Influence of incorporation or dual cropping of Azolla on methane
emission from a flooded alluvial soil planted to rice in eastern India
K. Bharati, S.R. Mohanty, D.P. Singh, V.R. Rao, T.K. Adhya
∗Laboratory of Soil Microbiology, Division of Soil Science and Microbiology, Central Rice Research Institute, Cuttack - 753006, India
Received 15 December 1998; received in revised form 17 August 1999; accepted 8 November 1999
Abstract
Green manures are widely used in rice production and may influence methane efflux (CH4). Influence of application of
Azolla (A. caroliniana Wild.), a widely used biofertilizer for rice (Oryza sativa L.), on CH4efflux from a flooded alluvial
soil planted to rice, and select soil and plant variables were investigated in a field experiment at Cuttack, India. Azolla was either incorporated as green manure at the beginning of the experiment or grown as dual crop in the standing water along with the rice crop. Dual cropping of Azolla (equivalent to 30 kg N ha−1) in conjunction with urea (30 kg N ha−1) effected
lowest CH4flux (89.29 kg CH4ha−1). Cumulative CH4flux followed the order of urea > Azolla (incorporated)+urea > Azolla
(incorporated+dual crop) > no N control > urea+Azolla (dual crop). Growing Azolla had a moderating effect on CH4efflux
from flooded soil through an increase in the dissolved oxygen concentration at the soil–floodwater interface. Among the different soil and plant variables studied, soil redox potential, dissolved oxygen concentration at the soil–floodwater interface
anda-naphthylamine oxidase activity of root base exhibited significant negative relationship with CH4flux. In addition, Fe2+
and ninhydrin reactive nitrogen (NRN) contents of the flooded soil exhibited significant positive relationship with CH4flux.
Results indicated that, dual cropping of Azolla in conjunction with urea considerably reduced CH4efflux without affecting
the rice yields and can be used as a practical mitigation option for minimizing CH4flux from flooded paddy. © 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Methane efflux; Flooded soil; Azolla application; Rice plants; Grain yield
1. Introduction
Methane (CH4), the most abundant gaseous hydro-carbon in the atmosphere, is an important greenhouse gas that may account for approximately 15–20% of the total current increase in global warming (Rodhe, 1990). Flooded rice paddy has been identified as one of the important sources of anthropogenic CH4 with estimates of annual emission ranging between 47
∗Corresponding author. Tel.:+91-671-642445;
fax:+91-671-641744.
E-mail address: crriinfo@ori.nic.in (T.K. Adhya)
and 60 Tg per year, representing 8.5–10.9% of total emission from all sources (Crutzen, 1995; Houghton et al., 1995). With the intensification of rice culti-vation to meet the needs for rising population, CH4 emission from this important ecosystem is likely to increase (Anastasi et al., 1992). However, a reduction of 15–20% would result in stabilization of the CH4 concentration in the atmosphere to that of 1990 levels (Watson et al., 1995).
Flooded soils planted to rice are conducive to the production and emission of CH4due to the presence of methanogenic bacteria that utilize readily decompos-able organic compounds under anaerobic soil
tion. Both CH4production 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 CH4efflux from flooded rice soils (Neue et al., 1997). However, the relationship between fertilizer application and CH4 efflux from flooded rice system is far from clear and available lit-erature on the effect of fertilizers on CH4 emission is often contradictory (Minami, 1995). While organic matter amendment generally increases CH4 emission (Wassmann et al., 1996; Neue et al., 1997), CH4 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 N2-fixing cyanobac-terial symbiont Anabaena Azollae Stras., can fix 30–60 kg N ha−1in 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 – – 0
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
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 CH4efflux 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 CH4emission 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.
