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Abstract [Objectives] This study was conducted to analyze the genetic toxicity of Solidago decurrens， with a view to scientifically and safely using S. decurrens. [Methods] Using commercially available S. decurrens as an experimental material， according to Vicia faba root tip micronucleus technique， V. faba root tips were treated with different doses of aqueous extract from S. decurrens for different times， and the mitotic index， frequency of micronucleus and chromosomal aberration of root tips in V. faba were detected， so as to reveal the allelopathic potential and the potential ecological risks of S. decurrens at the cellular level . [Results] The decrease in the mitotic index caused by the aqueous extract of S. decurrens was positively related to the concentration of the aqueous extract and the treatment time. The aqueous extract of S. decurrens increased the frequency of micronucleus in root tip cells of V. faba， and the increase was positively related to the concentration of the aqueous extract and the treatment time. The aqueous extract of S. decurrens caused abnormal phenomena such as chromosome bridges， chromosome fragmentation， and chromosome lag in V. faba root tip cells， and the effect of the aqueous extract on V. faba root tips had significant effects dependent on time and concentration. Above results indicate that the aqueous extract of S. decurrens inhibited and damaged the mitosis of V. faba root tip cells to some degrees， had certain genetic toxicity and had the ability to rapidly spread. [Conclusions] This study provides a theoretical basis for scientifically and rationally using S. decurrens and preventing the spread of S. decurrens.
Key words Solidago decurrens; Genetic toxicity; Vicia faba root tip; Micronucleus
Solidago decurrens is a perennial herbaceous plant belonging to the Asteraceae family. It mainly grows in East China， Central South， Southwest China， Shaanxi， Taiwan and other places. As a very widely used material for commercial flowers， because it belongs to the same genus as the well-known foreign malignant weed S. canadensis， the ecological safety of its application has been controversial. Zhang et al.[1] made a comparative study on the allelopathy of S. decurrens and S. canadensis， and the results showed that the aqueousextracts of the two solidago plants had allelopathic effects on the tested plants， but the allelopathy of the underground part of S. canadensis was stronger than its aboveground part， while the local S. decurrens was the opposite. In order to use S. decurrens scientifically and safely， it is necessary to extensively study the genetic toxicity of the plant. At present， it is generally believed that allelopathy is a "novel weapon" for the successful invasion of exotic plants[2]. Vicia faba root tip micronucleus test （V. faba-MCN） is an effective method to detect cell genetic damage. It is widely used in the detection of environmental teratogenic and mutagenic compounds due to its high efficiency， sensitivity and simplicity[3]. It is also widely used to detect the genetic toxicity of invasive alien plants[4-9]. In this study， according to V. faba root tip micronucleus technique， V. faba root tips were treated with different doses of S. decurrens aqueous extract for different times， and the mitotic index， frequency of micronucleus and chromosomal aberration of V. faba root tips were detected， so as to reveal the allelopathic potential of S. decurrens at the cellular level and the potential ecological risks brought by planting S. decurrens， which provide a theoretical basis for scientific and reasonable utilization and prevention of its spread.
Materials and Methods
Experimental materials
The donor plant S. decurrens was purchased from the Huangzhou Flower Market， and the recipient plant V. faba seeds were purchased from the Huangzhou Seed Market.
Experimental methods
Preparation of water extract
The fresh stems and leaves of S. decurrens were rinsed with water， dried， and pulverized with a micro plant sample grinder （1 400 r/min）. The sample was put in a conical flask， and added with distilled water at a ratio of 1∶4 （W/V）， followed by mixing well. The mixture was extracted on a shaker （130 r/min， 23 ℃） for 48 h. After filtering the extract twice with 2 layers of gauze， 0.25 g/ml mother liquor of aqueous extract was obtained and stored in a 4 ℃ refrigerator for later use.
Material cultivation and treatment
The cultivation and treatment of materials and data processing were carried out using the experimental methods of Hu et al.[9].
