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Determination of bacterial and fungal fumigation κc factors across a soil moisture gradient
Soil E&l. Lfiochem. Vol. 22, No. 6. pp.
81 I-816, 1990 Printed in Great Britain. All rights rcscrvcd
Copyright 0
0038-0717/90 53.00 + 0.00 1990 Pcrgamon Press plc
DETERMINATION OF BACTERIAL AND FUNGAL FUMIGATION kc FACTORS ACROSS A SOIL MOISTURE GRADIENT D. A.
WARDLE’ *
and D. PARKINSON~
‘Department of Biological Sciences and ‘Kananaskis Centre for Environmental Research, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N lN4 (Accepred 10 March 1990)
Summary-Six samples of agricultural soil were maintained at five separate soil moisture contents (15, 25,35,45 or 55%) for 6 h or 3, 10 or 30 days. These samples were then amended with “C-labelled fungal or bacterial tissue, and the k, values determined following chloroform fumigation. Soil moisture effects explained 12.5% of the variation in the data set for fungal k, factors, and 9.5% in the bacterial data set. The 15% moisture treatment always had lower k, values than for at least some of the other moisture contents, in both the fungal and bacterial data sets but the differcnccs were usually small. The 25.35.45 and 55% treatments did not usually differ from each other. Duration of incubation had a much stronger effect, explaining 51.6 and 35.8% of variation in the fungal and bacterial data sets, with prolonged incubation depressing k, values. The significance of blocking effects in each case indicated that the six samples (blocking units) had different k, values. Analysis of k, values,determined for the entire microbial biomass using a bacterial:fungal ratio of 25: 75, showed essentially similar responses to the treatment effects tested for. Values of k.. determined using a different baeterial:fungal ratio determined for each subsample, were almost identical to those using a bacterial:fungal ratio of 25:75.
INTRODUCTION
The soil microbial biomass exerts an important influence on soil carbon and nutrient cycling, both through the oxidation of soil organic matter, and the storage of carbon and mineral nutrients (Anderson and Domsch, 1980; Juma and Paul, 1984). Therefore, environmental factors which control turnover of the microbial biomass exert an important in!Iuencc on nutrient cycling (McGill et al., 1986; van Veen et al., 1987). Soil moisture has often been demonstrated to exert an important or dominating effect on the magnitude of the soil microbial biomass, both in laboratory investigations (Bottner, 1985; Kieft et al., 1987) and in the field (Ross et al., 1981; McGill et al., 1986; Schnurer et al., 1986; Wardle and Parkinson, 1990a). Drying-rewetting cycles also result in substantially enhanced respiration (Lund and Gorksoyr, 1980; Orchard and Cook, 1983), and “C studies have been used to demonstrate that this is derived from the decomposition of microbial cells (Shields et al., 1974; Sorenson, 1974). The fumigation-incubation method (Jenkinson, 1966; Jenkinson and Powlson, 1976) has often been used for assessing microbial biomass response to soil moisture, usually using a constant value for the decomposition constant k, [e.g. Bottner (1985); McGill et al. (1986)]. However, this method has sometimes demonstrated uncertain relationships with other proposed methods for estimating microbial biomass in dry soils, or when soil moisture gradients *To whom all correspondence should be addressed at: MAF Technology, Ruakura Agricultural Centre. Private Bag, Hamilton, New Zealand.
are involved (Ross et (II., 1984; Sparling ef al., 1986; West er al., 1986, 1988). Fumigation k, values determined by Ross (1987) were substantially depressed by waterlogging, and even when the fumigationincubation technique was used with separate k, values for seasonally different moisture conditions, it was concluded that fumigation-incubation underestimated microbial biomass in waterlogged conditions. In addition to soil moisture, k, values have also been demonstrated to vary with age of microbial tissue (Ross et al., 1987), soil texture and microbial species composition (Nicolardot et al., 1984) and soil pH (Vance et al., 1987). Bacterial : fungal ratios may also a&t the k, factor because dead tissue of the two groups of organisms appear to mineralize at different rates (Anderson and Domsch, 1978). Our purpose was to determine whether maintaining soil samples over a range of soil moisture contents for varying periods influences the subsequent mineralization of “C-labelled bacteria and fungi in an agricultural soil. These values were used to determine the ways in which varying k, factors for different moisture contents influence estimates of soil microbial biomass (Wardle and Parkinson, 199Oc).
