Biol Fertil Soils (1996) 21:284-292

9 Springer-Verlag 1996

ORIGINAL PAPER

N . S . B o l a n 9 L . D . Currie 9 S. Baskaran

Assessment of the influence of phosphate fertilizers on the microbial activity of pasture soils

Received: 24 May 1994

The objective o f the present work was to examine the effects of phosphate fertilizers on the microbial activity of pasture soils. Various microbial characteristics were measured using soils from an existing long-term phosphate fertilizer field trial and a short-term incubation experiment. The measurements included basal respiration, substrate induced respiration, inhibition of substrate-induced respiration by streptomycin sulphate (fungal activity) and actidione (bacterial activity) and microbial biomass C. The long-term field trials was initiated during 1985 to examine the effectiveness of different sources of phosphate fertilizers (single superphosphate, North Carolina phosphate rock, partially acidulated North Carolina phosphate rock, and diammonium phosphate) on pasture yield. The incubation experiment was conducted for 8 weeks using the same soil and the sources of phosphate fertilizers used in the field trial. In the incubation experiment the fertilizer addition caused an initial decrease in basal and substrate-induced respiration but had no effect on total microbial biomass. The initial decline in basal and substrate-induced respiration with the fertilizer addition was restored within 8 weeks after incubation. In the field experiment the fertilizer addition had no significant effect on basal respiration but increased substrate-induced respiration and microbial biomass C. The short-term and the long-term effects of phosphate fertilizer addition on the microbial characteristics of the soils are discussed in relation to its effects on pH, salt concentration, and the nutrient status of the soils. Abstract

Key words Basal respiration 9 Long-term effect 9 Metabolic quotient 9 Microbial biomass 9 Osmotic potential 9 Pasture soil 9 Phosphate fertilizers 9 Substrate-induced respiration (SIR)

N.S. Bolan (~) L.D. Currie 9 S. Baskaran Fertilizer and Lime Research Centre, Massey University, Palmerston North, New Zealand

Introduction Fertilizers are added to soils mainly to replace the nutrients removed by plant uptake and lost through processes such as produce removal, leaching, runoff, and volatilization. Fertilizers not only increase the nutrient status but also influence the biological activity of the soils (Russel 1973; Marshall 1977) through an increase in nutrient status and/or an increase in plant growth (Kirchner et al. 1993). Fertilizers sometimes influence the composition of specific microbial communities (e.g. nitrifying organisms) in soils through effects on soil chemical characteristics, such as p H and the ionic composition of the soil solution (Kaufman and Williams 1964; Khonje et al. 1989). There is growing interest in alternate farming systems, such as organic and biodynamic farming systems. These alternative farming systems may differ in their basic philosophies and recommended cultural and management practices, but have in common a total or partial prohibition on the use of soluble chemical fertilizers and chemical pesticides. One of the reasons for the prohibition o f chemical fertilizers in alternate farming is that the addition of fertilizer in conventional farming systems has been claimed to harm soil microorganisms and earthworm activity (Marshall 1977; Arden-Clarke and Hodges 1988). However, the effects of fertilizer on biology activity vary between pasture and cropping soils. In highly fertile pasture soils regular fertilizer addition has been shown to have little effect on soil microbial activity, and the shortterm changes due to climatic variations (seasonal effects) were greater than the long-term changes caused by fertilizer (Sarathchandra et al. 1988, 1993). The activity of soil microorganisms is measured either by the conventional plate-count technique or by soil respiration and microbial biomass (Jenkinson 1988). The plate-count technique gives the number or the mass of microorganisms, which may not necessarily indicate activity. According to Russel (1973) "the activity of the population is not a concept that can be given a quantitative definition, but for many purposes it can be measured by the amount of either CO: (respiration) or heat evolved

285 by the p o p u l a t i o n , t h a t is, by the rate at w h i c h either the oxidisable C c o m p o u n d s or the energy available for o r g a n i c g r o w t h are being dissipated". Various m e a s u r e m e n t s have b e e n u s e d as indices o f mic r o b i o l o g i c a l activity in soils ( N a n n i p i e r i et al. 1978). These m e a s u r e m e n t s include b a s a l respiration, substrate ( g l u c o s e ) - i n d u c e d r e s p i r a t i o n , m e t a b o l i c q u o t i e n t , microbial b i o m a s s , i n h i b i t i o n o f s u b s t r a t e - i n d u c e d r e s p i r a t i o n by s t r e p t o m y c i n s u l p h a t e a n d actidione, v a r i o u s e n z y m e activities a n d A T P contents. I n h i b i t i o n o f s t r e p t o m y c i n s u l p h a t e a n d a c t i d i o n e gives a m e a s u r e o f the relative activity o f fungi a n d b a c t e r i a , respectively. T h e m e t a b o l i c q u o t i e n t (or specific r e s p i r a t i o n rate) is d e f i n e d as the a m o u n t o f C respired as C O 2 p e r unit m i c r o b i a l b i o m a s s ( A n d e r s o n a n d D o m s c h 1986) a n d is c o n s i d e r e d to be a m o r e s e n s i t i v e index o f e n v i r o n m e n t a l a n d c h e m i c a l stress to soil m i c r o o r g a n i s m s t h a n r e s p i r a t i o n a l o n e (Wolters a n d J o e r g e n s e n 1991; A n d e r s o n a n d D o m s c h 1993). Since s u b s t r a t e - i n d u c e d r e s p i r a t i o n gives a close e s t i m a t e o f the active m i c r o b i a l b i o m a s s ( S p a r l i n g et al. 1981), the ratio b e t w e e n b a s a l r e s p i r a t i o n a n d s u b s t r a t e - i n d u c e d respirat i o n is likely to be p r o p o r t i o n a l l y related to the m e t a b o l i c q u o t i e n t (Wardle a n d P a r k i n s o n 1991). O n this basis, the ratio o f b a s a l r e s p i r a t i o n to s u b s t r a t e - i n d u c e d r e s p i r a t i o n is u s e d as an index o f the m e t a b o l i c q u o t i e n t to assess the effect o f fertilizer a d d i t i o n o n m i c r o b i a l activity. A l t h o u g h v a r i o u s studies have e x a m i n e d the effects o f a specific fertilizer a d d i t i o n o n the m i c r o b i a l b i o m a s s (Boltan et al. 1985; P e r r o t t a n d S a r a t h c h a n d r a 1989; Bristow a n d Jarvis 1991; Singh a n d Singh 1993) a n d resp i r a t i o n ( N a n n i p i e r i et al. 1979; K a n a z a w a et al. 1988; A m a d o r a n d Jones 1993) very few a t t e m p t s have b e e n m a d e to i d e n t i f y the factors which i n f l u e n c e the longt e r m effects o f fertilizer on m i c r o b i a l activity. T h e objective o f the present w o r k was to examine the s h o r t - t e r m a n d l o n g - t e r m effects o f p h o s p h a t e fertilizers o n m i c r o b i al activity as m e a s u r e d b y r e s p i r a t i o n a n d m i c r o b i a l biom a s s C. T h e a d d i t i o n o f p h o s p h a t e fertilizers n o t o n l y increases n u t r i e n t c o n c e n t r a t i o n s in soils b u t also influences o t h e r soil characteristics such as p H a n d ionic strength. T h e effect o f P fertilizers o n m i c r o b i a l activity is discussed in r e l a t i o n to the effects o f p H a n d ionic strength on m i c r o b i a l activity.

