Allelopathic Effects of Extracts from Three Asteraceae Plants on Seed Germination and Seedling Growth of Four Plants

Zhijia TANG　Guofu FU　Min GUO　Chenzhong JIN　Xiu LIU　Di ZOU　Yunyun ZHOU

Abstract[Objectives] This study was conducted to investigate the allelopathic effects of the extracts of Asteraceae plants on plant seeds and seedlings. [Methods]The effects of the extracts of three Asteraceae plants （Erigeron annuus， Bidens pilosa and Inula japonica） on the seed germination and seedling growth of four plants （Setaria viridis， Erigeron canadensis， Chenopodium album and Lactuca sativa） were studied by the Petri dish filter paper method and bioassay method. [Results] Different concentrations （10， 20， 40， 100， 200 mg/ml） of the extracts of E. annuus， B. pilosa and I. japonica had different allelopathic effects on the seed germination of the same recipient plant， and with the increase of the concentrations of the extracts， the inhibitory effects were more significant. The extract of B. pilosa had the strongest inhibitory effect. The extracts at the low concentration （10 mg/ml） had no significant effects on the germination rates and germination energy of the four kinds of plant seeds. In terms of root growth and biomass， the extracts of E. annuus and I. japonica showed the effect of low promotion and high inhibition. Under the treatment with high concentrations （100， 200 mg/ml） of the extracts from the three Asteraceae plants， the germination of plant seeds， plant root length， stem height and biomass were inhibited. With the concentrations of the extracts of the three Compositae plants increasing， the chlorophyll contents of the four plants showed a downward trend， and the activity of SOD， POD， CAT and other antioxidant protective enzymes showed a trend of first increasing and then decreasing. [Conclusions]These three species of Asteraceae plants had certain allelopathic inhibitory effects on the germination and growth of the four kinds of plant seeds， including S. viridis， E. canadensis， C. album and L. sativa， and they have potential in the development of botanical herbicides or plant protection.

Key wordsAsteraceae; Extract; Seed germination; Seedling growth; Allelopathy

Allelopathy exists widely in nature. Allelopathy of plants refers to the unfavorable or favorable effects of secondary metabolites produced by plants into the environment through leaching， volatilization， decomposition of residues and root excretion on other adjacent plants or themselves[1]. Asteraceae is the largest family of angiosperms， widely distributed all over the world， and there are about more than 2 300 species of 230 genera in China， which are widely distributed in farmland and have high ornamental and economic value. It has been reported in the literature that many genera and species of this family generally have allelopathy， especially the plants of genera Solidago， Helianthus， Ageratum， Parthenium， Artemisia， and the identified allelochemicals are mostly terpenes， polyacetylenes， phenols and organic acids， which have different degrees of inhibitory or promoting effects on a variety of receptor plants[2-5]. The water extracts from the leaves of Ageratina adenophora had allelopathic effects on the seed germination rate and seedling growth of six leguminous plants[6]. Different water extracts of Erigeron canadensis had different intensities of allelopathic effect on the germination and seedling growth of Agrostis matsumurae and Lactuca sativa[7]. Four kinds of invasive plants of Asteraceae （A. adenophora， E. canadensis， Eupatorium odoratum and Tithonia diversifolia） had strong inhibitory effects on Cynodon dactylon， and it is suggested that this factor should be fully considered when applying C. dactylon for substitution control and ecological restoration， and corresponding measures should be taken to deal with it[3]. The study of plant allelopathy has become one of the most active fields of chemical ecology， and the allelopathy of Asteraceae and the application of their allelochemicals in weed control and sustainable agriculture have become a new research hotspot. In this study， the allelopathic effects of the extracts of three Asteraceae plants， including Erigeron annuus， Bidens pilosa and Inula japonica， on the seed germination and seedling growth of Setaria viridis， E. canadensis， Chenopodium album and L. sativa， hoping to provide a basis for further discussion on the comprehensive and effective control of the three species of Asteraceae plants.

Materials and Methods

Experimental materials

The test plants， E. annuus and B. pilosa， were collected in Loudi City， Xiangxiang City， Lianyuan City and other places in central Hunan from August to October 2020， and the test plant， I. japonica， was collected in Sangzhi County， Hunan Province from August to September 2020.

The recipient plants included S. viridis， E. canadensis， C. album and L. sativa. The seeds of the first three were collected from the wild around Loudi， and L. sativa was purchased from a private farmer market in Loudi City.

