded by Bardgett et al. (1993a). Fauna were added to microcosms to achieve the following treatments: (1) microbes only (CONTROL); (2) microbes and nematodes (N); (3) microbes and Collembola (C), and: (4) microbes, nematodes and Collembola (N + C). Each treatment was replicated three times, and sucient microcosms were established to allow four destructive samplings over the experimental period. The establishment of nematode populations was checked by extracting nematodes from 10 g fresh soil at each sample date using the methodology of Whitehead and Hemming (1965). We were unable to check population numbers of Collembola due to the small amounts of soil available for extraction (Bardgett et al., 1998). However, their survival was con®rmed by visual inspection. Microcosms were arranged in a randomized block design and incubated for 60 d in a controlled environment facility at 158C, with a light±dark period of 16±8 h. After 15, 30, 45 and 60 d, microcosms of each treatment were destructively sampled. Soil moisture contents were maintained throughout the experiment by adding distilled water to a pre-determined mass. At each sample date, soils and plant material were analyzed. 2.3. Total microbial biomass The extractable microbial C of 10 g dry weight equivalent samples was measured using the fumigation±extraction technique (Vance et al., 1987). Organic C in ®ltered extracts was determined by the acid dichromate oxidation method (Tate et al., 1988). Microbial C (dierence between extractable C from fumigated and unfumigated samples) was converted to microbial biomass C using a kEC-factor of 0.35 (Sparling et al., 1990).
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2.4. Nutrient mineralization Nutrient mineralization was measured as the release of mineral N (NO3ÿ ±N and NH4+ ±N) and PO3ÿ 4 -P from microcosm chambers (Ineson et al., 1982). This was done by repeatedly leaching (3 times) microcosms with 50 ml distilled water and analysing the leachates for concentrations of mineral-N using a Skalar continuous ¯ow autoanalyser (Ineson et al., 1982; Bardgett et al., 1993c). 2.5. Plant growth and nutrient content At each sample date, root and shoot biomass (fresh weight) was determined for individual microcosms. Shoot tissue was then oven-dried for 24 h at 808C, ground and weighed into digestion tubes. Shoot tissue N and P contents were determined for d 30, 45 and 60 using the methodology of Thomas et al. (1967). Values are expressed as mg g ÿ 1 dry plant material. 2.6. Statistical analysis Data were checked for normality and the eects of animal treatments and time on each of the variables were determined using analysis of variance (ANOVA). Dierences between means were tested using Fisher's PLSD test. All data are presented as arithmetic means 2 standard errors. 3. Results 3.1. Establishment of faunal populations The number of nematodes in the N and N + C treatment varied signi®cantly (F = 30.1, P < 0.0001) over the experimental period, being lowest at d 45 and highest at d 60 (Fig. 1). There was no signi®cant dierence in numbers of nematodes between the two inoculation treatments on any sample date (Fig. 1). The addition of Collembola (the N + C treatment) had no eect on numbers of nematodes. 3.2. Soil microbial biomass In all treatments, there was a strong temporal trend of increasing soil microbial biomass (F = 35.6, P < 0.0001) over the experimental period (Table 1). At d 30, the independent treatments of nematodes (N) and Collembola (C) increased soil microbial biomass by 62% and 28%, respectively, relative to the control (F for the treatmenttime interaction = 4.0, P = 0.0017: Fig. 2A). At d 60, the presence of both nematodes and Collembola (N + C) increased soil microbial biomass by 26%, relative to the control
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high in the N + C treatment than in the other treatments, this eect was not signi®cant (Fig. 3). The leaching of inorganic N and P was negatively correlated with soil microbial biomass (for NH4+ ±N: r =ÿ 0.34, P = 0.018; for NO3ÿ ±N r =ÿ 0.35, P = 0.014; for total mineral±N r =ÿ 0.42, P = 0.003, and; for PO3ÿ 4 ±P r =ÿ 0.45, P = 0.0013).
3.4. Plant biomass and nutrient content Plant biomass (shoot and root) and nutrient content (N and P) increased over the experimental period in all treatments (Table 1). Over all sample dates, shoot biomass was signi®cantly (F = 4.9, P = 0.006) aected by animal treatment, being reduced in both the Collembola (C) (28%) and the nematode and Collembola (N + C) (32%) treatments, relative to the control (Fig. 4). The presence of nematodes alone had no eect on shoot or root biomass. Root biomass was signi®cantly (F = 4.1, P = 0.013) aected by the animal treatments, being reduced by both the Collembola (C) (39%) and the combined nematode and Collembola (N + C) treatments (43%), relative to the control (Fig. 4). None of the treatments aected plant shoot:root biomass ratios (data not shown: F = 2.4, P = 0.082). Shoot N and P contents were signi®cantly (F = 6.6, P = 0.0003 and F = 8.8, P < 0.0001, respectively; Fig. 5) aected by the animal manipulation treatments. The presence of individual animal groups had no eect on shoot N content. However, the presence of nematodes and Collembola (N + C) signi®cantly increased shoot N content by 54% relative to the control. This eect was most pronounced at d 45 (F for the treatmenttime interaction = 3.7, P = 0.002; Fig. 5). Shoot P content also only increased, relative to the control, in the presence of both nematodes and Collembola (N + C). This eect was most pronounced at d 45 (F
Fig. 1. Temporal changes in numbers of nematodes recovered from microcosms of the nematode (N) and nematode and Collembola (N + C) treatments. Values are means2 S.E.
