Heavy Metals Tolerance in an Invasive Weed (Fallopia japonica) under Different Levels of Soils Contamination

In order to assess the tolerance of the highly invasive weed Fallopia japonica to heavy metals, a greenhouse experiment was conducted in which this plant was cultivated in control soil and in the soils polluted by different levels of Cd, Cr, Cu, Pb and Zn. The content of heavy metals in soil did not eliminate the F. japonica rhizome’s capacity to regenerate. However, at the beginning of the experiment, the presence of some metal doses: Cd (100, 200 mg·kg-1), Pb (200 mg·kg-1) and Zn (300 mg·kg-1) delayed the rhizome regeneration compared to the control plants. In the soils contaminated with any level of Cr or Pb, shoots grew with similar vigour to the control plants. Only the high doses of Cd (100, 200 mg·kg-1), Cu (300 mg·kg-1) and Zn (300 mg·kg-1) significantly delayed the plants’ growth. The morphological features of F. japonica from the soils polluted with Cr and Pb were not significantly different from the control plants. Among the tested heavy metals that had the greatest impact on the morphology of F. japonica were Cd (100, 200 mg·kg-1), Cu (300 mg·kg-1) and Zn (300 mg·kg-1). A chemical analysis indicated that this weed accumulated large quantities of metals when cultivated in the contaminated soil. Particular attention was paid to its relatively high Cd uptake. In the variant where a dose of 100 mg Cd·kg-1 was applied, the plants (aboveground part) accumulated more than 630 times the amount of cadmium found in the control. The abilities of F. japonica to regenerate from rhizome fragments, to grow and develop under the stress conditions created by heavy metals, and to take up metals are evidence that this plant is characterised by metal tolerance.


INTRODUCTION
Biological invasions represent one of the main problems of contemporary ecology and are considered a significant component of the global change connected with human activity (Vilá et al. 2007). On the basis of the knowledge about the invasive plant species accrued from many studies, it can be concluded that the phenomenon of their invasiveness is determined by several characteristics that give the invader an advantage. These characteristics can be summarised as vigorous and rapid growth, high fecundity (Bradley et al. 2010), strong competing abilities (Sharma et al. 2005) and sexual reproduction by hybridisation, which may serve as a stimulus for the evolution of invasiveness (Ellstrand and Schierenbeck 2000). It has also been noted that in order to become a successful invader, a plant must possess wide environmental tolerances and be well-suited to various conditions (Bradley et al. 2010). A typical example of a plant that possesses such characteristics is the invasive weed Fallopia japonica from the Fallopia genus (Polygonaceae). It is native to East Asia, with a natural range that mainly encompasses China, Japan, North and South Korea, and Taiwan (Bailey 2003, CABI 2020. Fallopia japonica was deliberately introduced to Europe in the 19 th century as an ornamental plant. Today, F. japonica is viewed as Europe's worst invasive alien plant species (Nentwig et al. 2018) because it is spreading in almost all European countries. Globally, it is also now common in North America Australia, and New Zealand (Anderson and Hayley 2012). Apart from the natural areas, F. japonica occurs frequently in the man-made habitats such as roads, highways and railways (Tokarska-Guzik et al. 2017). The species is often observed in the places with extremely high levels of anthropogenic pollution; for instance, it can inhabit heavy-metal-polluted soils. In Poland, F. japonica was noted on a zinc smelter (Chmura et al. 2013), coal mining spoil heaps (Rahmonov et al. 2014) and urban parks with different levels of disturbance (Rahmonov et al. 2019). Preliminary field studies in the city of Wrocław, Poland proved that an increased content of heavy metals in soil does not deter F. japonica invasion-on the contrary, it can even enhance the invader's competitiveness in relation to other plant species (Sołtysiak et al. 2014). Taking into account the field observations in the literature, it was hypothesised that F. japonica has some tolerance to toxic metals. For this reason, a greenhouse experiment was conducted and the reaction of F. japonica to the stress conditions created by the presence of different levels of heavy metals in the soil was investigated. The aim of this research was to determine: 1) The ability of the highly invasive weed F. japonica to regenerate from rhizome segments in the soils polluted by various doses of Cd, Cr, Cu, Pb, and Zn; 2) The toxic effects of these metals on the growth and development of the tested plant; 3) The possibility of F. japonica to accumulate heavy metals in its tissues.
Cd, Cr, Cu, Pb, and Zn were chosen as the stressors because they are widespread pollutants of soils in Central Europe, where the Fallopia japonica scrubs were noted (Sołtysiak et al. 2011, Sołtysiak and Brej 2014, Rahmonov et al. 2014, Rahmonov et al. 2019).  Bailey et al. 2008). The Fallopia japonica studied in the greenhouse experiment reported here was collected from the city of Wrocław (SW Poland). It was a female clone with 88 chromosomes. The species identification was based on the morphological features of the leaves (shape, size, hairiness of leaf blade) and a cytological examination in order to determine the number of chromosomes. Fresh rhizomes were collected from one cluster of a natural population of Fallopia japonica that grows along a bank of the Oder River (51°07.404'N and 17°04.146'E). In the laboratory, the rhizomes of F. japonica were cleaned with distilled water and cut into fragments of a similar weight (12.5 g).

