Suppressive effect of rice bran incorporation in paddy soil on germination of Monochoria vaginalis and its relationship with electric conductivity

Abstract

Laboratory experiments were conducted to investigate (1) the effects of the addition of rice (Oryza sativa. L.) bran to paddy soil on the germination of Monochoria vaginalis (Burm. f.) Kunth, and (2) the relationship between the electric conductivity (EC) of the soil solution and germination. Soil samples were collected at 4 sites in Japan. After flooded soils with rice bran had been incubated for 7 d at 30°C, the soil solution was collected using a porous cup and the EC of the soil solution was measured. The amounts of rice bran added to the soil were 0%, 0.3%, 0.6% and 0.9% (weight(w)/w). In the soil solution obtained, seeds of M. vaginalis were incubated for 3 d at 30°C, and the germination percentage was then analyzed. The addition of rice bran suppressed germination, and the degree of suppression increased with increasing content of rice bran. Although the same amount of rice bran was applied to each soil, the degree of growth suppression by rice bran as well as the EC of the soil solution differed among the soils. In each soil, there was a positive correlation between the amount of rice bran and EC, and the degree of growth suppression significantly increased with an increase in EC. When EC was higher than 150 mS m⁻¹, seeds of M. vaginalis hardly germinated. There was no significant correlation between the oxidation-reduction potential (Eh) of soil and seed germination, suggesting that EC is a more reliable and convenient indicator than Eh for evaluating the relationship between the addition of organic material and seed germination. In conclusion, the addition of rice bran to soil increases the EC of the soil solution, and EC is one of the factors that suppress the germination of M. vaginalis. The suppressive effect of rice bran on germination is different among soils. This fact is attributed to the difference in EC due to the addition of rice bran. Thus, it is expected that EC can be used as an indicator for determining how much rice bran to add.

Keywords:

• electric conductivity (EC)
• Monochoria vaginalis
• paddy soil
• rice bran
• soil solution.

INTRODUCTION

Weed control via the application of rice (Oryza sativa. L.) bran to paddy fields in Japan has recently received a great deal of attention. While there have been very few reports published in English on weed control by organic materials in soil, many reports published in Japanese have shown that the application of rice bran has a certain effect on weed control. For example, Fukushima and Uchikawa (2002) showed that the application of 2 t ha⁻¹ of rice bran to soil enabled weed control without reducing rice yield. Although this method is expected to be useful in organic agriculture, its use has been limited because its effect fluctuates for unknown reasons, especially for the weed Monochoria vaginalis (Burm. f.) Kunth (Shimamune and Suzuki 2006). According to Nakai and Toritsuka (2009), M. vaginalis remained in the paddy field despite the application of 1.5 t ha⁻¹ of rice bran to the field. The application of organic material decreased the redox potential of flooded soil (Inubushi et al. 1984). It has been suggested that the predominance of M. vaginalis is associated with strong tolerance of this species to soil reduction (Nakai and Toritsuka 2009). However, the mechanisms of this reduction tolerance are not well understood.

Under field conditions, it is difficult to determine the oxidation-reduction potential (Eh) of soil because its value fluctuates according to soil conditions. Thus, we have embarked on the present study to evaluate an indicator other than Eh to investigate the effect of rice bran on the germination of M. vaginalis. In flooded soil, electric conductivity (EC) increases with decreasing soil Eh (Inubushi et al. 1984). Kawaguchi et al. (1997) reported that incubation of M. vaginalis with aqueous extracts of rice husk (10% weight(w)/volume(v)) promoted the germination of M. vaginalis. On the basis of an experiment using sodium chloride (NaCl) solution, however, an NaCl solution having an EC of 300 mS m⁻¹ suppressed germination in the light, while germination was not affected in the dark. Therefore, the promotive activity in the presence of aqueous extracts of rice husk was not due to any change in EC, although both the germination percentage and EC increased with the increase in the amount of extract. Muroi et al. (2005) reported that the EC of paddy water varied in the range of less than 50 mS m⁻¹ in a Japanese field and that this value of EC was too small to suppress the germination of paddy weeds. However, M. vaginalis usually germinates in the paddy soil (Chen and Kuo 1999), and the effect of the soil solution on germination has not been elucidated.

In this study, laboratory experiments were conducted to investigate: first, the effects of the addition of rice bran to paddy soil on the germination of M. vaginalis, and second, the relationship between the EC of the soil solution and the percentage of germination.

