Soil Biol. Biorkm. Vol. 27, No. 11, pp.

1431-1443, 1995
Copyright ,D 1995 Elsevier ScienceLtd
Pergamon
0038-0717(95)00069-0 Printed in Great Britain. All rights reserved
0038-0717195 $9.50 + 0.00

SOIL :MICROBIAL BIOMASS, C AND N MINERALIZATION

AND ENZYME ACTIVITIES IN A HILL PASTURE:
INFLrUENCE OF SEASON AND SLOW-RELEASE P AND S
FERTILIZER
1). J. ROSS,‘* T. W. SPEIR,‘? H. A. KETTLES’ and A. D. MACKAY3
‘Manaaki-Whenua Landcare Research New Zealand, Private Bag 11052, Palmerston North,
New Zealand, 2Landcare Research New Zealand, Private Bag 3 127, Hamilton, New Zealand and
“AgResearch Sustainable Production Systems Division, Private Bag 11008, Palmerston North,
New Zealand

(Accepted 16 April 1995)

Summary-Hill pastures are widely used in New Zealand for sheep and cattle production. The influence
of season and added slow-release fertilizer (rock phosphate and elemental S) on soil microbial pools and
biochemical activities in a low-fertility hill pasture was investigated during 2 y. The soil was a Typic
Dystrochrept. Specifically determined &and kEpl-factors were used in the fumigation-extraction
procedures for measuring microbial C and N. An appropriate factor for converting substrate-induced
respiration values to microbial C was also determined. Fertilizer tended to increase dry matter production
and the legume component ofthe sward in the second year of the trial, but variability was high and differences
were generally non-significant. Fertilizer increased extractable soil inorganic P and S, but had no consistent,
if any, effect on soil biochemical properties. Soil moisture content was generally high at all sampling times.
Amounts of microbial biomass, and the percentages of microbial C and N in total C and N, respectively,
were similar to those in more fertile lowland pastures. Temporal fluctuations in microbial C, N and P
concentrations were small and mainly non-significant, and were related to variability in total C and N
concentrations. Temporal fluctuations were also mainly non-significant for invertase and sulphatase
activities, but were very marked for phosphodiesterase activity. CO?-C production under standardized
conditions was highest in spring samples and significantly correlated with extractable C concentration.
Min-N was generally immobilized initially on incubation at 2X, but net N mineralization increased over
14-56 d with maxima also in spring; NH4+-N predominated throughout. Overall, potential rates of C and
N mineralization were more susceptible to seasonal effects than were the microbial biomass pools.

INTRODUCTION determined under standardized conditions

The key role of the soil microbial biomass in the
cycling of C, N and P and other nutrient elements in
terrestrial ecosystems is now well recognized (Jenkin-
son and Ladd, 1981; Duxbury et al., 1989; Smith and
Paul, 1990). Changes in pool sizes and activities of the
component microorganisms can be influenced by
many environmental factors, including climate and
soil nutrient status, and, in turn, have important
implications for plant growth (Sarathchandra et al.,
1988).
Marked temporad effects on the amounts of soil
microbial biomass have been found during the growth
of annual crops (Lynch and Panting, 1980; Ritz and
Robinson, 1988; Kaiser and Heinemeyer, 1993) and in
some, but not all, grasslands (Sarathchandra et al.,
1988; Patraetal., 1990; Perrottetal., 1990,1992; Ross,
1990b; Bristow and Jarvis, 1991; Hill et al., 1993;
DeLuca and Keeney, 1994). Respiratory activities,
*Author for correspondence.
tPresent address: Envl ronmental Science and Research Ltd,
P.O. Box 30547, Lower Hutt, New Zealand.
(Sarathchandra et al., 1988; Patra et al., 1990; Ross,
1990b) and enzyme activities (Ross and Speir, 1984;
Perrott et al., 1990; Hill et al., 1993) of grassland soils
can also vary seasonally in a temperate climate.
The effects of added fertilizer on soil biochemical
properties are, in part, dependent on the prevailing
nutrient status of the soil, and were found by
Sarathchandra et al. (1988) to be mainly negligible in
an already-fertile pasture. Although microbial
biomass indices (mineral-N flush and ATP values)
were likewise similar in two pastures of different
P-fertility status, net N mineralization was markedly
lower in the soil that had been unfertilized for over
30 y (Tate et al., 1991).
We here examine the influence of seasons, and
slow-release P and S fertilizer addition, on extractable
and microbial C, N and P concentrations, invertase,
phosphodiesterase and sulphatase activities, and C
and N mineralization in the soil of a low-fertility hill
pasture. Such pastures are widely used in New Zealand
for sheep and cattle production and are dependent on
P and S fertilizers to sustain legume growth and N