Greenhouse experiment procedure
The greenhouse experiment procedure was conducted according to a study by Sołtysiak and Brej (2019), where the effect of different doses of Pb on invasive Fallopia x bohemica (a hybrid of F. japonica and F. sachalinensis) was examined. The prepared plant materials (rhizome fragments of F. japonica) were introduced into plastic pots (one rhizome in each pot) with a capacity of 5 kg of soil. The soil with pH of 5.3 (determined in KCl) and 1.2% organic matter was used in the experiments. The macronutrient contents were as follows: K 2 O -26.7 mg·100g -1 , P 2 O 5 -11.6 mg·100g -1 and Mg -4.0 mg·100g -1 , while the baseline heavy metal concentrations were Cd -0.1 mg·kg -1 , Cr -6.7 mg·kg -1 , Cu -8.4 mg·kg -1 , Pb -10.2 mg·kg -1 and Zn -27.1 mg·kg -1 . The experiment was a completely randomized twofactorial design with five heavy metals (Cd, Cr, Cu, Pb and Zn) and three different levels of each metal: Cd -20, 100, 200 mg·kg -1 , Cr -10, 20, 100 mg·kg -1 , Cu -100, 200, 300 mg·kg -1 , Pb -50, 100, 200 mg·kg -1 and Zn -100, 200, 300 mg·kg -1 . The individual metal solutions were applied directly into the pots. The pots with no addition of a heavy metal were treated as controls. Four replicates were created for each metal treatment and the control group. The greenhouse experiment was conducted during one growing season, from early June to September. The plants were grown under natural light and temperature conditions (varied from 20 to 30°C) and watered every day.

Plant observations, measurements, and heavy metal concentration analysis
During the experiment, the regeneration, growth and development of F. japonica were observed every day. At the beginning, the ability of F. japonica rhizomes to regenerate in the soils polluted by various doses of Cd, Cr, Cu, Pb and Zn was monitored for 3 weeks. Next, after the rhizome regeneration, the dynamic of F. japonica growth was noted. The height of the highest areal shoots of plants for each treatment group was measured on a centimetre scale once a week for 14 weeks. Afterwards, the morphological traits of plants, such as the length and width of the leaf blade (cm), fresh and dry weights of aboveground shoots (g), were measured in a laboratory. Additionally, the concentrations of heavy metals in the aboveground organs (leaves) and underground organs (rhizomes) of F. japonica were analysed. For this purpose, the plant material was washed with distilled water. Next, it was dried at 80ºC for 48 h, milled with a stainless-steel grinder and digested in a mixture of HCl and HNO 3 (at a ratio of 3:1). The contents of metals in the leaves and rhizomes were analysed (each sample in four replications) with the ICP-OES (Inductively Coupled Plasma Optical Emission Spectroscopy) technique using a Varian Liberty Series 220 spectrometer. The analyses were performed in a laboratory of the Centre of Analysis of Environmental Quality at the Wrocław University of Life Sciences.