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. CH4flux measurements
Plant-mediated CH4 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 CH4 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 CH4 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 (M/s 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 (M/s Aerovironment Inc.) at fixed intervals of 0, 15 and 30 min. The air samples from the sampling bags were analyzed for CH4
2.3. CH4estimation
The CH4 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 CH4was 0.65 min. The GC was calibrated before and after each set of mea-surements using 5.38, 9.03 and 10.8ml CH4ml−1 in N2(Scotty® II Analyzed gases, M/s Altech associates Inc., USA) as primary standard and 2.14ml CH4ml−1 in air as secondary standard to provide a standard curve linear over the concentration ranges used. The minimum detectable limit for CH4 was 0.5ml ml−1
and the normal measurements of gas samples from the field lay within the lower range (2–6ml CH4ml−1)
of the standard curve. CH4 was determined by peak area and CH4flux was expressed as mg m−2h−1.
2.4. Soil analyses
Measurements for redox potential and dissolved oxygen concentration were done with each set of CH4 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 Fe2+, readily mineralizable carbon (RMC) and ninhydrin re-active nitrogen (NRN) contents. The Fe2+content was measured by agitating fresh soil samples (5 g) with 50 ml of NH4OAC : HCl (pH 2.8) for 1 h, and deter-mining Fe2+ colorimetrically after reaction with or-thophenanthroline (Pal et al., 1979) and expressed as
mg Fe2+g−1 soil. The RMC content was measured
titrat-ing the extract with ferrous ammonium sulfate after wet digestion with chromic acid (Mishra et al., 1997) and expressed asmg C g−1soil. The NRN content of
flooded soil was estimated colorimetrically following the method of Amato and Ladd (1988) and expressed asmg NRN g−1soil.
2.5. Plant parameters
Mean aerial biomass (fresh and dry weights) was measured by harvesting above-ground portions on each day of CH4sampling. Thea-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 (20mg ml−1)
within 10 min of collection of roots. The root oxidase activity was expressed asmg ofa-naphthylamine
ox-idized g−1dry root h−1. Grain and straw yields from individual replicated treatments were measured at maturity and the harvest index was calculated using the formula:
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 CH4 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 CH4emission.
3. Results
3.1. Methane flux
Methane flux varied considerably among differ-ent treatmdiffer-ents with one peak each at vegetative and maturity stages of the rice crop. CH4 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 CH4 flux during the first 20 days after transplantation. Interestingly, CH4 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). Applicaincorpora-tion of urea alone at 60 kg N ha−1 also stimulated CH
4 efflux from flooded fields planted to rice. Thus, the mean CH4 emission followed the order of urea at 60 kg N ha−1 (8.15 mg
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 CH4flux 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 CH4flux from a flooded field planted to rice and the relationship between CH4flux 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 CH4flux indicated a significant negative relationship (Table 3).
3.2. Soil and plant parameters
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, bars/half-bars indicate the SD.
(r= −0.337*, n=40) was observed between DO2 and CH4emission.
Thea-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 highera-naphthylamine oxidase activity than the
root base. Thea-naphthylamine oxidase activity of the
Table 2
Variation in redox potential in the root region of rice plants under the influence of Azolla and ureaa
Treatment Redox potential (mV)
Days after transplantation
10 20 30 40 50 60 70 80
Control (no N) −40b −78a −106a −135b −137a −134a −140b −108a Urea (60 kg N) −54c −172d −173c −186d −193c −189c −144b −156b
Azolla incorporated (30 kg N)+urea (30 kg N) −69d −135b −135b −157c −185c −182c −147b −112a
Azolla dual cropping (30 kg N)+ urea (30 kg N) −17a −106b −105a −149c −156b −154b −104a −107a
Azolla incorporated (30 kg N)+dual cropping (30 kg N) −58c −97b −104a −122a −127a −133a −146b −103a aMean 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 CH4 flux and select plant and soil parameters
Parameter CH4 flux Eh Dissolved oxygen Fe2+ RMC NRN Root oxidase of root tip
Eh (40)a −0.366∗ – – – – – –
Dissolved O2 (40) −0.337∗ −0.579∗∗ – – – – –
Fe2+ (35) 0.520∗∗ −0.195 0.027 – – – –
RMCb (35) 0.065 −0.702∗∗ 0.370∗ 0.179 – – –
NRNc (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∗
aValues in parenthesis indicate the number of observations. bRMC: Readily mineralizable carbon.
cNRN: 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 CH4 flux indicating a direct or indirect role of root oxidizing power on CH4flux.