1431
 1432 D. J. Ross ef al.
fixation (Lambert et al., 1983). The extent to which
these biochemical properties can change seasonally is
of interest from several viewpoints, including their
possible value as indicators of soil quality (Doran and
Parkin, 1994) and their relationships with plant
growth. Invertase and sulphatase activities, for
example, can reflect pasture productivity (Ross et al.,
1982) while phosphatase activity can vary with P
availability (Speir and Ross, 1978). This particular
investigation formed part of a wider study examining
the effects of fallowing, with or without added
fertilizer, on aspects of pasture dynamics and
biochemical and biophysical characteristics of the soil
(Mackay et al., 1991; Ross et al., 1995).
MATERIALS AND METHODS
Site
The site was located on a south-facing slope in
Matai paddock at AgResearch Ballantrae Hill
Country Research Station, 20 km north-east of
Palmerston North and situated on the southern slopes
of the Ruahine Range, New Zealand. The mean
annual rainfall is ca. 1200 mm, and is evenly
distributed throughout the year. The mean annual
temperature is 12.2”C.
The experimental area was subdivided into three
horizontal blocks, each containing six fully fenced
plots of 0.05-0.08 ha. One plot in each block was
selected at random for each of the fertilizer and
no-fertilizer treatments; the remaining plots were used
for an associated fallowing study (Mackay et al.,
1991). The pasture had not been fertilized for more
than 10 y and was dominated by low-fertility plant
species, including browntop (Agrostis capillaris L.),
sweet vernal (Anthoxanthum odoratum L.), suckling
clover (Trifolium dubium Sibth.) and Lotuspeduncula-
tus Cav. The soil was predominantly Mangaweka silt
loam, a Typic Dystrochrept, with an Ah horizon at
about O-13 cm depth and AB horizon at about
13-25 cm depth; [horizon designations are according
to Hewitt (1992)]. Some mottling and gleying occurred
at greater depths.
Fertilizer, consisting of North Carolina reactive
phosphate rock (35 kg P ha-’ y-‘) and elemental S
(14 kg ha-’ y-l), was applied by hand to the
appropriate plots in early spring (September 1989 and
September 1990). Both the fertilized and non-fertilized
plots were rotationally grazed by sheep and cattle. The
trial itself commenced in September 1989, after
preliminary investigations had been made in July of
the same year.
Soil sampling
Estimation of spatial variability. A preliminary
assessment of soil chemical and biochemical variabil-
ity within each plot was carried out in July 1989. In
each plot, 10 pegs were placed as distant as possible
from each other, ca. 3-15 m apart, on suitable 12-20”
slopes. Two cores (2.5 cm dia, O-5 cm depth) were
taken within a radius of 25 cm of each peg, and the 20
cores from each plot then pooled and sieved ( < 2 mm
mesh). Subsequent data (not shown) indicated that
between-plot differences of ca. 10% or more could be
needed to detect significant treatment effects. The
values of (LSD)/(grand mean) in this assessment
averaged 10% (range 8-13%) for moisture, total C
and N, and extractable-C flush, 15% for substrate-in-
duced respiration (SIR) and 29% for extractable C
values.
Routine procedure. At each sampling time, three
cores (2.5 cm dia, O-5 cm depth) were taken within
25 cm of the above 10 pegs in each plot, to provide
sufficient soil for the measurements made. The 30 cores
from each plot were pooled and the sieved (< 2 mm),
field-moist soil was stored at 4°C. Three replicate
samples were thereby obtained for each of the fertilizer
and no-fertilizer treatments.
Herbage sampling
Exclusion cages (0.5 m’) were used to measure
pasture production, with one cage being rotated
around the 10 pegs used for soil sampling in each plot.
Herbage was cut l-2 cm above ground level, at
intervals that coincided with critical periods in the
associated fallowing trial (Mackay et al., 1991). After
dissection into different components, the samples were
dried at 65°C.
Analytical methods
Results are expressed on the basis of oven-dry
(105C) weight of material, unless otherwise stated.
Soil biochemical analyses commenced within 7 d of
sampling and were mainly made in duplicate. Three
replicates were, however, used for the determinations
of extractable P, microbial P and phosphodiesterase
and sulphatase activities, and four replicates for SIR.
Soil moisture, pH (in water) and total C and N
concentrations were determined according to Blake-
more et al. (1987). Preliminary estimates of soil water
content at - 5 kPa were made with separate, pooled
samples from each horizontal block by the tension-
table method (Gradwell, 1972). Water-holding
capacity (WHC) was measured according to Harding
and Ross (1964).
E.xtractable C’, N and P. Extractable C and N were
determined by shaking 10.0 g samples, that had been
adjusted to -5 kPa (ca. 65% of WHC), in an
end-over-end shaker for 30 min with 25 ml 0.5 M
