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(American Journal of Botany. 2001;88:975-979.)
© 2001 Botanical Society of America, Inc.

Physiology and Biochemistry

Effect of aquatic weeds on methane emission from submerged paddy soil1

Kazuyuki Inubushi2,3, Hotaka Sugii2,4, Shouji Nishino2,5 and Eisho Nishino2

2Faculty of Horticulture, Chiba University, Matsudo 648, Chiba 271-8510 Japan

Received for publication March 7, 2000. Accepted for publication September 14, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Paddy fields are one of the dominant anthropogenic sources of methane emission to the atmosphere, and the main passageway of methane from paddy soil is through the rice plant. However, the effect of aquatic weeds on methane emission from rice paddies has not been properly evaluated yet. Methane emission from weeded pots and unweeded ones with anaerobic paddy soil was measured throughout the period of rice growth. More than double the amount of methane was emitted from weeded pots compared with unweeded ones. Peroxidase activity of rice root was not different between weeded and unweeded pots. However, methanogenic bacteria populations were higher in weeded pots than in unweeded ones, while methane oxidation activity, measured by the propylene oxidation technique, was higher in unweeded pots than in weeded ones. Methane oxidation activity of roots from three typical aquatic weeds in paddy fields, Lipocarpha sp., Rotala indica, and Ludwigia epilobioides, was higher than that of rice plants, while lower stems of these aquatic plants showed similar or lower activity compared with the same areas of rice plants. These results indicate that the role of aquatic weeds in paddy soil in methane emission should not be overlooked in evaluating mitigation options for reducing methane emission from paddy fields.

Key Words: aquatic weeds • methane emission • methane oxidation • paddy soil • propylene oxidation • rhizosphere


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Paddy fields are one of the dominant anthropogenic sources of methane to the atmosphere (estimated as 15% of global methane emission; IPCC, 1994 ). The main passageway of methane emission from anaerobic paddy soils to the atmosphere is through the arenchyma of the rice plant (Oryza sativa) (Cicerone and Shetter, 1981 ; Inubushi et al., 1989 ; Nouchi, Mariko, and Aoki, 1990 ). One possible mitigation option to reduce methane emission from paddy fields is water management, such as percolation, mid-summer drainage, and intermittent drainage (Inubushi, Muramatsu, and Umebayashi, 1992 ; Wassmann, Papen, and Rennenberg, 1993 ; Yagi, Tsuruta, and Minami, 1997 ). This would introduce oxygen into anaerobic soil, rendering it aerobic, and in so doing reduce the formation of methane by enhancing methanotrophic (methane oxidizing) bacteria in soil (Bosse and Frenzel, 1997 ; Gilbert and Frenzel, 1998 ). Organic farming, which involves the use of organic matter instead of chemical fertilizer and without herbicide application, has become popular in Japan (AFFRC, 2000 ). Both drainage and organic farming may enhance the growth of weeds in paddy fields. However, the effect of aquatic weeds in paddy fields on methane emission has not been properly evaluated yet. Therefore it is important to investigate whether such feedback would exacerbate methane emission by introducing more organic matter as weeds into the soil or mitigate methane emission by methane oxidation.

In this paper, methane emission from weeded and unweeded pots with paddy soils were compared in order to estimate the effect of aquatic weeds on methane flux from paddy soils throughout the period of rice growth. Microbial activities related to methane production and oxidation in the soil with rice plants and typical aquatic weeds in paddy soils were also measured during crop season to examine possible mitigation options to reduce methane emission from paddy fields.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Soils and pot experiments
Sandy gley soil was taken from paddy fields in Kuju-kuri, Chiba prefecture, Central Japan, and 3.5 kg of the moist sieved (<7.5 mm) soil was transferred into 18 plastic pots (0.02 m2 x 20 cm depth). Total carbon, total nitrogen, cation exchange capacity (CEC), and pH(H2O) of the soil samples were quantified by standard soil analytical method (Bremner and Mulvaney, 1982 ). Mixed chemical fertilizers were applied as basal and were equivalent to 100 kg N/ha, 25 kg P/ha, and 42 kg K/ha. Chopped rice straw (<1 cm length) was applied at 4000 kg/ha on 27 May 1997. Thirty-five-day-old rice seedlings (Oryza sativa, ‘Nihonbare’) were transplanted 2 d after fertilizer application and soil submergence. A second fertilizer application was made as top dressing at the rate of 20 kg N/ha and 42 kg K/ha at maximum tillering stage on 25 July. All pots were maintained under flooded conditions by being placed in a large plastic water pool until harvest on 25 September. All weeds were manually removed weekly from nine pots (weeded pots), while aquatic weeds, mainly Lipocarpha sp., Rotala indica, and Ludwigia epilobioides, all typical paddy weeds in Japan, were allowed to grow with rice plants in the other nine pots (unweeded pots). Height and tiller numbers of rice plants were measured weekly. Soil Eh (redox potential) was monitored to see the effect of weeds on the anaerobic status in soil at 5 cm depth by Pt electrodes and a pH/Eh meter (Toa Electronics, RM-12P, 29-10-1 Takadanobaba, Shinjuku-ku, Tokyo, Japan). Root, shoot, and the lower parts of rice stems (below soil surface and above the root system) were separated after removal from treatment pots, washed thoroughly, and weighed after drying at 80°C for 24 h on 50, 82, and 111 d after transplanting (DAT). Similarly, corresponding parts of the aquatic weeds were also taken from the unweeded pots on the same dates and treated in the same way as the rice plants.