MATERIALS AND METHODS
Soil and sire description The soil used was a brown Chemozem with 7.0% C, a pH of 5.9, and a sand : silt : clay ratio of 52 : 30 : 18. It was collected from farmland in south-central Alberta, Canada which had been planted in hay for the previous 5 yr. 811
812
D. A. WARDLE and D. PARKINSON
Growth and harvest of microbial cells Seven species of fungi were grown, i.e. Mucor hiemalis, Mortierella alpina, Trichoderma harzianum, Arthrinium sphaerospermum, Cladosporium cladosporioides, Fusarium oxysporum and Penicillium nigricans. These had been found to be the most abundant fungal species in this soil (Wardle and Parkinson, 1990b). Cultures of these fungi were maintained on malt extract agar. Three 1 g samples of soil were diluted to lo-’ in 100 ml 0.1% peptone solution. A 0.1 ml aliquot of this dilution was spread on 5 plates of nutrient agar, for each sample. Sixteen bacterial (including actinomycete) colonies were randomly selected, and maintained on nutrient agar. The liquid medium used for growing “C-1abelled micro-organisms was based on that of Anderson and Domsch (1978) and Ross (1987). This consisted (per 1 water) of 10 g U”C-labelled D-glucose (sp. act. 370 Bq mg-’ glucose), 1 gNH,NO,, 1 g KHrP04, 1 g K,HPO,, OSg MgSO,, 50mg CaClr and 20mg FeCl,. In addition media for fungi and bacteria contained respectively 1 and 3 g of yeast extract (DIFCO). The media were autoclaved prior to addition of the labelled glucose. The bacterial and fungal colonies were cultured in 50 ml liquid medium in 125 ml Erlenmeyer flasks, on a culture shaker at 22”C, until harvest. Fungi were harvested, using filtration, at the late linear phase, as determined by preliminary experimentation. They were rinsed by deionized water, air-dried and ground to 0.5 mm using a mortar and pestle. The bacteria were harvested at the late logarithmic growth phase by centrifugation at 15,000 rev min-’ for 20min. These were then airdried, rinsed, air-dried again and ground to 0.5 mm. Ail tire fungal material was then mixed together, as was all the bacterial tissue. Determination of “C content of tissue For both the bacterial and fungal tissue, six replicate 50 mg subsamples were dissolved using 1 ml of the tissue solvent protosol (Du Pont). This was left for 2 days, and 15 ml of the scintillation fluid BIOFLUOR (Du Pont) was added. This was left for a further day and the “C content was determined using a liquid scintillation counter (2200CA Tricarb Packard). Pretreatment of soil samples Six soil samples (mean field moisture content of 48% on dry wt basis) were sieved (~4 mm) and held at 4°C for a maximum of 4 months. These were then remoistened to 55% moisture content (dry wt basis) using an atomizer, and various subsamples of these were air-dried at 22°C until they reached one of five different moisture contents, i.e. 15, 25, 35.45 or 55% H,O (dry wt basis). These moisture contents corresponded to - 13000, - 1500, -400, - 30 and -6 kPa. soil moisture potential respectively. For each subsample described above, 20 subsamples (each 15 g dry wt) were prepared, i.e. the 5 moisture contents outlined above x 4 incubation periods (6 h or 3,10 or 30 days). The moisture contents used have been found to be the normal range of moisture
contents in the field soil during the growing season (Wardle and Parkinson, 199Oa). The above procedure was performed twice, once for each of the bacterial and fungal k, determinations described below. Determination of ‘C release from tissue following fwnigation Tests demonstrated that the soil used in our study responded in the classical manner to chloroform fumigation (Jenkinson and Powlson, 1976). This was performed by measuring the CO& release from both fumigated and non-fumigated soils hourly using an i.r. gas analyser (continuous flow rate = 178 ml min-‘). Within 5 days the respiratory difference between control and fumigated soils was slight; within 8 days this difference was non-existent. At the end of the 4 incubation periods, each subsample of soil was amended with 40 mg of either bacterial or fungal tissue, and thoroughly mixed. This was then fumigated with chloroform (alcohol-free) for 18-24 h (Jenkinson and Powlson, 1976). Subsequently the soil was removed, remoistened to 55% moisture content and reinnoculated with 1.67 g dry wt soil from the same sample (without prior incubation) so as to provide a 9: 1 ratio of fumigated soil to re-inoculant (Chapman, 1987). The subsamples were kept in airtight Qorpak jars (5OOml) together with a 50 ml beaker containing 15 ml of 1.OM NaOH. After 10 days, 1.5 ml NaOH was removed from each beaker and added to 15 ml BIOFLUOR scintillation fluid. The “CO& content of each sample was then determined using liquid scintillation counting. Data analysis For each subsample, k, factors were determined separately for fungi and bacteria, as a ratio of the amount of “CO& evolved from the tissue in 10 days to the total “C content of the tissue at the beginning of the incubation. In order to test whether the k, factors were influenced by the treatments used in this study, data were analysed using 3-way analysis of variance, testing for the blocking, moisture and duration of prior incubation effects, as well as the time x moisture interaction. When significant moisture effects were detected, multiple comparison analysis was performed within each prior incubation period across the soil moisture gradient, using Tukey’s test, following two-way ANOVA (moisture content and blocking). RESULTS
For the 120 fungal k, factors determined, the values ranged from 0.219 to 0.449, with a mean of 0.365. This k, factor was significantly influenced by soil moisture, duration of prior incubation and blocking, although time effects were the strongest (Tables 1 and 4). The k, factors for the 15% moisture treatment were always significantly lower than for the 35% treatment, as well as below the 45% treatment (10 days) and 55% treatment (6 h). The differences were, however, not large. The 25, 35, 45 and 55% treatments were never significantly different from each other. Duration of prior incubation exerted strong effects on the k, factor, with soil incubated for 30 days
Soil moisture effects of k, factor estimates Table 1. Fungal k, factors determined f~&wnaistun mcubation times
813
contents and four prior
Initial soil moisture content (%) Duration of prior incubation 6h 3 days lOdays 30 days
I5
25
35
45
55
0.3Sga 0.379a 0.335a 0.296a
0.379ab 0.396ab 0.352ab 0.319ab
0.4OOb 0.41lb 0.378b 0.35Ob
0.39Oab 0.389ab 0.371b 0.328ab
0.403b 0.382ab 0.35&b 0.316ab
Within each row. numbers followed by different letters arc significantly different at P = 0.05.