(Fig. 2A). At this time the other treatments had no eect on soil microbial biomass. 3.3. Nutrient mineralization For all inorganic nutrients measured there was a distinct temporal trend of declining concentration in leachates over the experiment period (for NH4+ ±N, NO3ÿ ±N and PO3ÿ 4 ±P, F = 80.5, 11.9 and 13.9, respectively, and P < 0.0001 in all cases: Table 1). The release of NH4+ ±N (F = 11.5, P < 0.0001: Fig. 2B) and NO3ÿ ±N, F = 3.3, P < 0.03: Fig. 3) in leachates was increased by the presence of both nematodes and Collembola (N + C), relative to the control. However, for NH4+ ±N this eect was only evident at d 30 (F for the treatmenttime interaction = 6.5, P < 0.0001: Fig. 2B). The presence of Collembola (C) also signi®cantly increased NH4+ ±N release relative to the control and the nematode only (N) treatment at d 30, 45 and 60 (Fig. 2B). Collembola had a similar eect on NO3ÿ ±N, however, this eect was not signi®cant (Fig. 3). Although PO3ÿ 4 ±P release was over twice as Table 1 Temporal eects of animal treatments Sample date (d)
Microbial biomass (mg C g ÿ 1 dry soil) NH4+ ±N leachates (mg g ÿ 1) NO3ÿ ±N leachates (mg g ÿ 1) ÿ1 PO3ÿ ) 4 ±P leachates (mg g Shoot biomass (mg fresh weight) Root biomass (mg fresh weight) Shoot N (mg g ÿ 1 dry wt) Shoot P (mg g ÿ 1 dry wt)
15
30
45
60
LSD
5586 (405)a 12.1 (0.5)a 2.2 (0.1)a 36.7 (6.3)a 10 (4)a 5 (1)a
9470 (731)b 14.5 (2.4)b 1.9 (0.1)ab 7.5 (6.6)b 26 (2)b 14 (1)a 10.1 (1.0)a 1.0 (0.2)a
7029 (646)c 3.9 (0.6)c 1.6 (0.2)b 0.0 (0.0)b 77 (7)c 38 (4)b 20.8 (1.5)b 1.9 (0.2)b
11990 (547)d 0.4 (0.3)d 0.7 (0.3)c 0.0 (0.0)b 114 (9)d 97 (12)c 9.1 (1.2)a 0.4 (0.0)c
1359 2.14 0.5 13.5 14 16 3.1 0.4
Data are means (standard errors) and values with the same letter are not signi®cantly dierent at the P < 0.05 level. LSD = least signi®cant dierence between means.
R.D. Bardgett, K.F. Chan / Soil Biology and Biochemistry 31 (1999) 1007±1014
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Fig. 2. Temporal changes in (A) soil microbial biomass, and (B) the amount of NH4+ ±N leached from microcosms in the dierent animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola). Values are means 2S.E. For soil microbial biomass, F = 3.98 and P = 0.0017 for the interaction between treatmenttime; For NH4+ ±N leaching, F = 6.5 and P < 0.0001 for the interaction between treatmenttime.
Our data con®rm the hypothesis that in montane grassland ecosystems, soil fauna can enhance nutrient mineralization and nutrient uptake by indigenous grass species. We have shown that in the presence of fungalfeeding Collembola, and when nematodes (largely bacterial-feeders) and Collembola are combined, N mineralization (measured as mineral N release) and shoot N
content of N. stricta was signi®cantly higher than in a defaunated control. We also showed that P mineralization (measured as P03ÿ 4 ±P release) increased Ð although not signi®cantly Ð in the presence of both nematodes and Collembola, resulting in a signi®cant increase in shoot P content of N. stricta. These data cast doubt over the ecological value of results from previous `plant-free' microcosm studies of soils and litters from similar ecosystems. Such studies showed that in the absence of plants, the same fauna had no eect on N or P mineralization, due presumably to the reutilization of nutrients by the microbial biomass of soils and litters with high C-to-N ratio's (Bardgett et
Fig. 3. Eects of animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola) on the amount of N03ÿ ±N (mg g ÿ 1 soil) and PO3ÿ 4 ±P, leached from microcosms. Values are means2S.E. For each measure, values with the same letter are not signi®cantly dierent.
Fig. 4. Eects of animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola) on shoot and root biomass. Values are means2S.E. For each measure, values with the same letter are not signi®cantly dierent.
for the treatmenttime interaction = 2.4, P = 0.03; Fig. 5). 4. Discussion
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Fig. 5. Temporal changes in (A) shoot N content, and (B) shoot P content in the dierent animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola). Values are means 2S.E. For shoot N, F = 3.7 and P = 0.0017 for the interaction between treatmenttime; For shoot P, F = 2.4 and P = 0.03 for the interaction between treatmenttime.