Experimental methods

Preparation of extracts from the three Asteraceae plantsWhole test plants in the breeding period were randomly taken in the field， dried after cleaning， and cut into 3-4 cm segments， which were placed in an oven， dried at 50 ℃ to a constant temperature， and stored in storage bags for later use. Under the condition of room temperature， the dried E. annuus， B. pilosa and I. japonica materials were pulverized with a pulverizer for 15 min， respectively. Each powder was extracted with shaking at a constant speed in distilled water at a materialtoliquid ratio of 1∶5 （g∶ml） under a constant temperature of 25 ℃ for 48 h， and the extraction system was fully filtered with double layers of gauze to obtain a 200 g/L mother liquor of extract， which was stored in a refrigerator at 4 ℃ for later use. Each mother liquor was diluted with distilled water to 10， 20， 40， 100， and 200 mg/ml water extracts， which were prepared for use freshly.

Effects of extracts from the three Asteraceae plants on crop seed germination and seedling growthBefore the experiment， the seeds were soaked and disinfected with 0.5% potassium permanganate solution， and rinsed 3 times with distilled water， and 50 seeds of S. viridis， E. canadensis， C. album and L. sativa with uniform size were taken and placed in petri dishes with a diameter of 90 mm， respectively， and two layers of filter paper were laid in each petri dish. Different concentrations （10， 20， 40， 100， 200 mg/ml） E. annuus or B. pilosa or I. japonica extract （treatments of different concentrations were marked as T， T， T， T， T， respectively） were added to the petri dishes， respectively， with distilled water as the control （CK）. The seeds were cultured at 25 ℃ with 12 h of light （4 000 lx）， and supplemented with corresponding extract every day. Each treatment was repeated 3 times.

With the radicle breaking through the seed coat by 1-2 mm as the standard， the number of germinated seeds was observed and recorded every 24 h. The germination energy （GE） was calculated on the 5 d， and the germination rate （GR）， the germination index （G） and vigor index （V） were calculated on the 7 d [8]. After the 15 d， 5 plant seedlings were randomly taken from each petri dish， and the surface water was gently absorbed with filter paper. Then， the root length and seedling height of the seedlings were measured with a ruler， and the fresh weight of the seedlings was measured with an electronic balance. The calculation formulas for various indexes were as follows[9]：

Germination rate （GR）=Number of seeds normally germinated within 7 d/Number of seeds tested×100%

Germination energy （GE）=Number of seeds germinated within 5 d/Number of seeds tested×100

Germination index （G）=∑ （G/D）

Vigor index （V）=S∑ （G/D）=S×G

In the formulas： G is the number of seeds germinated on the "t" day; D is the "t" day of the seed germination test; S is the growth （weight） of the seedlings; and G is the seed germination index.

On the 30 d， the physiological and biochemical indexes of the seedlings were determined. The chlorophyll content was determined by the ethanol and acetone extraction method[10]， and the activity of superoxide dismutase （SOD）， peroxidase （POD）， catalase （CAT） and other indexes was detected by ultraviolet spectrophotometry[10].

Data statistics and processing

Microsoft Excel 2019 software and SPSS 22.0 software were used for data statistics， analysis and graphing.

Results and Analysis

Effects of different concentrations of extracts from three Asteraceae plants on seed germination of four plant seeds

The extracts of three different Asteraceae plants had different allelopathic effects on the germination rates， germination energy， germination indexes and vigor indexes of the seeds of the four plants. The germination rates of plants in the control group were nearly 100%， and the seeds germinated within five days. With the increase of the concentrations of the extracts， various indexes of plant seed germination were affected to different degrees， and after the treatment with high concentrations of extracts， the germination rates of plant seeds were inhibited， and the germination energy， germination indexes and vigor indexes were significantly reduced. Comparing the three different extracts， the extract of B. pilosa had relatively strong allelopathic inhibitory effects on the four species of plants， and with the concentration increasing， the inhibitory effects were more significant.

Effects of different concentrations of E. annuus extract on seed germination of four plantsThe four kinds of plant seeds were treated with different concentrations of E. annuus extract， and the germination process of the seeds was affected differently. With the increase of the concentration of the extract， the germination of the seeds was inhibited to a certain extent， and the inhibitory effect was significant in treatments T4 and T5. The germination energy decreased with the increase of the concentration of the extract， and the germination speed was slowed down while the germination of plant seeds was inhibited. The germination indexes and vigor indexes of S. viridis， E. canadensis and C. album showed a trend of first increasing and then decreasing with the increase of the concentration， that is， the dual effect of "low promotion and high inhibition"， while the germination index and vigor index of L. sativa showed a downward trend with the increase of the concentration （Fig. 1a-Fig. 1d）.