MATERIALS AND METHODS

Seeds

Seeds of M. vaginalis were collected from plants grown in concrete pots (50 cm × 50 cm × 15 cm height) at the National Agricultural Research Center (NARC) for Tohoku Region, Daisen City, Japan (39°29′N, 140°29′E). Collected seeds were air-dried and stored in a refrigerator at 5°C for more than six months, and were then stored in tap water for more than two months in a refrigerator at 5°C.

Soil and rice bran

Soil samples were collected from the plow layer of a paddy field at 4 sites: NARC for Hokkaido Region, Sapporo City, Japan (43°00′N, 141°24′E) (soil A); NARC at Yawara experimental paddy field, Tsukubamirai City, Japan (36°00′N, 140°01′E) (soil B); Mie Prefecture Agricultural Research Institute, Iga City, Japan (34°42′N, 136°08′E) (soil C), and Miyagi Prefectural Furukawa Agricultural Experiment Station, Osaki City, Japan (38°35′N, 140°54′E) (soil D). The soil samples were air-dried and passed through a 2-mm mesh sieve. The values of pH [soil:water (w/v) = 1:2.5] (Thomas 1996), total carbon (C) and total nitrogen (N) were determined using a CN analyzer (varioMAX CNS; Elementar, Hanau, Germany) and are shown in Table 1. Commercial rice bran was passed through a 2-mm mesh sieve. Total N and total C determined using the same CN analyzer were 36.4 (g kg⁻¹) and 376.4 (g kg⁻¹), respectively, and the C:N ratio was 10.3.

Table 1. Soil description. N = nitrogen; C = carbon

Name	Soil type	Soil property	pH (H2O)	Total N (g kg^−1)	Total C (g kg^−1)
A	black volcanic	loam	6.2	4.9	64.9
B	brown volcanic	clay loam	6.0	1.8	25.2
C	alluvial	loam	5.0	1.2	14.5
D	alluvial	loam	5.2	2.1	22.7

Germination in soil solution

The apparatus used for collecting soil solution is shown in Fig. 1. One hundred grams of soil with rice bran was put into a polyethylene vessel (60 mm × 60 mm × 85 mm in height). The ratios of rice bran to soil were 0%, 0.3%, 0.6% and 0.9% (w/w). As mentioned above, there were 4 soil treatments, and each treatment had 5 replications. So, 80 vessels were used in the experiment (4 soil types × 4 rice-bran ratios × 5 replications). Distilled water was added to the vessel, followed by thorough stirring to remove the air in the soil. The depth of flooded water was maintained at 3–4 cm throughout the incubation. The vessels were incubated at 30°C under a 12-h light/12-h dark condition. A small hole was made in the side of each vessel 2 cm below the soil surface. A porous cup (3 mm in diameter and 9 cm in length) was inserted into the hole and set horizontally to the soil surface. The space between the vessel and the porous cup was sealed with adhesive. The porous cup was connected to a flexible plastic tube, and the soil solution obtained was introduced into a 10-mL evacuated test tube. After a 7-d period of incubation, the soil solution was collected. The tube was opened and the EC of the soil solution was measured using a compact EC meter (B-173; HORIBA, Kyoto, Japan). Thirty seeds of M. vaginalis were put into the soil solution in the tube, and the tube was sealed with a flexible film. The test tubes were incubated at 30°C under a 12-h light/12-h dark condition. After a 3-d period of incubation, the germination percentage was determined. In each treatment, the germination percentage was calculated on the basis of the presence or absence of germination in 30 seeds. The values of germination percentage and EC were expressed as the means of 5 replications.

Figure 1. Apparatus used for collecting the soil solution.

Changes in Eh

One hundred grams of soil A with rice bran was put into the same vessel as described above. The ratios of rice bran to soil were 0%, 0.3%, 0.6% and 0.9% (w/w) with 5 replications. Twenty vessels were used in the experiment (1 soil type × 4 rice-bran ratios × 5 replications). Distilled water was added to the vessel, followed by thorough stirring. To measure Eh, a glass electrode (9300-10D, HORIBA, Kyoto, Japan) and an ORP (oxidation-reduction potential) meter (D-54, HORIBA, Kyoto, Japan) were used. The electrode was inserted vertically into the soil, and the tip of the electrode was set at 3 cm below the soil surface. The vessel was left open and allowed to stand for 21 d at 30°C under a 12-h light/12-h dark condition. The depth of flooded water was maintained at 3–4 cm throughout the incubation. The values of Eh were measured every 2, 3 or 4-d for the 21-d period of incubation. The number of replications was 5.