Materials and methods Field experiment The field experiment was initiated on 26 March I985 at the Soil Science Department Research Area, Massey University. The soil at the field site is Tokomaru silt loam, which is loess-derived (Typic Fragiaqualf) and has been described in detail elsewhere (Bolan et al. 1986). In summary, the soil has a pH (1 : 2.5 soil: water) of 5.8, an organic C content of 5.2%, NaHCO3-extractable P (Olsen P) of 12mgkg -1, and vermiculite and mica are the predominant clay minerals. The original objective of the trial was to compare the agronomic effectiveness of various phosphate fertilizer sources on the dry matter yield and botanical composition of the pasture. The experiment was a random block design with a plot size of 3 m • 2 m. There were eight treatments, each replicated five times. The treat-

ments included control (no P applied), four levels of single superphosphate (15, 30, 45, and 60kgP ha-l), North Carolina phosphate rock, partially acidulated phosphate rock, and diammonium phosphate, all added annually at 30 kg P ha -~. The partially acidulated sock was prepared by acidulating North Carolina phosphate rock with less (30%) acid than that required for complete acidulation to produce superphosphate. Sulphate-S addition was balanced over all treatments, and K and Mo were added to all plots. The soil samples were collected from a depth of 0 - 5 cm on 22 November 1993. The soil moisture content ( 0 - 5 cm depth) was 0.27 gg l, which is equivalent to 66% of the field capacity. Ten soil samples were collected from each plot and bulked together. The samples were stored at <4 ~ and the analyses were carried out within a week after collection.

Short-term incubation experiments The short-term effects of phosphate fertilizers, pH, and ionic strength on the microbial activity of soil were examined under laboratory conditions using unfertilized soil from the field experiment. Fresh soil samples from an unfertilized area near the long-term field experiment (Tokomaru silt loam) were collected, sieved to pass 2 mm, and stored in a cold room (< 4 ~ The phosphate fertilizer sources used in the field experiment were mixed with the soil and the samples were incubated at field capacity at room temperature (20 ~ The rates of fertilizer applied were equivalent to 15, 30, 45, and 60 kg P ha -1 for single superphosphate and 30 kg P ha -1 for North Carolina phosphate rock, partially acidulated phosphate rock, and diammonium phosphate. The fertilizer samples used were in a powder form (< 250 mm). Soil samples were withdrawn after 1 and 8 weeks of incubation and analysed for pH, electrical conductivity, basal respiration, substrate-induced respiration, inhibition of substrate-induced respiration by streptomycin sulphate and actidione, and microbial biomass C. The ionic strength of the soil solution was calculated from the electrical conductivity using the following equation (Black and Campbell 1982): Ionic strength (g)= 0.014 EC-0.0002 (1) where EC is electrical conductivity in mS cm -I. Unfertilized soil samples used in the incubation experiment were amended to different pH values using 0.1 M N a O H or 0.1 M HC1 solutions. The soil samples were incubated at field capacity for 1 week and then the microbial activity measurements were taken. The addition of increasing amounts of NaOH or HC1 not only alters the pH of the soil, but also results in an increase in the concentration of Na + and CI-, which may interfere with the effects of pH on microbial activity; hence it may not be possible to separate the effects of these two variables (pH and Na § and CI-) on microbial activity. However, the maximum concentration of Na + and CIwas 115 and 155 mg kg -1, respectively, and was expected to have a negligible effect on microbial activity (Killham and Firestone 1984). Another set of the unfertilized soil samples used in the incubation experiment were incubated with different levels of CaC12 solution. The soil samples were incubated for 1 week and the chemical (pH and electrical conductivity) and microbial characteristics were measured. The ionic strength (g) of the soil solution was calculated using Eq. 1.