K?SO+ Organic C in the filtrates was measured by
dichromate oxidation (Jenkinson and Powlson,
1976a), and total N by persulphate oxidation (Ross,
1992). The determination of inorganic P (Pi)
extractable in 0.5 M NaHCO, was based on the
procedure of Brookes et al. (1982). Extractable total
P (Pt) was determined by treating 5.0 ml of the 0.5 M
NaHCO, extract with 1.O ml 2.5 M H?SO+ followed by
3.0 ml concentrated H$04 and a Kjeldahl copper
 Pasture soil: seasonal and fertilizer effects
catalyst tablet (BDH). The mixture was evaporated
and digested as described by Blakemore et al. (1987).
After diluting the digest to 50 ml with water, 5.0 ml of
the resultant supernatant solution were neutralized
with 5.0 ml 1 M NaOH. P was then measured with an
ammonium molybclate-malachite green reagent (Mo-
tomizu et al., 1983). Extractable organic P (PO) was
calculated as the dilference between extractable P, and
P, values.
CO1 production. CO2 production was measured in
Biometer flasks (Bartha and Pramer, 1965) with 10.0 g
soil at -5 kPa incubated for 14 d at 25°C. CO* was
absorbed in 100 rnM KOH and estimated, after the
addition of 1 M BaClr, by titration of excess alkali with
50 mM HCl, using phenolphthalein as an indicator.
SIR. SIR was determined according to West and
Sparling (1986) by adding water (to a total volume of
2.0 ml) and glucose (30 mg ml-‘) to the equivalent of
1.0 g oven-dry soil. COZ production over 2 h,
commencing 30 min after the glucose addition, was
measured by gas chromatography.
Extractable-Cjush and extractable-Nflush. These
indices of microbial C (Vance et al., 1987) and N
(Brookes et al., 1985) were determined by chloroform
fumigation-extraction methods, as described by Ross
and Tate (1993).
C02-C@ush. CO*-C flush was measured in only two
sets of samples to estimate microbial C, and thereby
derive &-factors for converting extractable-C flush to
microbial C values. The chloroform fumigation-incu-
bation procedure for determining CO*-C flush
(Jenkinson and Powlson, 1976b) is described by Ross
and Tate (1993), and used 20.0 g soil at - 5 kPa and
a small inoculum (ca. 25 mg) of unfumigated soil.
Various controls were employed for determining
CO*-C flush values; the &factor of 0.45 (Jenkinson,
1988) was used for subsequent conversion of the flush
to microbial C.
MicrobialP. The fumigation-extractionmethod for
determining microbial P was based upon that of
Brookes et al. (1982); it used field-moist soil
(equivalent to 4.0 g oven-dry wt), 80 ml 0.5 M
NaHCOs extractant, and an extraction time of 2 h in
an end-over-end shaker. Before filtration, ca. 1 ml
aqueous polyacryla:mide (BDH) solution (0.2% w/v)
was added. The filtcates (10 ml) were slightly acidified
by adding 1.0 ml 5.2 M HCl, and shaken for ca.
0.5 min with 0.2 g activated charcoal (Sigma); blanks
and standard solutions were treated in the same way.
A further 1.0 ml 5.2 M HCl was then added and the
suspensions were filtered immediately through a GFC
paper. Pi in the subsequent filtrates was measured by
the method of Murphy and Riley (1962). The P, flush,
viz. P, extracted from fumigated soil minus Pi extracted
from unfumigated soil, was converted to microbial P
by using the kp-factor of 0.40 and correcting for P,
adsorption from an added KHzPOd-P (250 pg) spike
(Brookes et al., 1!)82). Recovery of the P, spike
averaged 48 +_ 3(SD)%.
N mineralization., nitriJication and min-N flush.
SBBZf/ll-F
1433
Min-N (NHt-N + NO, -N) in 2 M KC1 extracts was
determined by autoanalyser procedures (Blakemore
et al., 1987). Net N mineralization was estimated by
incubating 10.0 g soil at - 5 kPa for 14 d and for 56 d
at 25°C; the min-N produced was extracted with
100 ml 2 M KC1 for 1 h. The percentage of min-N
present as NO, -N after incubation was calculated for
both unamended soil and for soil treated initially with
a solution of (NH&S04-N (300 pg; equivalent to ca.
50 pg N g-r oven-dry soil).
Min-N flush was determined by the fumigation-in-
cubation procedure (Ayanaba et al., 1976), using the
above weight of sample, a small (ca. 12 mg) inoculum
of unfumigated soil and an added aqueous solution of
(NH& SO,-N (300 pg) (Ross, 1990~). The influence of
this added (NH&S04-N on min-N flush values was
determined in a separate experiment, with pooled
subsamples of soil collected in April 1991. Min-N flush
was calculated as the difference between the net
amount of min-N produced by fumigated and
unfumigated samples incubated for 14 d at 25°C.
Invertase, phosphodiesterase and sulphatase activi-
ties. Invertase activity was measured at 30°C
according to Ross (1987). Field-moist soil (1.0 g),
0.15 ml toluene and 4.0 ml potassium phosphate
buffer (0.5 M; pH 5.0) were dispersed with a glass rod,
and 4.0 ml aqueous sucrose solution (5% w/v) then
added. The pH at the end of the 24-h assay was
essentially unchanged, and ranged between 5.0-5.1,
Phosphodiesterase and sulphatase activities were
determined according to Sparling et al. (1986) and