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 ureaa
Treatment Dissolved oxygen (mg l−1 water)
Days after transplantation
10 20 30 40 50 60 70 80 Mean
Control (no N) 2.90a 1.46a 1.55b 1.48bc 1.06a 0.85b 1.02b 1.46b 1.47 Urea (60 kg N) 1.35d 0.41c 1.04c 1.66b 0.97ab 0.65c 1.80a 1.79a 1.21
Azolla incorporated (30 kg N)+urea (30 kg N) 1.40d 0.34c 1.68ab 1.39bc 0.84b 0.87ab 1.74a 1.43b 1.21
Azolla dual cropping (30 kg N)+urea (30 kg N) 2.45b 1.32a 1.84a 1.82a 1.03a 1.00a 1.12b 1.52b 1.51
Azolla incorporated (30 kg N)+dual cropping (30 kg N) 2.05c 1.10b 1.71a 1.52bc 1.02a 0.95ab 1.74a 1.50b 1.45 aMean 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 Fe2+ content was high in urea-amended and
Azolla-incorporated soils which increased
Table 5
Thea-naphthylamine oxidase activity of roots of rice plants under the influence of Azolla and ureaa
Treatment mga-naphthylamine oxidized g−1dry 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) 206b 205ab 1075a 218c 1556a 200c 290a 57a
Urea (60 kg N) 697a 159b 878ab 272abc 1200b 295ab 250a 39a
Azolla incorporated (30 kg N)+ urea (30 kg N)
408ab 247a 846ab 250bc 1093b 348a 167a 57a
Azolla dual cropping (30 kg N)+ urea (30 kg N)
383ab 237ab 741b 298ab 1000b 293ab 353a 44a
Azolla incorporated (30 kg N)+ dual cropping (30 kg N)
250b 233ab 693b 340a 556c 267bc 500a 100a aMean 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 CH4 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 amendmdiffer-ents, mean NRN contdiffer-ent was lowest in field plots where Azolla was dual cropped in conjunc-tion with urea. A simple correlaconjunc-tion analysis between
Table 6
Variation in the Fe2+content of a flooded alluvial soil planted to rice under the influence of Azolla and ureaa
Treatment mg Fe2+recovered g−1dry soil
Days after transplantation
10 20 30 40 50 60 70 Mean
Control (no N) 2694a 670b 1835b 1379c 3200b 2998a 486a 1895
Urea (60 kg N) 2552a 1718a 2256a 3076a 3249b 3077a 712a 2377
Azolla incorporated (30 kg N)+urea (30 kg N) 2333a 917b 2348a 2870a 3670a 2861a 556a 2222
Azolla dual cropping (30 kg N)+urea (30 kg N) 2483a 952b 2431a 2501b 3294b 2282b 559a 2072
Azolla incorporated (30 kg N)+dual cropping (30 kg N) 2511a 1406a 1957b 2248b 2871b 2233b 653a 1983
aMean 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 CH4emission under the influence of Azolla application indicated a highly significant positive relationship.