Analytical measurements Soil respiration was measured from the amount of 02 consumed by a sample of soil using a Gilson differential respirometer (Macgregor and Naylor 1982). Under normal conditions, soils simultaneously generate CO 2 while consuming 02. To measure 02 consumption alone, the CO 2 evolved is removed by absorption onto KOH-impregnated filter paper. Both basal respiration and substrate-induced respiration were measured. A soil slurry was prepared by shaking 20 g moist soil with 40 cm 3 deionised water. Ap-

286 proximately 5 cm3 of freshly prepared slurry was used for the respiration measurement. To measure substrate-induced respiration, 0.5 cm3 of 1~ (w/v) glucose solution was added to the slurry. To absorb any CO 2 released during the respiration, a small filter paper was placed in the centre well of the respiration flask and soaked with 0.3 cm3 of 20o7o (w/v) KOH solution. The flasks were shaken for 4 h in a water bath maintained at 30 ~ and the amount of 02 consumed was monitored from the manometer at 30-min intervals. In order to obtain uniform sampling, the soil sample was made into a slurry before being used for respiration measurements. Soil respiration is generally measured at a moisture content close to field capacity (West and Sparling I986). The use of a soil slurry is likely to cause anaerobic conditions and also reduces the rate of diffusion of O2 and thereby affects the respiration measurement. However, a preliminary study showed that 02 consumption increased linearly with time for approximately the first 10 h, suggesting that anaerobic conditions did not occur within the period of respiration measurements. The continued shaking of the respiration flasks helps to increase the diffusion of O2. The metabolically active bacterial and fungal biomass was measured by the inhibition of substrate-induced respiration by streptomycin sulphate and actidione, respectively (Wardle and Parkinson 1990). Inhibition of respiration was assessed from the 02 uptake measurements using the Gitson respirometer in the presence of 10 mg of either streptomycin sulphate or actidione. A solution of 0.5 cm3 streptomycin sulphate or actidione was added to the soil slurry along with glucose solution, and the uptake of 02 was measured as described above. Microbial biomass was measured following the fumigation-extraction technique (Vance et al. 1987b). Six subsamples of fieldmoist soils, each equivalent to 25 g oven-dry soil, were weighed into 100-cm3 glass beakers. Three of these samples were placed in a desiccator containing about 25 cm3 ethanol-free CHC13 in a small beaker. The desiccator was evacuated until the CHC13 had boiled for 2 min and then kept in the dark at 20~ After 24 h of fumigation the beaker of CHCI 3 was removed and the residual CHC13 vapour in the soil was removed by repeated evacuations. The fumigated and the non-fumigated (control) soil samples were extracted with 0.5 MK2SO 4 at a solid: solution ratio of 1 : 4 for 30 rain in an endover-end shaker. The extract was filtered (Whatman no. 42) and the organic C in the extract was measured by K2Cr207 digestion. The microbial biomass was calculated from the following equation (Vance et al. 1987b): Microbial biomass C = Eoc/Kec

(2)

where Eoc is the increase in 0.5 M-K2SO4 extractable organic C in the fumigated soil over the non-fumigated (control) soil and K~r represents the efficiency of organic C extraction (Sparling and West 1988).

Table 1 Chemical characteristics of soil samples from the field experiment. Within a column, means followed by the same letter are not significantly different at the 5% level. Treatments: SSP single superphosphate, NCPR North Carolina phosphate rock, PAPR partially acidulated phosphate rock, DAP diammonium phosphate, applied at 15-60 kg P ha-l; pH was measured in water at a

Microbial biomass C was also estimated from the 0 z consumption data obtained during the substrate-induced respiration measurements using the following equation (Van de Waft and Verstraete 1987): Microbial biomass C = 11.261 Y-2.784

(3)

where Y is 02 consumption during substrate-induced respiration measurements (cm3 kg -1 h-l).

Statistical analysis The data were statistically analysed using Duncan's multiple range test (Gomez and Gomez 1984).

Results and discussion Field experiment T h e general characteristics of the soil samples collected from the field trial are presented i n Table 1. I n the first 3 years there was evidence of a dry m a t t e r response to P application a n d there was some difference between the fertilizer sources (data n o t shown). I n later years, a l t h o u g h there was a yield response to P a p p l i c a t i o n over the control, there was n o significant difference between the various fertilizer sources. The p H of the soils treated with single superphosphate, partially acidulated p h o s p h a t e rock, a n d d i a m m o n i u m p h o s p h a t e was slightly lower t h a n that o f control a n d N o r t h C a r o l i n a p h o s p h a t e rock-treated soils. C o n t i n u o u s a p p l i c a t i o n of P fertilizers to legume-based pastures has been shown to accelerate the rate o f soil acidification (Williams 1980). The increased rate of acidification with P fertilizer a p p l i c a t i o n has been att r i b u t e d to the acidity released from the direct dissolution of the m o n o c a l c i u m p h o s p h a t e c o m p o n e n t of the superp h o s p h a t e granules a n d a n increase in N2 fixation (Bolan et al. 1991). F i x a t i o n of a t m o s p h e r i c N 2 a n d the subseq u e n t leaching of NO~- f o r m e d from the m i n e r a l i z a t i o n o f fixed N result in the acidification of soils (Helyar

soil: solution ratio of 1:2.5; Olsen P was extracted by alkaline 0.1 M NaHCO 3 solution (pH 8.3) at a soil : solution ratio of 1 : 10; mineral N is NH4~-N+NO;-N extracted by 2 M KCI at a soil: solution ratio of 1 : i0. Ionic strength (Ix) = 0.014 EC-0.0002 where EC is electrical conductivity (mS cm-1)

Treatment

pH (H20)

Organic C (%)

Total P (mg kg 1)

Olsen P (mg kg- 1)

Total N (mg kg- l)

Mineral N (mg kg- l)