Speir et al. (1984) respectively, except for the use of
only 0.5 g field-moist soil and, for phosphodiesterase,
an incubation period of 1 h.
Statistical analyses
The significance of differences between sampling
times was determined by analysis of variance and
Fisher’s LSD test (Steel and Torrie, 1980), and
between treatments at each sampling time by t-tests or
Fisher’s LSD test. The significance of differences in
ratios was assessed by paired t-tests, using log-trans-
formed data. Linear correlations were used to estimate
relationships between properties.
RESULTS
Herbage yields
Growth rates of pasture herbage varied from as little
as 6 kg dry matter(DM) ha-’ d-r during winter to
74 kg DM ha-l d-r in late spring (Fig. 1). Legume
growth rates varied from 0.2 to 7.5 kg DM ha-’ d-‘;
in 1990-1991, they were consistently higher in the
fertilized plots, but differed significantly from those in
the non-fertilized plots on only one sampling date in
summer when growth rates were ~2 kg ha-Id-’
(Fig. 1).
Annual production of herbage dry matter in the
fertilized treatment ranged from 9380 to
1434 D. J. Ross et al.
10,440 kg ha-‘, but variability was high and differ-
ences from the yields of 8630-9050 kg ha-’ in the
non-fertilized treatment were again not significant.
Soil physical and chemical properties
Dry bulk density of O-5 cm depth soil, sampled in
September 1989, was similar in all plots and averaged
0.78 t m-3. Soil moisture content at -5 kPa was
determined with July 1989 samples and averaged
74 f 2(SD)%.
All samples were moderately acid, averaging
pH 5.54kO.09. Total C and N concentrations were
similar at most sampling times, but were consistently,
and often significantly, higher in the non-fertilized soil
than in the fertilized soil (Fig. 2). C-to-N ratios differed
little between the two treatments, and averaged
13.9 + 0.7.
Extractable C concentration tended to be highest in
early spring (September) and some winter samples. It
was initially highest in soil from the fertilized plots, but
similar in both treatments over the last six samplings
(Fig. 2).
Extractable Pi increased markedly in the fertilized
soil between autumn (May) and winter (July) and was
then, and subsequently, higher than in the non-fertil-
ized soil (Fig. 3). Extractable Pt fluctuated appreciably
with sampling time, but was always higher in fertilized
than in non-fertilized soil. Extractable organic P (PO)
was also consistently higher in the fertilized soil (data
not shown).
Soil biochemical properties
COz-C production was comparatively high in the
early spring (September) samples of both years, and in
the late spring (November) samples in 1990 (Fig. 2).
Although several of the between-treatment differences
a-
Legumes
6-
4
4- *.
:
SNJMMJSN
1989 1990
Fig. 1. Rates of dry matter production of total herbage and the legume component: A- - -A, fertilizer added;
a--_O, fertilizer not added. Significant differences between fertilizer treatments at each sampling time are
indicated by * (P < 0.05).
were significant, there was no consistent fertilizer
effect.
Extractable-C flush was generally similar in both
treatments and, with few exceptions, similar at
different sampling times (Fig. 4). SIR values were more
variable with sampling time and, as the trial
progressed, tended to be higher in the fertilized soil.
Values of C02-C flush were very dependent on the
particular control used, and were highest with a
control of fumigated soil incubated for 10-20 d
(Table 1). A detailed sampling-time study was not
made, but values with a fumigated soil control were
statistically indistinguishable in summer (February)
and autumn (April) samples. No fertilizer effects were
then apparent.
Neither extractable-N flush nor min-N flush varied
markedly at different sampling times, although a
few significant differences did occur in min-N flush
values (Fig. 4). Generally, values of both properties,
especially min-N flush, were higher in the fertilized
soil. Min-N flush estimates were significantly greater,
by ca. 5-lo%, in the absence of added NH4+-N in the
incubation systems (data not shown).
Sampling-time changes in microbial P were likewise
small (Fig. 4). Although initially highest in non-fertil-
ized soil, microbial P tended to be higher in the
fertilized soil as the trial progressed.
The min-N concentration of non-incubated soil
(min-N, 0 d) differed significantly at some sampling
times, but no clear seasonal trends were apparent
(Fig. 5). Treatment effects also varied irregularly.
Except for the first two samplings, net immobiliz-
ation of min-N resulted on incubation of the samples
for 14 d, with values fluctuating considerably at
different sampling times (Fig. 5). Net mineralization
occurred in all samples during the incubation period
of 14-56 d, with values tending to be highest in spring,
A
: :
,,’ ‘.*
I I I I I I ,
J M M J S
1991
 Pasture soil: seasonal and fertilizer effects 1435