Effects of different concentrations of B. pilosa extract on seed germination of four plantsThe four kinds of plant seeds were treated with different concentrations of B. pilosa extract. With the increase of the concentration of the extract， the germination of seeds was inhibited to a certain extent， and the germination rates and germination energy of plant seeds were both in a downward trend; and the germination indexes and the viability indexes also showed a decreasing trend with the concentration increasing （Fig. 2a-Fig. 2d）.

Effects of different concentrations of I. japonica extract on seed germination of four plantsThe four kinds of plant seeds were treated with different concentrations of I. japonica extract. When in the lowconcentration （10， 20 mg/ml， T， T） treatments， their germination rates and germination energy suffered from little effect. With the increase of the concentration of the extract， the germination of seeds was inhibited to a certain extent， and the germination rates and germination energy of the seeds decreased. The vigor index showed a trend of first increasing and then decreasing with the increase of the concentration for S. viridis， E. canadensis and C. album， that is， the dual effect of "low promotion and high inhibition"， while the seed vigor index of L. sativa showed a downward trend with the increase of the concentration （Fig. 3a-Fig. 3d）.

Effects of different concentrations of extracts from three Asteraceae plants on seedling growth and physiological characteristics of four plants

Effects of different concentrations of extracts from three Asteraceae plants on seedling growth of four plantsThe allelopathic effects of different concentrations of extracts from the three Asteraceae plants on the growth of the four kinds of plant seedlings were different， and different concentrations of different extracts showed different effects on the biological indexes of different plant seedlings （see Table 1）. The extracts of E. annuus and I. japonica showed a promoting effect on the growth of the four kinds of plant seedlings at low concentrations， and the stem heights， root lengths and fresh weight indexes of the seedlings were all higher than those of the control group， but with the increase of the concentrations， the growth of seedlings was inhibited， and their various indexes decreased， which meant that the dual effect of "low promotion and high inhibition" existed， especially in the high concentration treatment （T5）， in which their various indexes were significantly different from the control group. The lowconcentration extract （T1） of B. pilosa had no significant effects on the seedlings of the four plants， but had a certain promoting effect on the stem height， root length and fresh weight of the seedlings of E. canadensis and C. album， and with the concentration increasing， the allelopathic inhibition on the seedlings was enhanced. The allelopathic inhibitory effects of the extract of B. pilosa on the seedlings of the four species of plants were slightly stronger than those of the extracts of E. annuus， and various indexes of the four species of plants in treatment T4 and T5 were significant different from the control group.

Effects of different concentrations of extracts from three Asteraceae plants on seedling physiological characteristics of four plantsThe chlorophyll contents and antioxidant enzyme activity of the four species of plants were affected by the extracts of the three Asteraceae plants. With the increase of the concentrations of the extracts， the chlorophyll contents of the plants showed a decreasing trend; and with the increase of the concentrations of the extracts， the activity of the three antioxidant protective enzymes in the four plants showed a trend of increasing or first increasing and then decreasing.

Effects of different concentrations of E. annuus extract on seedling physiological characteristics of four plantsThe chlorophyll contents of the four plant seedlings showed a decreasing trend overall after treatment with different concentrations of E. annuus extract， but the sensitivity of different plants to the treatment of E. annuus was different. The chlorophyll contents of S. viridis and C. album seedlings treated with T1 were slightly higher than those of the control group; and the chlorophyll contents of the seedlings of the four plants treated with T4 and T5 were significantly lower than those of the control group， indicating that the reduction range of chlorophyll content increased under the stress of high concentrations of the extract. When plants respond to external stress， they can increase the activity of antioxidant enzymes to resist the adversity. When the extract reached a certain concentration， the activity of enzymes decreased， and the plants also showed decreases in biological characteristics. After various treatments， the activity of SOD， POD and CAT first increased with the increase of the concentration of the extract， and then decreased with the increase of the concentration. In treatment T1， the activity of SOD， POD， CAT and other antioxidant enzymes were significantly improved， especially for the activity of antioxidant enzymes in L. sativa seedlings， which changed significantly; and in treatments T4 and T5， the antioxidant enzyme activity of some plant seedlings showed a downward trend.

Effects of different concentrations of B. pilosa extract on seedling physiological characteristics of four plantsThe chlorophyll contents of the four kinds of plant seedlings showed a decreasing trend overall after treatment with different concentrations of B. pilosa extract. Under different concentrations of treatments， the activity of SOD， POD and CAT of various plant seedlings first increased with the increase of the concentration of the extract， and then decreased with the concentration increasing. The activity of antioxidant protective enzymes such as SOD， POD and CAT of the seedlings treated with high concentrations of extract was reduced， and even lower than the enzyme activity indexes of the control， indicating that the plants were under the stress of high concentrations of extract， and their enzyme activity decreased.