al., 1993a, 1998). The data presented here suggest that in montane grasslands, plants roots can compete very eectively with soil microbes for these nutrients which have been released by the feeding activities of fauna Ð via the microbial loop (Clarholm, 1985). However, we have also shown that large quantities of these nutrients released by faunal grazing are not taken up by plants or immobilized by microbes, but remain weakly bound to colloidal surfaces or in soil solution, where they are susceptible to leaching. The ability of plant roots to compete with microbes for available nutrients is of particular note in the light of the extremely large microbial biomass, and hence potential nutrient sink, in these montane soils. Soil microbial biomass values ranged from 5,400±14,000 mg g ÿ 1 soil, representing some 2±6% of the total organic C in the soil. These values are in line with estimates from ®eld studies of sub-montane and montane grassland soils (Bardgett and Leemans, 1996; Bardgett et al., 1997) and are three-to-four times higher than would be expected in less organic, lowland grassland soils (Lovell et al., 1995). Combined with the very high C-to-N ratio of these montane soils (approximately 40) one would expect these conditions to lead to net N immobilization by the large and extremely N limited soil microbial community. The presence of a nematode community alone, which was dominated by bacterial-feeders (Bardgett et al., 1998), had no eect on the mineralization of N or P, or shoot nutrient content. This ®nding is contrary to many studies which have shown that the rate of net soil N mineralization is greater in the presence of bacterial-feeding nematodes (Coleman et al., 1977; Anderson et al., 1981; Woods et al., 1982), resulting in increased soil N availability and N uptake by plants (Ingham et al., 1985). Our data also do not support the hypothesis of Vreeken-Buijs et al. (1997) that most
nutrient mineralization in soils is facilitated by the grazing of microorganisms by microfauna, and that microoarthropods only in¯uence these processes when the activities of microfauna are inhibited in some way. In contrast, we have shown that in this montane soil the positive eects of bacterial-feeding nematodes on N mineralization only occur when fungal-feeding microarthropods are present. This synergistic eect, which was most apparent at d 30, may be related to the increased grazing pressure on the soil microbial community in the combined nematode and Collembola treatment, which may have reduced microbial growth and hence the immobilization of N. Indeed, at this date the presence of nematodes alone doubled the size of the soil microbial biomass relative to the defaunated control, presumably leading to increased microbial immobilization of N. In the nematode and Collembola treatment, however, increased grazing pressures kept the soil microbial biomass at a similar level to the defaunated control, possibly leading to reduced N microbial immobilization and increased cycling of N through the animal excretion pathway. Additional support for this thesis comes from the ®nding that over the experimental period there was signi®cant negative correlation between soil microbial biomass and the leaching of NH4+ ±N, NO3ÿ ±N and PO3ÿ 4 ±P from microcosm chambers. The ®nding that Collembola had a greater eect on N mineralization than did nematodes, is likely to be related to the make-up of the soil microbial community and the feeding strategies of the animals added to the microcosms. As already stated, previous studies of similar soils showed that the nematode community which develops after the addition of a mixed inoculum to defaunated soils is dominated by fast-growing species which tend to be bacterial-feeders (Bardgett et al., 1998). In contrast, the collembolan (O. procampa-
R.D. Bardgett, K.F. Chan / Soil Biology and Biochemistry 31 (1999) 1007±1014
tus) used in these experiments is a known specialist fungal-feeder (Bardgett et al., 1993a,b,c). Since the microbial community of these acid, organic soils is largely fungal-dominated (Bardgett, 1996; Bardgett et al., 1997), and that NH4+ ±N Ð the main mineral N form in these soils Ð is preferentially used by fungi (Bardgett et al., 1993c), it is likely that the feeding activities of Collembola will have a greater eect on N mineralization. In the nematode only treatment, therefore, any N released through bacterial grazing is likely to have been re-utilized by the ungrazed, and hence growing fungal biomass. This argument could also be extended to the combined nematode and Collembola treatment, where the grazing activities of the Collembola may have suppressed fungal growth, allowing bacteria to proliferate. This may have provided an increased food resource for bacterial-feeding nematodes, which in turn may have increased N release through the nematode excretion pathway. Clearly, to test these ideas it would be necessary to examine, in future experiments, changes in the biomass and structure of the soil microbial community. Surprisingly, the animal treatments that increased N mineralization and plant nutrient content (N and P) also signi®cantly reduced plant growth (shoot and root). This ®nding is contrary to several studies which have shown fauna to increase plant productivity, an eect ascribed to enhanced nutrient mineralization (Ingham et al., 1985; Alphei et al., 1996). The most plausible explanation is that increased NH4+ ±N availability in the soil of the two Collembola treatments (C and N + C) restricted the growth of N. stricta. Indeed, several studies have reported restricted growth of N. stricta when grown in solution culture at high NH4+ ±N concentrations (Bradshaw et al., 1964; Gigon and Rorison, 1972; Troelstra et al., 1995). As in this study, restricted plant growth has also been reported to be associated with an increased N content of shoot material (Troelstra et al., 1995). We therefore suggest that high NH4+ ±N concentrations in the soil solution of Collembola treatments and its subsequent uptake inhibited the growth of N. stricta. In conclusion, our study supports the thesis that the activities of soil fauna enhance nutrient mineralization in montane grassland ecosystems. We also have some evidence to suggest that this increase in soil nutrient availability leads to an increase in nutrient leaching from soil and enhanced nutrient uptake by an indigenous grass species. The ®nding that these positive eects of fauna on soil nutrient turnover had a negative eect on plant productivity is likely to be a species-speci®c response of N. stricta. It is probable that in montane grasslands the productivity of other more N responsive grasses, such as Agrostis capillaris and Festuca ovina (Bradshaw et al., 1964), will increase as a result of faunal-mediated increases in soil nutrient availability. Our
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data also show that dierent faunal groups, and their combinations, can alter these processes of nutrient ¯ux in upland grasslands, due presumably to dierential eects on the size, structure and dynamics of the soil microbial community. Further studies need to explore the eects of more complex trophic interactions, for example with the inclusion of predators (sensu Mikola and SetaÈlaÈ, 1998), to fully understand the role of soil fauna in montane and upland grassland ecosystems.
Acknowledgements We thank the Peak District National Park for permission to collect soils from the study site. We also thank Gary Porteous, Angela Jones, Lisa Cole, Pisit Chareonsudjai, Harriet Waters and Alissar Chaker for general help in the laboratory.