Moisture

i &~!~,

Total C

Extractable C

CO& production

I I I I I I , I I , I I I I I I I I I I , I
SNJMMJSNJMMJ
1989 1990 1991

Fig. 2. Influence of sampling times and fertilizer treatment on moisture, total C, total N and 0.5 M
KSOa-extractable C contents, and COAZ production (14 d at 25°C): A- - -A, fertilizer added; ? -? _O,
fertilizer not added. Vertical bars represent LSD values; significant differences between fertilizer treatments
at each sampling time are indicated by (*),* (P < 0.10,0.05), with symbols above or below the graphs when
values from fertilized soil were higher or lower, respectively, than those from non-fertilized soil.

and in the 1991 late summer samples. Values were (Fig. 6). Phosphodiesterase activity, in contrast, varied
initially highest in the samples of non-fertilized soil markedly at different sampling times, and tended to be
but, as the trial progressed, tended to be significantly highest in autumn (May) and winter (July). Values
higher in the samples of fertilized soil. were consistently highest in the non-fertilized soil.
Over the period of the trial, NO<-N averaged ca.
10% of the min-K present in non-incubated soil. Relationships between properties
NO< -N comprised ca. 35 + 15% of the min-N in soil All of the biochemical properties except min-N flush
incubated with added NH:-N for 14 d, and 51+ 7% and phosphodiesterase activity showed some signifi-
of the mm-N in unamended fertilized soil and cant correlations with soil moisture, total C or total N
42 + 10% of the mm-N in unamended unfertilized soil “.concentrations (Table 2).
after incubation for 56 d. Replicate variability was The percentages of microbial C and N in total C and
high and no markled seasonal trends in nitrifying N, and microbial C-to-N and C-to-P ratios, are given
activity were apparent (data not shown). in Table 3. None of these ratios differed significantly
Invertase and sulphatase activities were similar at all with either sampling time or fertilizer treatment (the
sampling times and, with only two exceptions, did not only exceptions were one of the microbial N-to-total
differ significantly between fertilizer treatments N and one of the microbial C-to-N ratios).
1436 D. J. Ross et al.
C02-C production was correlated significantly with
extractable C, but not with extractable-C flush or SIR
values (Table 4).
Phosphodiesterase activity showed no significant
relationships with either extractable or microbial P, or
with invertase or sulphatase activity (Table 4), which
were significantly correlated with each other (r, 0.60;
P < 0.001). Phosphodiesterase activity was, however,
significantly correlated with extractable Pi (r, -0.466;
P < O.Ol), P, (r, 0.419; P < 0.01) and extractable P, (r,
0.552; P < 0.001) when the winter (July 1990) values
were omitted; in the fertilizer treatment alone, r values
for the correlations of phosphodiesterase activity with
extractable Pi, Pt and P, were then -0.506 (P < O.OS),
0.607 (P < 0.01) and 0.757 (P < 0.001) respectively.
DISCLESION
Accumulation of fixed C in pasture herbage and the
legume component showed the expected seasonal
variability, with maxima in late spring or early
summer. Although the application of fertilizer P and
S appears to have had some stimulatory effects in the
second year of the trial, differences from the
non-fertilized treatment were generally not significant
because of high plot variability. The proportionately
greater response of legumes to added fertilizer,
compared to the other pasture components, redects
both the greater sensitivity of legumes to P availability
(Jackman and Mouat, 1972) and the P-deficient nature
of the soil; at the beginning of the trial, the Olsen P
value (Blakemore et al., 1987) was only 7 mg kg-‘.
Legume production in 199&l 99 1 was almost doubled
by the added fertilizer, while annual N fixation
increased over 3-fold, from 13 kg ha-’ in the
unfertilized plots to 46 kg ha-l in the fertilized plots
(Mackay et al., 1991).
Although the increased legume growth and N
01 I I I 1 1 I
S N J M
1989
Fig. 3. Influence of sampling times and fertilizer treatment on 0.5 MNaHCO,-extractable P, and P, contents:
A- - -h, fertilizer added; O-0, fertilizer not added. Symbols as for Fig. 2.
fixation would eventually result in an additional input
of N in the fertilizer treatment, the effect on other soil
properties is likely to have been slight over the period
of the trial. The pronounced seasonal fluctuations in
the amount of C fixed could, however, have influenced
soil biochemical properties through associated
changes in both above-and below-ground organic
matter inputs. In comparison, the fertilizer influences
on overall pasture production were small and unlikely
to have had any major effects.
The difficulty of making treatment comparisons in
a hill pasture is highlighted by the systematic
differences found between plots for several of the soil
properties (Figs 24). These inherent plot differences
may have obscured possible fertilizer effects, particu-
larly where soil property values were initially higher in
the fertilized than in the non-fertilized treatment, but
are unlikely to have prevented the detection of any
seasonal changes.
Increases in the amounts of extractable Pi (Fig. 3)
and sulphate (Searle et al., 1991) during the course of
the trial showed that P and S had become more
available in the fertilized plots. As in our unfertilized
soil, a winter peak in extractable Pi was also found in
a fertile pasture soil by Perrott et al. (1990), and its
agronomic significance is discussed by Searle et al.
(1991). Th;: marked decline of extractable P, in spring
and late autumn could be indicative of the importance
of this P fraction for pasture plants during spring and
autumn flushes of growth (Searle et al., 1991).
The temporal changes observed in total C and N
could have resulted mainly from spatial variability or
sampling errors. For example, the increase in the C
concentration of non-fertilized soil in 1990 from 6.3%
in late autumn (May) to 6.8% in winter (July) (Fig. 2)
would have required an unlikely input of ca.
1950 kg C ha-’ in O-5 cm depth soil over 10 weeks.
Some accumulation of organic matter during the
1 , 1 I I I I r
M J S N
1990
 Pasture soil: seasonal and fertilizer effects 1437

Extractable-C flush

_ 400
‘i
r 30 -
‘i
A
: 20- I
*
b
8 10
h 300 - Min-N flush
7
0)
9 200 -
z
100
Extractable-N flush

Microbial P

5011, , IIIlll,,, 1 I I 1 I I I I I
SNJMMJS “N J M M J
1989 1990 1991

Fig. 4. Influence of sampling times and fertilizer treatment on extractable-C flush, SIR, min-N flush,
extractable-N flush and microbial P: A- - -A, fertilizer added; ? -?
-_O, fertilizer not added. Symbols as for
Fig. 2.

