Allelopathy Journal 21 (2): 389-395 (2008) Tables: 1, Figs : 2
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Allelopathic potential of barnyard grass on rice and soil microbes in paddy 1
Y. GU1,2, H.B. LI1,2 and C. H. KONG1* Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China 2 Graduate School, Chinese Academy of Sciences, Beijing 100039, China E.mail:
[email protected] (Received in revised form: January 9, 2008)
ABSTRACT Allelopathic potentials of barnyard grass (Echinochloa crus-galli) on rice (Oryza sativa) and soil microbes in paddy were investigated. In the barnyard grass infested soil, the growth of non-allelopathic rice variety Liaojing-9 was considerably reduced than allelopathic rice variety PI 312777. Further experiments showed that barnyard grass germinating seeds exudates and their one component phydroxymandelic acid suppressed the germination and growth of rice. The inhibitory effects depended on concentrations, the maximum inhibition occurred at 2000 barnyard grass seeds/ 10 ml water per dish (barnyard grass germinating seeds exudates) and 500 μg/g soil of p-hydroxymandelic acid. Similarly, soil microbial populations in paddy soil varied with concentrations of barnyard grass germinating seeds exudates and p-hydroxymandelic acid. Actinomycetes and fungi were less sensitive than bacteria to the exogenous application of barnyard grass germinating seeds exudates, but fungi were more sensitive to p-hydroxymandelic acid. These results suggested that allelopathic interference could occur from barnyard grass on rice growth and soil microbial population in paddy ecosystem. Key words: Allelopathy, Echinochloa crus-galli L, germinating seeds exudates, phydroxymandelic acid, Oryza sativa L, soil microbe.
INTRODUCTION Barnyard grass (Echinochloa crus-galli) is a major weed in paddy fields and drastically reduces the rice (Oryza sativa) yields (12). The competition from 25 barnyard grass plants/m2, reduced the rice yields by 50% (3). Besides competition, it has long been suspected of using allelopathy to interfere with the growth and establishment of rice in paddy ecosystems. Yamamoto et al. (18) identified p-hydroxymandelic acid, a growthinhibiting allelochemical releasing from the germinating barnyard grass roots and it inhibited the root elongation and shoot growth of rice. Xuan et al. (16) suggested that barnyard grass released toxic compounds during germination, which strongly inhibited the germination, root length and shoot length of rice, lettuce and monochoria (16). These * Correspondence author
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studies indicated that barnyard grass could inhibit the growth of rice through releasing allelochemicals, but these experiments were done without the soil. Actually, the allelochemicals have to be available to exert an allelopathic effect in soil under natural condition (10, 11). Furthermore, many kinds of allelochemicals may be modulated by soil microbes (1). Therefore, alleochemicals and mechanism involved in barnyard grass against rice are largely unknown. The objectives of this study were (a) to evaluate inhibitory effects of the barnyard grass infested soils on rice growth and (b) to assess the effects of barnyard grass germinating seeds exudates and their one component (p-hydroxymandelic acid) on rice growth and soil microbial populations, which provide evidence of barnyard grass interference with rice via allelopathy. .
MATERIALS AND METHODS Barnyard grass seeds and soils were collected from the Experimental Station of Ecology, Chinese Academy of Sciences, Shenyang (Northeast China, N 41º31´, E 123º24´). Soil was Albic luvisols with pH of 5.75±0.28, organic matter content of 1.31±0.22% and medium fertility, [total N 0.97±0.12 g kg-1; available N 108.64±19.12 mg kg-1; total P 0.41±0.09 g kg-1; available P 29.76±9.21 mg kg-1; total K 0.81±0.17 g.kg-1; available K 77.85±12.43 mg kg-1]. Soils were air-dried & sieved (2 mm mesh) to remove plant tissues. A putative allelopathic rice variety PI 312777 and another non-allelopathic rice variety Liaojing-9 were selected from USDA-ARS rice germplasm collection (4) and popular commercial cultivars in Northeast China, respectively. The p-hydroxymandelic acid was purchased from Sigma Co. (USA). All seeds of barnyard grass or rice were sterilized with 1% sodium hypochlorite for 30 min. The dormancy of barnyard grass seeds was broken before tests with 1N HNO3, then seeds were rinsed many times with water. Bioassays The first experiment was done to determine whether barnyard grass infested soil caused phytotoxicity to the emergence and growth of rice. Pre-germinated seeds of barnyard grass (10,50,100,200 seeds/pot) were uniformly sown in the pots (6.5 cm × 8.0 cm) containing 100 g soils described above. The pots without barnyard grass served as control. At the 3rd leaf stage, barnyard grasses were removed and then 5 pre-germinated rice seeds were sown in these pots. Rice shoots were clipped at the point of first root, dried for 48 h at 800C, and shoot dry weights of rice were determined at the 3rd leaf stage (12). The second experiment was conducted to determine the effect of barnyard grass germinating seeds exudates and p-hydroxymandelic acid on rice emergence and growth. Twenty rice seeds were sown into each of pots containing soils mentioned above. The following two manipulations were conducted: (i) the exudates of germinating barnyard grass seeds were applied to the pots. The exudates were obtained from the sterilized barnyard grass seeds incubated in a Petri dish (9 cm dia) with sterilized water in the dark at 25±10C for 5 days (100, 500, 1000, 2000 seeds/10 ml water per dish). (ii) P-hydroxymandelic acid with different concentrations (25, 50, 100, 300, 500 μg/g soil) was added to each treated pots. Control pot received only water. The emergence (%) of rice was recorded after 7 days and shoot dry weights at the 3rd leaf stage, respectively.