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Bardgett, R.D., Whittaker, J.B., Frankland, J.C., 1993c. The eect of collembolan grazing on fungal activity in dierently managed upland pastures: a microcosm study. Biology and Fertility of Soils 16, 255±262. Bradshaw, A.D., Chadwick, M.J., Jowett, D., Snaydon, R.W., 1964. Experimental investigations into the mineral nutrition of several grass species. Journal of Ecology 52, 665±676. Coleman, D.C., Cole, C.V., Anderson, R.V., Blaha, M., Campion, M.K., Clarholm, M., Elliot, E.T., Hunt, H.W., Schaefer, B., Sinclair, J., 1977. Analysis of rhizosphere±saprophage interactions in terrestrial ecosystems. Ecological Bulletins 25, 299±309. Clarholm, M., 1985. Interactions of bacteria, protozoa and plants leading to mineralization of soil nitrogen. Soil Biology & Biochemistry 17, 181±187. Gigon, A., Rorison, I.H., 1972. The response of some ecologically distinct plant species to nitrate- and to ammonium±nitrogen. Journal of Ecology 60, 93±102. Ineson, P., Leonard, M.A., Anderson, J.M., 1982. Eect of collembolan grazing upon nitrogen and cation leaching from decomposing leaf litter. Soil Biology & Biochemistry 14, 601±605. Ingham, R.E., Trofymow, J.A., Ingham, E.R., Coleman, D.C., 1985. Interactions of bacteria, fungi, and their nematode grazers: eects on nutrient cycling and plant growth. Ecological Monographs 55, 119±140. Lovell, R.D., Jarvis, S.C., Bardgett, R.D., 1995. Soil microbial biomass and activity in long-term grassland: eects of management change. Soil Biology & Biochemistry 27, 969±975. Mikola, J., SetaÈlaÈ, H., 1998. No evidence of trophic cascades in an experimental microbial-based soil food web. Ecology 79, 153±164. Rodwell, J.S., 1992. Grassland and Montane Communities. British Plant Communities, Vol. 3. Cambridge University Press, Cambridge.
Sparling, G.P., West, A.W., Feltham, C.W., Reynolds, J., 1990. Estimation of soil microbial C by a fumigation±extraction method: use on soils of high organic matter content and reassessment of the kEC-factor. Soil Biology & Biochemistry 22, 301±307. Tate, K.R., Ross, D.J., Feltham, C.W., 1988. A direct method to estimate soil microbial biomass C: eects of experimental variables and some dierent calibration procedures. Soil Biology & Biochemistry 20, 329±335. Thomas, R.L., Sheard, R.W., Myer, J.R., 1967. Comparison of conventional and automated procedures for nitrogen, phosphorus and potassium analysis of plant material using a single digestion. Agronomy Journal 59, 240±243. Troelstra, S.R., Wagenaar, R., Smant, W., 1995. Nitrogen utilization by plant species from acid heathland soils. 1. Comparison between nitrate and ammonium nutrition at constant low pH. Journal of Experimental Botany 46, 1103±1112. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology & Biochemistry 19, 703±707. Visser, S., Whittaker, J.B., Parkinson, D., 1981. Eects of collembolan grazing on nutrient release and respiration of a leaf litter inhabiting fungus. Soil Biology & Biochemistry 13, 215±218. Vreeken-Buijs, M.J., Geurs, M., De Ruiter, P.C., Brussard, L., 1997. The eects of bacterivorous mites and amoebae on mineralization in a detrital based below-ground food web ± microcosm exepriment and simulation of interactions. Pedobiologia 41, 481±493. Whitehead, A.G., Hemming, J.R., 1965. A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Annals of Applied Biology 55, 25±38. Woods, L.E., Cole, C.V., Elliot, E.T., Anderson, R.V., Coleman, D.C., 1982. Nitrogen transformations in soil as aected by bacterial:microfaunal interactions. Soil Biology & Biochemistry 14, 93± 98.
Soil Biology and Biochemistry 31 (1999) 1007±1014
Experimental evidence that soil fauna enhance nutrient mineralization and plant nutrient uptake in montane grassland ecosystems Richard D. Bardgett *, Kin F. Chan School of Biological Sciences, 3.614 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK Accepted 9 December 1998
Abstract This microcosm study is concerned with understanding those factors which regulate ecosystem processes of nutrient cycling and plant productivity in a montane grassland ecosystem. We examined the eects of dierent groups of soil fauna, namely bacterial-feeding nematodes and Collembola, on nutrient mineralization (N and P) in an acid, organic soil taken from a montane grassland in the Peak District National Park, United Kingdom. We also examined whether faunal in¯uences on nutrient release, a measure of nutrient mineralization, resulted in changes in nutrient uptake and biomass production of an indigenous montane grass species (Nardus stricta (L.)). We found that in the presence of Collembola, and when nematodes and Collembola were combined, N mineralization, nutrient leaching and shoot N contents of N. stricta was signi®cantly increased relative to a defaunated control. We also found that net P mineralization and leaching increased (although not signi®cantly) in the presence of both nematodes and Collembola, resulting in a signi®cant increase in shoot P content of N. stricta. The presence of nematodes alone, which were largely bacterial-feeders, had no eect on the mineralization of N or P, or shoot nutrient content. We suggest that dierences in the eect of faunal treatments on nutrient mineralization are related to the feeding strategies of the added fauna, and to their consequent eect on the size of the soil microbial biomass. The treatments that increased N mineralization and plant nutrient content (N and P) also signi®cantly reduced plant growth (shoot and root). We suggest that high NH4+ ±N concentrations in the soil solution of Collembola treatments inhibited the growth of N. stricta and that the growth of other grassland species may bene®t from this improvement in nutrient availability. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction It is becoming increasingly apparent that soil fauna play a signi®cant role in soil processes through their in¯uence on the quantity, composition and activity of soil micro¯ora. Soil animals may aect microbial communities directly, by feeding on various microorganisms, or indirectly, by mixing and channelling detritus, dispersing microbial propagules and excreting nutrient* Tel.: +44-161-2755959; fax: +44-161-2753938; e-mail: rbardget @fs1.scg.man.ac.uk. Present address: Department of Biological Sciences, Institute of Environmental and Natural Sciences, University of Lancaster, Lancaster LA1 4YQ, UK.
