American Journal of Botany 88(6): 975–979. 2001.

EFFECT OF AQUATIC WEEDS ON METHANE EMISSION

FROM SUBMERGED PADDY SOIL1

KAZUYUKI INUBUSHI,2,3 HOTAKA SUGII,2,4 SHOUJI NISHINO,2,5 AND

EISHO NISHINO2
2
Faculty of Horticulture, Chiba University, Matsudo 648, Chiba 271-8510 Japan

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 popu-
lations 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.

Paddy fields are one of the dominant anthropogenic sources pots with paddy soils were compared in order to estimate the
of methane to the atmosphere (estimated as 15% of global effect of aquatic weeds on methane flux from paddy soils
methane emission; IPCC, 1994). The main passageway of throughout the period of rice growth. Microbial activities re-
methane emission from anaerobic paddy soils to the atmo- lated to methane production and oxidation in the soil with rice
sphere is through the arenchyma of the rice plant (Oryza sa- plants and typical aquatic weeds in paddy soils were also mea-
tiva) (Cicerone and Shetter, 1981; Inubushi et al., 1989; Nou- sured during crop season to examine possible mitigation op-
chi, Mariko, and Aoki, 1990). One possible mitigation option tions to reduce methane emission from paddy fields.
to reduce methane emission from paddy fields is water man-
agement, such as percolation, mid-summer drainage, and in- MATERIALS AND METHODS
termittent drainage (Inubushi, Muramatsu, and Umebayashi,
Soils and pot experiments—Sandy gley soil was taken from paddy fields
1992; Wassmann, Papen, and Rennenberg, 1993; Yagi, Tsu-
in Kuju-kuri, Chiba prefecture, Central Japan, and 3.5 kg of the moist sieved
ruta, and Minami, 1997). This would introduce oxygen into (,7.5 mm) soil was transferred into 18 plastic pots (0.02 m2 3 20 cm depth).
anaerobic soil, rendering it aerobic, and in so doing reduce the Total carbon, total nitrogen, cation exchange capacity (CEC), and pH(H2O)
formation of methane by enhancing methanotrophic (methane of the soil samples were quantified by standard soil analytical method (Brem-
oxidizing) bacteria in soil (Bosse and Frenzel, 1997; Gilbert ner and Mulvaney, 1982). Mixed chemical fertilizers were applied as basal
and Frenzel, 1998). Organic farming, which involves the use and were equivalent to 100 kg N/ha, 25 kg P/ha, and 42 kg K/ha. Chopped
of organic matter instead of chemical fertilizer and without rice straw (,1 cm length) was applied at 4000 kg/ha on 27 May 1997. Thirty-
herbicide application, has become popular in Japan (AFFRC, five-day-old rice seedlings (Oryza sativa, ‘Nihonbare’) were transplanted 2 d
2000). Both drainage and organic farming may enhance the after fertilizer application and soil submergence. A second fertilizer applica-
growth of weeds in paddy fields. However, the effect of aquat- tion was made as top dressing at the rate of 20 kg N/ha and 42 kg K/ha at
ic weeds in paddy fields on methane emission has not been maximum tillering stage on 25 July. All pots were maintained under flooded
properly evaluated yet. Therefore it is important to investigate conditions by being placed in a large plastic water pool until harvest on 25
whether such feedback would exacerbate methane emission by September. All weeds were manually removed weekly from nine pots (weeded
introducing more organic matter as weeds into the soil or mit- pots), while aquatic weeds, mainly Lipocarpha sp., Rotala indica, and Lud-
igate methane emission by methane oxidation. wigia epilobioides, all typical paddy weeds in Japan, were allowed to grow
In this paper, methane emission from weeded and unweeded 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
1
Manuscript received 7 March 2000; revision accepted 14 September 2000. monitored to see the effect of weeds on the anaerobic status in soil at 5 cm
The authors thank ex Prof. Yasuji Fukuda for botanical identification. This depth by Pt electrodes and a pH/Eh meter (Toa Electronics, RM-12P, 29-10-
work was supported by the Ministry of Agriculture, Fishery and Forestry, 1 Takadanobaba, Shinjuku-ku, Tokyo, Japan). Root, shoot, and the lower parts
Japan, Research Fund on Agroecological Technology for Controlling Factors of rice stems (below soil surface and above the root system) were separated
of Global Environmental Changes, and Research Institute of Innovative Tech- after removal from treatment pots, washed thoroughly, and weighed after dry-
nology for The Earth Fund on Excellent Research Project headed by Prof. ing at 808C for 24 h on 50, 82, and 111 d after transplanting (DAT). Similarly,
Emer. Iwao Watanabe.
corresponding parts of the aquatic weeds were also taken from the unweeded
2
Author for reprint requests (Tel 181-47-308-8816, Fax 181-47-308-8720,
e-mail inubushi@midori.h.chiba-u.ac.jp). pots on the same dates and treated in the same way as the rice plants.
3
Current address: Yoshitomi Pharmaceutical Industries Ltd., Yoshiki 3224-
3-508, Yamaguchi 753-0811 Japan. Methane emission—During rice growth, methane emission was measured
4
Current address: Kimitsu Agroforestry Highschool, Aoyagi 48, Kimitsu, almost weekly using the closed chamber method (Inubushi et al., 1989). A
Chiba, 292-0454 Japan. cylindrical acrylic chamber (15 cm diameter 3 1 m height) was placed on