Effects of different concentrations of I. japonica extract on seedling physiological characteristics of four plantsThe chlorophyll contents of S. viridis and E. canadensis seedlings was slightly increased at first and then decreased after treatment with different concentrations of I. japonica extract， while the chlorophyll contents of C. album and L. sativa seedlings showed an overall decreasing trend.  After treatment with different concentrations of extract， the activity of SOD， POD and CAT on the four kinds of plant seedlings generally first increased with the increase of the concentration of the extract， and then decreased with the increase of the concentration， but the allelopathic effects of I. japonica on different plants were different. The activity of the three antioxidant protective enzymes of S. viridis increased with the increase of the concentration of the extract， but decreased in the T5 treatment. SOD， CAT and other indexes of E. annuus seedlings increased with the increase of the concentration of the extract， and the activity of POD increased with the increase of the concentration of the extract， but decreased in the T treatment. The indexes such as POD and CAT of C. album seedlings increased with the increase in the concentration of the extract， while the SOD enzyme activity decreased in the T treatment. The activity of the three enzymes in L. sativa increased with the increase of the concentration of the extract， reached the highest in treatment T， and then decreased in treatments T and T.

Discussion and Conclusions

Plant allelopathy is an important scientific issue in the field of ecology. It is of great significance in the succession and restoration of plant communities， rational planting of crops and medicinal materials， weed control， pest control and reduction of continuous cropping obstacles and autotoxic hazards. Plant allelochemicals are a common ecological defense mechanism in plants or chemical means to improve their own survival competitiveness， which can promote or inhibit seed germination and seedling growth of their own or neighboring plants[9]. The water extracts （0.25-2.00 g/L） of four species of Asteraceae plants， Ageratina adenophora， E. canadensis， Eupatorium odoratum and Tithonia diversifolia， had a significant inhibitory effect on the germination of Cynodon dactylon seeds. With the concentrations increasing， the inhibitory effects of the leaf water extracts on the germination rate and germination energy of C. dactylon seeds and the allelopathic effects on C. dactylon seeds were enhanced in turn. When treating at the high concentration （2.00 g/L）， E. odoratum had the strongest inhibitory effect， and there were no significant differences between other three plants[3]. In the study of the effects of A. adenophora on the germination characteristics of Eucalyptus globulus and Cunninghamia lanceolata and their seedlings， it was found that the germination energy， germination rate， germination speed and germination index of C. lanceolata seeds and radicle length， hypocotyl length， fresh weight and fresh weight of C. lanceolata seedlings were all significantly affected by different treatments， and all germination characteristics and seedling indexes were significantly negatively correlated with the treatment concentration[11]. In the study which explored whether the exotic plant Cyclachaena xanthiifolia has allelopathic effects on the seed germination and seedling growth of indigenous plants， the germination of S. viridis and Amaranthus tricolor seeds were both sensitive to the extracts of different parts of C. xanthiifolia， and Artemisia sieversiana， C. album and Echinochloa crusgalli showed a trend of low stimulation and high inhibition to the extracts of different parts of C. xanthiifolia[12]. The results of this study showed that the allelopathic effects of different concentrations of E. annuus， B. pilosa and I. japonica extracts on the seed germination of the same recipient plant were different， and as the concentrations of the extracts increased， the inhibitory effects were more significant. Among them， the extract of B. pilosa had the most inhibitory effect. In the treatments with the extracts of three species of Asteraceae plants， when the concentrations of the extracts were 20 mg/ml and above， the germination rates， germination indexes and vigor indexes of the seeds of the four plants and the root lengths and biomass of the seedlings decreased with the concentration increasing.