Methane emission
During rice growth, methane emission was measured almost weekly using the closed chamber method (Inubushi et al., 1989 ). A cylindrical acrylic chamber (15 cm diameter x 1 m height) was placed on the pot with rice for 30 min in the morning (between 1000 and 1200). The methane mixing ratio inside the chamber was measured at 0, 10, 20, and 30 min after placing the chamber. The gas inside the chamber was sampled with a syringe, and the mixing ratio of methane in it was quantified by injecting it into an FID-GC (gas chromatograph with frame ionized detector; Shimadzu GC-7A, Nishinokyou-kuwahara, Nakagyouku, Kyoto, Japan). The emission rate was calculated by taking into account the increase in the methane mixing ratio, the volume of the chamber, and the temperature inside the chamber.

Methanogenic bacteria and their activity in soil
When rice plants and aquatic weeds were collected, soil samples were taken by truncated plastic syringe to a depth of 1–5 cm from a spot between the rice hill and inner surface of the pot. In order to avoid exposing soil samples to the air, they were immediately transferred into glass bottles and homogeneously mixed with oxygen-free water. Subsamples of soil, equivalent to 10 g wet mass, were either incubated anaerobically under N2 headspace at 30°C in the dark in a closed flask to measure methanogenic activity (Chidthaisong, Inubushi, and Watanabe, 1996 ; Chidthaisong et al., 1996 ) or further diluted to estimate populations of methanogenic bacteria by the most probable number method (Asakawa and Hayano, 1995 ).

Methane oxidation activities in soil and plant samples
Soil subsamples (5 g wet mass) or samples of root, stem, and shoots of rice and aquatic weeds (2 g wet mass) were transferred into 30-mL Erlenmeyer flasks. To measure methane oxidation activity, the propylene oxidation method was used (Watanabe et al., 1995 ). Thirty milliliters of headspace in the flasks was replaced with pure propylene and methane to give final concentrations of 20 and 10% v/v, respectively. The flasks were then incubated for 12 h at 30°C in the dark. At 0, 6, and 12 h of incubation, 0.5 mL of headspace was taken to determine propylene oxide concentration by FID-GC with Tenax TA® (GL Science, 6-22-1, Nishishinjuku, Tokyo, 163-1130 Japan).

Peroxidase activity of rice plant and weeds
The peroxidase activity of roots of rice and aquatic weeds was measured according to Futami (1990) . Briefly, the plant part, equivalent to 2 g wet mass, was placed in a 100-mL flask and incubated with 50 mL of 40 mg/L alpha-naphthylamine solution and 0.1 mol/L sodium phosphate buffer solution mixture (1:1) for 6 h at 30°C in the dark. After the incubation, 2 mL of the solution in the flask was mixed with 10 mL distilled water, 1 mL of 1% sulfanil acid, and 10 ppm sodium nitrite solutions. The optical absorption at 510 nm was read and calibrated with standard alpha-naphthylamine solutions as a blank test. All the results were expressed on a dry matter (DM) basis and analyzed statistically by SYSTAT® at 5% significance level using a t test for comparison of the treatment means.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Methane flux from weeded and unweeded plots
The soil had total carbon = 7.9 g/kg, total nitrogen = 0.8 g/kg, cation exchange capacity (CEC) = 76.4 mmol/kg and pH(H2O) = 6.36. Methane flux through rice plants in weeded pots peaked around 30 DAT and was at a level similar to those in unweeded pots until 50 DAT (Fig. 1). However, methane flux increased rapidly to reach the maximum on 78 DAT in weeded pots, and this was almost three times higher than in unweeded plots. As a result, more than double the amount of methane was emitted during the crop season from weeded pots compared with unweeded ones. Soil Eh was significantly lower in weeded pots than unweeded pots after 40 DAT except 82 DAT (Fig. 2), indicating that more oxidized conditions were probably provided by aquatic weeds.