975
976 AMERICAN JOURNAL OF BOTANY [Vol. 88

Fig. 1. Temporal changes in methane flux from weeded (SP) and un- Fig. 2. Temporal changes in soil redox potential (Eh) (5 cm) in weeded
weeded plots (SW). Bars indicate SD of the mean. (SP) and unweeded plots (SW). Bars indicate SD of the mean.

exchange capacity (CEC) 5 76.4 mmol/kg and pH(H2O) 5

the pot with rice for 30 min in the morning (between 1000 and 1200). The
6.36. Methane flux through rice plants in weeded pots peaked
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
around 30 DAT and was at a level similar to those in un-
a syringe, and the mixing ratio of methane in it was quantified by injecting weeded pots until 50 DAT (Fig. 1). However, methane flux
it into an FID-GC (gas chromatograph with frame ionized detector; Shimadzu increased rapidly to reach the maximum on 78 DAT in weeded
GC-7A, Nishinokyou-kuwahara, Nakagyouku, Kyoto, Japan). The emission pots, and this was almost three times higher than in unweeded
rate was calculated by taking into account the increase in the methane mixing plots. As a result, more than double the amount of methane
ratio, the volume of the chamber, and the temperature inside the chamber. was emitted during the crop season from weeded pots com-
pared with unweeded ones. Soil Eh was significantly lower in
Methanogenic bacteria and their activity in soil—When rice plants and weeded pots than unweeded pots after 40 DAT except 82 DAT
aquatic weeds were collected, soil samples were taken by truncated plastic (Fig. 2), indicating that more oxidized conditions were prob-
syringe to a depth of 1–5 cm from a spot between the rice hill and inner ably provided by aquatic weeds.
surface of the pot. In order to avoid exposing soil samples to the air, they
were immediately transferred into glass bottles and homogeneously mixed Plant growth and peroxidase activity of rice roots—Rice
with oxygen-free water. Subsamples of soil, equivalent to 10 g wet mass, plant height and tiller number were not significantly different
were either incubated anaerobically under N2 headspace at 308C in the dark between weeded and unweeded pots (Fig. 3). Dry matter mas-
in a closed flask to measure methanogenic activity (Chidthaisong, Inubushi, ses of shoots and roots of rice showed no significant difference
and Watanabe, 1996; Chidthaisong et al., 1996) or further diluted to estimate between the two treatments, except being higher in unweeded
populations of methanogenic bacteria by the most probable number method pots on 82 DAT, in which roots of weeds were present (Fig.
(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 (Wa-
tanabe 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 308C 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 TAt (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 308C 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
SYSTATt at 5% significance level using a t test for comparison of the treat-
ment means.