rich wastes (Bardgett and Griths, 1997). Generally, microcosm studies suggest that these soil faunal:microbial interactions result in increased rates of soil nutrient mineralization (Ineson et al., 1982; Bardgett et al., 1998) and an increased uptake of available inorganic nutrients by plants (Ingham et al., 1985). Montane ecosystems in Britain are dominated by soils of high organic matter status and low pH. Primary productivity in these ecosystems is almost entirely dependent on nutrients released by decomposition and, therefore, the activities and interactions of the soil biota (Bardgett and Leemans, 1996). Previous studies have provided evidence that the soil fauna is an important factor regulating soil microbial abundance and community structure, and hence processes
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 1 4 - 0
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of decomposition and nutrient release in these harsh ecosystems. A laboratory study by Bardgett et al. (1993a) showed that the feeding activities of an abundant Collembola (Onychiurus procampatus) in¯uenced the growth and activity of a decomposer fungus common to sub-montane grasslands. Likewise, in a study of montane grasslands, Bardgett et al. (1998) showed that both fungal-feeding Collembola and a largely bacterial-feeding nematode community increased the total biomass and activity of micoorganisms in two soils of dierent organic matter content. These biotic interactions, however, were shown to have no eect on net nutrient mineralization (N and P), presumably due to the re-utilization Ð or immobilization Ð of inorganic nutrients by the larger microbial biomass (Visser et al., 1981; Bardgett et al., 1993a; Bardgett et al., 1998). The immobilization of nutrients is likely to be exacerbated by the high C-to-N ratio of these organic soils (Bardgett et al., 1993a). What is less clear, is how these soil biotic interactions in¯uence net nutrient mineralization in the face of competition from plants. Indeed, most previous microcosm experiments on soil biotic interactions in grasslands have been conducted in the absence of plants (e.g. Woods et al., 1982; Bakonyi, 1989; Bardgett et al., 1993a; Bardgett et al., 1998) where nutrient availability will be regulated largely by microbial mineralization±immobilization dynamics. In the ®eld, plant roots may compete eectively with soil microorganism for inorganic nutrients that have been excreted by the microbial-feeding fauna (Ingham et al., 1985). Therefore, the question remains, does faunal grazing on microorganisms in organic, montane grassland soils translate into (a) increased nutrient mineralization and, therefore, (b) increased plant nutrient uptake and growth of indigenous grassland plants? An additional question is whether dierent groups of soil fauna have a greater in¯uence on nutrient mineralization when combined, or when added to microcosms independently. It has been hypothesized that most mineralization of nutrients in soil is facilitated by the action of the most ecient microbial grazers, the microfauna, and that microarthropods only contribute signi®cantly to these processes when the activities of microfauna are inhibited in some way (Vreeken-Buijs et al., 1997). To test these hypotheses, we selected the fungal-feeding Collembola O. procampatus, which makes-up some 95% of total numbers of Collembola in these montane soils (Bardgett et al., 1993a), and a mixed community of nematodes, dominated by bacterial-feeders, taken from the ®eld site (Bardgett et al., 1998). The nematode community that develops in these microcosm systems has been characterized by Bardgett et al. (1998). The microcosm study focuses on N and P release and their uptake by Nardus stricta (L.), a ubiquitous grass species of sheep-grazed mon-
tane grasslands in the United Kingdom (Rodwell, 1992). 2. Methods and materials 2.1. Sites and soils In June 1997 soil was collected from the surface horizon (to 10 cm depth) of a grassland site in the Peak District National Park, roughly 500 m altitude and 24 km east of Manchester, United Kingdom. The site is a Nardus stricta dominated grassland with a humic stagnopodzol soil (Hafren series; pH 4.4, organic matter 47%, C-to-N ratio 40). Soil was passed through a 6 mm sieve and stored at 48C until use. In order to eliminate the fauna, the sieved soil from both sites was subjected to air drying, followed by freezing at ÿ808C for 24 h, and then further air drying for 24 h (Bardgett et al., 1998). Subsequent samples taken from the defaunated soil revealed that the procedure had completely eliminated the multicellular fauna (nematodes, microarthropods and larger fauna). Sheep dung collected from the same grassland site, was defaunated by freezing for 24 h at ÿ808C. 2.2. Microcosms The experiments were carried out in microcosms, as described by Anderson and Ineson (1982). Microcosms were prepared by weighing 40 g (dry mass) of defaunated soil into opaque plastic containers (3.5 cm dia., 10 cm height). To all microcosm chambers, 0.6 g dry mass defaunated sheep dung, collected from the ®eld site, was added to the surface of the soil. Our previous studies have shown that the addition of sheep dung to these soils is essential to ensure rapid colonisation and reproduction of added fauna (Bardgett et al., 1998). Thereafter, microcosms were reinoculated with micro¯ora by adding 10 ml of a soil suspension made from the appropriate soil type; the suspension was made by mixing 1 kg of sieved soil in 2 l of sterile distilled water which was then centrifuged at 2500 rpm for 5 min to obtain a supernatant that was ®ltered through a 10 mm sieve. Soils were adjusted to moisture contents of 60% (w/w) (equivalent to a water potential of ÿ5 kPa) by adding sterile distilled water. Microcosms were then incubated at 158C (average summer 0±5 cm soil depth temperature; Bardgett and Leemans, 1996) for 10 d to allow establishment of micro¯ora. Pots were then planted with ®ve seedlings of N. stricta which had been germinated from surface sterilized seeds obtained from Emorsgate Seeds, King's Lynn, UK. Soil fauna used in the experiment were collected as described by Bardgett et al. (1998). Brie¯y, nematodes
R.D. Bardgett, K.F. Chan / Soil Biology and Biochemistry 31 (1999) 1007±1014