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  		Fig. 1. Temporal changes in methane flux from weeded (SP) and unweeded plots (SW). Bars indicate SD of the mean

 


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  		Fig. 2. Temporal changes in soil redox potential (Eh) (5 cm) in weeded (SP) and unweeded plots (SW). Bars indicate SD of the mean

 
Plant growth and peroxidase activity of rice roots
Rice plant height and tiller number were not significantly different between weeded and unweeded pots (Fig. 3). Dry matter masses of shoots and roots of rice showed no significant difference between the two treatments, except being higher in unweeded pots on 82 DAT, in which roots of weeds were present (Fig. 4). Similarly, the peroxidase activity of rice roots showed no difference between these pots, although it decreased gradually from 50 DAT to harvest (Fig. 5).



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  		Fig. 3. Temporal changes in plant height (a) and tiller number (b) of rice planted in weeded (SP) and unweeded plots (SW). Bars indicate SD of the mean

 


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  		Fig. 4. Dry mass of shoot (upper) and root (lower) of rice (R) and weed (W) in weeded (SP) and unweeded plots (SW). Bars indicate SD of the mean

 


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  		Fig. 5. Peroxidase activity (µg alpha-naphthylamine · g DM–1 · h–1) of rice root in weeded (SP) and unweeded plots (SW). Bars indicate SD of the mean

 
Methanogenic bacteria and methane production activity in soil
Populations of methanogenic bacteria in soil was 4–7 times higher in weeded pots than in unweeded ones (Fig. 6). Methane production activity was also higher in weeded pots than in unweeded ones on 82 and 111 DAT.



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  		Fig. 6. Populations of methanogenic bacteria (upper) and their activity (lower) in soil of weeded (SP) and unweeded plots (SW). Bars indicate SD of the mean. DAT = days after transplanting, MPN = most probable number

 
Methane oxidation activity of soil
Methane oxidation activity measured by the propylene oxidation technique was higher in unweeded pots than in weeded ones, particularly during the later part of the croping season (Fig. 7).



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  		Fig. 7. Methane oxidation activity in soil of weeded (SP) and unweeded plots (SW). Bars indicate SD of the mean. PPO = Propylene oxide, DM = dry matter

 
Methane oxidation activity of roots of three aquatic weeds
Roots of Lipocarpha sp., Rotala indica, and Ludwigia epilobioides, typical aquatic weeds in paddy fields in central Japan, showed higher methane oxidation activity than those of rice plants. The methane oxidation activity was rated in the order of Ludwigia > Lipocarpha > Rotala > rice (Fig. 8). On the other hand, lower parts of the stem of rice plants showed much higher methane oxidation activity than roots. The other parts of the aquatic plants showed almost the same methane oxidation activity as the roots of each plant, and this was similar or lower as compared with the same parts of rice plants.



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  		Fig. 8. Potential methane oxidation activity of plant parts in rice (Oryza) and three species of weeds (Rotala, Lipocarpha, and Ludwigia) growing in flooded paddy soil. Bars indicate SD of the mean. DM = dry matter

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The significance of rice plants in methane emission from paddy soils to the atmosphere has been recognized since Cicerone and Shetter (1981) . Inubushi et al. (1989) estimated that >90 % of methane flux was through rice plants. Nouchi, Mariko, and Aoki (1990) examined in detail methane passage through rice plants. A similar movement of methane from soil to the atmosphere was also reported for other hygrophytes (Wagatsuma et al., 1992 ; Frenzel and Rudolph, 1998 ). During methane transport from soil to the atmosphere, 20–80% of methane was oxidized in rhizosphere soil with rice plants (Conrad and Rothfuss, 1991 ; Sass et al., 1991 ). In natural wetlands, aquatic plants were also important in methane oxidation. King (1994) and Calhoun and King (1998) demonstrated methane oxidation by methanotrophic bacteria in the rhizosphere of aquatic plants. Gilbert and Frenzel (1995) , Bosse and Frenzel (1997) , and Gilbert et al. (1998) found similar phenomena in rice plants.