Results and Analysis
Effects of S. decurrens extract on mitosis of root tip cells in V. faba
From Table 1 and Fig. 1， it can be seen that the aqueous extract of S. decurrens has a significant inhibitory effect on the mitotic index of V. faba root tip cells. The inhibitory effect of the extract on mitosis was positively correlated with the treatment time and the mass concentration of the extract.More details， with the increase of the mass concentration and the extension of the treatment time， the inhibitory effect was enhanced. Compared with the control group， the differences in the mitotic index of V. faba root tip cells between various treatment groups reached an extremely significant level （P<0.01）; and at the late treatment period （72 h）， the mitotic index was slightly higher in the 0.025 and 0.050 g/ml treatments than the control group， which might be due to that in the presence of certain allelochemicals from S. decurrens ， the osmotic potential of the extract was small， which reduced the water stress to a certain extent. However， with the increase in the mass concentration of allelochemicals， such relief effect was lost. The RI value of the aqueous extract of S. decurrens on the mitotic index also reflected that the aqueous extract of S. decurrens had a significant inhibitory effect on the mitotic index of V. faba root tip cells.
Effect of S. decurrens extract on the frequency of micronucleus of root tip cellsin V. faba
The aqueous extract of S. decurrens induced a higher frequency of micronucleus （Table 2 and Fig. 2）， and there was also a dual dependence on the treatment time and the concentration of the extract. That is， with the prolongation of the treatment time and the increase of the mass concentration， the micronucleus cells in every 1 000 root tip cells had an obvious increasing trend. Compared with the control group， the differences in the frequency of micronucleus of V. faba root tip cells between various treatment groups reached an extremely significant level （P<0.01）.
Conclusions and Discussion
Allelopathic effect of the aqueous extract of S. decurrens on V. faba root growth
The apical meristem of plants is the most sensitive area of plants to adverse factors， so the allelopathy of S. decurrens can be studied by detecting the mitotic activity of root tip cells. The results of this study showed that with the increase in the concentration of the aqueous extract of S. decurrens， the mitotic index of V. faba root tip cells decreased significantly. It could be seen that the water-soluble allelochemicals of S. decurrens entered the soil through leaching or root secretion， thereby disturbing the roots of local plants， resulting in poor growth and even death of other plants.
Genetic toxicity of the aqueous extract of S. decurrens
The results of this study showed that the aqueous extraction of S. decurrens could induce higher frequency of micronucleus， and the micronucleus cells in every 1 000 root tip cells in the water extract treatment groups were significantly higher than that of the control group. This effect had time and concentration effects， which is consistent with the results of Fan et al.[5] on Galinsoga parviflora Cav.， Du et al.[6] on Bidens pilosa L.， and Gao et al.[7] on Flaveria bidentis. Meanwhile， under the action of the water extract of V. faba root tip cells， various chromosomal aberrations appeared in V. faba root tip cells， indicated that that the water-soluble allelochemicals from S. decurrens destroyed the normal structure and function of the spindle filaments of V. faba root tip cells. Because dicentric and acentric chromosomal fragments are formed due to chromosome breaks and fusions and then bridges are formed， the formation of all bridges is accompanied with the appearance of chromosome fragments[8]. In addition， water-soluble allelochemicals in S. decurrens might interfere with the normal repair of DNA damage， so the formed chromosomal fragments could not move to the polar， and formed micronuclei free from the main nuclei during the rebuilding of daughter nuclei， resulting in various aberrations and distortion. References
[1] ZHANG ZX， ZHANG XP， LIU HJ， et al. Comparative study on the allelopathy of invasive species Solidago canadensis L. and native species Solidago decurrens Lour.[J]. Journal of Wuhan Botanical Research， 2010， 28（2）： 191-198. （in Chinese）
[2] CALLAWAY RM， RIDENOUR WM. Novel weapons： invasive success and the evolution of increased competitive ability[J]. Frontiers in Ecology Environment， 2004（2） ： 436443.
[3] MA TH. Vicia faba cytogenetic test for environmental mutagen. a report of the U.S. environmental protection agency gene-tox program[J]. Mutat Res， 1982（99）： 257-271.
[4] HENG WJ， HE XJ. Allelopathic potential of the invasive plant Ageratum conyzoides with Vicia-micronucleus test[J]. Plant Protection，2012，38（1）： 24-30. （in Chinese）
[5] FAN XT， MA DW， LIU S， et al. Assessment of alleopathy of Galinsoga parviflora Cav. using Vicia-micronucleus test[J]. Ecology and Environment， 2008， 17（1）： 323-326. （in Chinese）
[6] DU FY， YU SH， MA DW， et al. The genetic toxicity of Bidens pilosa L. to Vicia faba root tip cells[J]. Ecology and Environment， 2007， 16（3）： 944-949. （in Chinese）
[7] GAO RY， GAO XG， LI HF， et al. Genetic toxicity of Flaveria bidentis to wheat root tip cells[J]. Journal of Triticeae Crops，2013， 33（5）： 1035-1038. （in Chinese）
[8] LUO MC， YANG B， HAO YJ， et al. Genetic toxicity of invasive alien plant Oxalis corymbosa[J]. Molecular Plant Breeding，2016，14（12）： 3544-3547. （in Chinese）
[9] HU WJ， MA DW， WANG YN， et al. Allelopathicpotential of volatile oil from Chenopodium ambrosioides L. on root tip cells of Vicia faba[J]. Acta Ecologica Sinica， 2011， 31（13）： 3684-3690. （in Chinese）
Key words Solidago decurrens; Genetic toxicity; Vicia faba root tip; Micronucleus
Solidago decurrens is a perennial herbaceous plant belonging to the Asteraceae family. It mainly grows in East China， Central South， Southwest China， Shaanxi， Taiwan and other places. As a very widely used material for commercial flowers， because it belongs to the same genus as the well-known foreign malignant weed S. canadensis， the ecological safety of its application has been controversial. Zhang et al.[1] made a comparative study on the allelopathy of S. decurrens and S. canadensis， and the results showed that the aqueousextracts of the two solidago plants had allelopathic effects on the tested plants， but the allelopathy of the underground part of S. canadensis was stronger than its aboveground part， while the local S. decurrens was the opposite. In order to use S. decurrens scientifically and safely， it is necessary to extensively study the genetic toxicity of the plant. At present， it is generally believed that allelopathy is a "novel weapon" for the successful invasion of exotic plants[2]. Vicia faba root tip micronucleus test （V. faba-MCN） is an effective method to detect cell genetic damage. It is widely used in the detection of environmental teratogenic and mutagenic compounds due to its high efficiency， sensitivity and simplicity[3]. It is also widely used to detect the genetic toxicity of invasive alien plants[4-9]. In this study， according to V. faba root tip micronucleus technique， V. faba root tips were treated with different doses of S. decurrens aqueous extract for different times， and the mitotic index， frequency of micronucleus and chromosomal aberration of V. faba root tips were detected， so as to reveal the allelopathic potential of S. decurrens at the cellular level and the potential ecological risks brought by planting S. decurrens， which provide a theoretical basis for scientific and reasonable utilization and prevention of its spread.
Materials and Methods
Experimental materials
The donor plant S. decurrens was purchased from the Huangzhou Flower Market， and the recipient plant V. faba seeds were purchased from the Huangzhou Seed Market.
Experimental methods
Preparation of water extract
The fresh stems and leaves of S. decurrens were rinsed with water， dried， and pulverized with a micro plant sample grinder （1 400 r/min）. The sample was put in a conical flask， and added with distilled water at a ratio of 1∶4 （W/V）， followed by mixing well. The mixture was extracted on a shaker （130 r/min， 23 ℃） for 48 h. After filtering the extract twice with 2 layers of gauze， 0.25 g/ml mother liquor of aqueous extract was obtained and stored in a 4 ℃ refrigerator for later use.
Material cultivation and treatment
The cultivation and treatment of materials and data processing were carried out using the experimental methods of Hu et al.[9].
Results and Analysis
Effects of S. decurrens extract on mitosis of root tip cells in V. faba
From Table 1 and Fig. 1， it can be seen that the aqueous extract of S. decurrens has a significant inhibitory effect on the mitotic index of V. faba root tip cells. The inhibitory effect of the extract on mitosis was positively correlated with the treatment time and the mass concentration of the extract.More details， with the increase of the mass concentration and the extension of the treatment time， the inhibitory effect was enhanced. Compared with the control group， the differences in the mitotic index of V. faba root tip cells between various treatment groups reached an extremely significant level （P<0.01）; and at the late treatment period （72 h）， the mitotic index was slightly higher in the 0.025 and 0.050 g/ml treatments than the control group， which might be due to that in the presence of certain allelochemicals from S. decurrens ， the osmotic potential of the extract was small， which reduced the water stress to a certain extent. However， with the increase in the mass concentration of allelochemicals， such relief effect was lost. The RI value of the aqueous extract of S. decurrens on the mitotic index also reflected that the aqueous extract of S. decurrens had a significant inhibitory effect on the mitotic index of V. faba root tip cells.