Statistical analyses
The statistical analyses were carried out using STATISTICA v. 13.3 (StatSoft Inc., 2017). The normality of the data distribution was checked using the Shapiro-Wilk test. The homogeneity of variance was checked using Levene's test. The morphological characteristics of the plants grown under different conditions (i.e., in the pots with soil contaminated by various doses of Cd, Cr, Cu, Zn, or Pb or in the control soils) were compared using one-way ANOVA. Homogeneous subsets were designated using the post-hoc testing of significance of differences with Tukey's HSD test. In order to determine the relationship between various heavy metal doses and their contents in the aboveground and underground parts of the plants, the correlation coefficients were determined: Pearson's correlation coefficient (p) in the case of the data for which a normal distribution was found and Spearman's rank correlation coefficient (Rs) for the data with a non-normal distribution.

The influence of heavy metals on regeneration ability and growth of Fallopia japonica
The results confirmed the ability of Fallopia japonica rhizomes to regenerate in the soils polluted by heavy metals (Table 1). However, at the beginning of the experiment, the presence of Cd (100, 200 mg·kg -1 ), Pb (200 mg·kg -1 ) and (Zn (300 mg·kg -1 ) delayed rhizome regeneration compared to the control plants. After the three weeks of cultivation, the highest regeneration (100%) rates were exhibited by the plants from the control pots and the pots polluted by Cd (20, 100, 200 mg·kg -1 ), Cr (10, 20, 100 mg·kg -1 ), Cu (100, 200 mg·kg -1 ) and Zn (100, 200 mg·kg -1 ). The fewest rhizomes (75%) showed active regeneration in the pots with addition of Cu and Zn in a doses of 300 mg·kg -1 and all Pb doses (50, 100, 200 mg·kg -1 ).
The study showed that Cr (10, 20, 100 mg·kg -1 ) and Pb (50, 100, 200 mg·kg -1 ) did not inhibit the growth of the aerial shoots of Fallopia japonica. The plant heights obtained in soils where Cr or Pb  (Figure 1a).

Heavy metal contents in the aboveand underground parts of plants
The analysis of metal concentrations in Fallopia japonica confirmed a high potential of this plant for heavy metal accumulation (Table 2). An increased concentration of metals in soil stimulated their increase in Fallopia japonica. The positive Pearson and Spearman's correlation coefficients (Table 3)  this element was more than 10 times higher than in the control. Cu and Pb were mostly accumulated in the underground parts of Fallopia japonica, while the leaves contained smaller amounts of those metals. The plants from the soil polluted with lead (200 mg·kg -1 ) contained 11 times more of this element than the control plants. In the underground parts of the plants from the pots with the addition of Cu at 300 mg·kg -1 , 14 times more Cu was found in comparison with the control. Chromium accumulated in both the underground and aboveground parts of Fallopia japonica. The addition of Cr to the soil at 100 mg·kg -1 resulted in a more than fourfold increase in the concentration of chromium in the underground and aboveground parts of the plants from the contaminated pots compared to the control.   All of these possess strong competitive abilities, but it is less known whether they are resistant to heavy metals or whether the soil contamination by heavy metals impacts the invasion of these exotic weeds. The plants from polluted soils have physiological strategies that allow them to exist under extreme conditions. One such strategy is the resistance to the toxic levels of heavy metals in soils. In order to achieve this, plants can rely on either avoidance or tolerance of heavy metals (Punz and Sieghardt 1993). The first of these concerns the cellular level as well as the level of the whole plant. Avoidance of the stress can refer to the sequestration of toxic metals from the protoplasm and the elimination of heavy metals or avoidance of the uptake of heavy metals from the soil solution. Such a strategy is represented by the invasive Solidago canadensis, which spreads on the soils contaminated by lead (Yang et al. 2007). The metal tolerance strategy of a plant species is a result of genetic, physiological, and ecological evolution. This process unfolded on natural metalliferous areas over a long time.