autumn-winter period is, however, possible and
higher amounts of organic C during winter have been
observed in two Canadian soils (Dormaar et al., 1984).
Extractable C and COTC
The apparent maxima in extractable C concen-
trations in late-autumn to early spring samples may
have been more a function of sieving and smearing of
the soil (Ross, 1992) than a seasonal effect. The use of
a larger-mesh sieve, or initial partial drying of samples
such as these, would partly overcome this problem
(Ross, 1992). Some authentic sampling-time differ-
ences are, nevertheless, suggested as neither soil
moisture nor total C concentration could explain most
of the variations in extractable C values (Table 2).
Winter maxima in water-extractable C content have
also been recorded for a more coarsely sieved
(<4 mm) soil (Sarathchandra et al., 1988), and for a
Canadian grassland soil (Dormaar et al., 1984).
The accumulation of some organic matter fractions
over winter is strongly suggested by the CO*-C
production values which, as in other pasture soils
(Sarathchandra et al., 1988; Ross, 199Oc), showed
pronounced spring maxima under standardized
laboratory conditions. The balance between the
production of labile compounds in a perennial
grassland soil and their rate of decomposition by the
soil biota would be expected to vary under different
climatic conditions. In an English grassland soil,
potential CO2 production was highest in mid-summer,
although less marked maxima also occurred in the late
winter and early spring samples (Patra et al., 1990). In
our samples, CO?-C production was correlated
significantly with extractable C content and a close
relationship between COrC production and soluble
1438 D. J. Ross et al.

Table 1. Influence of control in the fumigation-incubation procedure on COtC flush values, extractable-C flush and derived &c-factors
COK flush (gg g-‘)t; control and
incubation oeriod used
Fumigated
Unfumigated soil soil
Sampling Fertilizer Extractable-C Derived kEr
time treatment O-10 d 1&2Od l&20 d flush (fig g-‘) factorf
February 199 1 Not added 296-z@ 368b 663a 659~ 0.447(x)
Added 266c 381b 694a 620q 0.402(y)
April 1991 Not added 246c 414b 627a 601(p) 0.431x
Added 222c 400b 663a 577(q) 0.392~
tcalculated as COK produced by fumigated soil over the incubation period CL10 d minus the control.
fCalculated as extractable-C flush/microbial C [estimated from the CO>-C flush using a fumigated control and a kc-factor of 0.45 (Jenkinson,
1988)].
§For each sampling period, values of CO*-C flush, extractable-C flush and k EC not marked with the same letter are significantly different
(P -c 0.05; in parentheses, P -: 0.10).

organic C has likewise been found for a cropping soil
(Wang and Bettany, 1993).
Microbial biomass
As found in other pasture soils (Ross, 1990a) CO*-C
flush, and hence microbial C values, were highest when
a control of fumigated soil was used in the
fumigation-incubation procedures. Calibration of
extractable-C flush with these microbial C estimates
gave &-factors that ranged from 0.39 to 0.45; these
were higher than those found for several other soils
under pasture (Ross, 1990b; Sparling et al., 1990).
High RECvalues were, however, also obtained for
another low-fertility soil (Ross, 1990b) and raise the
possibility of under-estimation of microbial C by use
of an inappropriate &factor in the fumigation-incu-
bation method. A &factor of 0.39 was, nevertheless,
used to estimate microbial C for subsequent
calculation of microbial C-to-N ratios (Table 3).
The estimates of microbial C thereby obtained were
consistently and appreciably higher than those
calculated by the SIR method using the conversion
factor, viz. microbial C (Pegg-‘) = 50
(pl CO? g-l h-l), proposed by Sparling et al. (1990).
Mean microbial C values for both fertilizer treatments
at all sampling times were then 1600 /lg g-’ by the
fumigation+xtraction method and 1140 pg g-’ by the
SIR method. For this particular soil, a conversion
factor of at least 70 (~1 CO2 g-’ h-‘) would be more
suitable to calculate microbial C (pg g-l) by SIR.
A &factor of 0.57 (Jenkinson, 1988) was used to
provide an approximate measure of microbial N from

AMin-N (0 d)
60- *

_v- --

(*I
0
AMin-N (O-14 d)

o!, , , lb I I I I I I I I I I I I I 1 I I I
SNJMMJSNJMMJ
1989 1990 1991

Fig. 5. Inhence of sampling times and fertilizer treatment on min-N content of non-incubated (0 d) soil
and net N mineralization (Amin-N, O-14 and 14-56 d): A- - -A, fertilizer added; O----O, fertilizer not
added. Symbols as for Fig. 2.
 Pasture soil: seasonal and fertilizer effects 1439

1 lnvertase
3000
43

2000
tJ
I
1000
? Phosphodiesterase

Sulphatase

800 I

600 1 , , , I 1 I I I 1 I I I 1 I 1 I I I 1 I I 1
SNJMMJSNJMMJ
1989 1990 1991

Fig. 6. Influence of sampling times and fertilizer treatment on invertase, phosphodiesterase and sulphatase
activities (units: invertase, pmol “glucose” formed g- ’soil s-l; phosphodiesterase and sulphatase, pmol
p-nitrophenol formed g- ’soil s-l): A- - -A, fertilizer added; O-0, fertilizer not added. Symbols as for
Fig. 2.

min-N flush values. Calibration of extractable-N flush
against these microbial N estimates at different
sampling times gave a mean &factor of 0.32 f 0.03
for non-fertilized soil and 0.29 k 0.02 for fertilized
soil. In some other pasture soils also, &-factors
appeared to be lowler (Ross, 1992; Sparling and Zhu,
1993) than the value of 0.45 proposed by Jenkinson
(1988).
One of the most striking results of this study has
been the generally small temporal fluctuations in
microbial biomass indices (extractable-C and -N flush,
and microbial P) compared with seasonal variations in
C fixation by pasture plants. The fluctuations that did
occur were partly associated with variations in soil
total C and N concentrations. Amounts of soil
microbial biomass at this site were also largely
unaffected by large differences in inputs of plant
material over a period of ca. 6 months (Ross et al.,
1995). In other pasture soils with greater seasonal
fluctuations in moisture content only relatively small
biomass fluctuations have likewise been observed
(Ross, 1990b). Patra et al. (1990) similarly found only

