Allelopathy is defined as chemical interaction(s) between plants that interfere with plant growth, mediated by the release of plant-produced bioactive secondary metabolites, referred to allelochemicals [1
]. Allelochemical production represents a form of chemical warfare between plants competing for light, water, nutrients and other resources [2
]. Numerous allelochemicals derived from plants have been described to stimulate and/or inhibit other species’ germination and growth. Intense scientific efforts have focused in the past on describing the mechanisms used by plants to self-regulate their own and other species’ densities and distribution or those found at the basis of biological invasions via allelopathic interactions [4
]. Allelopathic crop varieties can release their own “phytotoxins” as allelochemicals to reduce the growth of weeds, thus permitting ecological weed management in cropping systems [6
Rice (Oryza sativa
L.) is the major food crop in Asia, providing daily sustenance to half of the global population [8
]. However, under the current pressures of rapid global population growth and adverse climate change, increased attention should be directed towards new approaches in improving both crop quality and yield for sustainable rice production. For more than four decades, substantial research effort has been invested in studying rice allelopathy, finding that allelopathic activity of rice is variety and origin dependent. For example, Japonica rice shows stronger allelopathic properties than Indica or Japonica–Indica hybrid [9
]. Numerous growth inhibitor allelochemicals belonging to the group of phenolic acids, fatty acids, diterpenoids, momilactones and others have been reported [10
In nature, flowering plants in general, rice included, are faced with a plethora of antagonists, and have evolved myriad defense mechanisms by which they are able to deal with various kind of abiotic and biotic stresses [13
]. Therefore, the level of allelopathic activity and allelochemical concentration plants release into the environment, including root exudates, are species specific and dependent on biotic and abiotic stress factors [1
]. In particular, climate change is expected to adversely affect allelochemical production, and has aroused considerable interest in this area of research in recent years [15
]. Previous studies have reported that in plants, produced allelochemicals, such as phenolic acids or other compounds upon biotic or abiotic stress exposure, such as cold, heat, submergence, and others are produced for protection and survival of the species under field conditions [12
Although the numerous studies focusing on rice allelopathy mechanisms and their agricultural relevance, the allelopathic responses of rice under the specific abiotic stresses are poorly understood. Therefore, the objectives of this study were to assess the allelopathic responses of the extracts and root exudates of rice seedlings under complete submergence and temperature stress. The results of this study provide useful information for better understanding the allelopathic responses of rice seedlings under multiple stresses, expected to occur concurrently in a changing climate.
2. Materials and Methods
2.1. Rice Seeds and Indicator Plants
Seeds of two rice varieties, Koshihikari (Japonica) and Jasmine (Indica) were kindly provided by the Laboratory of Plant Physiology and Biochemistry, Graduate School for International Development and Cooperation (IDEC), Hiroshima University, Japan. Seeds of lettuce (Lactuca sativa
L.) and radish (Raphanus sativus
L.) were used as indicator plants because they are sensitive to allelochemicals at low concentrations [20
]. Barnyardgrass (Echinochloa crus-galli
L.) seeds were provided by Department of Genetic Engineering, Agricultural Genetics Institute, Vietnam. The germination rate of indicator plants was randomly tested in distilled water before conducting experiment and showed >95%.
2.2. Paddy Soils Preparation
The paddy soils (pH: 6.3, total C: 2.22%, total N: 0.18%, CEC: 8.8 meq per 100 g soil; CaO: 91, MgO: 13, K2O: 17, K2O5: 18, SiO2: 25 mg per 100 g soil) were collected randomly from the experimental farm of Hiroshima University, Hiroshima city, Japan, where early-matured rice (Koshihikari variety) had been grown. The soils were collected to a depth of 10 cm in summer 2015. After that, the soils were dried and mixed until treatment was conducted.
2.3. Phenolic Standards and Reagents
The standard compounds for validating the phenolic compounds, including gallic acid, protocatechuic acid, catechol, chlorogenic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, vanillin, ferulic acid, sinapic acid, p-coumaric acid, benzoic acid, ellagic acid, and cinnamic acid, were purchased from Kanto Chemical Co., Inc., Tokyo, Japan.
2.4. Other Chemicals
The collected extracts and root exudates of rice seedlings were diluted by 1.0% Dimethyl sulfoxide (DMSO) in distilled water. Before carrying out experiments, the treatments using DMSO 1.0% were applied to evaluate the effect of DMSO 1.0% on the germination and growth of indicator plants and natural weeds. The control treatment consisted of distilled water only. The results showed that DMSO 1.0% had no effect on indicator plants and natural weeds to compare with the controls.