RESULTS
Fig. 3. Temporal changes in plant height (a) and tiller number (b) of rice
Methane flux from weeded and unweeded plots—The soil planted in weeded (SP) and unweeded plots (SW). Bars indicate SD of the
had total carbon 5 7.9 g/kg, total nitrogen 5 0.8 g/kg, cation mean.
June 2001] INUBUSHI ET AL.—EFFECT OF AQUATIC WEEDS ON CH4 EMISSION 977

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.

4). Similarly, the peroxidase activity of rice roots showed no Fig. 6. Populations of methanogenic bacteria (upper) and their activity
difference between these pots, although it decreased gradually (lower) in soil of weeded (SP) and unweeded plots (SW). Bars indicate SD
of the mean. DAT 5 days after transplanting, MPN 5 most probable number.
from 50 DAT to harvest (Fig. 5).

Methanogenic bacteria and methane production activity in Methane oxidation activity of roots of three aquatic
soil—Populations of methanogenic bacteria in soil was 4–7 weeds—Roots of Lipocarpha sp., Rotala indica, and Ludwigia
times higher in weeded pots than in unweeded ones (Fig. 6). epilobioides, typical aquatic weeds in paddy fields in central
Methane production activity was also higher in weeded pots Japan, showed higher methane oxidation activity than those of
than in unweeded ones on 82 and 111 DAT. rice plants. The methane oxidation activity was rated in the
order of Ludwigia . Lipocarpha . Rotala . rice (Fig. 8).
Methane oxidation activity of soil—Methane oxidation ac- On the other hand, lower parts of the stem of rice plants
tivity measured by the propylene oxidation technique was showed much higher methane oxidation activity than roots.
higher in unweeded pots than in weeded ones, particularly The other parts of the aquatic plants showed almost the same
during the later part of the croping season (Fig. 7). methane oxidation activity as the roots of each plant, and this

Fig. 5. Peroxidase activity (mg alpha-naphthylamine · g DM21 · h21) of Fig. 7. Methane oxidation activity in soil of weeded (SP) and unweeded
rice root in weeded (SP) and unweeded plots (SW). Bars indicate SD of the plots (SW). Bars indicate SD of the mean. PPO 5 Propylene oxide, DM 5
mean. dry matter.
978 AMERICAN JOURNAL OF BOTANY [Vol. 88

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 5 dry matter.

was similar or lower as compared with the same parts of rice garded as the difference between methane production and
plants. methane oxidation. Both activities in this study were measured
separately in soil and plant parts under laboratory conditions.
DISCUSSION Therefore, in situ activity in the soil–plant system should also
be examined more carefully in estimating methane oxidation.
The significance of rice plants in methane emission from Weeds did not reduce the growth of rice plants under these
paddy soils to the atmosphere has been recognized since Ci- experimental conditions (Fig. 3), even though the dry mass of
cerone and Shetter (1981). Inubushi et al. (1989) estimated weed shoots was ;14–19% of those of rice shoots (Fig. 4).
that .90 % of methane flux was through rice plants. Nouchi, However, these results should be examined in detail under var-
Mariko, and Aoki (1990) examined in detail methane passage ious conditions. Food production could be sustained and meth-
through rice plants. A similar movement of methane from soil ane emissions might be decreased by reducing the application
to the atmosphere was also reported for other hygrophytes of agrochemicals, such as herbicides and by maintaining or
(Wagatsuma et al., 1992; Frenzel and Rudolph, 1998). During increasing organic matter application to soil. Moreover, meth-
methane transport from soil to the atmosphere, 20–80% of ane emission from paddy soil can also be mitigated by water
methane was oxidized in rhizosphere soil with rice plants management, such as mid-season drainage and other options.
(Conrad and Rothfuss, 1991; Sass et al., 1991). In natural wet- The results in this study indicate that the role of aquatic weeds
lands, aquatic plants were also important in methane oxidation. in paddy soils in relation to methane emission should not be
King (1994) and Calhoun and King (1998) demonstrated overlooked in evaluating mitigation options to reducing meth-
methane oxidation by methanotrophic bacteria in the rhizo- ane emission from paddy fields.
sphere of aquatic plants. Gilbert and Frenzel (1995), Bosse
and Frenzel (1997), and Gilbert et al. (1998) found similar
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