Effect of S. decurrens extract on the frequency of micronucleus of root tip cellsin V. faba
The aqueous extract of S. decurrens induced a higher frequency of micronucleus （Table 2 and Fig. 2）， and there was also a dual dependence on the treatment time and the concentration of the extract. That is， with the prolongation of the treatment time and the increase of the mass concentration， the micronucleus cells in every 1 000 root tip cells had an obvious increasing trend. Compared with the control group， the differences in the frequency of micronucleus of V. faba root tip cells between various treatment groups reached an extremely significant level （P<0.01）.
Conclusions and Discussion
Allelopathic effect of the aqueous extract of S. decurrens on V. faba root growth
The apical meristem of plants is the most sensitive area of plants to adverse factors， so the allelopathy of S. decurrens can be studied by detecting the mitotic activity of root tip cells. The results of this study showed that with the increase in the concentration of the aqueous extract of S. decurrens， the mitotic index of V. faba root tip cells decreased significantly. It could be seen that the water-soluble allelochemicals of S. decurrens entered the soil through leaching or root secretion， thereby disturbing the roots of local plants， resulting in poor growth and even death of other plants.
Genetic toxicity of the aqueous extract of S. decurrens
The results of this study showed that the aqueous extraction of S. decurrens could induce higher frequency of micronucleus， and the micronucleus cells in every 1 000 root tip cells in the water extract treatment groups were significantly higher than that of the control group. This effect had time and concentration effects， which is consistent with the results of Fan et al.[5] on Galinsoga parviflora Cav.， Du et al.[6] on Bidens pilosa L.， and Gao et al.[7] on Flaveria bidentis. Meanwhile， under the action of the water extract of V. faba root tip cells， various chromosomal aberrations appeared in V. faba root tip cells， indicated that that the water-soluble allelochemicals from S. decurrens destroyed the normal structure and function of the spindle filaments of V. faba root tip cells. Because dicentric and acentric chromosomal fragments are formed due to chromosome breaks and fusions and then bridges are formed， the formation of all bridges is accompanied with the appearance of chromosome fragments[8]. In addition， water-soluble allelochemicals in S. decurrens might interfere with the normal repair of DNA damage， so the formed chromosomal fragments could not move to the polar， and formed micronuclei free from the main nuclei during the rebuilding of daughter nuclei， resulting in various aberrations and distortion. References
[1] ZHANG ZX， ZHANG XP， LIU HJ， et al. Comparative study on the allelopathy of invasive species Solidago canadensis L. and native species Solidago decurrens Lour.[J]. Journal of Wuhan Botanical Research， 2010， 28（2）： 191-198. （in Chinese）
[2] CALLAWAY RM， RIDENOUR WM. Novel weapons： invasive success and the evolution of increased competitive ability[J]. Frontiers in Ecology Environment， 2004（2） ： 436443.
[3] MA TH. Vicia faba cytogenetic test for environmental mutagen. a report of the U.S. environmental protection agency gene-tox program[J]. Mutat Res， 1982（99）： 257-271.
[4] HENG WJ， HE XJ. Allelopathic potential of the invasive plant Ageratum conyzoides with Vicia-micronucleus test[J]. Plant Protection，2012，38（1）： 24-30. （in Chinese）
[5] FAN XT， MA DW， LIU S， et al. Assessment of alleopathy of Galinsoga parviflora Cav. using Vicia-micronucleus test[J]. Ecology and Environment， 2008， 17（1）： 323-326. （in Chinese）
[6] DU FY， YU SH， MA DW， et al. The genetic toxicity of Bidens pilosa L. to Vicia faba root tip cells[J]. Ecology and Environment， 2007， 16（3）： 944-949. （in Chinese）
[7] GAO RY， GAO XG， LI HF， et al. Genetic toxicity of Flaveria bidentis to wheat root tip cells[J]. Journal of Triticeae Crops，2013， 33（5）： 1035-1038. （in Chinese）
[8] LUO MC， YANG B， HAO YJ， et al. Genetic toxicity of invasive alien plant Oxalis corymbosa[J]. Molecular Plant Breeding，2016，14（12）： 3544-3547. （in Chinese）
[9] HU WJ， MA DW， WANG YN， et al. Allelopathicpotential of volatile oil from Chenopodium ambrosioides L. on root tip cells of Vicia faba[J]. Acta Ecologica Sinica， 2011， 31（13）： 3684-3690. （in Chinese）