Ionic strength

Control SSP-15 SSP-30 SSP-45 SSP-60 NCPR PAPR DAP

5.86ab 5.72ab 5.76ab 5.72ab 5.61a 5.93ab 5.81ab 5.52a

5.7a 5.8ab 5.8ab 5.9ab 6.2b 5.9ab 6.0ab 6.lab

670a 760ab 929cd 1066d 1039d 970cd 845bc 965cd

18.6a 22.8ab 42.4c 61.4d 71.9d 30.5b 28,lab 45.6c

4689a 4400a 4838a 4678a 5098a 4885a 4993a 5079a

13a 29a 15a 53a 12a 23a 5a 56a

0,002a 0,009b 0,015cd 0,011bc 0.019d 0.007ab 0,015cd 0.014c

287 1976). T h e amount of acidity produced indirectly by N fixation depends mainly on the extent of N leaching and is much higher than the acidity produced directly from the dissolution of superphosphate granules. The addition of reactive phosphate rock such as North Carolina phosphate rock is likely to reduce the rate of soil acidification. The liming action of reactive phosphate rock occurs through two processes. Firstly, most reactive phosphate rock contains some free CaCO3, which can act as a liming agent. Secondly, the dissolution process of a phosphate mineral component (apatite) in soils consumes acid and thereby reduces soil acidity (Wright et al. 1991). The ionic strength of the soil solution ranged from 0.002 to 0.019, and generally increased with fertilizer application. Edmeades et al. (1985) have attributed the increase in ionic strength with superphosphate fertilizer application mainly to the increase in the concentration of Ca in the soil solution. The increase in ionic strength of the soil solution is likely to influence the microbial activity in soils (see below). The organic C values were greater in the fertilizer-treated soils than in the control soil and there was no difference between the fertilizer treatments. Sarathchandra et al. (1988) observed an increase of approximately 0.3% organic C in pasture soils due to fertilizer application in a long-term (8 years) field trial. The increases in organic C in fertilized soils are caused by the incorporation of plant residues and the release of root exudates due to an increase in plant growth. Both total and Olsen (available) P values were higher in the fertilized soils than the control soil and increased with increasing single superphosphate application. At 30 kg P ha -1, total soil P did not differ between the various P sources, available P was less in soils treated with North Carolina phosphate rock or partially acidulated phosphate rock than in those treated with single superphosphate or diammonium phosphate. The Olsen reagent (NaHCO3) extracts less P from reactive phosphate rocktreated soils than from soils treated with soluble P sources (Perrott et al. 1993). Dissolution of reactive p h o s p h a t e rock requires acidic conditions. Unless the added reactive phosphate rock dissolves in soils, the alkaline Olsen ex-

tract is unable to extract P directly from the reactive phosphate rock particles. There was no difference in concentration of mineral or total N between the various treatments. Mineral N contributes less than 2% of the total N, and hence it is not surprising that there was no difference in mineral N between the treatments. However, the addition of P fertilizer was expected to increase N fixation by legumes and thereby increase soil N. The absence o f any difference in total soil N between the fertilizer treatments may indicate the occurrence of a spatial redistribution of fixed N from high-fertility to low-fertility areas through the transfer of dung and urine. The effects of long-term fertilizer application on basal respiration, substrate-induced respiration, and bacterial and fungal activity are presented in Table 2. There was no significant fertilizer effect on basal respiration. The addition of single superphosphate at higher rates (30, 45, and 6 0 k g h a -1) and diammonium phosphate slightly increased substrate-induced respiration over the control treatment. The application o f single superphosphate (45 k g h a -1) and diammonium phosphate also slightly decreased the metabolic quotient, indicating less stress in these treatments. Insam et al. (1991) found no relationship between basal respiration and the increases in soybean yield from fertilizer application. However, the metabolic quotient was negatively correlated with the soybean yield. A high metabolic quotient means that the nutrient turnover is accompanied at great C expense. If more C is lost by respiration with less C input more care needs to be taken to maintain the organic C content. In accord with substrate-induced respiration both the bacterial and the fungal activity increased with the application of single superphosphate (30, 45, and 60kg h a - i ) and diammonium phosphate (Table 2). The bacterial: fungal activity ratio varied between 0.56 and 0.92. The application of diammonium phosphate resulted in the lowest bacterial: fungal activity ratio, which may be related to the increased acidification caused by this treatment. Both the selective inhibition and the plate-count techniques have shown that the fungal biomass is often

Table 2 Microbial characteristics of soil samples from the field experiment (SIR substrate-induced respiration; for other explanations see Table 1) Treatments

Respiration (cm3 kg- 1h- t) Basal

Control SSP-15 SSP-30 SSP-45 SSP-60 NCPR-30 PAPR-30 DAP-30

36a 29a 41a 36a 50a 38a 36a 20a

SIR

100ab 85a 145bc 220d 195cd ll4ab 125ab 200cd

Bacteria : fungi activity ratio

Metabolic quotient

Selectiveinhibition Bacteria

Fungi

60ab 45a 78bc 105cd 120d 69ab 57a 85bc

70ab 50a 95b 150c i43c 76ab 60a 151c

0.86ab 0.90b 0.82ab 0.70ab 0.84ab 0.91b 0.95b 0.56a

0.36c 0.34c 0.28bc 0.16ab 0.26bc 0.33c 0.24abc 0.10a

Biomass carbon (mg kg- 1)