2.5. Extracts and Root Exudates of Rice Seedlings under the Stress Conditions
The seeds of two rice varieties (Koshihikari and Jasmine) were germinated in seed beds filled with moist sterilized soil for 7 days. After that, the seedlings of each variety were placed into a test tube (five individuals of each variety per tube) and were treated in complete submergence [21
] and placed in dark conditions at the different specific temperatures (10, 25, 32, 37 °C) for 7 days. After that, the rice seedlings and water in each tube were collected to obtain extracts and root exudates for further experiments. A total of eight treatments with six replications were implemented in this study. All the treatments are shown in Table 1
The extracts were obtained by grinding fresh rice seedlings and stirring in 80% MeOH + 0.1% HCl (20 mL) for 24 h at room temperature. After that, the extracts were centrifuged at 10,000 rpm for 10 min at 10 °C, followed by filtration. The combined supernatants were dried under vacuum in a rotary evaporator (SB-350-EYALA, Tokyo, Japan) at 35 °C. The obtained dried extracts were dissolved in 1.0% dimethyl sulfoxide (DMSO) with the concentration of 10 mg/mL. Finally, they were diluted with distilled water to adjust the concentration to 1 mg/mL for allelopathic evaluation against the indicator plants and paddy weeds.
The root exudates of rice seedlings were attained from the water of all the treatments, which were then extracted by using 70% EtOH (200 mL) for 10 min at room temperature, followed by filtration. Subsequently, the solvent was removed using a rotary evaporator (SB-350-EYALA, Tokyo, Japan) at 40 °C. The root exudates were dissolved in 1.0% DMSO to 10 mL and kept at 4 °C for further experiments. After that, they were diluted by 50% using distilled water to evaluate the allelopathic activity against the indicator plants and paddy weeds.
2.6. Allelopathic Response of the Extracts and Root Exudates from Rice Seedlings
2.6.1. Evaluating Allelopathic Response of the Extracts and Root Exudates in Laboratory Condition
Allelopathic effects on the germination and growth of indicator plants, lettuce, radish and barnyargrass in the laboratory condition were evaluated. The seeds of indicator plants were rinsed several times with distilled water. Subsequently, they were surface-sterilized by immersion in 0.1% sodium hypochlorite (NaOCl) for 30 min. Finally, they were continuously rinsed several times with distilled water. Ten seeds of each type of indicator plants were placed in a sterilized beaker, then was added by 3 mL (1 mg/mL) of solution of rice seedling extracts. Similarly, another treatment was added by 3 mL of solution (50%) of root exudates, respectively. All the treatments were placed in a growth chamber for a 16 h photoperiod at 28/25 °C day/night temperature. After 7 days, the germination rate (GR), survival rate (SR), shoot height (SH), root length (RL) and dry weight (DW) of the indicator plants were recorded. The control treatment was used with distilled water only. The inhibition or stimulation percentages of the treatments were calculated over the controls.
To assess allelopathic responses on the emergence and growth of natural weeds in paddy soils, one hundred grams of paddy soils was placed in Petri dishes (diameter: 9 cm) and saturated with distilled water. After that, 3 mL solution (1 mg/mL) of the rice seedlings-extracts were added. Similarly, 3 mL solution (50%) of root exudates-extracts were directly added to the paddy soils as the separate treatment. The controlled treatments were used with distilled water only. All experiments were conducted with at least 3 replications. All Petri dishes were placed in a growth chamber with 16 h photoperiod at 28/25 °C day/night temperature. After 20 days, the number of weeds and the weeds’ DW were determined. The inhibition or stimulation percentages of the treatments were calculated.
2.6.2. Evaluating Allelopathic Responses of the Extracts and Root Exudates against the Growth of Barnyardgrass in Greenhouse
Ten healthy seeds of barnyardgrass were germinated in seed trays, which were filled with sterilized soils as described above. After 14 days of growth, 3 mL solution (1 mg/mL) of the extracts and 3 mL solution (50%) of the root exudates were directly and separately sprayed onto the barnyardgrass leaves, stems and soil surface of each treatment. The control treatments were performed with distilled water only. After 1 week, the SH (cm), RL (cm) and DW (g) of the plants were recorded. The inhibition or stimulation percentages of the treatments were determined over the controls.
2.7. Determination of Total Phenolic and Flavonoid Contents
The total phenolic contents of the extracts and root exudates were determined by using the Folin-Cicalteau method of Medini et al. [22
]. The solutions were mixed, including 0.125 mL of the extracts or root exudates, 0.5 mL of distilled water and 0.125 mL of 10% Folin-Ciocalteu reagent (in distilled water). After 6 min, an amount of 1.25 mL of 7.5% aqueous Na2
solution (in distilled water) was added. Subsequently, distilled water was added for adjustment to final volume (3 mL). The absorbance was recorded at 760 nm by using spectrophotometer after incubation for 30 min at room temperature. Finally, the milligram of gallic acid equivalent (GAE)/mL was identified as an expression of total phenolic content.
