F.P. Vinther, E.S. Jensen Agriculture, Ecosystems and Environment 78 2000 139–147 141
growing seasons to a height of 2–3 cm above soil surface, separated into grass and clover, and dried at
80
◦
C overnight. 2.3. Nitrogen incorporated into the soil
Shortly before the second year grass-clover sward was ploughed under in the spring of 1995, 1996 and
1997, samples were taken for determination of dry matter and total N. Four turves from each of the two
animal manure treatments were taken. Each turf mea- sured 20 cm × 20 cm and included soil to approxi-
mately 20 cm depth.
Soil was washed from the turves, which were then separated into above-ground plant material and roots.
It was not possible to separate grass and clover roots. The dry matter production and total N content was
determined by elemental analysis after drying the fractions at 80
◦
C overnight. 2.4. Analysis and calculations
SNF was determined using the
15
N isotope dilu- tion method with ryegrass from the grass-clover mix-
ture as the reference plant. The content of total N and
15
N in the dried plant material was determined on a N-analyzer coupled online to an isotope ratio mass
spectrometer Carlo Erba-Finnigan as described by Jensen 1991. The
15
N natural abundance of the soil was 0.3663 at.
15
N and this value was used for cor- recting the
15
N abundance of plant samples for back- ground. The proportion of N derived from the atmo-
sphere was calculated using the following equation Fried and Middelboe, 1977:
pNdfa = 1 − atom
15
N excess
clover
atom
15
N excess
grass
Atom
15
N excess =
atom
15
N
clover or grass
− 0.3663
The amount of N
2
fixation was estimated as SNF = total clover N × pNdfa.
Analysis of variance was carried out using the GLM procedure in SAS SAS Institute, 1989. Least signif-
icant difference LSD was used to compare means, when differences were found to be significant at the
P = 0.05 level.
Table 1 Soil temperature and precipitation during the 3 years of the inves-
tigation Months
Soil temperature at Precipitation
10 cm
◦
C mm
1994 1995
1996 1994
1995 1996
January 2.3
1.3 0.5
82.1 72.0
2.7 February
0.9 3.2
0.1 21.0
77.6 13.8
March 2.5
3.4 0.3
74.6 62.8
0.7 April
7.2 6.6
5.7 20.1
35.9 5.3
May 11.3
10.8 9.5
10.2 49.8
51.7 June
13.4 14.8
14.8 58.5
45.0 14.8
July 18.8
17.4 15.4
10.2 18.4
27.6 August
17.3 18.6
17.0 133.0
14.1 48.2
September 13.5
14.1 12.2
171.8 83.4
58.7 October
9.2 11.3
9.8 43.9
28.6 62.6
November 7.1
5.6 5.7
55.0 50.5
99.7 December
4.6 1.9
1.9 73.9
5.0 34.7
Mean Total
9.0 9.1
7.7 754.3
543.1 420.5
3. Results
3.1. Climatic conditions The 3 years of investigation varied considerably
with respect to both soil temperature and precipitation Table 1. The average soil temperature was 1.3–1.4
◦
C lower in 1996 than in the previous 2 years. The lower
average temperature in 1996 was primarily because of a cold spring with monthly temperatures 1–3
◦
C lower than in 1994 and 1995. The total precipitation as well
as the distribution differed significantly among the 3 years of investigation. The total precipitation in 1996
comprised only 56 of that in 1994, and in 1996 nearly 25 of the total precipitation fell in the month
of November, whereas nearly 50 of the total precip- itation in 1994 fell during the months of August and
September. Furthermore, the first 4 months of 1996 were very dry. During these 4 months, the accumu-
lated precipitation was 22.5 mm compared with 198.3 and 248.3 mm during the same periods of 1994 and
1995, respectively.
3.2. Effects of manure application The level of manure application 0.8 and 1.2 a.u.
did not influence the dry matter production, total N accumulated or N
2
fixation. Levels of significance
142 F.P. Vinther, E.S. Jensen Agriculture, Ecosystems and Environment 78 2000 139–147
P-value ranged from 0.23 to 0.45. As a consequence, the results from these two treatments were pooled at
each harvest.
3.3. Dry matter production of grass-clover mixtures The total dry matter TDM production varied
considerably during the 3 years of investigation. The annual dry matter production ranged from 618 to
1206 g m
− 2
and it was greatest in 1995 Table 2. The time course of the dry matter production
followed a similar pattern during the three grow- ing seasons. The highest rates of DM production
of the grass-clover mixtures were found during May–July ranging between 4 and 12 g m
− 2
per day, followed by rates between 3 and 7 g m
− 2
per day during July–September. Rates were lowest in
September–November 4 to 1 g m
− 2
per day. The proportion of clover in the harvested crop grad-
ually increased during the growing season to reach maximum values in August and then declined in sub-
Table 2 Total dry matter TDM production g m
− 2
and proportion of clover during the years 1994–1996. Data represent mean of 8 replicates ± s.d. in parenthesis
Date of harvest TDM g m
− 2
Clover TDM
Clover g m
− 2
TDM g m
− 2
Clover Undersown grass-white clover
1st year grass-white clover 2nd year grass-red clover
100594 88 20
18 4 53 9
64 13 240594
99 9 27 5
93 9 64 14
090694 58 14
40 6 66 9
56 11 050794
173 17 70 5
211 39 65 8
030894 207 27
78 3 225 38
80 3 010994
170 23 76 4
170 30 77 5
021194 162 35
41 5 145 15
26 1 –
– Total 1994
162 35 940 65
818 44 230595
296 61 42 12
342 49 38 5
150695 199 26
49 5 187 14
54 14 190795
338 34 66 7
246 39 59 10
170895 177 68
60 7 120 28
60 13 101095
154 31 41 12
121 35 41 9
161195 129 45
24 19 42 11
18 6 32 8
16 6 Total 1995
129 45 1206 83
1048 34 230596
147 38 20 16
150 33 42 13
130696 118 18
30 15 95 26
46 12 030796
77 2 52 21
81 22 61 10
010896 156 50
67 24 102 33
65 8 270896
232 45 68 8
107 9 35 7
260996 56 15
44 12 46 8
12 4 291096
72 7 34 13
37 5 5 2
Total 1996 858 78
618 76
sequent harvests to reach lowest values at the end of October and early November Table 2.
3.4. Nitrogen in above ground biomass of grass-clover
As with dry matter production rates, there was a seasonal variation in the concentration of total N Fig.
1 with greatest values early in the growing season fol- lowed by a decrease during the summer months, and
an increase again during the autumn resulting in values similar to the contents early in the growing season. The
concentration of total N in the clover ranged between 3.0 and 5.9, and there was no significant difference
between the first and second year grass-clover. In the grass the average N concentration was 2.6 ± 0.7 and
ranged between 1.6 and 4.6. The average concen- trations were 2.5 in the first and 2.8 in the second
production year, but the difference was not statistically significant.
F.P. Vinther, E.S. Jensen Agriculture, Ecosystems and Environment 78 2000 139–147 143
Fig. 1. Concentration of total N in clover
d
and grass
j
during 1994 to 1996. Data represent mean of eight replicates ± s.d.
3.5. Method for
15
N labelling of the soil inorganic N pool
In 1995 and 1996 the two methods for labelling the plant available N with
15
N were compared. The
15
N enrichment of the grass differed significantly depend-
ing on the labelling method. The grass in pre-labelled plots had an enrichment of 0.68 ± 0.13 at.
15
N excess in November 1994. In May 1995 the grass
enrichment was 0.42 ± 0.04 at.
15
N excess and the enrichment remained at a relatively high level through-
out the following two growing seasons Fig. 2. The
15
N enrichment in the grass was 0.14 ± 0.02 at.
15
N excess in November 1996 32 months after applica-
tion. In the grass-white clover mixture, in which the soil was labelled with inorganic
15
N in the spring of 1995 without the immobilising agent sucrose, the
15
N enrichment in the grass declined rapidly and 2 months after application, it was 1.20 ± 0.04 at.
15
N excess Fig. 2. The decrease continued and at the end
of the second year 1996, the enrichment was only slightly greater 0.019 ± 0.002 at.
15
N excess than the enrichment of the clover 0.0017 ± 0.0007 at.
15
N excess. Similarly, the atom
15
N excess in the grass of the first year grass-white clover in 1996,
showed a rapid decline when the soil was labelled with
15
N without immobilisation, and a more slow decline when labelled with
15
N + sucrose Fig. 2. A half-life of atom
15
N excess in the soil inorganic N pool following labelling with or without sucrose could
be estimated to approximately 1 year and 1 month, respectively.
In spite of these differences between the two la- belling methods, there were no significant differences
between the pNdfa Fig. 3. The pNdfa from both methods showed similar seasonal variations with
the lowest values early and late in the growing sea- sons. However, the small differences between the
15
N enrichment in the grass and in the clover in the second production year resulted in relatively large
standard deviations of the pNdfa Fig. 2, when cal- culated from the treatment, where the
15
N was added
144 F.P. Vinther, E.S. Jensen Agriculture, Ecosystems and Environment 78 2000 139–147
Fig. 2. Atom
15
N excess during the growing seasons of 1995 and 1996 in grass labelled with
15
N-labelled ammonium sulphate only
j
or labelled with
15
N-labelled ammonium sulphate + sucrose
15
N-immobilisation method
d
. Data represent mean of eight replicates ± s.d.
without sucrose Fig. 3. The average coefficient of variance of the pNdfa-values determined using the
15
N-immobilisation method was 2.0 which is sig- nificantly P ≤ 0.05 lower than 3.3 found in the
treatment without sucrose amendment. Because the two methods with and without su-
crose resulted in similar estimates, the average pNdfa-value was used for calculations of the amount
of N
2
fixed. 3.6. The pNdfa and amount of N
2
fixed in grass-clover mixtures
The pNdfa-values ranged between 0.70 and 0.99, with the lowest values in the beginning and at the end
of the growing season Table 3. The lowest values of pNdfa, 0.70 and 0.74, were measured at the first
harvest in 1994 in the first year grass-white clover and in the second year grass-red clover, respectively.
Fig. 3. Proportion of N derived from the atmosphere pNdfa during the growing seasons of 1995 and 1996 of clover using either
labelling with
15
N-labelled ammonium sulphate only
d
or with
15
N-labelled ammonium sulphate + sucrose
15
N-immobilisation method
s
. Data represent mean of eight replicates ± s.d.
The total amount of N
2
fixed in the undersown grass-white clover was 2.9 and 1.5 g N m
− 2
in 1994 and 1995, respectively, and ranged between 10.0 and
23.5 g N m
− 2
per year in the grass-clover mixtures dur- ing the 3 years of investigation Table 3. The rates
of N
2
fixation calculated in terms of mg N fixed per gram of clover shoot dry weight was 38.6 ± 1.3 for
the first and second year mixtures, and 45.0 ± 1.4 for the undersown grass-clover.
3.7. Nitrogen in the grass-clover sward when ploughed under
The amounts of dry matter and total N in the grass-clover sward was determined at the end of the
second year growing period Table 4. The total dry matter of the ryegrass-red clover was almost equally
distributed between roots and above ground material, whereas for the ryegrass-white clover mixture the
F.P. Vinther, E.S. Jensen Agriculture, Ecosystems and Environment 78 2000 139–147 145
Table 3 Proportion of N derived from the atmosphere pNdfa and amount of N
2
fixed g N m
− 2
during the years 1994–1996. Data represent mean of 8 replicates ± s.d. in parenthesis
Date of harvest pNdfa
N
2
-fixation g N m
− 2
pNdfa N
2
-fixation g N m
− 2
pNdfa N
2
-fixation g N m
− 2
undersown grass-white clover 1st year grass-white clover
2nd year grass-red clover 100594
0.74 0.09 0.5 0.2
0.70 0.05 1.2 0.5
240594 0.92 0.01
1.4 0.3 0.84 0.03
2.4 0.5 090694
0.96 0.01 1.0 0.3
0.89 0.07 1.4 0.5
050794 0.99 0.01
4.1 0.5 0.93 0.02
5.3 1.4 030894
0.98 0.01 6.1 1.1
0.94 0.04 5.4 0.8
010994 0.96 0.00
6.3 1.1 0.90 0.05
6.0 0.9 021194
0.94 0.01 2.9 0.8
0.83 0.00 1.5 0.1
– –
Total 1994 2.9 0.8
20.9 3.6 21.7 4.6
230595 0.92 0.02
5.1 0.8 0.90 0.01
6.1 1.3 150695
0.95 0.01 4.2 0.5
0.94 0.01 6.7 1.2
190795 0.94 0.02
7.0 1.1 0.95 0.01
3.9 2.6 170895
0.95 0.01 4.1 1.1
0.95 0.01 3.1 1.5
101095 0.94 0.02
2.8 1.1 0.90 0.02
1.9 0.3 161195
0.91 0.0 1.5 1.7
0.86 0.03 0.3 0.1
0.82 0.03 0.2 0.1
Total 1995 1.5 1.7
23.5 2.5 21.9 2.5
230596 0.90 0.02
1.0 0.6 0.84 0.02
2.5 0.6 130696
0.95 0.01 1.5 0.9
0.87 0.01 1.7 0.9
030796 0.96 0.01
1.5 0.7 0.89 0.03
1.6 0.4 010896
0.98 0.01 4.5 2.6
0.93 0.02 2.6 1.1
270896 0.94 0.01
6.1 2.2 0.93 0.02
1.3 0.3 260996
0.85 0.04 1.0 0.4
0.91 0.03 0.2 0.1
291096 0.90 0.02
1.1 0.4 0.92 0.02
0.1 0.0 Total 1996
16.7 6.1 10.0 2.3
approximate ratio between root and above ground dry matter was 10 : 1. The concentration of total N was
slightly higher in the roots of the grass-red clover sward than in roots of the grass-white clover sward,
resulting in total amounts of N incorporated into the soil ranging between 19.6 and 29.6 g N m
− 2
.
4. Discussion