having k, factors well below the other prior incubation times. The results suggest that the kc factors vary between soils of different moisture content, between soils incubated at a fixed moisture content for different periods, and between different samples of the same soil type. There was no significant interaction between incubation time and soil moisture content, indicating that moisture effects are essentially similar regardless of incubation time. The values for the 120 bacterial k, factors were slightly higher than those for the fungi. The factors ranged from 0.243 to 0.484 with a mean of 0.398. However, the treatment effects wefe essentially similar (Tables 2 and 4). The kc values for the 15% treatment were always significantly lower than for at least one of the other moisture. treatments but these differences were usually relatively small. The 25, 35, 45 and 55% treatments were never significantly different from each other. Prolonged prior incubation reduced the kc factor for all moisture contents, with 30-&y samples having kc factors that were the lowest. The kc factors varied significantly across soil moisture contents, different incubation periods and different samples. When microbial kc factors were determined for the microbial biomass as a whole, essentially similar trends were found. Using a constant bacterial : fungal ratio of 25: 75 (Anderson and Domsch, 1975, 1978), kc values of the 120 subsamples ranged from 0.250 to 0.450 with a mean of 0.373. Essentially similar treatment effects were found as for the bacterial and fungal kc factors (Tables 3 and 4). The 15% moisture treatment was significantly different to the 55% one at 6 h, and to the 35% treatment at days 3, 10 and 30. Again the 25,35,45 and 55% treatments were not significantly different from each other. The kc factor was significantly reduced by prolonged prior incubation and also differed significantly among the six samples. When a separate bacterial:fungal ratio was determined for each subsample a very similar pattern was found. Bacterial:fungal ratios were determined separately for each of the 120 subsamples using the
selective inhibition technique of Anderson and Domsch (1973, 1975) in which 1Omg streptomycin sulphate g-l soil and 15 mg actidione g-* soil were added to inhibit glucose-stimulated respiration of bacteria and fungi, respectively. These ratios are presented in Wardle and Parkinson (199t.M). Although selective inhibition does not appear to inhibit much of the microbial respiration even when both inhibitors are added together (West, 1986; Wardle and Parkinson, 199Od), these values were used as in indicator in our study to determine what effects variation in bacterial : fungal ratios might exert on the factor. Using kc factors based on separate ratios for each subsample, kc values ranged from 0.246 to 0.448 with a mean of 0.374. Virtually identical trends were found by using varying bacterial:fungal ratios compared with using a constant ratio of 25:75, with regard to moisture effects, incubation time and blocking (Table 4). For all 120 subsamples, the k, factor determined using a ratio of 25 : 75 never differed by > 3% from the factors determined using a varying ratio. DISCUSSiON
Soil moisture effects on k, values The fumigation-incubation technique is frequently used with the assumption that k, values vary little between soil types (Jenkinson, 1976) or between treatments (Bottner, 1985; Duah-Yentumi and Johnson, 1986; Powlson et al., 1987). However Jenkinson (1988) summarixed information indicating variations in k, values under different soil conditions (particularly pH). Our study indicated significant variations in k, values with soil moisture content. Relatively dry soil (15%) displayed reduced values of k, even when kept at 55% moisture for the 10 days following fumigation like all the other treatments. Soil moisture contents of lO-20% were typical for this soil in the field during June and July 1988; during such conditions microbial biomass would be underestimated by using a constant k, factor relative to soils with a more optimal mois-
Table 2. Bacterial k, factors determined for five moisture contents and four incubation times
prior
Initial soil moisture content (%) Duration of prior incubation 6h 3 days 10 days 30 days
I5 0.4028 0.391a 0.358a 0.347a
25
35
45
55
0.40&b 0.415ab 0.417b 0.357ab
0.422ab 0.435b 0.391ab 0.389b
0.421ab 0.421ab 0.396ab 0.37Oab
0.435b 0.44Oab 0.4OOb 0.346ab
Within each row, numbers followed by different ktters arc significantly diffcwnt at P = 0.05. SIB 22,%-F
814
D. A.
WARDS and D. PARKINSON
Table 3. Microbial kc factors determined for five moisture contents and four prior incubation times. assuming a bacterial fungal ratio of 25:75 Initial soil moisture content (%) Duration of __._..... _~ prior incubation 6h 3 days 10 days 30 days
I5
25
35
45
55
0.37Oa 0.382a 0.34la 0.309a
0.385ab 0.4OOab 0.37Oab 0.328ab
0.405ab 0.417b 0.381b 0.36Ob
0.397ab 0.397ab 0.377ab 0.338ab
0.41 lb 0.396ab 0.368ab 0.324ab
Within each row, numbers followed by different ktters are significantly ditkrent at P - 0.05.
ture status. The depression of the k, factor caused by low moisture contents was not large, causing it to be reduced by ~20% (relative to optimal moisture conditions) in all cases. Nevertheless, this could exert an effect on the results of studies conducted across a gradient of moisture contents. Because incubation conditions (including the moisture status) for the 10 days following fumigation were identical for all soils, there is no obvious explanation as to why microorganisms in soils previously held at 15% of soil moisture (even for only 6 h) should demonstrate a reduced k, factor relative to other treatments. It is most likely, however, that soils at 15% moisture are in some way physically or chemically different to those at other moisture contents, and even when they are brought up to 55% moisture these differences are maintained for some time. This presumably results in the decomposition of added substrates being reduced over 10 days. We found the reverse trend to that of Ross (1987) with regard to soil moisture effects. In that study, higher soil moisture contents (above 60% of field capacity) resulted in reduced mineralization of added microbes, especially if moister soils had been smeared and compacted, kc values were also reduced if soils had been incubated at 60% field capacity for 7 days. In contrast, mineralization of microorganisms in our study was not significantly reduced by higher moisture, probably because smearing or compacting was not observed even at the highest moisture content which represented field capacity. Both our study, and that of Ross (1987) indicate that the use of the same kc value across a soil moisture gradient may be problematic. In our study, there was considerable variation in kc between different soil samples. This can be explained in terms of chemical or physical differences amongst the six samples (or blocking units) used in this study. Nicolardot er al. (1984) demonstrated that different soil types can have different microbial kc factors.