Table 2. Correlations ofsoil biochemical properties with soil moisture, total C and total N contents
-
Correlation coefficient (r)
Prooertv Moisture Total C Total N
Extractable C 0.65”’ 0.41*** 0.05
Extractable min-N 0.51*** 0.37** 0.22
Extractable Pi 0.63**’ 0.26 0.06
Extractable P, 0.409’ 0.17 0.16
Total C 0.34” 1.00 0.73***
Total N 0.03 0.73”’ 1.00

Extractable-C flush 0.29’ 0.53.’ 0.51***

Extractable-N flush 0.20 0.49”’ 0.64***
mill-N flush 0.01 0.16 0.15
Microbial P 0.06 0.67*** 0.72**’

C&C production 0.58’** 0.48”’ 0.21

CCkC production (SIR) 0.41.. 0.06 0.13
Amin-N (O--l4 d) 0.48*** 0.06 0.18
Amin-N (14-56 d) 0.04 0.30’ 0.49***

Invertase 0.18 0.61*** 0.52”’

Phosphodiesterase 0.11 0.28 0.02
0.47,’ 0.799’9 0.72***
*, I’*, ***P < 0.05, 0.01, 0.001, respectively; degrees of freedom range from 34 to 62.
1440 D. J. Ross et al.

Table 3. Microbial biomass ratios (values are means f SD from all
samoline times)
Non-fertilized
Ratio soil
Microbial C-to-% of total CT 2.5 * 0.3
Microbial N-to-% of total N$ 7.4 * 0.3
Microbial C-to-N 4.6 + 0.3
Microbial C-to-P 14.9 * 1.0
tMicrobia1 C was calculated from extractable-C flush, using a
/w-factor of 0.39.
iMicrobial N was calculated from min-N flush, using a &factor
0.57 (Jenkinson, 1988).
small temporal changes in an old grassland soil,
whereas Bristow and Jarvis (199 1) recorded somewhat
greater fluctuations in a grazed grass-clover pasture.
The smallness of these fluctuations is consistent with
the estimated turnover times of microbial biomass in
temperate soils under uniform management for several
years (Patra et al., 1990). Appreciable temporal
fluctuations have, however, been observed in prairie
soils in a more extreme climatic environment (DeLuca
and Keeney, 1994).
Soil microbial biomass contents were comparable to
those found in other more fertile lowland pastures
(Ross, 1990b; Sparling, 1992), and are in accord with
the high organic matter contents at this site.
Confirmation of the close relationship between
microbial biomass and soil organic matter contents is
provided by the similar microbial C-to-total C and
microbial N-to-total N ratios found at all samplings.
Overall, the percentages of microbial C in total C were
similar to, but the percentages of microbial N in total
N were somewhat higher than, those reported
elsewhere (Smith and Paul, 1990). These data indicate
that such hill pastures are a favourable environment
for microbial growth.
The values of the microbial C-to-N and C-to-P
ratios obtained for this soil are obviously dependent
on the &c-factor used for converting extractable-C
flush to microbial C. Because of some calibration
uncertainty and possible under-estimation of micro-
bial C, these ratios may be unduly low. However, the
mean microbial C-to-N ratios of 4.6 and 4.0 in the
non-fertilized and fertilized soils, respectively, were
very similar to those in an old grassland soil (Patra
et al., 1990), although lower than those calculated for
some other New Zealand pasture soils (Ross, 1990a).
The average microbial P content of 42 + 2 kg ha-’
(O-5 cm depth) over the trial period was similar to the
54 kg ha-’ of microbial P found by Seeling and
Zasoski (1993) for a grassland topsoil (O-5 cm depth)
Fertilized
soil
and the mean content of 32 kg ha-’ (c7.5 cm depth)
recorded by Perrott and Sarathchandra (1989) for a
2.6 k 0.2
8.9 f 0.4 number of high-producing soils under pasture.
4.0 f 0.4 Microbial C-to-P ratios averaged 14.9 and 14.4 in the
14.4 + 1.1
non-fertilized and fertilized soils, respectively
(Table 3). Ratios were somewhat lower in the fertilized
of soil than in the non-fertilized soil at the last four
samplings (data not shown), and suggest that more P
was incorporated into the microbial biomass as
available P, increased with time after fertilizer
application. A much lower ratio of 8.5 was found by
Patra et al. (1990) for a grassland soil that had been
regularly fertilized with P and N for > 100 y.
N mineralization
The tendency for soil min-N content to be higher in
the wetter samples (Table 2) would, to some extent,
have been a sieving artefact (Ross, 1992). In the field,
soil min-N contents in other trials were very low at the
Ballantrae site and, as found here, consisted
predominantly of NH?-N, even when comparatively
high amounts of superphosphate had been applied for
several years (Sakadevan et al., 1993a,b).
The net N immobilization that generally occurred
during the initial stages of incubation (O-14 d) also
tended to be higher in the wettest samples (Table 2).
This, too, may have been partly a consequence of
sieving, with the N released being immobilized during
microbial metabolism of the labile C produced. The
appreciable net N mineralization found on further
incubation (1456 d) was broadly related to total N
content, as observed by Sakadevan et al. (1993b) in
other Ballantrae trials. Min-N production generally
appeared to be comparatively high in the spring
samples in each of the 3 y and indicative of increased
N availability for the subsequent flush in herbage
production (Fig. 1). Continuing N availability in
summer (February) is also suggested by the Amin-N
(1456 d) data (Fig. 5). A trend for relatively high
min-N production in spring-collected soil was also
found in another low-fertility pasture (Tate et al.,
1991), whereas no well-defined seasonal changes were
apparent in more fertile pastures (Ross and Speir,
1984; Sarathchandra et al., 1988; Tate et al., 1991).
Although P availability can influence the relative
proportions of NH,+-N and NO< -N produced during
N mineralization (Hue and Adams, 1984; Pastor et al.,