The present study showed active methane oxidation in rhizosphere and lower stem parts of typical aquatic weeds as well as rice plants in paddy fields in Japan. Although the mechanism employed by these weeds to reduce methane emission is still not clear, peroxidase activity of rice roots was not influenced by weeds (Fig. 5). Reduction of methane flux by weeds was more likely due to enhancing methanotrophic bacteria (Fig. 7) or repression of methanogenic bacteria in the rhizosphere (Fig. 6). Both contributions to the reduction of methane emissions should be evaluated. Since the potential methane oxidation activity was expressed on a dry matter basis (Fig. 8), the product of (dry matter) x (average activity) of weed roots were estimated to be about twice that of rice roots. However, rice shoots (lower columns and base of stem) also showed high methane oxidation activity. Therefore, such estimation should include these parts of rice plants. Methane emission from anaerobic soil to the atmosphere could be regarded as the difference between methane production and methane oxidation. Both activities in this study were measured separately in soil and plant parts under laboratory conditions. Therefore, in situ activity in the soil–plant system should also be examined more carefully in estimating methane oxidation.

Weeds did not reduce the growth of rice plants under these experimental conditions (Fig. 3), even though the dry mass of weed shoots was ~14–19% of those of rice shoots (Fig. 4). However, these results should be examined in detail under various conditions. Food production could be sustained and methane emissions might be decreased by reducing the application of agrochemicals, such as herbicides and by maintaining or increasing organic matter application to soil. Moreover, methane emission from paddy soil can also be mitigated by water management, such as mid-season drainage and other options. The results in this study indicate that the role of aquatic weeds in paddy soils in relation to methane emission should not be overlooked in evaluating mitigation options to reducing methane emission from paddy fields.


   FOOTNOTES
 
1 The authors thank ex Prof. Yasuji Fukuda for botanical identification. This work was supported by the Ministry of Agriculture, Fishery and Forestry, Japan, Research Fund on Agroecological Technology for Controlling Factors of Global Environmental Changes, and Research Institute of Innovative Technology for The Earth Fund on Excellent Research Project headed by Prof. Emer. Iwao Watanabe. Back

2 Author for reprint requests (Tel +81-47-308-8816, Fax +81-47-308-8720, e-mail inubushi{at}midori.h.chiba-u.ac.jp ). Back

3 Current address: Yoshitomi Pharmaceutical Industries Ltd., Yoshiki 3224-3-508, Yamaguchi 753-0811 Japan. Back

4 Current address: Kimitsu Agroforestry Highschool, Aoyagi 48, Kimitsu, Chiba, 292-0454 Japan. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
AFFRC (Agriculture, Forestry and Fisheries Research Council, Japan). 2000 http://www.maff.go.jp

Asakawa S. K. Hayano 1995 Populations of methanogenic bacteria in paddy field soil under double cropping conditions (rice–wheat). Biology and Fertility of Soils 20: 113-117

Bosse U. P. Frenzel 1997 Activity and distribution of methane-oxidizing bacteria in flooded rice soil microcosms and in rice plants (Oryza sativa). Applied and Environmental Microbiology 63: 1199-1207[Abstract]

Bremner J. M. C. S. Mulvaney 1982 Nitrogen-total. In Methods of soil analysis, Part 2, Agronomy monograph 9, 2nd ed., 595–624. American Society of Agronomy—Soil Science Society of America, Madison, Wisconsin, USA

Calhoun A. G. M. King 1998 Characterization of root-associated methanotrophs from three freshwater macrophytes: Pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia. Applied and Environmental Microbiology 64: 1099-1105[Abstract/Free Full Text]

Chidthaisong A. K. Inubushi Y. Muramatsu I. Watanabe 1996 Production potential and emission of methane in flooded rice soil microcosms after continuous application of straws. Microbes and Environments 11: 73-78

———, ———, and I. Watanabe 1996 Methanogenic characteristics of flooded rice soils in response to glucose amendment. Soil Science and Plant Nutrition 42: 645-649[ISI]

Cicerone R. J. J. D. Shetter 1981 Sources of atmospheric methane: measurements in rice paddies and a discussion. Journal of Geophysics Research 86C: 7203-7209

Conrad R. F. Rothfuss 1991 Methane oxidation in the soil surface layer of a flooded rice field and the effect of ammonium. Biology and Fertility of Soils 12: 28-32[CrossRef][ISI]

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Sass R. F. M. Fisher P. A. Harcombe F. T. Turner 1991 Methane emission from rice fields as influenced by solar radiation, temperature and straw incorporation. Global Biogeochemical Cycles 5: 335-350

Wagatsuma T. K. Jujo K. Tawaraya T. Sato A. Ueki 1992 Decrease of methane concentration and increase of nitrogen gas concentration in the rhizosphere by hygrophytes. Soil Science and Plant Nutrition 38: 467-476[ISI]

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Watanabe I. G. Takada T. Hashimoto K. Inubushi 1995 Evaluation of alternative substrates for determining methane-oxidizing activities and methanotrophic populations in soils. Biology and Fertility of Soils 20: 101-106

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