Variations in soil pH may also be important especially if soils of low pH are considered (Vance et al., 1987; Jenkinson, 1988). Also, our results suggest that there are important variations in kc between replicate samples collected within one soil type, as indicated by the blocking effect. This spatial variation of kc within a given soil type is consistent with temporal changes within a given soil, as reported by Ross (1987). Bacterial vs fungai k, values
Comparison of the bacterial and fungal kc factors indicates that a higher proportion of bacterial than fungal C is released as CO2 over 10 days. This was observed in all treatments, i.e. 36.5% of fungal ‘*C was released vs 39.8% of bacterial *‘C (mean of all 120 samples). Whether these differences are truly significant or not is unclear because tissue from all 7 fungal species were pooled as was tissue from all 16 bacterial colonies, preventing assessment of variability amongst species within each of the two microbial groups. In contrast, Anderson and Domsch (1978) found that fungal kc factors were significantly higher than bacterial kc factors (43.7% vs 33.3%). In our study varying the bacterial : fungal ratios did not greatly influence the microbial kc factors compared with using a constant ratio of 25:75. This is partly because the bacterial and fungal kc factors were not very different to each other, nor did the bacterial to fungal ratio differ greatly from a 25: 75 ratio (Wardle and Parkinson, 1990d). If the soils in our study had a ratio which differed greatly from 25: 75, then differences between k, values obtained using a 25 : 75 ratio, and values obtained using a different kc value for each subsample would have been greater also. In studies where the bacterial:fungal ratio is shifted by soil moisture [e.g. reduced since fungi are often more resistant to desiccation than are bacteria: Nakas and Klein (1979); Schnurer er al. (1986); West el al. (1987)] then the k, value may also be affected substantially.
Table 4. Relative importance of treatment effects on data presented in Tables l-3 Treatment efTect Soil moisture (A ) Duration of prior incubation (8) A x B interaction Blocking effect Unexplained
Fungal kc factor % of SS’
Bacterial kc factor % of ss
Microbial k, factor % of SSb
Microbial k, factor % of ss
12.7***
9.5”’
13.4***
14.0***
51.6*** 2.9 NS 7.5*** 25.5
35.a***
55.6**’ 2.8 NS 10.0*** 18.3
54.8*‘* 3.1 NS 9.8*** 18.2
,;.::.. 33:s
“Percentage of total sums of squares (SS) in data set explained by each effect. ‘kc factor using bacterial:fungal ratio of 25:75 (Tabk 3). ‘k, factor using bacterial: fungal rate separately determined for each sample. l, l*, *‘* Effect is significant at P =0.05, 0.01 and 0.001 respectively. NS, Effect not significant at 0.05.
Soil moisture effects of k, factor estimates
Soil moisture can exert an influence on microbial community structure (Kjoller and Struwe, 1982), and this may indicate that use of a constant k, value may be problematic, even if the bacterial:fungal ratios remains largely unchanged by soil moisture variation, such as we have observed. Data provided by Anderson and Domsch (1978) indicate that mineralization of the carbon in some fungal species in some soils was nearly twice as great, over 10 days, as that of other species. For the bacteria in that study, some species decomposed over five times more quickly than other species during that time. It is possible that microbial community structure may affect the k, factors of different soils or treatments, because some conditions may encourage microbial species which decompose faster in 10 days than do other conditions. Therefore, adding microbial tissues of the same species composition to different soils may not accurately reflect what happens to the native soil microbial biomass following fumigation, especially across a soil moisture gradient, although there is no effective way to test this. Our investigation demonstrated that the values of k, are significantly reduced in soils of low moisture content. Therefore, if the fumigation-incubation method is used with a constant kc value when responses of the microbial biomass to soil moisture are being considered (such as has been done in several studies), then the response of this biomass may be incorrectly assessed. Furthermore, the variation between k, values of soils held for different prior incubation times, and of replicate samples at each incubation time, indicate that the use of the fumigation-incubation method may be problematic if a constant k, value is used. In our study, variations in the bacterial:fungal ratios were not found to influence the k, factor significantly. However, this may be the case where bacterial: fungal ratios are observed to vary substantially across soil moisture gradients. Therefore, it can be recommended that, when the fumigation-incubation method is used to determine microbial biomass for soils of varying soil moisture content, efforts should be made to determine whether or not k, values are likely to remain independent of soil moisture values. Acknowledgements-We thank S. Visser for reviewing the manuscript, and D. M. Reid for the use of facilities for the work involving carbon-14. This work was supported by an NSERC grant (D. Parkinson) and a Canadian Commonwealth Scholarship (D. A. Wardle).