The total flavonoid contents were determined using the method described by Bueno-Costa et al. [23
], with some modifications. The extracts or root exudates (0.5 mL) were mixed with 0.5 mL of 2% aluminum chloride solution (in MeOH). The solutions were incubated at room temperature for 15 min, the absorption was measured at 430 nm by using spectrophotometer. Subsequently, the milligram of rutin equivalent (RE)/mL was determined as the expression of total flavonoid content.
2.8. Identification of Phenolic Contained in the Extracts and Root Exudates under the Stress Conditions
Phenolic profiles in the extracts and root exudates were analyzed following to the HPLC method of Xuan et al. [24
]. A HPLC system (JASCO, Tokyo, Japan) consisting of a LC-Net II/ADC, UV-2075 Plus and PU-2089 Plus detector, including RPC18 column (250 mm × 4.6 mm × 5 µm), was employed for the separation of phenolic constituents. The column temperature was 25 °C. An amount of 5 µL extracts or exudates were injected after filtration through a 0.45 µm filter membrane. The mobile phase consisted of 99.8% methanol (solvent A) and 0.1% acetic acid (v
) (solvent B). A gradient elution was run with a 1 mL/min flow-rate using the following time gradients: 5% A (0–5 min), 20% A (5–10 min), 50% A (10–20 min), 80% A (20–30 min), 100% A (30–40 min), 40–50 (100% A) min, 5% A (50–60 min). Phenolic standards (5–100 ppm) were injected into the HPLC in an amount of 5 µL. The peaks of the samples were identified and calculated based on the retention times and peak areas of phenolic standards.
2.9. Statistical Analyses
The data were analyzed by one-way ANOVA and two-way ANOVA using the Minitab 16.0 software for Windows. With regard to the significant differences, the means were separated using Tukey’s test at p < 0.05 with three replications and are expressed as the mean ± standard errors (SE).
Weeds are major constraints, reducing global rice production. Synthetic herbicides are able to manage infestations of weeds, but also exert an adverse influence on humans and environment. Much effort has been made to control weeds by exploiting the allelopathy of rice over the last four decades [9
]. More than 16,000 rice varieties collected from 99 countries have been evaluated for their allelopathic potential, and revealed that ≈4.0% of rice cultivars show weed-suppression of paddy weeds [4
]. Some reports have shown that Japonica rice cultivars have higher allelopathic potential than Indica rice varieties [26
]. Nevertheless, both Koshihikari and Jasmine cultivars have been demonstrated to be allelopathic varieties [27
]. In addition, Xuan et al. [28
] reported Japonica rice varieties show more abiotic tolerance than Indica rice. Notably, Khang et al. [29
] demonstrated that Koshihikari was a submergence-tolerant variety, while Jasmine was a susceptible variety.
Rice is a semi-aquatic crop and is typically grown under partially flooded conditions. Hence, submergence is considered to be one of the most harmful abiotic stresses, resulting in significant losses to rice yield [30
]. However, very little information is available that appraises allelopathic responses under the stress. Therefore, in this study, two rice varieties—Koshihikari (typical Japonica variety) and Jasmine (typical Indica variety)—were used to evaluate allelopathic responses, as well as to identify the correlation between allelopathic potential under the submergence and temperature stresses (10, 25, 32 and 37 °C). Rice seedlings were treated with complete submergence and different temperatures for 7 days under dark conditions. In nature, it is very difficult to distinguish allelopathic and competitive factors due to the multiple uncontrolled environment factors, which include substratum and climatic variations such as nutrients, light, moisture, temperature, etc. Therefore, in this study, rice seedlings were kept in the dark in order to accurately evaluate allelopathic responses under complete submergence and temperature stresses. In fact, dark conditions may not necessarily be a stress, as low-light shade conditions have been reported to enhance the production of allelopathic compounds [31
]. Rice plants react to abiotic and biotic stresses by releasing multiple secondary metabolites to activate defense and protect themselves [32
]. Hu et al. [34
] analyzed 51 Japonica and 49 Indica rice accessions based on 121 metabolites detected by using LC-tandem, GC-MS and reported that the differences in the metabolite abundance and correlations between the two typical rice varieties may reflect the degree of metabolic adaptation and the specific phenotypes in Japonica and Indica [33
]. Moreover, malic acid and long chain fatty acids of rice were enhanced under stagnant growing conditions [35
The results of the current study reveal that the allelopathic potential of extracts and root exudates from rice seedlings depend on a wide array of interactions between the different stressed conditions. Among these, the temperature is a key factor involved in allelopathic activity. Temperature has been reported to directly influence plant growth and to possibly enhance allelochemical production, which subsequently impacts the growth of associated plants [36
]. In this study, we found that the combined effect of temperature and submergence stresses was conducive to the allelopathic response, especially in allelopathic rice (Koshihikari) at 32 °C, which showed higher allelopathic activity in comparison with Jasmine. Allelopathic activity has been shown to decrease when the temperature increases to 37 °C. A plausible explanation is that Japonica samples exhibit their maximum allelopathic response under higher temperatures because of their adaptation to temperate regions with cooler climates, in contrast to the Indica sample. These results support the previous report by Xu et al. [37