were extracted from samples of bulked soil using Whitehead and Hemming (1965) trays to give an inoculum of 1200 per microcosm which was then applied in 2 ml of distilled water. The nematode community that develops in these montane soil microcosms has been shown to be dominated by bacterial-feeding nematodes (rhabditids, plectids and cephalobids), however, a few plant-feeding nematodes and fungal-feeding aphelenchids also persisted (Bardgett et al., 1998). For Collembola, the species Onychiurus procampatus was used, since it is a known fungal-feeder, and is present in large numbers in upland soils (Bardgett et al., 1993a,b,c, 1998). Brie¯y, specimens of O. procampatus were collected by gentle heat extraction from large soil samples taken from each of the sites, and were stored in Petri dishes with a base of moist plaster of Paris and fed on brewers' yeast extract. Seventeen O. procampatus individuals of sizes 2±3 mm were added to each of the Collembola treatments. Numbers of soil animals added were based on mean ®eld densities recorded by Bardgett et al. (1993a). Fauna were added to microcosms to achieve the following treatments: (1) microbes only (CONTROL); (2) microbes and nematodes (N); (3) microbes and Collembola (C), and: (4) microbes, nematodes and Collembola (N + C). Each treatment was replicated three times, and sucient microcosms were established to allow four destructive samplings over the experimental period. The establishment of nematode populations was checked by extracting nematodes from 10 g fresh soil at each sample date using the methodology of Whitehead and Hemming (1965). We were unable to check population numbers of Collembola due to the small amounts of soil available for extraction (Bardgett et al., 1998). However, their survival was con®rmed by visual inspection. Microcosms were arranged in a randomized block design and incubated for 60 d in a controlled environment facility at 158C, with a light±dark period of 16±8 h. After 15, 30, 45 and 60 d, microcosms of each treatment were destructively sampled. Soil moisture contents were maintained throughout the experiment by adding distilled water to a pre-determined mass. At each sample date, soils and plant material were analyzed. 2.3. Total microbial biomass The extractable microbial C of 10 g dry weight equivalent samples was measured using the fumigation±extraction technique (Vance et al., 1987). Organic C in ®ltered extracts was determined by the acid dichromate oxidation method (Tate et al., 1988). Microbial C (dierence between extractable C from fumigated and unfumigated samples) was converted to microbial biomass C using a kEC-factor of 0.35 (Sparling et al., 1990).
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2.4. Nutrient mineralization Nutrient mineralization was measured as the release of mineral N (NO3ÿ ±N and NH4+ ±N) and PO3ÿ 4 -P from microcosm chambers (Ineson et al., 1982). This was done by repeatedly leaching (3 times) microcosms with 50 ml distilled water and analysing the leachates for concentrations of mineral-N using a Skalar continuous ¯ow autoanalyser (Ineson et al., 1982; Bardgett et al., 1993c). 2.5. Plant growth and nutrient content At each sample date, root and shoot biomass (fresh weight) was determined for individual microcosms. Shoot tissue was then oven-dried for 24 h at 808C, ground and weighed into digestion tubes. Shoot tissue N and P contents were determined for d 30, 45 and 60 using the methodology of Thomas et al. (1967). Values are expressed as mg g ÿ 1 dry plant material. 2.6. Statistical analysis Data were checked for normality and the eects of animal treatments and time on each of the variables were determined using analysis of variance (ANOVA). Dierences between means were tested using Fisher's PLSD test. All data are presented as arithmetic means 2 standard errors. 3. Results 3.1. Establishment of faunal populations The number of nematodes in the N and N + C treatment varied signi®cantly (F = 30.1, P < 0.0001) over the experimental period, being lowest at d 45 and highest at d 60 (Fig. 1). There was no signi®cant dierence in numbers of nematodes between the two inoculation treatments on any sample date (Fig. 1). The addition of Collembola (the N + C treatment) had no eect on numbers of nematodes. 3.2. Soil microbial biomass In all treatments, there was a strong temporal trend of increasing soil microbial biomass (F = 35.6, P < 0.0001) over the experimental period (Table 1). At d 30, the independent treatments of nematodes (N) and Collembola (C) increased soil microbial biomass by 62% and 28%, respectively, relative to the control (F for the treatmenttime interaction = 4.0, P = 0.0017: Fig. 2A). At d 60, the presence of both nematodes and Collembola (N + C) increased soil microbial biomass by 26%, relative to the control
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high in the N + C treatment than in the other treatments, this eect was not signi®cant (Fig. 3). The leaching of inorganic N and P was negatively correlated with soil microbial biomass (for NH4+ ±N: r =ÿ 0.34, P = 0.018; for NO3ÿ ±N r =ÿ 0.35, P = 0.014; for total mineral±N r =ÿ 0.42, P = 0.003, and; for PO3ÿ 4 ±P r =ÿ 0.45, P = 0.0013).
3.4. Plant biomass and nutrient content Plant biomass (shoot and root) and nutrient content (N and P) increased over the experimental period in all treatments (Table 1). Over all sample dates, shoot biomass was signi®cantly (F = 4.9, P = 0.006) aected by animal treatment, being reduced in both the Collembola (C) (28%) and the nematode and Collembola (N + C) (32%) treatments, relative to the control (Fig. 4). The presence of nematodes alone had no eect on shoot or root biomass. Root biomass was signi®cantly (F = 4.1, P = 0.013) aected by the animal treatments, being reduced by both the Collembola (C) (39%) and the combined nematode and Collembola (N + C) treatments (43%), relative to the control (Fig. 4). None of the treatments aected plant shoot:root biomass ratios (data not shown: F = 2.4, P = 0.082). Shoot N and P contents were signi®cantly (F = 6.6, P = 0.0003 and F = 8.8, P < 0.0001, respectively; Fig. 5) aected by the animal manipulation treatments. The presence of individual animal groups had no eect on shoot N content. However, the presence of nematodes and Collembola (N + C) signi®cantly increased shoot N content by 54% relative to the control. This eect was most pronounced at d 45 (F for the treatmenttime interaction = 3.7, P = 0.002; Fig. 5). Shoot P content also only increased, relative to the control, in the presence of both nematodes and Collembola (N + C). This eect was most pronounced at d 45 (F
Fig. 1. Temporal changes in numbers of nematodes recovered from microcosms of the nematode (N) and nematode and Collembola (N + C) treatments. Values are means2 S.E.