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Wardle D. A. and Parkinson D. (199Oa) Interactions between microclimatic variables and the soil microbial biomass. Biology & Fertility of Soils (in press). Wardle D. A. and Parkinson D. (199Ob) Influence of the herbicide glyphosate on soil microbial community struc--_ ture. Plant & Soil 22, 29-37. Wardle D. A. and Parkinson D. (199Oc) Comnarison of physiological techniques for estitnating the response of the soil microbial biomass to soil moisture. Soil Biology & Biochemistry 22, 817-823. Wardle D. A. and Parkinson D. (199Od) Response of the soil microbial biomass to glucose, and selective inhibitors, across a soil moisture gradient. Soil Bio/ogy & Biochemistry 22, 825-834. West A. W. (1986) Improvements to the selective respiratory inhibition technique to meausre eukaryote:prokaryote ratios in soils. Journal of Microbiological Methods 5, 125438. West A. W., Sparling G. P. and Grant W. D. (1986) Correlation between four method to estimate total microbial biomass in stored, air dried, and glucose-amendment soils. Soil Biology & Biochemistry 18, 569-576. West A. W., Sparling G. P. and Grant W. D. (1987) Relationships between mycclial and bacterial populations in stored, air-dried and glucose-amended arable and grassland soils. Soil Biology & Biochemistry 19, 599401. West A. W., Sparling G. P., Speir T. W. and Wood J. M. (1988) Dynamics of microbial carbon, nitrogen flush and ATP, and enxyme activity of dried soils from a chmosequence. Australian Journal of Soil Research 2b, 519-530.
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0038-0717/90 53.00 + 0.00 1990 Pcrgamon Press plc
DETERMINATION OF BACTERIAL AND FUNGAL FUMIGATION kc FACTORS ACROSS A SOIL MOISTURE GRADIENT D. A.
WARDLE’ *
and D. PARKINSON~
‘Department of Biological Sciences and ‘Kananaskis Centre for Environmental Research, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N lN4 (Accepred 10 March 1990)
Summary-Six samples of agricultural soil were maintained at five separate soil moisture contents (15, 25,35,45 or 55%) for 6 h or 3, 10 or 30 days. These samples were then amended with “C-labelled fungal or bacterial tissue, and the k, values determined following chloroform fumigation. Soil moisture effects explained 12.5% of the variation in the data set for fungal k, factors, and 9.5% in the bacterial data set. The 15% moisture treatment always had lower k, values than for at least some of the other moisture contents, in both the fungal and bacterial data sets but the differcnccs were usually small. The 25.35.45 and 55% treatments did not usually differ from each other. Duration of incubation had a much stronger effect, explaining 51.6 and 35.8% of variation in the fungal and bacterial data sets, with prolonged incubation depressing k, values. The significance of blocking effects in each case indicated that the six samples (blocking units) had different k, values. Analysis of k, values,determined for the entire microbial biomass using a bacterial:fungal ratio of 25: 75, showed essentially similar responses to the treatment effects tested for. Values of k.. determined using a different baeterial:fungal ratio determined for each subsample, were almost identical to those using a bacterial:fungal ratio of 25:75.
INTRODUCTION
The soil microbial biomass exerts an important influence on soil carbon and nutrient cycling, both through the oxidation of soil organic matter, and the storage of carbon and mineral nutrients (Anderson and Domsch, 1980; Juma and Paul, 1984). Therefore, environmental factors which control turnover of the microbial biomass exert an important in!Iuencc on nutrient cycling (McGill et al., 1986; van Veen et al., 1987). Soil moisture has often been demonstrated to exert an important or dominating effect on the magnitude of the soil microbial biomass, both in laboratory investigations (Bottner, 1985; Kieft et al., 1987) and in the field (Ross et al., 1981; McGill et al., 1986; Schnurer et al., 1986; Wardle and Parkinson, 1990a). Drying-rewetting cycles also result in substantially enhanced respiration (Lund and Gorksoyr, 1980; Orchard and Cook, 1983), and “C studies have been used to demonstrate that this is derived from the decomposition of microbial cells (Shields et al., 1974; Sorenson, 1974). The fumigation-incubation method (Jenkinson, 1966; Jenkinson and Powlson, 1976) has often been used for assessing microbial biomass response to soil moisture, usually using a constant value for the decomposition constant k, [e.g. Bottner (1985); McGill et al. (1986)]. However, this method has sometimes demonstrated uncertain relationships with other proposed methods for estimating microbial biomass in dry soils, or when soil moisture gradients *To whom all correspondence should be addressed at: MAF Technology, Ruakura Agricultural Centre. Private Bag, Hamilton, New Zealand.
are involved (Ross et (II., 1984; Sparling ef al., 1986; West er al., 1986, 1988). Fumigation k, values determined by Ross (1987) were substantially depressed by waterlogging, and even when the fumigationincubation technique was used with separate k, values for seasonally different moisture conditions, it was concluded that fumigation-incubation underestimated microbial biomass in waterlogged conditions. In addition to soil moisture, k, values have also been demonstrated to vary with age of microbial tissue (Ross et al., 1987), soil texture and microbial species composition (Nicolardot et al., 1984) and soil pH (Vance et al., 1987). Bacterial : fungal ratios may also a&t the k, factor because dead tissue of the two groups of organisms appear to mineralize at different rates (Anderson and Domsch, 1978). Our purpose was to determine whether maintaining soil samples over a range of soil moisture contents for varying periods influences the subsequent mineralization of “C-labelled bacteria and fungi in an agricultural soil. These values were used to determine the ways in which varying k, factors for different moisture contents influence estimates of soil microbial biomass (Wardle and Parkinson, 199Oc).