Statistical analysis

The statistical software package SPSS16.0 J for Windows (SPSS Inc., Tokyo, Japan) was used for analysis of covariance (ANCOVA).

RESULTS AND DISCUSSION

Increase in EC with increase in rice bran

In this experiment, 0%, 0.3%, 0.6% or 0.9% rice bran was added to the soil. In Japanese paddy fields, the bulk density of soil is around 1 Mg m⁻³ (Shirato 2005). The width of the vessel used in this study was 0.36 cm² (60 mm × 60 mm). If the bulk density of soil is 1.00 Mg m⁻³, the soil depth in the vessel would be 2.7 cm. Based on this value of bulk density, the weight of a field with a plow layer of 2.7 cm in depth is calculated to be 270 t ha⁻¹. In this field, the application of 0.3%, 0.6% and 0.9% of rice bran to soil corresponds to 0.8, 1.6 and 2.4 t ha⁻¹, respectively. Fukushima and Uchikawa (2002) and Nakai and Toritsuka (2009) conducted field experiments with the application of at most 2.0 and 1.5 t ha⁻¹ of rice bran, respectively. Thus, the amounts of rice bran used in this experiment are comparable to those in their reports.

Tanji et al. (2003) reported that the EC of the soil solution under field conditions varied from plot to plot as well as within a plot, ranging from 60 to 360 mS m⁻¹. The lowest value was recorded with a straw-burned treatment with no winter flooding, and the highest was with a straw-returned treatment with winter flooding. In this study, the addition of rice bran increased the EC values significantly, with the maximum value of EC being 200 mS m⁻¹ (Fig. 2). Further work is needed to clarify the relationship between the amount of rice bran and the value of EC under field conditions.

Figure 2. Relationship between the germination percentage and the addition of rice bran. The broken lines indicate the regression lines of electric conductivity (EC). **: Significant at 1% level. Vertical bars indicate standard deviation (n = 5).

In this report, the EC values ranked in descending order were D > C > B > A (Fig. 2), while the pH values of the soil were A > B > D > C (Table 1). Sahrawat and Narteh (2002) reported that paddy soils with higher content of both organic C and reducible iron (Fe) had a higher EC of soil solution. During the soil reduction, insoluble Fe(III) oxides in soil will accept an electron from organic matter, and are reduced to soluble Fe²⁺ (Inubushi et al. 1984; Ponnamperuma 1985). A part of the organic matter changes to organic acids with the donation of these electrons. Thus, one of the factors that caused an increase in EC might have been an increase in the organic acids and Fe²⁺ contents of the soil solution. The amount of Fe²⁺ in soil solution tends to be high when the pH of the soil is low, because Fe²⁺ in soil solution precipitates as various oxides and hydroxides at high pH (Nozoe et al. 2008). The findings obtained in this study suggest that soil pH is one of factors responsible for EC.

Differences in the slopes shown in Fig. 2 were tested by ANCOVA using the test of the parallelism of regression lines. This test did not reject the null hypothesis that the slopes are the same (F = 11.10, P < 0.001), suggesting that the degree of increase in EC due to the addition of rice bran was different according to the soil. The inclination of soil A was the smallest (Fig. 2). This fact suggests that the rice bran in soil A decomposed more slowly than that in the others. During the reduction of paddy soil, organic matter in soil donates electrons to nitrate, manganese (Mn) (IV), Fe(III) and sulfate sequentially (Yao et al. 1999). The ratio of these electron acceptors to donors affects the decomposition of organic materials added to the soil. Although the amounts of electron acceptors were not determined in this report, the ratio of the electron acceptors to donors (rice bran) in soil A might have been greater than in the others.

Suppressive effect of soil solution on germination

Xuan et al. (2003) showed that the incorporation of by-products of rice (0.5 t ha⁻¹) in the paddy field suppressed the emergence of M. vaginalis. Under laboratory conditions, we confirmed that the addition of rice bran suppressed germination in soil solution, and the degree of suppression increased as the content of rice bran increased (Fig. 2). Although the same amount of rice bran was applied to each soil, growth suppression due to the addition of rice bran occurred in the order of soil D > C > B > A. An increase in the amount of rice bran increased the EC of the soil solution and decreased the germination percentage. To analyze the relationship between EC and germination percentages, the data shown in Fig. 2 were re-plotted in Fig. 3. When the EC was higher than 150 mS m⁻¹, there was little germination of seeds. This finding indicates that the amount of rice bran that enables effective control of M. vaginalis can be determined on the basis of EC with values higher than 150 mS m⁻¹. The amount of rice bran is not a useful index by which to judge whether the weed control is effective because the germination percentage changes according to the soil (Fig. 2). On the contrary, the value of EC can be used as a good indicator because it is well correlated with the germination percentages (Fig. 3).