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All pots were placed in a controlled environmental chamber (3 m3) with a 12 h day length and approximately 350 μmol/m2/s light intensity, 25-280C temperature and 70% relative humidity. The pots were watered and randomized once a day. The same manipulation was done three times for each determination under identical conditions. Inhibition (%) was obtained from the comparison of rice dry weights or emergence percentages between the treated and control pots, which was calculated by (1Treatment/control) ×%. Microbiological analysis The effects of barnyard grass germinating seeds exudates and p-hydroxymandelic acid on soil microbial numbers were studied with the soil dilution plate method (5). 50 g soils were placed in pots (6.5 cm × 8.0 cm) and then barnyard grass germinating seeds exudates mentioned above and p-hydroxymandelic acid with different concentrations (50, 100, 300, 500 μg/g soil) was added to treated pots, respectively. The control pots received water only. All pots were placed in dark environment growth chamber (1 m3) with 25 or 30 0C and 70% humidity until 5 days. 10 g soils were used to analyze the microbial communities (bacteria, actinomycetes and fungi). Nutrient agar medium for bacteria was beef extract-peptone medium and for actinomycetes was Gause-1 medium and for fungi was Martin’s medium containing rose Bengal 33 μg/ml and streptomycin 30μg/ml (19). All colonies we counted within 7 days and expressed as colony forming units (cfu) g-1 dry soil.
RESULTS AND DISCUSSION Rice growth was significantly inhibited in the barnyard grass infested soils, indicating that this soil was toxic to rice growth (Fig. 1). Furthermore, the growth of nonallelopathic rice variety Liaojing-9 was drastically reduced than allelopathic rice variety PI 312777. There was significant reduction of rice growth in the soils infested with high density of barnyard grass (200 seeds/100 g soil per pot), indicating that the growth reduction in rice was correlated with density of barnyard grass in soils. It is well known that barnyard grass interferes with the growth of rice through resources competition (9). This study showed that rice growth was inhibited also in the soils from which barnyard grass was removed and phytotoxins appears to be present in the barnyard grass infested soils. The presence of these phytotoxins in the barnyard grass infested soils may be involved in the inhibition of rice growth. Barnyard grass may exude allelochemicals that suppresses rice as well as other plants species growth in fields (14). Several compounds inhibiting the rice germination and growth are identified from germinating barnyard grass exudates (16,17,18). However, these experiments were done without soil media. It was important to design bioassays with soil to simulate field conditions to obtain growth inhibition of ecological relevance (6,7,8). In this study, the effects of barnyard grass germinating seeds exudates and their one component p-hydroxymandelic acid (different concentrations) on the emergence and growth of rice seedlings were determined. The emergence and dry weight of rice was inhibited by barnyard grass germinating seeds exudates and p- hydroxymandelic acid. There were significant differences in the shoot dry weight and emergence between
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Inhibition (%)
Liaojing-9
PI312777
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20
10
0 10
50
100
200
Density of barnyard grass (seeds/100 g soil per pot)
Figure 1. Inhibitory effects of barnyard grass infested soils on rice growth. The data are presented as means± SE from three independent experiments for each determination.