MATERIALS AND METHODS
Soil and sire description The soil used was a brown Chemozem with 7.0% C, a pH of 5.9, and a sand : silt : clay ratio of 52 : 30 : 18. It was collected from farmland in south-central Alberta, Canada which had been planted in hay for the previous 5 yr. 811
812
D. A. WARDLE and D. PARKINSON
Growth and harvest of microbial cells Seven species of fungi were grown, i.e. Mucor hiemalis, Mortierella alpina, Trichoderma harzianum, Arthrinium sphaerospermum, Cladosporium cladosporioides, Fusarium oxysporum and Penicillium nigricans. These had been found to be the most abundant fungal species in this soil (Wardle and Parkinson, 1990b). Cultures of these fungi were maintained on malt extract agar. Three 1 g samples of soil were diluted to lo-’ in 100 ml 0.1% peptone solution. A 0.1 ml aliquot of this dilution was spread on 5 plates of nutrient agar, for each sample. Sixteen bacterial (including actinomycete) colonies were randomly selected, and maintained on nutrient agar. The liquid medium used for growing “C-1abelled micro-organisms was based on that of Anderson and Domsch (1978) and Ross (1987). This consisted (per 1 water) of 10 g U”C-labelled D-glucose (sp. act. 370 Bq mg-’ glucose), 1 gNH,NO,, 1 g KHrP04, 1 g K,HPO,, OSg MgSO,, 50mg CaClr and 20mg FeCl,. In addition media for fungi and bacteria contained respectively 1 and 3 g of yeast extract (DIFCO). The media were autoclaved prior to addition of the labelled glucose. The bacterial and fungal colonies were cultured in 50 ml liquid medium in 125 ml Erlenmeyer flasks, on a culture shaker at 22”C, until harvest. Fungi were harvested, using filtration, at the late linear phase, as determined by preliminary experimentation. They were rinsed by deionized water, air-dried and ground to 0.5 mm using a mortar and pestle. The bacteria were harvested at the late logarithmic growth phase by centrifugation at 15,000 rev min-’ for 20min. These were then airdried, rinsed, air-dried again and ground to 0.5 mm. Ail tire fungal material was then mixed together, as was all the bacterial tissue. Determination of “C content of tissue For both the bacterial and fungal tissue, six replicate 50 mg subsamples were dissolved using 1 ml of the tissue solvent protosol (Du Pont). This was left for 2 days, and 15 ml of the scintillation fluid BIOFLUOR (Du Pont) was added. This was left for a further day and the “C content was determined using a liquid scintillation counter (2200CA Tricarb Packard). Pretreatment of soil samples Six soil samples (mean field moisture content of 48% on dry wt basis) were sieved (~4 mm) and held at 4°C for a maximum of 4 months. These were then remoistened to 55% moisture content (dry wt basis) using an atomizer, and various subsamples of these were air-dried at 22°C until they reached one of five different moisture contents, i.e. 15, 25, 35.45 or 55% H,O (dry wt basis). These moisture contents corresponded to - 13000, - 1500, -400, - 30 and -6 kPa. soil moisture potential respectively. For each subsample described above, 20 subsamples (each 15 g dry wt) were prepared, i.e. the 5 moisture contents outlined above x 4 incubation periods (6 h or 3,10 or 30 days). The moisture contents used have been found to be the normal range of moisture
contents in the field soil during the growing season (Wardle and Parkinson, 199Oa). The above procedure was performed twice, once for each of the bacterial and fungal k, determinations described below. Determination of ‘C release from tissue following fwnigation Tests demonstrated that the soil used in our study responded in the classical manner to chloroform fumigation (Jenkinson and Powlson, 1976). This was performed by measuring the CO& release from both fumigated and non-fumigated soils hourly using an i.r. gas analyser (continuous flow rate = 178 ml min-‘). Within 5 days the respiratory difference between control and fumigated soils was slight; within 8 days this difference was non-existent. At the end of the 4 incubation periods, each subsample of soil was amended with 40 mg of either bacterial or fungal tissue, and thoroughly mixed. This was then fumigated with chloroform (alcohol-free) for 18-24 h (Jenkinson and Powlson, 1976). Subsequently the soil was removed, remoistened to 55% moisture content and reinnoculated with 1.67 g dry wt soil from the same sample (without prior incubation) so as to provide a 9: 1 ratio of fumigated soil to re-inoculant (Chapman, 1987). The subsamples were kept in airtight Qorpak jars (5OOml) together with a 50 ml beaker containing 15 ml of 1.OM NaOH. After 10 days, 1.5 ml NaOH was removed from each beaker and added to 15 ml BIOFLUOR scintillation fluid. The “CO& content of each sample was then determined using liquid scintillation counting. Data analysis For each subsample, k, factors were determined separately for fungi and bacteria, as a ratio of the amount of “CO& evolved from the tissue in 10 days to the total “C content of the tissue at the beginning of the incubation. In order to test whether the k, factors were influenced by the treatments used in this study, data were analysed using 3-way analysis of variance, testing for the blocking, moisture and duration of prior incubation effects, as well as the time x moisture interaction. When significant moisture effects were detected, multiple comparison analysis was performed within each prior incubation period across the soil moisture gradient, using Tukey’s test, following two-way ANOVA (moisture content and blocking). RESULTS
For the 120 fungal k, factors determined, the values ranged from 0.219 to 0.449, with a mean of 0.365. This k, factor was significantly influenced by soil moisture, duration of prior incubation and blocking, although time effects were the strongest (Tables 1 and 4). The k, factors for the 15% moisture treatment were always significantly lower than for the 35% treatment, as well as below the 45% treatment (10 days) and 55% treatment (6 h). The differences were, however, not large. The 25, 35, 45 and 55% treatments were never significantly different from each other. Duration of prior incubation exerted strong effects on the k, factor, with soil incubated for 30 days
Soil moisture effects of k, factor estimates Table 1. Fungal k, factors determined f~&wnaistun mcubation times
813
contents and four prior
Initial soil moisture content (%) Duration of prior incubation 6h 3 days lOdays 30 days
I5
25
35
45
55
0.3Sga 0.379a 0.335a 0.296a
0.379ab 0.396ab 0.352ab 0.319ab
0.4OOb 0.41lb 0.378b 0.35Ob
0.39Oab 0.389ab 0.371b 0.328ab
0.403b 0.382ab 0.35&b 0.316ab
Within each row. numbers followed by different letters arc significantly different at P = 0.05.
having k, factors well below the other prior incubation times. The results suggest that the kc factors vary between soils of different moisture content, between soils incubated at a fixed moisture content for different periods, and between different samples of the same soil type. There was no significant interaction between incubation time and soil moisture content, indicating that moisture effects are essentially similar regardless of incubation time. The values for the 120 bacterial k, factors were slightly higher than those for the fungi. The factors ranged from 0.243 to 0.484 with a mean of 0.398. However, the treatment effects wefe essentially similar (Tables 2 and 4). The kc values for the 15% treatment were always significantly lower than for at least one of the other moisture. treatments but these differences were usually relatively small. The 25, 35, 45 and 55% treatments were never significantly different from each other. Prolonged prior incubation reduced the kc factor for all moisture contents, with 30-&y samples having kc factors that were the lowest. The kc factors varied significantly across soil moisture contents, different incubation periods and different samples. When microbial kc factors were determined for the microbial biomass as a whole, essentially similar trends were found. Using a constant bacterial : fungal ratio of 25: 75 (Anderson and Domsch, 1975, 1978), kc values of the 120 subsamples ranged from 0.250 to 0.450 with a mean of 0.373. Essentially similar treatment effects were found as for the bacterial and fungal kc factors (Tables 3 and 4). The 15% moisture treatment was significantly different to the 55% one at 6 h, and to the 35% treatment at days 3, 10 and 30. Again the 25,35,45 and 55% treatments were not significantly different from each other. The kc factor was significantly reduced by prolonged prior incubation and also differed significantly among the six samples. When a separate bacterial:fungal ratio was determined for each subsample a very similar pattern was found. Bacterial:fungal ratios were determined separately for each of the 120 subsamples using the
selective inhibition technique of Anderson and Domsch (1973, 1975) in which 1Omg streptomycin sulphate g-l soil and 15 mg actidione g-* soil were added to inhibit glucose-stimulated respiration of bacteria and fungi, respectively. These ratios are presented in Wardle and Parkinson (199t.M). Although selective inhibition does not appear to inhibit much of the microbial respiration even when both inhibitors are added together (West, 1986; Wardle and Parkinson, 199Od), these values were used as in indicator in our study to determine what effects variation in bacterial : fungal ratios might exert on the factor. Using kc factors based on separate ratios for each subsample, kc values ranged from 0.246 to 0.448 with a mean of 0.374. Virtually identical trends were found by using varying bacterial:fungal ratios compared with using a constant ratio of 25:75, with regard to moisture effects, incubation time and blocking (Table 4). For all 120 subsamples, the k, factor determined using a ratio of 25 : 75 never differed by > 3% from the factors determined using a varying ratio. DISCUSSiON
Soil moisture effects on k, values The fumigation-incubation technique is frequently used with the assumption that k, values vary little between soil types (Jenkinson, 1976) or between treatments (Bottner, 1985; Duah-Yentumi and Johnson, 1986; Powlson et al., 1987). However Jenkinson (1988) summarixed information indicating variations in k, values under different soil conditions (particularly pH). Our study indicated significant variations in k, values with soil moisture content. Relatively dry soil (15%) displayed reduced values of k, even when kept at 55% moisture for the 10 days following fumigation like all the other treatments. Soil moisture contents of lO-20% were typical for this soil in the field during June and July 1988; during such conditions microbial biomass would be underestimated by using a constant k, factor relative to soils with a more optimal mois-
Table 2. Bacterial k, factors determined for five moisture contents and four incubation times
prior
Initial soil moisture content (%) Duration of prior incubation 6h 3 days 10 days 30 days
I5 0.4028 0.391a 0.358a 0.347a
25
35
45
55
0.40&b 0.415ab 0.417b 0.357ab
0.422ab 0.435b 0.391ab 0.389b
0.421ab 0.421ab 0.396ab 0.37Oab
0.435b 0.44Oab 0.4OOb 0.346ab
Within each row, numbers followed by different ktters arc significantly diffcwnt at P = 0.05. SIB 22,%-F
814
D. A.
WARDS and D. PARKINSON
Table 3. Microbial kc factors determined for five moisture contents and four prior incubation times. assuming a bacterial fungal ratio of 25:75 Initial soil moisture content (%) Duration of __._..... _~ prior incubation 6h 3 days 10 days 30 days
I5
25
35
45
55
0.37Oa 0.382a 0.34la 0.309a
0.385ab 0.4OOab 0.37Oab 0.328ab
0.405ab 0.417b 0.381b 0.36Ob
0.397ab 0.397ab 0.377ab 0.338ab
0.41 lb 0.396ab 0.368ab 0.324ab
Within each row, numbers followed by different ktters are significantly ditkrent at P - 0.05.