Table 4. Correlation coefficients (r) for COK production and phosphodiesterase activity with
other soil properties
Property COK production Property Phosphodiesterase
Extractable C 0.55*** Extractable Pi -0.15
Extractable-C flush 0.04 Extractable P, 0.27
Microbial P 0.08
COrC production (SIR) 0.07 Invertase 0.22
Sulohatase 0.22
***p < 0.001
 Pasture soil: seasonal and fertilizer effects
1984), increasing amounts of extractable Pi in the
fertilized soil had here no detectable effect (data not
shown).
Enzyme activities
The small fluctuations that occurred in invertase
and sulphatase activities were related more to
variations in total C and N than soil moisture content
(Table 2). In a more fertile lowland pasture, invertase
activity increased appreciably under wet spring
conditions (Ross and Speir, 1984), whereas fluctu-
ations in sulphatase activity were again small. Because
of the many forms: in which enzymes can exist in soil
(Burns, 1982; Perrott et al., 1990) we cannot yet
account for these inter-site differences.
Phosphodiesterase activity fluctuated much more
widely at different sampling times than did phospho-
monoesterase activity in another low-fertility pasture
soil (Speir and Cowling, 1991). However, comparable
variations in phosphomonoesterase activity, with
some maxima in l,ate autumn and winter, have been
observed (Ross and Speir, 1984; Perrott et af., 1990;
Kirchner et al., 1’293). The fluctuations in our trial
were seemingly unrelated to changes in the other soil
properties examined, and a similar lack of relation-
ships has been found elsewhere (Speir and Cowling,
1991; Avidov et al., 1993). The significant relation-
ships between ph.osphodiesterase activity and ex-
tractable P fractions when the winter data were
omitted do, nevertheless, suggest that the amounts of
activity responded to these P fractions. A build-up in
phosphodiesterase activity could have been induced by
the seasonal accumulation of extractable P,. The
decline of extractable P, in spring and autumn could
also have been accompanied by a decline in
phosphodiesterase activity resulting from enzyme
denaturation through microbial metabolism, or from
inhibition by the Pi produced from the mineralization
of P, (Speir and Ross, 1978). The apparently
anomalous winter data may reflect differences in
metabolic rates, with the mineralization of extractable
P, being more rapid than the denaturation of
phosphodiesterase., or its inhibition by the increasing
amounts of extractable Pi. Although a decrease in soil
phosphatase activity was noted by Haynes and
Williams (1992) after long-term application of
phosphate to a lowland pasture, the treatment
differences observed here in phosphodiesterase
activity can be att.ributed to initial plot differences,
rather than short-term increases in extractable Pi after
the fertilizer additions.
Conclusions
Overall, it can be concluded that the pools of
microbial biomass :in this moist, hill soil were relatively
constant, whereas other labile organic pools and
phosphodiesterase activity were subject to greater
seasonal fluctuations. Potential rates of C and net N
mineralization were comparatively high in the soil in
soring and stronelv sueeest that labile organic C and
1441
N fractions had accumulated during winter. The
microbial biomass itself would also comprise an
appreciable pool of potentially available nutrients, but
further work is required to establish the major
mechanisms responsible for governing their release.
Although laboratory and field measurements of N
mineralization rates were not closely related at this
N-deficient site (Sakadevan et al., 1993a,b), our
incubation results confirm the tightness of the N cycle,
with net immobilization of min-N occurring initially
and NH$-N being the predominant form present.
P and S fertilizer generally had little, if any, effect on
these biochemical properties, possibly because of the
amounts used and the shortness of the time since their
application.
The data generally indicate that measurements at
any particular time would provide a reasonable
estimate of microbial biomass contents and invertase
and sulphatase activities in a moist hill pasture soil.
Acknowledgements-We thank Philip J. Budding and
Vanessa Pokaia for sampling assistance, Charles W. Feltham,
Lorraine Gilligan and Matthew D. Taylor for some total C
and N analyses and autoanalyser measurements of NHt-N
and NO,--N, John Reynolds and Deborah J. Donnell,
formerly Applied Mathematics Division, Department of
Scientific and Industrial Research, Wellington, and David J.
McQueen for statistical advice and assistance, and Phil B. S.
Hart for helping to initiate the trial.
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