(Fig. 2A). At this time the other treatments had no eect on soil microbial biomass. 3.3. Nutrient mineralization For all inorganic nutrients measured there was a distinct temporal trend of declining concentration in leachates over the experiment period (for NH4+ ±N, NO3ÿ ±N and PO3ÿ 4 ±P, F = 80.5, 11.9 and 13.9, respectively, and P < 0.0001 in all cases: Table 1). The release of NH4+ ±N (F = 11.5, P < 0.0001: Fig. 2B) and NO3ÿ ±N, F = 3.3, P < 0.03: Fig. 3) in leachates was increased by the presence of both nematodes and Collembola (N + C), relative to the control. However, for NH4+ ±N this eect was only evident at d 30 (F for the treatmenttime interaction = 6.5, P < 0.0001: Fig. 2B). The presence of Collembola (C) also signi®cantly increased NH4+ ±N release relative to the control and the nematode only (N) treatment at d 30, 45 and 60 (Fig. 2B). Collembola had a similar eect on NO3ÿ ±N, however, this eect was not signi®cant (Fig. 3). Although PO3ÿ 4 ±P release was over twice as Table 1 Temporal eects of animal treatments Sample date (d)
Microbial biomass (mg C g ÿ 1 dry soil) NH4+ ±N leachates (mg g ÿ 1) NO3ÿ ±N leachates (mg g ÿ 1) ÿ1 PO3ÿ ) 4 ±P leachates (mg g Shoot biomass (mg fresh weight) Root biomass (mg fresh weight) Shoot N (mg g ÿ 1 dry wt) Shoot P (mg g ÿ 1 dry wt)
15
30
45
60
LSD
5586 (405)a 12.1 (0.5)a 2.2 (0.1)a 36.7 (6.3)a 10 (4)a 5 (1)a
9470 (731)b 14.5 (2.4)b 1.9 (0.1)ab 7.5 (6.6)b 26 (2)b 14 (1)a 10.1 (1.0)a 1.0 (0.2)a
7029 (646)c 3.9 (0.6)c 1.6 (0.2)b 0.0 (0.0)b 77 (7)c 38 (4)b 20.8 (1.5)b 1.9 (0.2)b
11990 (547)d 0.4 (0.3)d 0.7 (0.3)c 0.0 (0.0)b 114 (9)d 97 (12)c 9.1 (1.2)a 0.4 (0.0)c
1359 2.14 0.5 13.5 14 16 3.1 0.4
Data are means (standard errors) and values with the same letter are not signi®cantly dierent at the P < 0.05 level. LSD = least signi®cant dierence between means.
R.D. Bardgett, K.F. Chan / Soil Biology and Biochemistry 31 (1999) 1007±1014
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Fig. 2. Temporal changes in (A) soil microbial biomass, and (B) the amount of NH4+ ±N leached from microcosms in the dierent animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola). Values are means 2S.E. For soil microbial biomass, F = 3.98 and P = 0.0017 for the interaction between treatmenttime; For NH4+ ±N leaching, F = 6.5 and P < 0.0001 for the interaction between treatmenttime.
Our data con®rm the hypothesis that in montane grassland ecosystems, soil fauna can enhance nutrient mineralization and nutrient uptake by indigenous grass species. We have shown that in the presence of fungalfeeding Collembola, and when nematodes (largely bacterial-feeders) and Collembola are combined, N mineralization (measured as mineral N release) and shoot N
content of N. stricta was signi®cantly higher than in a defaunated control. We also showed that P mineralization (measured as P03ÿ 4 ±P release) increased Ð although not signi®cantly Ð in the presence of both nematodes and Collembola, resulting in a signi®cant increase in shoot P content of N. stricta. These data cast doubt over the ecological value of results from previous `plant-free' microcosm studies of soils and litters from similar ecosystems. Such studies showed that in the absence of plants, the same fauna had no eect on N or P mineralization, due presumably to the reutilization of nutrients by the microbial biomass of soils and litters with high C-to-N ratio's (Bardgett et
Fig. 3. Eects of animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola) on the amount of N03ÿ ±N (mg g ÿ 1 soil) and PO3ÿ 4 ±P, leached from microcosms. Values are means2S.E. For each measure, values with the same letter are not signi®cantly dierent.
Fig. 4. Eects of animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola) on shoot and root biomass. Values are means2S.E. For each measure, values with the same letter are not signi®cantly dierent.
for the treatmenttime interaction = 2.4, P = 0.03; Fig. 5). 4. Discussion
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Fig. 5. Temporal changes in (A) shoot N content, and (B) shoot P content in the dierent animal treatments (N = nematodes, C = Collembola, and N + C = nematodes and Collembola). Values are means 2S.E. For shoot N, F = 3.7 and P = 0.0017 for the interaction between treatmenttime; For shoot P, F = 2.4 and P = 0.03 for the interaction between treatmenttime.