Fumigation

02 consumption

626a 1067b 1010b 1201b 1176b 998b 1038b 1144b

l123ab 954a 1630bc 2474d 2193cd 1280ab 1405ab 2249cd

288

two or three times larger than the bacterial biomass (Wardle and Parkinson 1991; Kirchner et al. 1993). Since fungi are believed to use substrate C more efficiently (Alexander 1977), the ratio of bacterial : fungal activity, as measured by the inhibition of substrate-induced respiration, is expected to be much lower than that observed in the present experiment (0.54-0.92). Recently, Sakamoto and Oba (1994) reported a bacterial : fungal biomass ratio of approximately 0.61 in soils treated with different sources of organic materials. The sum of bacterial and fungal activity in the present study was, in general, greater than substrate-induced respiration values. Similarly, Wardle and Parkinson (1991) have observed that the combined effect of the inhibitors reduced substrate-induced respiration by approximately 40%. This suggests that neither streptomycin sulphate nor actidione achieved complete inhibition of the activity of bacteria and fungi, respectively. Wardle and Parkinson (1991) have suggested that the selective inhibition technique can be used to determine the relative activity of bacteria and fungi but only for those organisms undergoing protein synthesis following glucose addition. Therefore this technique is likely to measure only one component of the total biomass. There have been conflicting reports on the effects of fertilizer addition on the microbial activity of soils. The effects of inorganic fertilizers on microbial activity depend on the time-scale used to analyse microbial activity measurement after the imposition of fertilizer treatments. Kanazawa et al. (1988) have suggested that the application of chemical fertilizers does not result in large-scale changes in microbial populations. Nannipieri et al. (1979) have reported that biomass and respiration varied independently after nutrient amendment. Martyniuk and Wagner (1978) observed a positive fertilizer effect on the microbial population. Sarathchandra et al. (1993) found that fungal and Gram-negative bacteria were higher in phosphate-fertilized than in unfertilized soils, and suggested that in the short term the increased plant growth, and the associated increase in root growth and rhizosphere activity caused by the fertilizer may have led to increases in the bacterial populations. The application of inorganic fertilizers may modify the abundance of fungi,

Table 3 Basal respiration and substrate-induced respiration (SIR) characteristics of soil samples from the incubation experiment (for further explanations see Table 1)

Treatments

but such alterations are frequently more the result of acidification than of nutrient addition. For example, treatment with fertilizer Containing ammonium salts increases fungal numbers because microbial oxidation of the N leads to acidification which diminishes bacteria and actinomycetes and favours fungi (Alexander 1977). Microbial biomass C as measured by the fumigation technique ranged from 6 2 6 t o 1176 mg kg -1 (Table 2). This value was generally lower than that obtained from substrate-induced respiration. However, the two measurements (fumigation and substrate-induced respiration) were carried out under different soil moisture and temperature conditions and it may not be appropriate to compare the microbial biomass C values thus obtained. There was an increase in soil microbial biomass C in the fertilized soil over the control soil. But there was no significant difference between any of the fertilizer treatments. Sarathchandra et al. (1984) observed a similar range in microbial biomass C (540-1890 mg C kg -1) in pastoral soils, which are considered to be much higher in microbial C than cropping soils (Jenkinson and Ladd 1981). Microbial biomass C ranged from 1.2 to 2.7% of the total organic C in the soil. In a survey of 19 New Zealand soils under pasture, microbial biomass C values ranging from 1.3 to 3.9% of total C were obtained by fumigation-incubation (Sarathchandra et al. 1984). Fertilizer increases the soil microbial biomass by increasing the root biomass, root exudates, and crop residues, thus providing increased substrate for microbial growth (Campbell et al. 1991; Kirchner et al. 1993). Lynch and Panting (1980) reported that the microbial biomass increases as root growth increases. Increased plant growth due to fertilization will increase the root biomass and thus the quantities of root exudates. Anderson and Domsch (1989) have shown that in soils with less than 2.5% organic C, increasing C additions increased the microbial biomass. In an earlier study, Anderson and Domsch (1986) showed that when management of the soil is changed, the soil microbial biomass responds more quickly to the changes than soil organic matter does. Thus the ratio of microbial biomass C to total C will increase for a time if the input of fertilizer or organic matter to a soil is increased, and decreases for a time if the input is decreased.

Respiration (cm 3 k g - 1 h - a) SIR

Basal

Control SSP-15 SSP-30 SSP-45 SSP-60 NCPR-30 PAPR-30 DAP-30

Metabolic quotient

Week 1

Week 8

Week 1

Week 8

36b 2lab 12a 15a 12a 25ab 28b 14a

32a 35a 39a 27a 29a 33a 35a 27a

103c 78bc 42a 45a 35a 60b 52ab 25a

96ab 108abc ll5abc 146d 125bcd 136bcd 137cd 85a

Week 1

Week 8

0.35abc 0.27a 0.29ab 0.33abc 0.34abc 0.42abc 0.54bc 0.56c

0.33a 0.32a 0.34a 0.18a 0.23a 0.24a 0.26a 0.32a

289 Table 4 Bacterial and fungal activity of soil samples from the incubation experiment (for further explanations see Table 1)

Treatments

Control SSP-15 SSP-30 SSP-45 SSP-60 NCPR-30 PAPR-30 DAP-30

Bacterial activity (cm3kg lh-1)

Fungal activity (cm3 kg-: h - 1)

