76 K. Bharati et al. Agriculture, Ecosystems and Environment 79 2000 73–83
ing the extract with ferrous ammonium sulfate after wet digestion with chromic acid Mishra et al., 1997
and expressed as
m
g C g
− 1
soil. The NRN content of flooded soil was estimated colorimetrically following
the method of Amato and Ladd 1988 and expressed as
m
g NRN g
− 1
soil. 2.5. Plant parameters
Mean aerial biomass fresh and dry weights was measured by harvesting above-ground portions on
each day of CH
4
sampling. The
a
-naphthylamine oxi- dase activity of roots was measured via the method of
Ota 1970 as modified by Satpathy et al. 1997. Rep- resentative samples of roots were exposed to freshly
prepared solution of
a
-naphthylamine 20
m
g ml
− 1
within 10 min of collection of roots. The root oxidase activity was expressed as
m
g of
a
-naphthylamine ox- idized g
− 1
dry root h
− 1
. Grain and straw yields from individual replicated treatments were measured at
maturity and the harvest index was calculated using the formula:
Harvest Index = grain yield
grain + straw yield ×
100 2.6. Statistical analyses
Individual character data sets were statistically analyzed and the mean comparison between treat-
ments was established by Duncan’s multiple range test using statistical package IRRISTAT, version
3.1 : International Rice Research Institute, Philip- pines. Simple and multiple correlations between CH
4
flux and select soil and plant parameters were deter- mined using the variation at each time of observation,
to establish possible statistical relationship between changes in soil and plant characters among different
treatments and CH
4
emission.
3. Results
3.1. Methane flux Methane flux varied considerably among differ-
ent treatments with one peak each at vegetative and maturity stages of the rice crop. CH
4
emission was low in all the plots during the first 2 weeks after
transplantation with the exception of Azolla incor- porated plots Fig. 1. Incorporation of fresh Azolla
at the rate of 16 Mg ha
− 1
, to provide 30 kg N ha
− 1
, resulted in a high CH
4
flux during the first 20 days after transplantation. Interestingly, CH
4
flux was considerably low in treatments where Azolla was
grown as a dual crop — either in conjunction with urea Treatment IV or following Azolla incorpora-
tion Treatment V. Application of urea alone at 60 kg N ha
− 1
also stimulated CH
4
efflux from flooded fields planted to rice. Thus, the mean CH
4
emission followed the order of urea at 60 kg N ha
− 1
8.15 mg CH
4
m
− 2
h
− 1
Azolla incorporated + urea at 30 kg N ha
− 1
7.80 mg CH
4
m
− 2
h
− 1
no N control 5.80 mg CH
4
m
− 2
h
− 1
Azolla incorporated + dual crop 5.40 mg CH
4
m
− 2
h
− 1
Azolla dual crop + urea at 30 kg N ha
− 1
4.61 mg CH
4
m
− 2
h
− 1
. Consistently higher flux was observed in Azolla in-
corporated plots as compared to no N control plots and the emission differences were maintained up to
60 days, after which the second emission peak was recorded. This peak of CH
4
flux was observed in all the plots, albeit with varying degree depending upon
the treatment, during maturity period of the crop. How- ever, the initial priming effect provided by Azolla ap-
plication was persistent even at later stages.
The moderating effect of growing Azolla dual crop on CH
4
flux from a flooded field planted to rice and the relationship between CH
4
flux and select soil and plant variables were investigated. Redox potential dropped
with plant growth but was lowered faster and further in Azolla-incorporated soils Table 2. Interestingly,
soils from treatment with dual crop of Azolla registered comparatively higher redox potential. The correlation
analysis of redox potential with CH
4
flux indicated a significant negative relationship Table 3.
3.2. Soil and plant parameters Dissolved oxygen DO
2
concentration in the soil–floodwater interface was high during the ini-
tial growth stages when CH
4
flux was low. On the other hand, DO
2
concentrations reached low levels at maturity when CH
4
flux was high Table 4. Mean DO
2
concentration was higher in field plots with a dual crop of Azolla. A significant inverse relationship
K. Bharati et al. Agriculture, Ecosystems and Environment 79 2000 73–83 77
Fig. 1. Effect of Azolla and urea application on methane efflux from flooded alluvial field planted to rice cv. CR 749-20-2 [A. no N Control, B. urea 60 kg N ha
− 1
, C. Azolla incorporated 30 kg N ha
− 1
+ urea 30 kg N ha
− 1
, D. Azolla dual cropping 30 kg N ha
− 1
+ urea 30 kg N ha
− 1
, E. Azolla incorporated 30 kg N ha
− 1
+ dual cropping 30 kg N ha
− 1
]. Means of three replicate values plotted, barshalf-bars indicate the SD.
r = −0.337, n = 40 was observed between DO
2
and CH
4
emission. The
a
-naphthylamine oxidase activity of rice roots at various growth stages under the influence of Azolla
application is given in Table 5. Root oxidase activity, in general, increased with the growth of the rice plant
upto 50 days and declined thereafter. Root tips exhib- ited higher
a
-naphthylamine oxidase activity than the root base. The
a
-naphthylamine oxidase activity of the root base was high in field plots with dual crop of
78 K. Bharati et al. Agriculture, Ecosystems and Environment 79 2000 73–83
Table 2 Variation in redox potential in the root region of rice plants under the influence of Azolla and urea
a
Treatment Redox potential mV
Days after transplantation 10
20 30
40 50
60 70
80 Control no N
− 40
b
− 78
a
− 106
a
− 135
b
− 137
a
− 134
a
− 140
b
− 108
a
Urea 60 kg N −
54
c
− 172
d
− 173
c
− 186
d
− 193
c
− 189
c
− 144
b
− 156
b
Azolla incorporated 30 kg N + urea 30 kg N −
69
d
− 135
b
− 135
b
− 157
c
− 185
c
− 182
c
− 147
b
− 112
a
Azolla dual cropping 30 kg N + urea 30 kg N −
17
a
− 106
b
− 105
a
− 149
c
− 156
b
− 154
b
− 104
a
− 107
a
Azolla incorporated 30 kg N + dual cropping 30 kg N −
58
c
− 97
b
− 104
a
− 122
a
− 127
a
− 133
a
− 146
b
− 103
a a
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different at p 0.05 by Duncan’s Multiple Range test DMRT.
Table 3 Matrix of correlation r coefficients between CH
4
flux and select plant and soil parameters Parameter
CH
4
flux Eh
Dissolved oxygen Fe
2+
RMC NRN
Root oxidase of root tip Eh
40
a
− 0.366
∗
– –
– –
– –
Dissolved O
2
40 −
0.337
∗
− 0.579
∗∗
– –
– –
– Fe
2+
35 0.520
∗∗
− 0.195
0.027 –
– –
– RMC
b
35 0.065
− 0.702
∗∗
0.370
∗
0.179 –
– –
NRN
c
25 0.523
∗∗
− 0.292
0.089 0.582
∗∗
0.117 –
– Root oxidase of root tip
20 −
0.367 −
0.595
∗∗
0.180 0.850
∗∗
0.140 0.284
– Root oxidase of root base
20 −
0.484
∗
− 0.335
0.191 0.753
∗∗
0.236 0.535
∗
0.546
∗ a
Values in parenthesis indicate the number of observations.
b
RMC: Readily mineralizable carbon.
c
NRN: Ninhydrin reactive nitrogen.
∗
Significant at p 0.05.
∗∗
Significant at p 0.01.
Azolla. Simple correlation analysis provided a signif- icant negative relationship between
a
-naphthylamine oxidase activity of root base Table 3 and CH
4
flux indicating a direct or indirect role of root oxidizing
power on CH
4
flux.
Table 4 Changes in the dissolved oxygen concentration in the soil–floodwater interface in a flooded alluvial field planted to rice under the influence
of Azolla and urea
a
Treatment Dissolved oxygen mg l
− 1
water Days after transplantation
10 20
30 40
50 60
70 80
Mean Control no N
2.90
a
1.46
a
1.55
b
1.48
bc
1.06
a
0.85
b
1.02
b
1.46
b
1.47 Urea 60 kg N
1.35
d
0.41
c
1.04
c
1.66
b
0.97
ab
0.65
c
1.80
a
1.79
a
1.21 Azolla incorporated 30 kg N + urea 30 kg N
1.40
d
0.34
c
1.68
ab
1.39
bc
0.84
b
0.87
ab
1.74
a
1.43
b
1.21 Azolla dual cropping 30 kg N + urea 30 kg N
2.45
b
1.32
a
1.84
a
1.82
a
1.03
a
1.00
a
1.12
b
1.52
b
1.51 Azolla incorporated 30 kg N + dual cropping 30 kg N
2.05
c
1.10
b
1.71
a
1.52
bc
1.02
a
0.95
ab
1.74
a
1.50
b
1.45
a
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different at p 0.05 by Duncan’s Multiple Range test DMRT.
The Fe
2+
content was high in urea-amended and Azolla-incorporated soils which increased progres-
sively with flooding. Fe
2+
content was highest at the reproductive stage of the rice crop Table 6. Corre-
lation analysis between CH
4
flux and Fe
2+
content
K. Bharati et al. Agriculture, Ecosystems and Environment 79 2000 73–83 79
Table 5 The
a
-naphthylamine oxidase activity of roots of rice plants under the influence of Azolla and urea
a
Treatment
m
g
a
-naphthylamine oxidized g
− 1
dry root h
− 1
Days after transplantation 20
30 50
70 Root tip
Root base Root tip
Root base Root tip
Root base Root tip
Root base Control no N
206
b
205
ab
1075
a
218
c
1556
a
200
c
290
a
57
a
Urea 60 kg N 697
a
159
b
878
ab
272
abc
1200
b
295
ab
250
a
39
a
Azolla incorporated 30 kg N + urea 30 kg N
408
ab
247
a
846
ab
250
bc
1093
b
348
a
167
a
57
a
Azolla dual cropping 30 kg N + urea 30 kg N
383
ab
237
ab
741
b
298
ab
1000
b
293
ab
353
a
44
a
Azolla incorporated 30 kg N + dual cropping 30 kg N
250
b
233
ab
693
b
340
a
556
c
267
bc
500
a
100
a a
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different at p 0.05 by Duncan’s Multiple Range test DMRT.
indicated a highly significant positive relationship Table 3.
The RMC content was highest in urea-amended and Azolla-incorporated plots. The RMC content of
flooded soils, which was initially high, declined at maturity Table 7. Simple correlation analysis, how-
ever, indicated no significant relationship between RMC and CH
4
emission. NRN content of the rhi- zosphere soil was high in the initial stages of crop
growth, declined later and increased again during re- productive stages Table 8. Mean NRN content of the
rhizosphere soil was high in almost all the amended soils as compared to no N control. Among the differ-
ent amendments, mean NRN content was lowest in field plots where Azolla was dual cropped in conjunc-
tion with urea. A simple correlation analysis between
Table 6 Variation in the Fe
2+
content of a flooded alluvial soil planted to rice under the influence of Azolla and urea
a
Treatment
m
g Fe
2+
recovered g
− 1
dry soil Days after transplantation
10 20
30 40
50 60
70 Mean
Control no N 2694
a
670
b
1835
b
1379
c
3200
b
2998
a
486
a
1895 Urea 60 kg N
2552
a
1718
a
2256
a
3076
a
3249
b
3077
a
712
a
2377 Azolla incorporated 30 kg N + urea 30 kg N
2333
a
917
b
2348
a
2870
a
3670
a
2861
a
556
a
2222 Azolla dual cropping 30 kg N + urea 30 kg N
2483
a
952
b
2431
a
2501
b
3294
b
2282
b
559
a
2072 Azolla incorporated 30 kg N + dual cropping 30 kg N
2511
a
1406
a
1957
b
2248
b
2871
b
2233
b
653
a
1983
a
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different at p 0.05 by Duncan’s Multiple Range test DMRT.
NRN and CH
4
emission under the influence of Azolla application indicated a highly significant positive
relationship. Application of urea at 60 kg N ha
− 1
resulted in higher grain yield over that of no N control Table 9.
Application of Azolla either alone equivalent to 60 kg N ha
− 1
or in combination with urea 30 kg N ha
− 1
as Azolla and 30 kg N ha
− 1
as urea also resulted in a significant increase in grain yield which was statis-
tically at par with that of urea alone 60 kg N ha
− 1
. Interestingly, highest CH
4
emission occurred in the field plots amended with urea alone at 60 kg N ha
− 1
, followed by those with Azolla incorporated as green
manure. Computation of CH
4
release kg per ton of rice yield indicated that Azolla incorporation led to
maximum efflux of CH
4
Table 9 while lowest efflux
80 K. Bharati et al. Agriculture, Ecosystems and Environment 79 2000 73–83
Table 7 Variation in the readily mineralizable carbon RMC content of a flooded alluvial soil planted to rice under the influence of Azolla and urea
a
Treatment Readily mineralizable carbon
m
g g
− 1
dry soil Days after transplantation
10 20
30 40
50 60
70 Mean
Control no N 1259
b
1533
b
2501
a
821
a
309
a
763
a
821
a
1144 Urea 60 kg N
2631
a
785
d
1262
b
568
b
444
a
624
ab
722
a
1005 Azolla incorporated 30 kg N + urea 30 kg N
2128
a
1771
a
2387
a
508
b
312
a
934
a
927
a
1281 Azolla dual cropping 30 kg N + urea 30 kg N
2127
a
1537
b
1425
b
461
b
497
a
823
a
842
a
1102 Azolla incorporated 30 kg N + dual cropping 30 kg N
2027
a
1088
c
2273
b
888
a
308
a
496
b
876
a
1137
a
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different at p 0.05 by Duncan’s Multiple Range test DMRT.
Table 8 Variation in the ninhydrin reactive nitrogen NRN content of a flooded alluvial soil planted to rice under the influence of Azolla and urea
a
Treatment
m
g ninhydrin reactive N g
− 1
dry soil Days after transplantation
10 20
40 50
70 Mean
Control no N 2.00
c
1.53
a
2.80
b
3.51
ab
1.75
a
2.32 Urea 60 kg N
6.30
b
1.55
a
5.51
a
4.38
a
1.11
a
3.77 Azolla incorporated 30 kg N + urea 30 kg N
7.85
a
1.23
a
4.74
a
3.16
b
1.05
a
3.61 Azolla dual cropping 30 kg N + urea 30 kg N
6.61
ab
1.19
a
5.18
a
2.44
c
0.62
a
3.21 Azolla incorporated 30 kg N + dual cropping 30 kg N
7.37
a
1.38
a
5.17
a
4.14
a
0.74
a
3.76
a
Mean of three replicate observations. In a column, means followed by a common letter are not significantly different at p 0.05 by Duncan’s Multiple Range test DMRT.
20.62 kg CH
4
Mg
− 1
grain yield was recorded in the plots where Azolla was grown as dual crop.
4. Discussion