al., 1993a, 1998). The data presented here suggest that in montane grasslands, plants roots can compete very eectively with soil microbes for these nutrients which have been released by the feeding activities of fauna Ð via the microbial loop (Clarholm, 1985). However, we have also shown that large quantities of these nutrients released by faunal grazing are not taken up by plants or immobilized by microbes, but remain weakly bound to colloidal surfaces or in soil solution, where they are susceptible to leaching. The ability of plant roots to compete with microbes for available nutrients is of particular note in the light of the extremely large microbial biomass, and hence potential nutrient sink, in these montane soils. Soil microbial biomass values ranged from 5,400±14,000 mg g ÿ 1 soil, representing some 2±6% of the total organic C in the soil. These values are in line with estimates from ®eld studies of sub-montane and montane grassland soils (Bardgett and Leemans, 1996; Bardgett et al., 1997) and are three-to-four times higher than would be expected in less organic, lowland grassland soils (Lovell et al., 1995). Combined with the very high C-to-N ratio of these montane soils (approximately 40) one would expect these conditions to lead to net N immobilization by the large and extremely N limited soil microbial community. The presence of a nematode community alone, which was dominated by bacterial-feeders (Bardgett et al., 1998), had no eect on the mineralization of N or P, or shoot nutrient content. This ®nding is contrary to many studies which have shown that the rate of net soil N mineralization is greater in the presence of bacterial-feeding nematodes (Coleman et al., 1977; Anderson et al., 1981; Woods et al., 1982), resulting in increased soil N availability and N uptake by plants (Ingham et al., 1985). Our data also do not support the hypothesis of Vreeken-Buijs et al. (1997) that most
nutrient mineralization in soils is facilitated by the grazing of microorganisms by microfauna, and that microoarthropods only in¯uence these processes when the activities of microfauna are inhibited in some way. In contrast, we have shown that in this montane soil the positive eects of bacterial-feeding nematodes on N mineralization only occur when fungal-feeding microarthropods are present. This synergistic eect, which was most apparent at d 30, may be related to the increased grazing pressure on the soil microbial community in the combined nematode and Collembola treatment, which may have reduced microbial growth and hence the immobilization of N. Indeed, at this date the presence of nematodes alone doubled the size of the soil microbial biomass relative to the defaunated control, presumably leading to increased microbial immobilization of N. In the nematode and Collembola treatment, however, increased grazing pressures kept the soil microbial biomass at a similar level to the defaunated control, possibly leading to reduced N microbial immobilization and increased cycling of N through the animal excretion pathway. Additional support for this thesis comes from the ®nding that over the experimental period there was signi®cant negative correlation between soil microbial biomass and the leaching of NH4+ ±N, NO3ÿ ±N and PO3ÿ 4 ±P from microcosm chambers. The ®nding that Collembola had a greater eect on N mineralization than did nematodes, is likely to be related to the make-up of the soil microbial community and the feeding strategies of the animals added to the microcosms. As already stated, previous studies of similar soils showed that the nematode community which develops after the addition of a mixed inoculum to defaunated soils is dominated by fast-growing species which tend to be bacterial-feeders (Bardgett et al., 1998). In contrast, the collembolan (O. procampa-
R.D. Bardgett, K.F. Chan / Soil Biology and Biochemistry 31 (1999) 1007±1014
tus) used in these experiments is a known specialist fungal-feeder (Bardgett et al., 1993a,b,c). Since the microbial community of these acid, organic soils is largely fungal-dominated (Bardgett, 1996; Bardgett et al., 1997), and that NH4+ ±N Ð the main mineral N form in these soils Ð is preferentially used by fungi (Bardgett et al., 1993c), it is likely that the feeding activities of Collembola will have a greater eect on N mineralization. In the nematode only treatment, therefore, any N released through bacterial grazing is likely to have been re-utilized by the ungrazed, and hence growing fungal biomass. This argument could also be extended to the combined nematode and Collembola treatment, where the grazing activities of the Collembola may have suppressed fungal growth, allowing bacteria to proliferate. This may have provided an increased food resource for bacterial-feeding nematodes, which in turn may have increased N release through the nematode excretion pathway. Clearly, to test these ideas it would be necessary to examine, in future experiments, changes in the biomass and structure of the soil microbial community. Surprisingly, the animal treatments that increased N mineralization and plant nutrient content (N and P) also signi®cantly reduced plant growth (shoot and root). This ®nding is contrary to several studies which have shown fauna to increase plant productivity, an eect ascribed to enhanced nutrient mineralization (Ingham et al., 1985; Alphei et al., 1996). The most plausible explanation is that increased NH4+ ±N availability in the soil of the two Collembola treatments (C and N + C) restricted the growth of N. stricta. Indeed, several studies have reported restricted growth of N. stricta when grown in solution culture at high NH4+ ±N concentrations (Bradshaw et al., 1964; Gigon and Rorison, 1972; Troelstra et al., 1995). As in this study, restricted plant growth has also been reported to be associated with an increased N content of shoot material (Troelstra et al., 1995). We therefore suggest that high NH4+ ±N concentrations in the soil solution of Collembola treatments and its subsequent uptake inhibited the growth of N. stricta. In conclusion, our study supports the thesis that the activities of soil fauna enhance nutrient mineralization in montane grassland ecosystems. We also have some evidence to suggest that this increase in soil nutrient availability leads to an increase in nutrient leaching from soil and enhanced nutrient uptake by an indigenous grass species. The ®nding that these positive eects of fauna on soil nutrient turnover had a negative eect on plant productivity is likely to be a species-speci®c response of N. stricta. It is probable that in montane grasslands the productivity of other more N responsive grasses, such as Agrostis capillaris and Festuca ovina (Bradshaw et al., 1964), will increase as a result of faunal-mediated increases in soil nutrient availability. Our
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data also show that dierent faunal groups, and their combinations, can alter these processes of nutrient ¯ux in upland grasslands, due presumably to dierential eects on the size, structure and dynamics of the soil microbial community. Further studies need to explore the eects of more complex trophic interactions, for example with the inclusion of predators (sensu Mikola and SetaÈlaÈ, 1998), to fully understand the role of soil fauna in montane and upland grassland ecosystems.
Acknowledgements We thank the Peak District National Park for permission to collect soils from the study site. We also thank Gary Porteous, Angela Jones, Lisa Cole, Pisit Chareonsudjai, Harriet Waters and Alissar Chaker for general help in the laboratory.
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