Figure 3. Relationship between the germination percentage and electric conductivity (EC) of soil solution.

Kawaguchi et al. (1997) reported that incubation of M. vaginalis with aqueous extracts of rice husk promoted the germination of M. vaginalis from 4% in the water-only control to at most 62% in the presence of extracts. In the present study, however, the germination percentages of soils A and B in the absence of rice bran were about 80% (Fig. 2) suggesting that the seed had a high ability of germination. Therefore, the promoting effect of rice bran was not elucidated. The seeds were stored in cool water for more than two months prior to the experiments. This procedure presumably broke seed dormancy and might have increased the germination percentage.

As stated above, the important factors that caused EC to increase were increase in the organic acids and Fe²⁺ content in the soil solution. A high concentration of Fe²⁺ in soil solution sometimes suppresses the growth of paddy rice (Nozoe et al. 2008). In our previous papers, we suggested that the accumulation of Fe²⁺ in soil solution was one of the factors related to the growth suppression of paddy weeds (Nozoe et al. 2009; Nozoe et al. 2010). However, Fe²⁺ is not the main factor that suppresses the germination of M. vaginalis because this species has a strong tolerance for iron toxicity (Nozoe et al. 2009). In the process of soil reduction, some phenolic acids that suppressed the root elongation of rice were produced (Tanaka et al. 1990). However, Olofsdotter et al. (2002) reported that these acids were unlikely to be the main toxins in weeds because their concentrations were not of an inhibitory level. Although further work on organic acids is in progress, few have dealt with the relationship between the organic acids of paddy soils and weed growth. It remains to be determined what mechanisms are involved in the suppression of weed growth.

Fukushima and Uchikawa (2002) suggest that the decrease in soil Eh, which was attributed to the application of rice bran, suppressed the germination of M. vaginalis. In the current study, the addition of 0.3%, 0.6% and 0.9% rice bran to flooded soil A decreased the Eh of soil (Fig. 4). However, there was no significant difference between the values of Eh in the presence of 0.3%, 0.6% and 0.9% rice bran. Thus, the correlation between the seed germination in soil A (Fig. 2) and Eh (Fig. 4) was not significant.

Figure 4. Changes in the values of oxidation-reduction potential (Eh) of soil A. Vertical bars indicate standard deviation (n = 5).

In this study, the addition of rice bran to soil suppressed the germination of M. vaginalis. In relation to the instability of the weed control effect of rice bran, the results obtained in this study suggest that one of the factors associated with this instability is the difference in the EC of the soil solution. It has been reported that the application of an excess of rice bran suppresses the growth of rice (Nakai and Toritsuka 2009). When they used a young seedling, the yields reduced to 85%, 65% and 60% in the presence of 0.6, 1.0 and 1.5 t ha⁻¹ of rice bran, respectively. On the contrary, when a middle-aged seedling was used, the yields increased to 102%, 101%, and 112%, respectively. These facts indicate that, first, the presence of rice bran suppresses the growth of rice seedlings as well as that of M. vaginalis, and, second, the young seedling is more susceptible to suppression than the middle-aged seedling. For effective weed control via the application of rice bran, it is important to keep the EC of the soil solution higher than 150 mS m⁻¹. Although the value of EC increases with the increase in the amount of rice bran, it may suppress the growth of rice seedlings. Therefore, we should reduce this risk by using middle-aged seedlings.

In conclusion, the addition of rice bran to soil increases the EC of the soil solution, and EC is one of the factors that suppress the germination of M. vaginalis. The suppressive effect of rice bran on germination differs among soils. This fact is attributed to the difference in the EC of the soil resulting from the addition of rice bran. Actually, under field conditions, it is difficult to determine Eh of soil because its values fluctuate according to soil conditions. This fact and findings obtained in this study suggest that EC is a more reliable and convenient indicator than Eh for evaluating the relationship between the addition of organic material and seed germination. This information will be useful in determining the effective amount of rice bran to add to each soil without causing a reduction in rice yield.