Potential Phytotoxic Effect of Essential Oil of Non-Native Species Impatiens parviflora DC.

Impatiens parviflora is non-native invasive plant species occupying large areas all over the Europe and threatens native communities by altering their species composition and reducing native biodiversity. The factor responsible for its spreading could be explained by releasing biochemical to the environment. On the other hands, high demand on secondary metabolites as potential source of new ecofriendly biocides could be beneficial. The analysis of I. parviflora essential oil (EO) led us to identify more than 60 volatiles. The main compound was hexahydrofarnesyl acetone, other dominant components were phytol, carvacrol, germacra-4(15),5,10(14)-trien-1-α-ol, and pentacosane. The potential phytotoxic effect of I. parviflora EO collected in two vegetation periods (summer and autumn) was evaluated on seed germination and root elongation of three dicot species (Raphanus sativus, Lepidum sativum, and Lactuca sativa) and on one monocot species (Triticum aestivum). The seed germination of only one dicot species, L. sativa, was affected by both EOs. In contrast, seed germination of monocot species T. aestivum was influenced only by the highest doses of EOs isolated from I. parviflora in autumn. The root elongation of tested plant species was less influenced by I. parviflora EOs. L. sativum showed sensitivity to one dose of EOs hydrodistilled in summer, while the monocot species was influenced by both EOs samples in highest doses. Our findings revealed that I. parviflora contained phenolics that were phytotoxic to the germination of some plant species, mainly at higher EOs doses, while root elongation of tested plants was not suppressed by essential oils.


Introduction
Impatiens parviflora DC. (small balsam) belongs to the Balsaminaceae family and is native to Eastern Siberia. The species spread in many countries cross the Europe as well as in Canada [1][2][3]. Slovak State Nature Protection agency and the European Union categorize this species as a non-native species. Different studies focus on its distribution and ecology as invasive species [3][4][5][6][7][8]. I. parviflora undoubtedly has an environmental influence on native biodiversity through changes in community structure, nutrient cycles, trophic levels, hydrology, and competition act as an invasive species [6,9]. According to Slovak legislation, I. parviflora is considered as a dangerous species with the potential to adversely affect natural ecosystems. It is assumed that it was introduced to the Europe by trading as ornamental plant as most of the invasive species. First observation of the I. parviflora in Europe is The aim of the present work was to determine (1) the content and composition of EO of I. parviflora isolated from the population collected in Slovakia twice within vegetation season and (2) the effect of different EO doses on the seeds germination and root elongation of four model plant species (Raphanus sativus L., Lepidium sativum L., Lactuca sativa L., and Triticum aestivum L.). We hypothesized variability in chemical composition of EO hydrodistilled from Slovak and Poland population of I. parviflora due to different climatic conditions, extraction methods, growth stage, etc., as well as different impact of EO doses on seed germination and root elongation of some test plant species.

Phytotoxic Effect
The biological effect of EO hydrodistilled from I. parviflora was investigated in different months (June and September) from the same locality. There were effects on (a) seed germination and (b) root elongation of three dicot species (Raphanus sativus L., Lactuca sativa L., and Lepidium sativum L.) and one monocot species (Triticum aestivum L.). In general, no significant effect of all applied EOs concentrations of I. parviflora was found on seed germination of R. sativus and L. sativum (p = 0.05). In contrast, seed germination of L. sativa was significantly reduced by different concentrations of EOs in comparison to the untreated control. Significant effect of inhibition of seed germination was evident in all concentrations of EOs extracted from I. parviflora collected in June (IP1) (p = 0.05). The highest impact was found at the 0.125 µg/mL EO concentration, where the number of germinated seeds was 50% lower (5.0 ± 1.0 germinated seeds) in comparison to control. However, treatments with IP2 EOs significantly reduced L. sativa seed germination only at doses 0.125, 0.250, and 1.250 µg/mL (p = 0.05).
Each value is an average of 3 replications; SD = standard deviation; the letters a,b,c in each column present statistical differences according to Least Significant Difference Test (p = 0.05).
Root elongation of L. sativum was significantly reduced, but only at one applied dose (0.625 µg/mL) of IP1 EO. In contrast to negative impact of tested doses of EO on seed germination, no inhibitory effect on root elongation of L. sativa and R. sativus has been recorded (p = 0.05). The highest applied dose (2.5 µg/mL) of IP1 an IP2 EOs significantly inhibited root elongation of monocot plant seeds of T. aestivum (p = 0.05) ( Table 4). Besides the expected inhibitory effect of tested EOs on root elongation of tested plants, a moderate stimulatory effect of some EO concentrations was found, however without statistical differences. There have not been found significant influence of the seasonal changes in EO composition and germination or root elongation of model plants. Different EOs and different concentrations have variable influence on different plant species.

Discussion
To the best of our knowledge, this is the first study that has investigated the biological activity of EO and the second study to analyze the composition of the EO of I. parviflora. Our results can be useful to explain the ecological role of I. parviflora, and its EO could be considered as a potential factor contributing to its spread in new areas. Surprisingly, the content and composition of I. parviflora EO collected in Slovakia differs from Poland [28]. While the yield of EO (w/w relative to dry material weight) of I. parviflora from Poland was 0.24% [28], the amount of I. parviflora EO collected in Slovakia was much lower (0.11-0.14%). Species of the genus Impatiens are not essential oil bearing plant; they contain only traces of volatiles. Dominant compounds identified in Polish population of I. parviflora EO were (E)-hex-3-en-1-ol (16.8%), linalool (15.1%), benzaldehyde (10.2%), followed by hexanal (5.3%) and hexanol (4.9%); while in Slovak population only linalool (1.1% in IP1 and 0.2% in IP2) and hexanal (0.2% in sample IP2) were found, but in very small concentration. Interestingly, we revealed presence of safranal in I. parviflora essential oils, allelochemical mainly present in saffron plants [31].
Based on this and previous study [28] we can observe strong differences in essential oil composition not only between selected Impatiens species, but also within one species, growing in a different areas (Slovakia and Poland [28]). If we compare the volatiles detected in current and already published study [28], we can observe that the I. parviflora oils are composed mainly of oxygenated compounds; in the examined oils, mainly oxygenated derivatives of sesquiterpene and monoterpene hydrocarbons as well of C13-norisoprenides, while in a previous study of oxygenated compounds, aliphatic hydrocarbons (42.8%) and monoterpenes (24.7%). What is interesting is that some C13-norisoprenides, like β-ionone and it epoxide as well cisand trans-rose oxides, were identified in both oils. Moreover, hexahydrofarnesyl acetone-the main volatile in EO of current study (15.7% and 31.0%)-was also observed in oil isolated from plant growing in Poland, but in much lower quantity (0.4%). From the main compounds-hex-3E-en-1-ol (16.8%), linalool (15.1%), benzaldehyde (10.2%), hexanal (5.3%) and hexanol (4.9%) identified in a previous study [28]-only linalool and hexanal were detected in the examined essential oils. A common feature of I. parviflora oils is that numerous groups of n-alkanes, as well of non-terpene aliphatic alcohols, aldehydes, esters, and acids, were detected.
The dominant compound, hexahydrofarnesyl acetone, identified in our samples have had only 0.4% content in Poland population of I. parvilfora [28], but was dominant in EO hydrodistillated in Hippuris vulgaris, which is in high risk of extinction in Italy [32], as well as in medicinal species Impatiens balsamina [28]. As main components were identified also in other plant species, for example in the SDE extract from the flowers of Trifolium repens L. grown in Poland [33], in fresh needles and branches of Torus baccata L. in Serbia (Gymnospermae) [34], or in leaves and flowers of Zizyphus jujube from Egypt [35].
The composition of EO highly depends on climatic conditions, plant growth stage, collection time, drying conditions, distillation method, etc. [36]. Within two collections (IP1 and IP2) in the 2017 vegetation season there have been observed changes in quantity and quality of EO. Considerable differences in the qualitative and quantitative composition of the EOs obtained from the same plant species of different ages were also revealed in several previous studies [37][38][39]. However, the total character of EO is influenced by genetic factors confirming that the potential to produce a certain chemical pattern is genetically coded, but the gene expression will be introduced or also repressed by environmental factors [40,41].
The allelopathic effect of I. parviflora water extracts of the aerial plant parts was already observed in Sinapis alba seeds [1,30]. Similar, the water and methanol extracts from familiar species I. glandulifera and I. noli-tangere on seed germination of Brassica napus as well as S. alba was evaluated [30]. The effect of the water extract of I. glandulifera was also compared on germination and seedling growth in B. napus, dicot, and Triticum aestivum, monocot [17]. Inhibition by those extracts was recorded stronger for B. napus than that for T. aestivum. Previous findings are in accordance with our data when seed dicot species L. sativa in germination and R. sativus in root elongation were more sensitive than monocot T. aestivum. A possible reason for this difference is various seed coat anatomies and thus its permeability.
The phytotoxic effect of the EO of different plant species is generally attributed to the presence of the main components. As there is no evidence about the hexahydrofarnesyl acetone phytotoxicity, the results of several previous studies show the significant influence of carvacrol and linalool. Herbicidal properties of Zataria multiflora EO are attributed to their major components carvacrol and linalool [42] as well as carvacrol along with thymol tested as a standard compounds were suggested as alternative pesticides, herbicides, as well as insecticides [43]. Comparable studies were done to evaluate phytotoxic effect of EO of invasive or non-native plants. Five populations of invasive goldenrod species (Solidag gigantea and Solidago canadensis) were investigated in east Slovakia. The samples significantly inhibited seeds germination of L. sativa while adverse effect was observed when Solidago spp. EO significantly stimulated the radical elongation of R. sativus as well as L. sativa [44]. Another study evaluated phytotoxicity of volatiles produced by alien species H. mantegazzianum to model plants. EO hydrodistilled from the alien plant species presented biological activity on the dicot and monocot plant species. The most sensitive was Lactuca sativa compared to Lepidium sativum and Raphanus sativus in seed germination as well as in root length elongation. Stimulation effect was visible in both root length and root number at two or one highest doses, respectively in monocot species Triticum aestivum L. [45].
Besides the experiments which could collaborate to explain important ecological role of invasive or non-native plant species, there is also interest to study their pesticide effect as a great source for ecopesticides/ecoherbicides. In the same way, I. parviflora evaporated methanolic extract was tested against green peach aphid (Myzus perssicae), an important insect pest of many plants [46]. I. parviflora repelled the aphid strongly and significantly. Other examples are Solidago gigantea and Solidago canadensis, two invasive weeds, which EOs have been also exploited as a potential source of green pesticides against Culex quin quefasciatus, Spodoptera littoralis and Musca domestica. Deep research of natural-cides, which are now known to be effective, selective, biodegradable, and less toxic to environment, would help decrease the negative impact of synthetic agents in environmental pollution [43]. EOs had been recognized as prospective, environmentally acceptable and active ingredients employed in IPM [20].

Plant Material
Non-native species Impatiens parviflora DC. was collected from a productive, undisturbed deciduous forest~100 years old, near the village of Skároš in south eastern Slovakia, central Europe (48 • 36.16 Collection was done two times within the vegetation season 2017 in different vegetation period. First collection was done on June 1st (IP1) when the first flowers generally show up. Second collection was done on September 17th (IP2) when the flowering and ripening seeds finalize. Plant material was dried in laboratory condition on thin layer on filtrate paper for 2 weeks until crushing. Thereafter it was placed for EO isolation.

EO Isolation
Approximately 30 g of dried aerial plant parts of each sample were cut in small pieces and subjected to hydrodistillation for 3 h in a Clevenger-type apparatus. Essential oils were stored in dark vials until tested and analyzed.

GC-MS Analysis
Essential oils were analyzed by GC-MS-FID in Institute of General Food Chemistry, Lodz University of Technology, Poland). The analysis was performed on a Trace GC Ultra coupled with DSQII mass spectrometer (Thermo Electron, Waltham, MA, USA). A simultaneous GC-FID and MS analysis was performed using a MS-FID splitter (SGE Analytical Science, Ringwood Victoria, Australia). Mass range was 33 to 550 amu; ion source heating: 200 • C; ionization energy: 70 eV. One microliter of essential oil solution (80% v/v) diluted in pentane: diethyl ether was injected in split mode at split ratios (50:1). Operating conditions for capillary column Rtx-1 MS (60 m × 0.25 mm i.d., film thickness 0.25 µm), and temperature program: 50 (3 min)-300 • C (30 min) at 4 • C/min. Injector and detector temperatures were 280 • C and 300 • C, respectively. Carrier gas was helium (constant pressure: 300 kPa).

Compounds Identification
The identification of compounds was based on a comparison of their mass spectra (MS) and linear retention indices (RIs, nonpolar column), determined with reference to a series of alkanes C8-C26, by comparing with those in MassFinder 3 [47,48]  . EOs were dissolved in distilled water/acetone 99.5:0.5 and diluted to prepare the desired concentrations. Distilled water/acetone 99.5:0.5 was used as control. Test seeds were surface sterilized in 95% EtOH for 15 s and rinsed triplicate in distilled water. Ten sterilized seeds were sown into each Petri dish (90 mm diameter) containing 5 layers of Whattman filter paper. In each Petri dish 7 mL of EO solution of different concentration or distilled water/acetone 99.5:0.5 was added. Each treatment was triplicated. The Petri dishes were kept in growth chamber (20 ± 1 • C, natural photoperiod, Sanyo, MLR-351H). Evaluation of germination and the radicle length (cm) was measured after 120 h.

Statistical Analysis
Data from the experiment were subjected to analysis of variance (ANOVA) by Least Significant Difference Test. Statistical analyses were performed using the PlotIT ver. 3.2 program (Scientific Programming Enterpises, Haslett, MI, USA).

Conclusions
I. parviflora is one of the most widespread invasive plants in the temperate and northern regions of Europe, especially in forest habitats. Our results showed that content and chemical composition of EOs isolated from tissues of I. parviflora (Slovak population) varied within vegetation season and was considerably different from those revealed in population from Poland. The EOs treatments at different doses affected seed germination of selected plant species, while root elongation was considerable less affected. The presence of carvacrol, one of the main compounds identified in EOs, whose phytotoxic effect was already manifested, shows that EO can be attributed as a possible factor responsible for the easy spreading of this invasive plant species. On the other hand, invasive plant species could present great source of the chemicals potentially used as ecofriendly biopesticides (herbicides, insecticides, fungicides, and bactericides), e.g., hexahydrofarnesyl acetone found as dominant compound in our I. parviflora plants. Future studies with hexahydrofarnesyl acetone may hold the answer to this question.
Author Contributions: M.M., J.J., and V.S. performed essential oil isolation, established phytotoxic activity experiment, and its evaluation; A.W.-B., D.K., and Z.G. performed GC-MS analysis; M.R. and P.Š. analyzed the data and performed statistical analysis; D.G. and J.P. conceived, designed, and managed the experiments, evaluated data, and wrote the paper; D.K. provided project administration, consulted the data, and wrote the paper.