Bacterial : fungal activity ratio

Week 1

Week 8

Week 1

Week 8

Week 1

Week 8

60c 59c 27ab 24ab 25ab 50bc 39abc 1la

63ab 6lab 72ab 65ab 58ab 65ab 78b 45a

70cd 76d 37ab 3lab 29ab 48bcd 42abc 17a

75ab 72a 96abc 105bc 115c i10c 101bc 71a

0.86ab 0.78ab 0.73a 0.77ab 0.86ab 1.04b 0.93ab 0.65a

0.84b 0.85b 0.75ab 0.62ab 0.50a 0.59ab 0.77ab 0.63ab

Laboratory incubation The effects of phosphate fertilizer addition on basal respiration, substrate-induced respiration and bacterial and fungal activity under laboratory conditions are presented in Tables 3 and 4. Both basal and substrate-induced respiration measured 1 week after incubation were lower in the fertilized than in the untreated soil. However, both these values increased in the fertilized soils 8 weeks after incubation and the difference between the fertilized and the control soils decreased. The metabolic quotient was higher in soils sampled 1 week than in those sampled 8 weeks after incubation. This suggests that there may be more stress in the former than in the latter soil samples or that the microorganisms had adjusted to the new microenvironment in the fertilized soils by 8 weeks after incubation. Like substrate-induced respiration both the bacterial and the fungal activity decreased soon after fertilizer application (1 week after incubation) but were restored by 8 weeks after incubation. The stress on microbial activity with the addition of fertilizer may be caused by its effect on soil chemical characteristics, such as p H and ionic strength. The effect of these on microbial activity was examined in separate experiments (see below). There was no fertilizer effect on the soil microbial biomass after 1 week or after 8 weeks of incubation (data not shown). However, there was a slight decrease in the soil microbial biomass after 8 weeks compared to the 1-week period. In incubation experiments the microbial biomass often decreases as the incubation period increases, an effect attributed to the continuous release of microbial biomass C as CO2 through respiration (Inubushi et al. 1991). Both basal and substrate-induced respiration decreased with a decrease in soil pH, and the effect of p H was more pronounced on substrate-induced than on basal respiration (Fig. 1 a). The metabolic quotient reached a minimum between p H 4.5 and 6.6 (Fig. i b), indicating less stress within these p H values. Wolters and J0ergensen' (1991) found that in forest soils, which are subjected to acid rain, exchangeable Ca, which indicates the extent of soil acidity, was negatively correlated with metabolic quotient. Microflora in the more acid soils (with less exchangeable Ca) have a reduced ability to incorporate C from freshly fallen litter into the subsurface C cycle. Re-

cently, Anderson and Domsch (1993) observed that the metabolic quotient increased with a decrease in soil pH, and suggested that the metabolic quotient is a better measure of environmental stress on soil biological activity than soil respiration alone. They suggested that a high metabolic quotient at a low p H is an indication of terrestrial community stress, in accord with the O d u m (1985) theory that "repairing damage by disturbance or stress requires diverting energy from growth and production to maintenance". Anderson and Domsch (1993) suggested that the high metabolic quotient at a low p H reflects a higher maintenance energy requirement by the microbial community or indicates a shift in the bacteria: fungi ratio. Both bacterial and fungal activity decreased with a decrease in soil p H (Fig. I c). The p H effect on bacterial activity was stronger than that on fungal activity, which resuited in a decrease in the bacterial : fungal activity ratio with a decrease in soil p H (Fig. 1 d). Highly acidic or alkaline conditions tend to inhibit many c o m m o n bacteria as the o p t i m u m p H for most species is near neutral. Generally, the greater the H + concentration, the smaller is the size of the bacterial community. Alexander (1977) suggested that because many fungi are less sensitive to a high H + concentration the fungi make up a larger proportion of the microbial community and are likely to be responsible for a major portion of the microbial activity in acid soils. There was no effect of soil p H on microbial biomass C (data not shown), and the effect on the soil microbial biomass was often variable. Low microbial biomass C values have been recorded from sites with a low p H (Jenkinson et al. 1979; Vance et al. 1987a). Increased acidification of soil through SO2 emission and elemental S addition frequently reduces the microbial biomass (Bewley and Parkinson 1984; Gupta et al. 1988). However, a simulated addition of acid rain to forest soils had little impact on the microbial biomass until p H values were depressed as low as 2 or 3 (Killham et al. 1983; Wolters 1991). In a recent review Wardle (1992) reanalysed 39 data sets with a wide range of p H values and concluded that p H is substantially less important than organic C and N in influencing the total microbial biomass. Both the basal and substrate-induced respiration remained constant with an initial increase in ionic strength

290 Fig. 1 Effect of pH on soil respiration (a), metabolic quotient (b), inhibition of substrate-induced respiration (SIR) (c) by streptomycin sulphate (fungal activity) and actidione (bacterial activity) and bacterial: fungal activity ratio (d). Within a line, means indicated by a common letter are not significantly different at the 5% level

Soil respiration

c SIR inhibition

A

c 120

~..~ 10080

bo/9 ~t/

br

~k

r

100 ab

=0.

~

60

~

20

X

0

so 60

oo ==

a0

r

~' 0

20

'

3

I

'

I

4

5

'

I

'

6

I

'

7

I

8

0

'

3

l

4

'

I

'

I

5

6

~

I

'

I

7

8

Soil pH

IT B~ Fungus )

b Metabolic quotient

d Bacterial : fungal activity ratio

c

OJ

o

~

0.5

0.9

C

0.6

0 ..Q

d

1.2-

0.6-

b

0.4 a

ab

=E

~

0.3

a

0.3

0.0

' 1 ' 1 ' 1 ' 1 ' [

3

4

5

6

7

8

, I , l , l , l , !

3

4

5

6

7

8

Soil pH

(0.01) but decreased at higher ionic strengths of the soil solution (Fig. 2 a). The metabolic quotient also remained constant at a low ionic strength but increased at a higher ionic strength (Fig. 2b). Killham (1985) observed that the metabolic quotient increases with an increase in the salt concentration. An increased salt concentration, as measured by ionic strength, increases the osmotic potential of the soil solution and thereby influences microbial activity (Killham and Firestone 1984). In the present study both bacterial and fungal activity and the ratio of bacterial: fungal activity decreased with an increase in the ionic strength (Fig. 2c, d). At the highest ionic strength, greater amounts of C were extracted from both the fumigated and the non-fumigated soils than at the lower ionic strength (data not shown). However, the ionic strength of the soil solution had no significant effect on microbial biomass C.