], who researched the allelopathic response of wild rice accessions to different temperature conditions. It can be concluded that rice seedlings are sensitive to oxygen deficiency and are markedly dependent on elevated ambient temperatures [38
]. Moreover, Zobayed and Kozai [39
] reported that temperature stress induced or enhanced many secondary metabolites in plants. For example, high temperature treatment improved the leaf total peroxidase activity, hypericin, pseudohypericin and hyperforin concentrations in the shoot tissues [39
]. In fact, allelochemical responses may be significantly modified by environmental stress factors and may vary due to several intrinsic and extrinsic factor [40
Phenolic compounds are important in the response of plants to biotic and abiotic stresses, for example cold, salinity, drought, etc., by determining the antioxidant activity of plants against the accumulation of reactive oxygen species (ROS) caused by stress conditions, which harm plant cells [12
]. Numerous studies have reported the role of phenolic compounds in the tolerance of plants to stresses. For example, in submergence conditions, the total phenolic and flavonoid contents of rice were increased, and the occurrence of some phenolic acids probably plays a major role [29
]. Another study by Weidner et al. [43
] indicated that under cold stress, the levels of gallic and caffeic acids contained in extracts from germinating Vitis californica
seeds were increased. Phenolic compounds have been also been reported to be the compounds most commonly involved in allelochemicals and responsible for allelopathic activity [9
]. Therefore, determination of the total phenolic and flavonoid contents and the identification of the phenolic acids in extracts and root exudates from rice seedlings were performed in this study. Similar to the results of Wu et al. [45
], the total phenolic and flavonoid content of the extracts of wheat (Triticum aestivum
L.) showed an insignificant correlation with the total phenolic and flavonoid content in the root exudates.
In our study, the total phenolic and flavonoid contents were higher in the extracts and root exudates from rice seedlings under submergence and high temperature (37 °C) conditions. However, total contents of either the compounds or the released compounds from rice seedlings exhibited no correlation with allelopathic activity. These results are consistent with the study by Gautam et al. [30
], reported that the amount of a particular allelochemical may be enhanced by one kind of stress and reduced by another kind of stress [46
The identification of phenolic components showed that no particular phenolic acids were present in the samples that were strongly allelopathic against the growth of weeds. The content of individual phenolic acids in the samples that were strong against weeds was no higher than for the weak samples and was similar to the results reported by Olofsdotter et al. [11
]. An explanation for this is that allelopathic potential is determined by the interaction of phenolic combinations under stresses, rather than by individual compounds. In this study, the root exudates of the sample K32 exhibited significant inhibition of natural weeds >76.0%. The allelopathic potential of this sample might be dependent on the interaction of five phenolic acids: p-
hydroxybenzoic, vanillic, syringic, sinapic and benzoic acids, in the responded doses 0.360, 0.045, 3.052, 1.309 and 5.543 μg/mL, respectively. The study by Zeinali et al. [47
] indicated that when rice cultivars are affected by salt, salicylic acid 2.0% could be an effective treatment for rice seedlings and exudates of allelochemicals against barnyardgrass. In our study, the rice variety Koshihikari under submergence and temperature stresses at 32 °C may result in exuding of p-
hydroxybenzoic, vanillic, syringic, sinapic and benzoic acids, which possibly caused the suppression of the plants’ growth.
The obtained results indicated that this root exudation exhibited maximal allelopathic responses against barnyardgrass and natural weeds, but contrarily displayed negligible allelopathic effect on germination and growth of lettuce and radish in laboratory bioassays. This may be explained by the fact that in aquatic environments (complete submergence), nutrients, light and temperatures are important abiotic stress factors. In this study, we minimized the light and nutrients; therefore, the submergence and temperature were key factors, causing allelopathic activity as well as predisposing allelochemicals to inhibit the target indicator plants with either positive or partially antagonistic activities. The effects of allelochemicals in environments might be affected by lights, temperatures, and microbes [9
]. The root treatments and foliar application by allelochemicals enhanced the submergence and drought tolerances in rice, respectively, have been recently demonstrated [49
]. Therefore, further research on the correlation between allelopathic responses of plants and weeds under stress, as well as the identification of the responsible allelochemicals, should be comprehensively conducted, in order to demonstrate the mode of actions of plant defense against invasion, and utilize roles of allelochemicals in agricultural practice.