DISCUSSION
In anthropogenic habitats, the evolution of metal tolerance takes much less time; it depends on the metal levels and on the selection pressure in the environment. The results of this greenhouse experiment provided the evidence that F. japonica might have a tolerance to toxic metals. This evidence is summarised below: 1. First, this experiment proved that even high contents of toxic metals (Cd, Cr, Cu, Zn and Pb) in the soil did not completely eliminate the rhizome's ability to regenerate. However, the presence of some metals (Cd, Pb and Zn) delayed the rhizome regeneration at the beginning of the experiment. A similar result was observed by Michalet et al. (2017). They investigated the impact of heavy metals on the rhizome regeneration and growth of the Fallopia genus occurring in the early stages of vegetative propagation, as well as its impact on belowground secondary metabolism. According to Michalet et al. (2017), the delay in the rhizome regeneration of knotweeds induced by the heavy metals is due to plant investment in the production of secondary metabolites than in primary metabolites needed for cell and tissue multiplications.  metal in soils (20 mg·kg -1 ) did not have a toxic effect for plants (Fig. 3).

Third, the analysis of the metal concentrations
in plant tissues showed a high potential of F. japonica for heavy metal uptake and their intensive accumulation in the aboveground (in the case of Cd and Zn) and underground parts (Cr, Cu, and Pb). The capacities of Fallopia japonica to accumulate heavy metals (such as Cd, Pb, Zn) under field conditions were proven by Böhmová & Šoltes (2017) or Rahmonov et al. (2019). Metal accumulation has been recognized as an extreme type of physiological response to heavy metal tolerance (Shaw 1989). Comparing the results of this experiment with the data from the literature, cadmium was removed particularly efficiently in this experiment. In the leaves of plants from the soils polluted by 100 mg Cd·kg -1 , the concentration of this metal exceeded 700 mg·kg -1 (dry weight). This value is much higher than the range of critical concentrations in plant tissues of 5.0 to 30.0 mg Cd·kg -1 dry weight that has toxic effects in most vascular plants (Radojevic and Bashkin 2006). Especially high levels of cadmium were found in the aerial parts of Brassica juncea and Brassica napus. When exposed to 10 mg·kg -1 Cd, the shoot Cd concentrations were 238 and 239 mg·kg -1 for Brassica juncea and Brassica napus, respectively, which was approximately 23 times higher than that of plants grown in the control substrate (Alvarez et al. 2009).
The results of the presented greenhouse experiment arouse many questions connected with Fallopia japonica tolerance to heavy metals. At present, the most interesting of them is: how does this characteristic support intensive Fallopia japonica expansion on the metal-contaminated areas? It is suspected that it facilitates the ability of Fallopia japonica to compete with native species and promotes its invasion of such places. These issues must be supported by another years of field research, which are planned to be carried out in the future.

CONCLUSIONS
On the basis of the results of the presented research, the following conclusions can be drawn:
2. Metals such as Cr or Pb do not have a toxic influence on plants' development, which was observed in this experiment. In the soils contaminated with any level of Cr or Pb, the shoots of this plant grew with similar vigour to the control plants and had the same morphological features as control. High doses of Cd (100, 200 mg·kg -1 ), Cu (300 mg·kg -1 ) and Zn (300 mg·kg -1 ) can delay the rapid growth of Fallopia japonica. In the greenhouse experiment, the Cd, Cu and Zn toxicity had also a significant impact on the morphology of Fallopia japonica.
3. An increased concentration of all metals in soil stimulated their increase in plants. This fact proves that Fallopia japonica accumulates heavy metals effectively when grown in contaminated soil. It was particularly observed in the case of Cd. In the aboveground parts of the plants from the pots with the addition of Cd at 100 mg·kg -1 , 630 times more Cd was found in comparison with the control.
4. The capacities of this invasive weed to regenerate from rhizome fragments, to grow and develop under the stress conditions created by toxic doses of some metals, and to take up metals, all of which were shown in this study, are the evidence that Fallopia japonica has metal tolerance.