We conclude that application of the chemically processed and unprocessed phosphate fertilizers to soils resulted in a temporary inhibition of soil microbial activity as measured by soil respiration. This inhibition was a result of changes in soil pH and in the ionic concentration of the soil solution as a direct result of the fertilizer application. However, microbial activity was restored within a short period after the fertilizer application. Thus, in a long-term field trial, phosphate fertilizer addition has a negligible effect on soil microbial activity. Increased plant growth due to P fertilizer addition, however, increases the total microbial biomass C. We thank Dr. A.N. Macgregor for help in the respiration measurements and Drs. D.A. Wardle and S. S.S. Rajan for suggestions during the initial stages of the work. Acknowledgments

291

a Soil respiration

Fig. 2 Effect of ionic strength on soil respiration (a), metabolic quotient (b), inhibition of SIR (e) by streptomycin sulphate (fungal activity) and actidione (bacterial activity) and bacterial: fungal activity ratio (d). For further expIanations see Fig. 1

c SIR inhibition A '7,

A

e- 70

120

bc

c

2; E

=E

100

60

80

.o

40

bc

c

ab

E 30 e,-

o

o o

a

20

10

0

0

20

133

a

x

so

bc

60 40

==

E

I

I

I Illll

I

I

I

I llIll

0.010

0.001

~,

a

0

I

0.100

0

=

L

li=i=i

0.001

i

i

0.010

i

llINl

I

0.100

Ionic strength

Iv:::::: H.

I~__~_

Basal~ SIR

)

b Metabolic quotient

d Bacterial : fungal activity ratio

0.8

1.0 c

-~

C , II l l

g

0.7

b

==

0.9

0.8 0.6

0.7 -

13

ab

0.5

"E

-

m

0.4

0.6 L 0.5 0.4 -

0.3

........

0.001

I

0.010

. . . . .

0.3

'"1

0.100

q =111~11

0.001

0.010

J

= = |lllU

I

0.100

Ionic strength

References Alexander M (1977) Introduction to soil microbiology. Wiley and Sons, New York Amador JA, Jones RD (1993) Nutrient limitations on microbial respiration in peat soils with different total phosphorus content. Soil Biol Biochem 25:793-801 Anderson TH, Domsch KH (1986) Application of eco physiological quotients (qCO 2 and qD) on microbial biomass from soils of different cropping histories. Soil Biol Biochem 22:251-256 Anderson TH, Domsch KH (1989) Ratio of microbial biomass carbon to total organic carbon in arable soils. Soil Biol Biochem 21:471-479 Anderson TH, Domsch KH (1993) The metabolic quotient for CO 2 (qCO2) as a specific activity parameter to assess the effects of environmental conditions, such as pH, on the microbial biomass of forest soils. Soil Biol Biochem 25:393-395 Arden-Clarke C, Hodges RD (1988) The environmental effects of conventional and organic/biological farming systems. II. Soil

ecology, soil fertility and nutrient cycles. Biol Agric Hort 5:223- 287 Bewley RJF, Parkinson D (1984) Effects of sulphur dioxide pollution on forest soil microorganisms. Can J Microbiol 30: 179-185 Black AS, Campbell AS (1982) Ionic strength of soil solution and its effect on charge properties of some New Zealand soils. J Soil Sci 33:249-262 Bolan NS, Syers JK, Tillman RW (1986) Ionic strength effects on surface charge and adsorption of phosphate and sulphate by soils. J Soil Sci 37:379-388 Bolan NS, Hedley M J, White RE (1991) Processes of soil acidification during nitrogen fixation with special emphasis on legume based pastures. Plant and Soil 134:53-63 Boitan H Jr, Elliott LF, Papendick RI, Bezdicek DF (1985) Soit microbial biornass and selected soil enzyme activities; effect of fertilization and cropping practices. Soil Biol Biochem 17:297-302 Bristow AW, Jarvis SC (1991) Effects of grazing and nitrogen fertilizer on the soil microbial biomass under permanent pasture. J Sci Food Agric 54:9-21

292 Campbell CA, Lafond GP, Zentner RP, Biederbeck VO (1991) Influence of fertilizer and straw baling on soil organic matter in a thin black chernozen in Western Canada. Soil Biol Biochem 23:443 - 4 4 6 Edmeades DC, Wheeler DM, Clinton OE (1985) The chemical composition and ionic strength of soil solutions from New Zealand topsoils. Aust J Soil Res 23:151-165 Gomez KA, Gomez A A (1984) Statistical procedures for agricultural research, 2nd edn. Wiley and Sons, New York Gupta VVSR, Lawrence JR, Germida JJ (1988) Impact of elemental sulphur fertilization on agricultural soils. II. Effects on microbial biomass and enzyme activities. Can J Soil Sci 68:463 - 473 Helyar KR (1976) Nitrogen cycling and soil acidification. J Aust Inst Agric Sci 42:217-211 Insam H, Mitchell CC, Dormarr JF (1991) Relationship of soil microbial biomass and activity with fertilization practice and crop yield of three Utisols. Soil Biol Biochem 23:459-464 Inubushi K, Brookes PC, Jenkinson DS (1991) Soil microbial biomass C, N and ninhydrin-N in aerobic and anaerobic soils measured by fumigation-extraction method. Soil Biol Biochem 23:737 -741 Jenkinson DS (1988) Determination of microbial biomass carbon and nitrogen in soils. In: Wilson JR (ed) Advances in nitrogen cycling in agricultural ecosystems. CAB International, Wallingford, pp 368-386 Jenkinson DS, Ladd JN (1981) Microbial biomass in soil: Measurement and turnover. Soil Biochem 5:415-471 Jenkinson DS, Davidson SA, Powlson DS (1979) Adenosine triphosphate and microbial biomass in soils. Soil Biol Bioehem 11:521 - 527 Kanazawa S, Asakawa S, Takai Y (1988) Effect of fertilizer and manure application on microbial numbers, biomass, and enzyme activities in volcanic ash soits. I. Microbial numbers and biomass carbon. Soil Sci Plant Nutr 34:429-439 Kaufman DD, Williams LE (1964) Effect of mineral fertilization and soil reaction on soil fungi. Phytopathology 54:I34-139 Khonje DJ, Varsa EC, Klubek B (i989) The acidulation effects of nitrogenous fertilizers on selected chemical and microbiological properties of soil. Commun Soil Sci Plant Anal 20:1377-1395 Killham K (1985) A physiological determination of the impact of environmental stress on the activity of microbial biomass. Environ Pollut (Ser A) 38:283-294 Killham K, Firestone MK (1984) Salt stress control of intracellular solutes in streptomycetes indigenous to saline soils. Appl Environ Microbiol 47:301-306 Killham K, Firestone MK, McColl JG (1983) Acid rain and soil microbial activity: Effects and their mechanisms. J Environ Qual 12:133-137 Kirchner M J, Wollum AG II, King LD (1993) Soil microbial populations and activities in reduced chemical input agroecosystems. Soil Sci Soc Am J 57:1289-1295 Lynch JM, Panting LM (1980) Cultivation and the soil biomass. Soil Biol Biochem 12:29-33 Macgregor AN, Naylor LM (1982) Effect of municipal sludge on the respiratory activity of a cropland soil. Plant and Soil 65:149-I52 Marshall VG (1977) Effects manures and fertilizers on soil fauna: A review: Commonwealth Bureau of Soils, Spec Pubt 3, Commonwealth Bureau, London Martyniuk S, Wagner GH (1978) Quantitative and qualitative examination of soil microflora associated with different management systems. Soil Sci 125:343-351 Nannipieri P, Johnson RL, Paul EA (1978) Criteria for measurement of microbial growth and activity in soil. Soil Biol Biochem 10:223 - 2 2 9 Nannipieri P, Pedrazzini E, Arcara PG, Piovanelli C (t979) Changes in amino acids, enzyme activities and biomass during soil microbial growth. Soil Sci 127:26-34 Odum E (1985) Trends expected in stressed ecosystems. Bioscience 164:262- 270

