74 K. Bharati et al. Agriculture, Ecosystems and Environment 79 2000 73–83
tion. Both CH
4
production and emission from flooded rice soils are strongly influenced by several soil pro-
cesses including changes in soil redox status and pH, dynamics of substrate and nutrient availability and
textural stratification Bouwman, 1990. In addition, common cultivation practices such as application of
agrochemicals also affect CH
4
efflux from flooded rice soils Neue et al., 1997. However, the relationship
between fertilizer application and CH
4
efflux from flooded rice system is far from clear and available lit-
erature on the effect of fertilizers on CH
4
emission is often contradictory Minami, 1995. While organic
matter amendment generally increases CH
4
emission Wassmann et al., 1996; Neue et al., 1997, CH
4
ef- flux is also strongly influenced by the type, method
and rate of application of chemical fertilizer. Although urea remains the preferred chemical
N-fertilizer for rice cultivation Vlek and Byrnes, 1986, several organic sources including partially de-
composed and fresh organic matter and biofertilizers are widely used for maintaining the soil fertility and
sustained high yield in tropical rice fields Venkatara- man, 1984. Azolla, a free-floating aquatic fern having
symbiotic association with the N
2
-fixing cyanobac- terial symbiont Anabaena Azollae Stras., can fix
30–60 kg N ha
− 1
in 30 days. It is either incorporated as green manure at the beginning of the cropping
season or grown as a dual crop along with rice, in the standing water of flooded fields. The fern is used
to a great extent in China Liu and Zheng, 1992, India Singh and Singh, 1997, Bangladesh Islam
et al., 1984 and Vietnam Lumpkin and Plucknett, 1982 as an important biological source to improve
Table 1 Summary table of various experimental treatments on Azolla application at the Central Rice Research Institute, Cuttack, India
Treatment Treatment
Amendments Total N application
number details
kg N ha
− 1
Azolla application Urea amendment
I No N control
– –
II Urea-N
– Urea to provide 60 kg N ha
− 1
60 III
Azolla incorporation + urea Incorporated as green manure
at transplantation to provide 30 kg N ha
− 1
Urea to provide 30 kg N ha
− 1
60 IV
Urea + Azolla dual cropping Dual cropping to provide
30 kg N ha
− 1
Urea to provide 30 kg N ha
− 1
60 V
Azolla incorporation + Azolla dual cropping
Incorporated as green manure at transplantation to provide
30 kg N ha
− 1
+ dual cropping
to provide 30 kg N ha
− 1
– 60
the N balance of rice fields. The nitrogen fixed by the cyanobacterial symbiont is either released upon decay
of the incorporated Azolla Mian and Stewart, 1985 or leached into the standing water from the growing
Azolla Rains and Talley, 1979 and is available for uptake by the rice crop.
The objective of the study was to evaluate the effects of applying Azolla as green manure or dual cropping
it on CH
4
efflux from flooded alluvial soil planted to rice. In addition, the alterations in select soil and plant
parameters in Azolla applied soil and their relationship with CH
4
emission were investigated.
2. Materials and methods
2.1. Field experiment The field experiment was conducted in the ex-
perimental farm of the Central Rice Research Insti- tute, Cuttack 20
◦
N, 86
◦
E during the dry cropping season January–May of 1997 under irrigated con-
ditions. The soil was a typic haplaquept Fluvisol with sandy clay-loam texture clay 155 g kg
− 1
, silt 185 g kg
− 1
, sand 660 g kg
− 1
with the following chemical characteristics: pH 6.1, cation exchange
capacity 114 mMol Kg
− 1
soil, electrical conductiv- ity 0.36 dS m
− 1
, organic matter 7 g kg
− 1
and total N 0.8 g kg
− 1
. The field was ploughed, puddled thor- oughly, leveled and subdivided into plots 5 m × 5 m
separated by leeves. The experiment was laid out in a randomized block
design with five treatments Table 1, each with three
K. Bharati et al. Agriculture, Ecosystems and Environment 79 2000 73–83 75
replicates. A. caroliniana Wild., grown in multipli- cation blocks, was incorporated as green manure at
16 Mg ha
− 1
equivalent to 30 kg N ha
− 1
to field plots of third and fifth treatments, a day before transplant-
ing. For treatments IV and V, where it is grown as dual crop, Azolla was inoculated in field plots at 1 Mg ha
− 1
a week after transplantation of rice and allowed to grow. The biomass build-up over a period of 30 days,
which coincided with the peak vegetative stage tiller- ing stage of the rice crop, provided 30 kg N ha
− 1
. Rice plants 21-day old seedlings, cv. CR 749-20-2
were transplanted in the field-plots at a spacing of 15 cm × 20 cm with two seedlings per hill. A common
basal dose of 17.5 and 33.2 kg ha
− 1
of P and K, re- spectively, in the form of single superphosphate and
muriate of potash was applied to the crop at the time of transplantation. Fertilizer N as urea was applied in
two equal splits at 30 and 60 days after transplanta- tion DAT for all the treatments except the second
treatment. For the second treatment, 50 of fertilizer N was applied at the time of transplantation and 25
each in two equivalent splits at 30 and 60 DAT. All the field plots were kept continuously flooded to a water
depth of 10 ± 2 cm during the crop growth. The crop was grown without any application of pesticides and
harvested at maturity 100 DAT.
2.2. CH
4
flux measurements Plant-mediated CH
4
emission flux from the field plots planted to rice was measured by closed chamber
method of Adhya et al. 1994 at regular intervals from transplanting till 90 DAT. Samplings for CH
4
flux measurements were made at 09:00–09:30 hours and 15:00–15:30 hours, and the average of morning
and evening fluxes was used as the flux value for the day. For measuring CH
4
emission, six rice hills were covered with a locally-fabricated perspex cham-
ber 53 cm length × 37 cm width × 51 cm height. A battery-operated air circulation pump with air dis-
placement of 1.5 l min
− 1
Ms Aerovironment Inc., Monrovia, CA, USA, connected to polyethylene tub-
ing was used to mix the air inside the chamber and draw the air samples into Tedlar® air-sampling bags
Ms Aerovironment Inc. at fixed intervals of 0, 15 and 30 min. The air samples from the sampling bags
were analyzed for CH
4
2.3. CH
4
estimation The CH
4
was estimated in a Shimadzu GC-8A gas chromatograph equipped with FID Bharati et
al., 1999. The gas samples were injected through a sample loop 3 ml with the help of an on-column in-
jector. The retention time of CH
4
was 0.65 min. The GC was calibrated before and after each set of mea-
surements using 5.38, 9.03 and 10.8
m
l CH
4
ml
− 1
in N
2
Scotty® II Analyzed gases, Ms Altech associates Inc., USA as primary standard and 2.14
m
l CH
4
ml
− 1
in air as secondary standard to provide a standard curve linear over the concentration ranges used. The
minimum detectable limit for CH
4
was 0.5
m
l ml
− 1
and the normal measurements of gas samples from the field lay within the lower range 2–6
m
l CH
4
ml
− 1
of the standard curve. CH
4
was determined by peak area and CH
4
flux was expressed as mg m
− 2
h
− 1
. 2.4. Soil analyses
Measurements for redox potential and dissolved oxygen concentration were done with each set of
CH
4
flux measurement. The redox potential of the field soil was measured by inserting a combined
platinum–calomel electrode Barnant Co., IL, USA to the root region and measuring the potential differ-
ence in mV Satpathy et al., 1997. All the values were corrected to that of a hydrogen electrode by adding
+
240 mV to the redox readings. Dissolved oxygen concentration at the soil–floodwater interface was
measured using a portable oxymeter Model Oxi 320, WTW gmbH, Weilheim, Germany and expressed as
mg l
− 1
. Soil chemical components were analyzed from field
soils sampled by inserting a tube auger 2 cm diame- ter to a depth of 5–7 cm, in between two rice hills.
The soil samples, after draining excess of water, were immediately subsampled for measurement of Fe
2+
, readily mineralizable carbon RMC and ninhydrin re-
active nitrogen NRN contents. The Fe
2+
content was measured by agitating fresh soil samples 5 g with
50 ml of NH
4
OAC : HCl pH 2.8 for 1 h, and deter- mining Fe
2+
colorimetrically after reaction with or- thophenanthroline Pal et al., 1979 and expressed as
m
g Fe
2+
g
− 1
soil. The RMC content was measured by extracting soil samples with 0.5M K
2
SO
4
, titrat-
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