allelopathic rice PI 312777 (mean 16.9% and 10.8% inhibition) and non-allelopathic rice Liaojing-9 (mean 28.9% and 17.8% inhibition). Particularly, the dry weight and emergence of allelopathic rice PI 312777 was not significantly reduced as compared to the non-allelopathic rice Liaojing-9, (62.5% inhibition) at 2000 seeds/10 ml water per dish barnyard grass germinating seeds exudates. A concentration-dependent growth response was observed for p-hydroxymandelic acid, whose inhibitory effects increased with increasing concentrations (Table 1). However, no obvious differences were observed in the shoot dry weight and emergence between allelopathic rice PI 312777 (13.5% and 12.1% inhibition) and non-allelopathic rice Liaojing-9 (15.4% and 14.3% inhibition, respectively). The response of soil microbial population to barnyard grass germinating seeds exudates and p-hydroxymandelic acid was measured. The soil microbial populations varied with concentrations of barnyard grass germinating seeds exudates and phydroxymandelic acid (Fig. 2). The total soil microbial population was increased after applying p-hydroxymandelic acid and barnyard grass germinating seeds exudates within certain concentration. Bacteria showed the highest stimulation from application of p-hydroxymandelic acid (100 μg/g soil) and barnyard grass germinating seeds exudates (1000 seeds/10 ml water per dish), which grew to 11.9×108 and 10.5×108 cfu g-1 dry soil, respectively. However, bacterial population decreased in soils with higher rates of
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Table 1. Effects of barnyard grass germinating seeds exudates and p-hydroxymandelic acid on the emergence and growth of rice Concentrations
Inhibition (%)
Emergence PI312777
Liaojing-9 PI312777 Germinating seeds exudates (seeds/pot)
100 500 1000 2000 Mean
0.9±1.7a 5.0±3.6a 11.1±7.2b 26.1±1.8c 10.8
25 50 100 300 500 Mean
0.1±6.4a 1.6±2.7a 7.4±3.6a 21.1±0.8b 30.5±0.1b 12.1
1.4±2.2a 0.6±7.0a 9.8±1.4a 5.9±1.8a 26.4±1.7b 22.8±0.4b 33.5±5.4b 38.4±3.2c 17.8 16.9 p-Hydroxymandelic acid (μg/g soil) 0.7±7.6a 2.8±3.9a 12.5±8.2b 23.7±0.4c 31.9±2.4c 14.3
0.8±5.1a 1.8±2.6a 9.5±0.8a 24.0±6.4b 31.4±3.9b 13.5
Shoot dry weight Liaojing-9 10.7±1.9b 11.5±4.6b 30.7±0.9c 62.5±1.8d 28.9 1.2±4.0a 4.6±3.4a 12.7±1.3b 25.2±1.6c 33.2±5.4c 15.4
Means ±SE from three independent experiments for each determination are shown. Data in a column followed by the same letter are not significantly different at P=0.05, ANOVA with Duncan’s multiple-range test.
p-hydroxymandelic acid (500 μg/g soil) and barnyard grass germinating seeds exudates (2000 seeds/10 ml water per dish). Actinomycetes and fungi were less sensitive than bacteria, to the exogenously applied barnyard grass germinating seeds exudates. However, fungi were more sensitive to p-hydroxymandelic acid. The counts in the soil sample (50 μg/g soil) indicated that fungi population significantly decreased with concentration increase from 5.1×104 cfu g-1 dry in control to 1.2×104 cfu g-1 dry in 500 μg/g soil. The exogenous application of barnyard grass germinating seeds exudates and phydroxymandelic acid may stimulate the soil microbial populations within certain concentration and some soil microbial species may utilize the allelochemicals in the exudates as organic carbon (2,13,15). There are various chemicals interactions between the barnyard grass and rice (10,11). This study suggested that allelopathic interference may occur in barnyard grass against rice growth and soil microbial populations in paddy ecosystem, but key allelochemicals remained obscure. Besides inhibitory allelochemicals, there are potent growth-promoting substances such as a lepidmoide in the exudates of germinating barnyard grass (17,18). Allelochemicals involved in barnyard grass against rice growth are still a controversial issue (16). It is necessary to determine the allelochemicals and mechanism of barnyard grass interference with rice and soil microbes in paddy.
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B
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Numbers -1 (CFU g dry soil)
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(×106)
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(×108)
Actinomycetes
(×104)
Bacteria
(×106)
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Actinomycetes
Bacteria
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Barnyard grass germinating seeds exudates p- Hydroxymandelic acid
Figure 2. Effects of barnyard grass germinating seeds exudates and p-hydroxymandelic acid on soil microbial populations. Exudates concentrations: A: 100 seeds/10 ml water per dish, B: 500 seeds/10 ml water per dish, C: 1000 seeds/10 ml water per dish and D: 2000 seeds/10 ml water per dish. p-hydroxymandelic acid concentrations: A: 50 μg /g soil, B: 100 μg /g soil, C: 300 μg /g soil and D: 500 μg /g soil. The vertical lines at the top of bars show ± SE
ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of China (NSFC No.30430460) and the Eleventh Five-year Plan of Science & Tech Program of China (2006BAD08A09).
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