ture status. The depression of the k, factor caused by low moisture contents was not large, causing it to be reduced by ~20% (relative to optimal moisture conditions) in all cases. Nevertheless, this could exert an effect on the results of studies conducted across a gradient of moisture contents. Because incubation conditions (including the moisture status) for the 10 days following fumigation were identical for all soils, there is no obvious explanation as to why microorganisms in soils previously held at 15% of soil moisture (even for only 6 h) should demonstrate a reduced k, factor relative to other treatments. It is most likely, however, that soils at 15% moisture are in some way physically or chemically different to those at other moisture contents, and even when they are brought up to 55% moisture these differences are maintained for some time. This presumably results in the decomposition of added substrates being reduced over 10 days. We found the reverse trend to that of Ross (1987) with regard to soil moisture effects. In that study, higher soil moisture contents (above 60% of field capacity) resulted in reduced mineralization of added microbes, especially if moister soils had been smeared and compacted, kc values were also reduced if soils had been incubated at 60% field capacity for 7 days. In contrast, mineralization of microorganisms in our study was not significantly reduced by higher moisture, probably because smearing or compacting was not observed even at the highest moisture content which represented field capacity. Both our study, and that of Ross (1987) indicate that the use of the same kc value across a soil moisture gradient may be problematic. In our study, there was considerable variation in kc between different soil samples. This can be explained in terms of chemical or physical differences amongst the six samples (or blocking units) used in this study. Nicolardot er al. (1984) demonstrated that different soil types can have different microbial kc factors.
Variations in soil pH may also be important especially if soils of low pH are considered (Vance et al., 1987; Jenkinson, 1988). Also, our results suggest that there are important variations in kc between replicate samples collected within one soil type, as indicated by the blocking effect. This spatial variation of kc within a given soil type is consistent with temporal changes within a given soil, as reported by Ross (1987). Bacterial vs fungai k, values
Comparison of the bacterial and fungal kc factors indicates that a higher proportion of bacterial than fungal C is released as CO2 over 10 days. This was observed in all treatments, i.e. 36.5% of fungal ‘*C was released vs 39.8% of bacterial *‘C (mean of all 120 samples). Whether these differences are truly significant or not is unclear because tissue from all 7 fungal species were pooled as was tissue from all 16 bacterial colonies, preventing assessment of variability amongst species within each of the two microbial groups. In contrast, Anderson and Domsch (1978) found that fungal kc factors were significantly higher than bacterial kc factors (43.7% vs 33.3%). In our study varying the bacterial : fungal ratios did not greatly influence the microbial kc factors compared with using a constant ratio of 25:75. This is partly because the bacterial and fungal kc factors were not very different to each other, nor did the bacterial to fungal ratio differ greatly from a 25: 75 ratio (Wardle and Parkinson, 1990d). If the soils in our study had a ratio which differed greatly from 25: 75, then differences between k, values obtained using a 25 : 75 ratio, and values obtained using a different kc value for each subsample would have been greater also. In studies where the bacterial:fungal ratio is shifted by soil moisture [e.g. reduced since fungi are often more resistant to desiccation than are bacteria: Nakas and Klein (1979); Schnurer er al. (1986); West el al. (1987)] then the k, value may also be affected substantially.
Table 4. Relative importance of treatment effects on data presented in Tables l-3 Treatment efTect Soil moisture (A ) Duration of prior incubation (8) A x B interaction Blocking effect Unexplained
Fungal kc factor % of SS’
Bacterial kc factor % of ss
Microbial k, factor % of SSb
Microbial k, factor % of ss
12.7***
9.5”’
13.4***
14.0***
51.6*** 2.9 NS 7.5*** 25.5
35.a***
55.6**’ 2.8 NS 10.0*** 18.3
54.8*‘* 3.1 NS 9.8*** 18.2
,;.::.. 33:s
“Percentage of total sums of squares (SS) in data set explained by each effect. ‘kc factor using bacterial:fungal ratio of 25:75 (Tabk 3). ‘k, factor using bacterial: fungal rate separately determined for each sample. l, l*, *‘* Effect is significant at P =0.05, 0.01 and 0.001 respectively. NS, Effect not significant at 0.05.
Soil moisture effects of k, factor estimates
Soil moisture can exert an influence on microbial community structure (Kjoller and Struwe, 1982), and this may indicate that use of a constant k, value may be problematic, even if the bacterial:fungal ratios remains largely unchanged by soil moisture variation, such as we have observed. Data provided by Anderson and Domsch (1978) indicate that mineralization of the carbon in some fungal species in some soils was nearly twice as great, over 10 days, as that of other species. For the bacteria in that study, some species decomposed over five times more quickly than other species during that time. It is possible that microbial community structure may affect the k, factors of different soils or treatments, because some conditions may encourage microbial species which decompose faster in 10 days than do other conditions. Therefore, adding microbial tissues of the same species composition to different soils may not accurately reflect what happens to the native soil microbial biomass following fumigation, especially across a soil moisture gradient, although there is no effective way to test this. Our investigation demonstrated that the values of k, are significantly reduced in soils of low moisture content. Therefore, if the fumigation-incubation method is used with a constant kc value when responses of the microbial biomass to soil moisture are being considered (such as has been done in several studies), then the response of this biomass may be incorrectly assessed. Furthermore, the variation between k, values of soils held for different prior incubation times, and of replicate samples at each incubation time, indicate that the use of the fumigation-incubation method may be problematic if a constant k, value is used. In our study, variations in the bacterial:fungal ratios were not found to influence the k, factor significantly. However, this may be the case where bacterial: fungal ratios are observed to vary substantially across soil moisture gradients. Therefore, it can be recommended that, when the fumigation-incubation method is used to determine microbial biomass for soils of varying soil moisture content, efforts should be made to determine whether or not k, values are likely to remain independent of soil moisture values. Acknowledgements-We thank S. Visser for reviewing the manuscript, and D. M. Reid for the use of facilities for the work involving carbon-14. This work was supported by an NSERC grant (D. Parkinson) and a Canadian Commonwealth Scholarship (D. A. Wardle).
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