Table 7
Variation in the readily mineralizable carbon (RMC) content of a flooded alluvial soil planted to rice under the influence of Azolla and ureaa Treatment Readily mineralizable carbon (mg g−1 dry soil)
Days after transplantation
10 20 30 40 50 60 70 Mean
Control (no N) 1259b 1533b 2501a 821a 309a 763a 821a 1144
Urea (60 kg N) 2631a 785d 1262b 568b 444a 624ab 722a 1005
Azolla incorporated (30 kg N)+urea (30 kg N) 2128a 1771a 2387a 508b 312a 934a 927a 1281
Azolla dual cropping (30 kg N)+urea (30 kg N) 2127a 1537b 1425b 461b 497a 823a 842a 1102
Azolla incorporated (30 kg N)+dual cropping (30 kg N) 2027a 1088c 2273b 888a 308a 496b 876a 1137
aMean 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 ureaa
Treatment mg ninhydrin reactive N g−1 dry soil
Days after transplantation
10 20 40 50 70 Mean
Control (no N) 2.00c 1.53a 2.80b 3.51ab 1.75a 2.32
Urea (60 kg N) 6.30b 1.55a 5.51a 4.38a 1.11a 3.77
Azolla incorporated (30 kg N)+urea (30 kg N) 7.85a 1.23a 4.74a 3.16b 1.05a 3.61
Azolla dual cropping (30 kg N)+urea (30 kg N) 6.61ab 1.19a 5.18a 2.44c 0.62a 3.21
Azolla incorporated (30 kg N)+dual cropping (30 kg N) 7.37a 1.38a 5.17a 4.14a 0.74a 3.76
aMean 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 CH4Mg−1grain yield) was recorded in the plots where Azolla was grown as dual crop.
4. Discussion
4.1. Methane flux
Application of organic substrates, including green manure, often increased the CH4 flux from flooded
Table 9
Variations in select parameters of rice plants and cumulative CH4 efflux from a flooded rice paddy under the influence of Azolla and ureaa
Treatment Grain yield Straw yield Harvest Cumulative kg CH4Mg−1
(Mg ha−1) (Mg ha−1) index (%) CH4 (kg ha−1) grain yield
Control (no N) 3.58a 3.75a 48.84 94.94a 26.52
Urea (60 kg N) 4.58b 5.18b 46.93 155.28c 33.90
Azolla incorporated (30 kg N)+urea (30 kg N) 4.38b 4.93b 47.05 149.37c 34.10
Azolla dual cropping (30 kg N)+urea (30 kg N) 4.33b 4.94b 46.71 89.29a 20.62
Azolla incorporated (30 kg N)+dual cropping (30 kg N) 4.24b 4.78b 47.01 105.64b 24.92
aAverage 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).
consortia. Its incorporation as green manure (Treat-ment III) resulted in a high CH4flux during the first 20 DAT, which also coincided with the most active period of Azolla decomposition (Watanabe et al., 1989). Soil organic matter, both native and applied, is the main source of CH4emitted from the paddy soils during the initial stages of crop growth (Neue et al., 1997).
The situation was markedly different in treatments (IV and V) where Azolla was grown as dual crop, ei-ther in conjunction with urea or following incorpora-tion of Azolla as green manure. In both the treatments, CH4 flux was low, possibly because the growing
Azolla crop had a moderating effect on the CH4 flux from flooded soil as compared to the incorporated
Azolla. Application of urea stimulated CH4flux from flooded fields by causing increased plant growth and metabolic activity that perhaps contributed to a higher CH4 efflux. In an earlier greenhouse study, applica-tion of urea stimulated CH4production and emission through rice plants (Banik et al., 1996).
A consistently higher CH4 flux was observed in
Azolla incorporated plots as compared to no N control
plots. This difference in CH4 emission among treat-ments was maintained upto 60 days. A second emis-sion peak of CH4 flux was observed in all the plots, albeit with varying degree depending upon the treat-ment, during maturity period of the crop. The increase in emission during this period could be attributed to C supplied by root lysis or exudation from rice after flowering (Schutz et al., 1989; Lindau et al., 1991). However, the initial priming effect caused by Azolla incorporation was persistent even at later stages.
4.2. Redox status and oxygen diffusion
Redox status of a flooded soil is an indirect indi-cator of CH4flux pattern from rice ecosystem (Wang et al., 1993) and soils with lower redox potential are usually associated with high CH4 flux. Follow-ing floodFollow-ing, soil Eh decreases in a thermodynamic sequence and the progress of soil reduction is con-trolled by the relative abundance of electron donors and electron acceptors in the soil. The main electron donor in flooded rice soils is readily decomposable organic matter and in soils where Azolla was incor-porated, the enhanced reduction is probably due to the ready availability of organic matter from decom-posing Azolla. Interestingly, soil from treatments with
dual crop of Azolla registered a higher redox potential leading to low CH4flux.
Lowering of the Eh is negatively influenced by the diffusion of oxygen to the surface soil layer. High DO2 in the floodwater might retard CH4emission from rice field by promoting CH4 oxidation at the soil–water interface (Hanson and Hanson, 1996). Further, leach-ing of oxygenated water by percolation in flooded rice fields would inhibit methanogenesis by keeping the soil in a more oxidized state as well as stimu-late CH4oxidation in the reduced soil layer (Kimura et al., 1992). The mean DO2concentration was higher in field plots with a dual crop of Azolla indicating the role of Azolla in enriching the standing water with oxygen. In dual cropping of Azolla, the growing fern forms a mat above the standing water, but unlike many other aquatic plants is not particularly known to re-lease oxygen through its floating roots (Ashton and Walmsley, 1976). It is possible that the oxygen re-leased during active photosynthesis by Azolla in the standing water maintains more oxidized conditions af-fecting the emission of CH4from the flooded soil. In an earlier report, CH4 emission from a flooded field was low during the active growth of algae and this was attributed to the release of oxygen from the algal mat during photosynthesis (Wang et al., 1995).
4.3. Other soil and plant parameters
Thea-naphthylamine oxidase activity of rice roots,
another index of the oxidation status of the rhizosphere region (Ota, 1970), has been correlated well with CH4 efflux from different cultivars (Satpathy et al., 1998), growth stages (Adhya et al., 1994) and even diurnal variation in CH4flux (Satpathy et al., 1997) from rice plants. In this study,a-naphthylamine oxidase activity
of the root base was high in field plots with dual growth of Azolla indicating higher oxidation status in such field plots. Thus, low CH4flux from field plots with dual cropping of Azolla could be a combination of higher redox potential, high dissolved oxygen in the standing water and higher a-naphthylamine oxidase
high in Azolla-incorporated plots which also exhibited higher CH4 flux. NRN, an index of free amino and amide-N released from dead and decaying microbial cells, is another indicator of available substrates for microbial activity (Joergensen and Brookes, 1990). NRN content of the rhizosphere soil was high in almost all the amended soils as compared to no N control and had a statistically significant positive re-lationship with CH4 emission under the influence of
Azolla. Thus, RMC and NRN which are indicators of
substrate availability had direct or indirect effect on CH4efflux from Azolla-incorporated plots.
Azolla is used as a green manure or biofertilizer to
supplement the N demand of the rice crop and can partially replace the costly chemical N fertilizer under conditions of sustainable agriculture. In the present study, application of Azolla either alone or in combi-nation with urea resulted in a significant increase in grain yield that was statistically at par with that of urea alone. In an earlier study, similar grain yield increase was recorded due to Azolla application (Watanabe et al., 1989). What was interesting is that lowest value of CH4 release per ton of rice yield (20.62 kg CH4Mg−1 grain) was obtained in plots were Azolla was grown as a dual crop. Thus, a dual crop of Azolla results in a lowering of CH4 efflux while increasing the rice yield.
5. Conclusion
Azolla is used to supplement the N demand of the
rice crop. The present study indicated that dual crop-ping of Azolla reduced CH4 flux and yet increased grain yield similar to that of urea application. The de-crease in CH4efflux in plots with dual crop of Azolla could be related to the release of oxygen in the stand-ing water by the growstand-ing Azolla leadstand-ing to less reduced conditions in the soil. This suggests the possibility of practicing Azolla dual cropping for sustaining higher yields and protecting the environment by minimizing the CH4flux from flooded rice paddies.
Acknowledgements
The authors thank Dr. K.C. Mathur (Director) for permission to publish this work. This work was
supported, in part, by the IRRI-UNDP Interregional Research Program on Methane Emission in Rice Fields (GLO/91/G31).
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