Perrott KW, Sarathchandra SV (1989) Phosphorus in the microbial biomass of New Zealand soils under established pasture. NZJ Agric Res 32:409-413 Perrott KW, Saggar S, Menon RG (1993) Evaluation soil phosphate status where phosphate rock based fertilizers have been used. Fert Res 35:67-82 Russell EW (1973) Soil conditions and plant growth, 10th edn. Longman, London Sakamoto K, Oba Y (1994) Effect of fungal to bacterial biomass ratio on the relationship between CO 2 evolution and total soil microbial biomass. Biol Fertil Soils 17:39-44 Sarathchandra SU, Perrott KW, Upsdell MP (1984) Microbiological and biochemical characteristics of a range of New Zealand soils under established pasture. Soil Biol Biochem 16:177-183 Sarathchandra SU, Perrott KW, Boase MR, Waller JE (1988) Seasonal changes and the effect of fertilizers on some chemical, biochemical and microbiological characteristics of high producing pastoral soils. Biol Fertil Soils 6:328-335 Sarathchandra SU, Lee A, Perrott KW, Rajan SSS, Oliver EHA, Gravett IM (1993) Effects of phosphate fertilizer applications on microorganism in pastoral soil. Aust J Soil Res 31:299-309 Singh H, Singh KP (1993) Effect of residue placement and chemical fertilizer on soil microbial biomass under tropical dryland cultivation. Biol Fertil Soils 16:275-281 Sparling GP, West AW (1988) A direct extraction method to estimate soil microbial C: In situ calibration using microbial respiration and ~4C labelled cells. Soil Biol Biochem 20:337-343 Sparling GP, Ord BG, Vaughan D (1981) Microbial biomass and activity in soils amended with glucose. Soil Biol Biochem 13: 99 - 104 Van de Werf H, Verstraete W (1987) Estimation of active soil microbial biomass by mathematical analysis of respiration curves: Calibration of the test procedure. Soil Biol Biochem 19: 261-263 Vance ED, Brookes PC, Jenkinson DS (1987a) Microbial biomass measurements in forest soils: Determination of K c values and tests of hypothesis to explain the failure of the chloroform fumigation-incubation methods in acid soils. Soil Biol Biochem 19:689- 696 Vance ED, Brookes PC, Jenkinson DS (1987b) An extraction method for measuring soil microbial biomass. Soil Biol Biochem 19:703 - 7 0 7 Wardle DA (1992) A comparative assessment of factors which influence microbial biomass carbon and nitrogen levels in soil. Biol Rev 67:321-358 Wardle DA, Parkinson D (1990) Response of the soil microbial biomass to glucose, and selective inhibitors, across a soil moisture gradient. Soil Biol Biochem 22:825-834 Wardle DA, Parkinson D (1991) A statistical evaluation of equations for predicting total microbial biomass carbon using physiological and biochemical methods. Agric Ecosyst Environ 34:75 - 86 West AW, Sparling GP (1986) Modifications to the substrate-induced respiration method to permit measurement of microbial biomass in soils differing water contents. J Microbiol Methods 5:177-189 Williams CH (1980) Soil acidification under clover. Aust J Exp Agric Anim Husb 20:561-567 Wolters V (1991) Biological processes in two beech soils treated with simulated acid rain - a laboratory experiment with Isotoma tigrina (Insecta, Collembola). Soil Biol Biochem 23:381-390 Wolters V, Joergensen RG (1991) Microbial carbon turnover in beech forest soils at different stages of acidification. Soil Biol Biochem 23:897-902 Wright RJ, Baligar VC, Belesky DP, Snuffer JD (1991) The effect of phosphate rock dissolution on soil chemical properties and wheat seedling root elongation. In: Wright R J, Baligar VC, Murrmann RP (eds) Plant-soil interactions at low pH. Kluwer Academic Publishers, London, pp 281-290