Under adverse environmental conditions， plants respond to adverse environmental stresses by regulating gene expression， changing metabolic intensity or pathways， and coordinating the entire defense system to maintain the balance between antioxidant enzymes， thereby resisting stressinduced oxidative damage， reducing the accumulation of reactive oxygen species， reducing the degree of membrane damage and maintaining normal growth. In the study on the allelopathic effects of A. adenophora extract on different varieties of strawberries， the activity of CAT， POD， SOD and other antioxidant enzymes showed a trend of first increasing and then decreasing with the increase of the concentration of the extract[13]. In order to explore the potential allelopathic effect of Juglans sigillata and the feasibility of interplanting Sarcandra glabra under J. sigillata forests， the effects of J. sigillata leaf water extract on the activity of three protective enzymes of S. glabra seedlings were different. The activity of CAT decreased with the increase of the concentration of the extract， and the activity of POD and SOD first increased and then decreased with the increase of the concentration of the extract[14]. CAT， POD and SOD are the antioxidant protective enzymes of plants， which can work together to prevent the damage of ROS to plant cell membranes when plants are stressed. In this study， four species of plants were treated with different concentrations of extracts from three species of Asteraceae plants， and with the increase of the concentrations of the extracts from the three species of Asteraceae plants， the activity of SOD， POD， CAT and other antioxidant protective enzymes basically showed the same trend of rising first and then falling. Such phenomenon showed that when these plant seedlings were affected by low concentrations of extracts， SOD and POD in cells would first be activated to remove excess ROS， and resist the influence of external allelopathic substances， but when the concentrations of the extracts increased， the antioxidant enzyme system of cells could not clear excess ROS， which eventually led to cell damage， and the enzyme activity gradually decreased. The results also showed that the activity of POD and CAT enzyme did not decrease with the increase of concentration in E. annuus and C. album， which might be due to the relatively strong resistance of these two plants to the allelopathic inhibitory effects by the extracts. With the increase of the concentrations of the extracts of the three Asteraceae plants， the chlorophyll contents of the four plants showed a downward trend. Combined with the root lengths and biomass of the seedlings， it was shown that the extracts of the three Asteraceae plants had significant allelopathic inhibitory effects on the four plants.

References

[1] RICE EL. Allelopathy[M]. New York： Academic Press， 1984.

[2] ZHOU K， GUO WM， XU YC. Advances of research on allelopathic potential in Asteraceae [J]. Acta Ecologica Sinica， 2004（8）： 1780-1788. （in Chinese）.

[3] JIANG ZL， YANG LP， SHEN K， et al. Allopathic effects of aqueous extracts from leaves of four invasive Asteraceae plants on seed germination of Cynodon dactylon[J]. Southwest China Journal of Agricultural Sciences， 2020， 33（12）： 2943-2947. （in Chinese）.

[4] JABRAN K， MAHAJAN G， SARDANA V， et al. Allelopathy for weed control in agricultural systems[J]. Crop Protection， 2015（72）： 57-65.

[5] BENVENUTI S， CIONI PL， FLAMINI G， et al. Weeds for weed control： Asteraceae essential oils as natural herbicides[J]. Weed Research， 2017， 57（5）： 342-353.

[6] LI HM， CHEN LY， QIN XM. Allelopathic effects of Eupatorium adenophorum Spreng on six species of Leguminosae[J]. Hubei Agricultural Sciences， 2010， 49（8）： 1856-1858. （in Chinese）.

[7] ZHANG HY， ZHANG M， QI SS， et al. Allelopathy and active components of the invasive plant Conyza canadensis[J]. Weed Science， 2015， 33（4）： 5-9. （in Chinese）.

[8] LUO FC， BI YF， HUANG BZ. Practical teaching guidance for grass science[M]. Yunnan： Yunnan Science and Technology Press， 2008. （in Chinese）.

[9] YAO SK， LI FL， PENG LN. A study of the allelopathic effect of extracts from different parts of Iva xanthiifolia on five Brassicaceae species[J]. Acta Prataculturae Sinica， 2018， 27（9）： 56-66. （in Chinese）.

[10] CHEN JX， WANG XF. Plant physiology experiment guidance[M]. Guangzhou： South China University of Technology Press， 2002. （in Chinese）.

[11] CAO ZL， WANG YY， WANG XL， et al. Allelopathic effect of Eupatorium adenophorum on seed germination and seedling growth of Cunninghamia lanceolata[J]. Seed， 2017， 36（7）： 32-36. （in Chinese）.

[12] LI FL， WU JW， YAO SK， et al. A study of the allelopathic effect of extracts from different parts of Iva xanthiifolia on five native species[J]. Acta Prataculturae Sinica， 2020， 29（9）： 169-178. （in Chinese）.

[13] GUO Y， XIANG CL， YE YL， et al. Allelopathy of Eupatorium adenophorum extracts on seed germination and seedling growth of different strawberry varieties[J]. Seed， 2021， 40（6）： 96-101. （in Chinese）.

[14] PU XB， GUO S， LI ZL， et al. Allelopathy of aqueous extract from Juglans sigillata leaves on germination of Sarcandra glabra seeds and seedling growth[J]. Guangxi Forestry Science， 2021， 50（6）： 678-684. （in Chinese）.

Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU