Directory UMM :Data Elmu:jurnal:A:Agriculture, Ecosystems and Environment:Vol79.Issue1.Jun2000:

(1)

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

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 condi-0167-8809/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.

(2)

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−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 – – 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

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 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.

The experiment was laid out in a randomized block design with five treatments (Table 1), each with three

(3)

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

(4)

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−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 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 CH4m−2h−1) > Azolla (incorporated)+urea at 30 kg N ha−1 (7.80 mg CH4m−2h−1) > no N control (5.80 mg CH4m−2h−1) > Azolla (incorporated+dual crop) (5.40 mg CH4m−2h−1) > Azolla (dual crop)+urea at 30 kg N ha−1(4.61 mg CH4m−2h−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 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

Dissolved oxygen (DO2) concentration in the soil–floodwater interface was high during the ini-tial growth stages when CH4 flux was low. On the other hand, DO2concentrations reached low levels at maturity when CH4 flux was high (Table 4). Mean DO2 concentration was higher in field plots with a dual crop of Azolla. A significant inverse relationship

(5)

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

(6)

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 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 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∗

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 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 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 Fe2+ content was high in urea-amended and

Azolla-incorporated soils which increased

progres-sively with flooding. Fe2+_{content was highest at the} reproductive stage of the rice crop (Table 6). Corre-lation analysis between CH4 flux and Fe2+ content

(7)

Table 5

Thea-naphthylamine oxidase activity of roots of rice plants under the influence of Azolla and ureaa

Treatment mga-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) 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 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 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

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 CH4emission 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 CH4 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 CH4 release (kg) per ton of rice yield indicated that Azolla incorporation led to maximum efflux of CH4(Table 9) while lowest efflux

(8)

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

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 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

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 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

a_{Average 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).

rice paddy (Yagi and Minami, 1990; Denier van der Gon and Neue, 1995; Wassmann et al., 1996). Azolla, which is used to supplement the N requirement of the growing rice crop, is either incorporated in flooded paddy as green manure or grown as a dual crop. In the present study, CH4emission was low in all the plots during the first 2 weeks after transplantation with the exception of Azolla incorporated plots. Fresh Azolla is easily degradable and upon decomposition, adds to the soil pool of fermentable substrates for methanogenic

(9)

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

activity indicating an overall higher oxidation status. Higher rate of soil organic matter decomposition is known to accelerate CH4 production by supplying large amount of substrates for methanogenesis. The RMC content of the soil, an indicator of available sub-strate for methanogenesis (Mishra et al., 1997) was

(10)

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).

References

Adhya, T.K., Rath, A.K., Gupta, P.K., Rao, V.R., Das, S.N., Parida, K., Parashar, D.C., Sethunathan, N., 1994. Methane emission from flooded rice fields under irrigated conditions. Biol. Fertil. Soils 18, 245–248.

Amato, M., Ladd, J.N., 1988. Assay for microbial biomass based on ninhydrin reactive nitrogen in extracts of fumigated soils. Soil Biol. Biochem. 20, 107–114.

Anastasi, C., Dowding, M., Simpson, V.J., 1992. Future CH4 emissions from rice production. J. Geophys. Res. 97, 7521– 7525.

Ashton, P.J., Walmsley, R.D., 1976. The aquatic fern Azolla and its Anabaena symbiont. Endeavor 35, 39–43.

Banik, A., Sen, M., Sen, S.P., 1996. Effects of inorganic fertilizers and micronutrients on methane production from wetland rice (Oryza sativa L.). Biol. Fertil. Soils 21, 319–322.

Bharati, K., Mohanty, S.R., Adhya, T.K., Banerjee, A., Rao, V.R., Sethunathan, N., 1999. Influence of a commercial formulation of tridemorph on methane production and oxidation in a tropical rice soil. Chemosphere 39, 933–943.

Bouwman, A.F., 1990. Exchange of greenhouse gases between terrestrial ecosystems and the atmosphere. In: Bouwman, A.F. (Ed.), Soils and the Greenhouse Effect, Wiley, New York, pp. 61–192.

Crutzen, P.J., 1995. On the role of CH4in atmospheric chemistry: sources, sinks and possible reductions in anthropogenic sources. Ambio 24, 52–55.

Denier van der Gon, H.A.C., Neue, H.U., 1995. Influence of organic matter incorporation on the methane emission from a wetland rice field. Global Biogeochem. Cycles 9, 11–22. Hanson, R.S., Hanson, T.E., 1996. Methanotrophic bacteria.

Microbiol. Rev. 60, 439–471.

Houghton, J.T., Meira Filho, L.G., Bruce, J., Lee, H., Callander, B.A., Haites, E., Harris, N., Maskell, K., 1995. Climate change 1994: Radiative Forcing and an Evaluation of the IPCC IS92 Emission Scenarios. Cambridge University Press, Cambridge. Islam, A., Molla, A.L., Haque, S., 1984. Azolla and blue-green

algae as alternative source of nitrogen for rice and their mineralization in soils of Bangladesh. Ind. J. Agric. Sci. 54, 1056–1060.

Joergensen, R.G., Brookes, P.C., 1990. Ninhydrin reactive nitrogen measurements of microbial biomass based on ninhydrin reactive nitrogen in extracts of fumigated soils. Soil Biol. Biochem. 22, 1023–1027.

Kimura, M., Miura, Y., Watanabe, A., Murase, J., Kuwatsuka, S., 1992. Methane production and its fate in rice paddies I. Effects of rice straw application and percolation rate on the leaching into subsoil of methane and other soil components. Soil Sci. Plant Nutr. 38, 665–672.

(11)

Lindau, C.W., Bollich, P.K., DeLaune, R.D., Patrick Jr, W.H., Law, V.J., 1991. Effect of urea fertilizer and environmental factors on CH4 emission from a Louisiana USA rice field. Plant Soil 136, 195–203.

Liu, Z.Z., Zheng, W.W., 1992. Nitrogen fixation of Azolla and its utilization in agriculture in China. In: Hong, G.F. (Ed.). The Nitrogen Fixation and its Research in China. Springer, Berlin, pp. 526–537.

Lumpkin, T.A., Plucknett, D.L., 1982. Azolla as a Green Manure: Use and Management in Crop Production. West view Press, Boulder, CO, USA.

Mian, M.H., Stewart, W.D.P., 1985. Fate of fertilizer nitrogen applied as Azolla and blue green algae (Cyanobacteria) in water-logged rice soils —15_{N tracer study. Plant Soil 83, 363–370.} Minami, K., 1995. The effect of nitrogen fertilizer use and other practices on methane emission from flooded rice. Fert. Res. 40, 71–84.

Mishra, S., Rath, A.K., Adhya, T.K., Rao, V.R., Sethunathan, N., 1997. Effect of continuous and alternate water regimes on methane efflux from rice under greenhouse conditions. Biol. Fertil. Soils 24, 399–405.

Neue, H.U., Wassmann, R., Kludze, H.K., Wang, B.J., Lantin, R.S., 1997. Factors and processes controlling methane emissions from rice fields. Nutr. Cycling Agroecosyst. 49, 111–117. Ota, Y., 1970. Diagnostic method for measurement of root activity

in rice plant. Jap. Agric. Res. Q. 5, 1–6.

Pal, S.S., Barik, S., Sethunathan, N., 1979. Effects of benomyl on iron and managanese reduction and redox potential in flooded soil. J. Soil Sci. 30, 155–159.

Rains, D.W., Talley, S.N., 1979. Use of Azolla in North America. Nitrogen and Rice. International Rice Research Institute, Philippines, pp. 419–433.

Rodhe, H., 1990. A comparison of the contribution of various gases to the greenhouse effect. Science 248, 1217–1219. Satpathy, S.N., Mishra, S., Adhya, T.K., Ramakrishnan, B., Rao,

V.R., Sethunathan, N., 1998. Cultivar variation in methane efflux from tropical rice. Plant Soil 202, 223–229.

Satpathy, S.N., Rath, A.K., Ramakrishnan, B., Rao, V.R., Adhya, T.K., Sethunathan, N., 1997. Diurnal variation in methane efflux

at different growth stages of tropical rice. Plant Soil 195, 267– 271.

Schutz, H., Seiler, W., Conrad, R., 1989. Processes involved in the formation and emission of methane in rice paddies. Biogeochem. 7, 33–53.

Singh, P.K., Singh, D.P., 1997. Azolla-Anabaena symbiosis. In: Dadarwal, K.R. (Ed.), Biotechnological Approaches in Soil Microorganisms for Sustainable Crop Production. Scientific Publishers, Jodhpur, India, pp. 93–107.

Venkataraman, A., 1984. Development of organic matter-based agricultural system in south Asia. In: Banta, S., Mendoza, C.V., (Eds.), Organic Matter and Rice, International Rice Research Institute, Philippines, pp. 57–70.

Vlek, P.L.G., Byrnes, B.H., 1986. The efficacy and loss of fertilizer N in lowland rice. Fertil. Res. 9, 131–147.

Wang, Z.P., Crozier, C.R., Patrick Jr., W.H., 1995. Methane emission in a flooded rice soil with and without algae. In: Lal, R., Kimble, J., Levine, E., Stewart, B.A. (Eds.), Soil Management and Greenhouse Effect. CRC Press, Boca Raton, USA, pp. 245-250.

Wang, Z.P., DeLaune, R.D., Masscheleyn, P.H., Patrick Jr, W.H., 1993. Soil redox and pH effects on methane production in a flooded rice soil. Soil Sci. Soc. Am. J. 57, 382– 385.

Wassmann, R., Shangguan, X.J., Tolg, M., Cheng, D.X., Wang, M.X., Papen, H., Rennenberg, H., Seiler, W., 1996. Spatial and seasonal distribution of organic amendments affecting methane emission from Chinese rice fields. Biol. Fertil. Soils 22, 191– 195.

Watanabe, I., Ventura, W., Mascarina, G., Eskew, D.I., 1989. Fate of Azolla spp. and urea nitrogen applied to wetland rice (Oryza

sativa L.). Biol. Fertil. Soils 8, 102–110.

Watson, R.T., Zinyowera, H.C., Moss, R.H., 1995. Climate Change 1995. Impacts, Adaptation and Mitigation of Climate Change: Scientific-Technical Analysis. Cambridge University Press, Cambridge.

Yagi, K., Minami, K., 1990. Effect of organic matter application on methane emission from some Japanese paddy fields. Soil Sci. Plant Nutr. 36, 599–610.

(1)

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

<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∗

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

flux

indicating a direct or indirect role of root oxidizing

power on CH

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 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

<0.05 by

Duncan’s Multiple Range test (DMRT).

The Fe

content was high in urea-amended and

Azolla-incorporated soils which increased

progres-sively with flooding. Fe

_{content was highest at the}

reproductive stage of the rice crop (Table 6).

Corre-lation analysis between CH

flux and Fe

content

(2)

Table 5

The

a-naphthylamine oxidase activity of roots of rice plants under the influence of Azolla and urea

Treatment

mg

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) 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

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

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 Fe

2+_{recovered g}−1_{dry 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 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

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

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

release (kg) per ton of

rice yield indicated that Azolla incorporation led to

maximum efflux of CH

(Table 9) while lowest efflux

(3)

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 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 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 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

Mg

−1

grain 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 CH

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 a_{Average 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).

rice paddy (Yagi and Minami, 1990; Denier van der

Gon and Neue, 1995; Wassmann et al., 1996). Azolla,

which is used to supplement the N requirement of the

growing rice crop, is either incorporated in flooded

paddy as green manure or grown as a dual crop. In the

present study, CH

emission was low in all the plots

during the first 2 weeks after transplantation with the

exception of Azolla incorporated plots. Fresh Azolla is

easily degradable and upon decomposition, adds to the

soil pool of fermentable substrates for methanogenic

(4)

consortia. Its incorporation as green manure

(Treat-ment III) resulted in a high CH

flux 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 CH

emitted 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,

CH

flux was low, possibly because the growing

Azolla crop had a moderating effect on the CH

flux

from flooded soil as compared to the incorporated

Azolla. Application of urea stimulated CH

flux from

flooded fields by causing increased plant growth and

metabolic activity that perhaps contributed to a higher

CH

efflux. In an earlier greenhouse study,

applica-tion of urea stimulated CH

production and emission

through rice plants (Banik et al., 1996).

A consistently higher CH

flux was observed in

Azolla incorporated plots as compared to no N control

plots. This difference in CH

emission among

treat-ments was maintained upto 60 days. A second

emis-sion peak of CH

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 CH

flux pattern from rice ecosystem (Wang

et al., 1993) and soils with lower redox potential

are usually associated with high CH

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 CH

flux.

Lowering of the Eh is negatively influenced by the

diffusion of oxygen to the surface soil layer. High DO

in the floodwater might retard CH

emission from rice

field by promoting CH

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 CH

oxidation in the reduced soil layer (Kimura

et al., 1992). The mean DO

concentration 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 CH

from the flooded soil. In

an earlier report, CH

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

The

a

-naphthylamine oxidase activity of rice roots,

another index of the oxidation status of the rhizosphere

region (Ota, 1970), has been correlated well with CH

efflux from different cultivars (Satpathy et al., 1998),

growth stages (Adhya et al., 1994) and even diurnal

variation in CH

flux (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 CH

flux 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

activity indicating an overall higher oxidation status.

Higher rate of soil organic matter decomposition

is known to accelerate CH

production by supplying

large amount of substrates for methanogenesis. The

RMC content of the soil, an indicator of available

sub-strate for methanogenesis (Mishra et al., 1997) was

(5)

high in Azolla-incorporated plots which also exhibited

higher CH

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 CH

emission under the influence of

Azolla. Thus, RMC and NRN which are indicators of

substrate availability had direct or indirect effect on

CH

efflux 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 CH

release per ton of rice yield (20.62 kg

CH

Mg

−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 CH

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 CH

flux and yet increased

grain yield similar to that of urea application. The

de-crease in CH

efflux 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 CH

flux 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).

References