Allelopathic Activity of Canadian Goldenrod (Solidago canadensis L.) Extracts on Seed Germination and Growth of Lettuce (Lactuca sativa L.) and Garden Pepper Cress (Lepidium sativum L.)

Native to N. America, Canadian goldenrod (Solidago canadensis L.) was introduced to Europe as an ornamental plant and quickly spread here and in other parts of the world. The rapid spread of the plant is due to several reasons: phenotypic plasticity, broad climatic tolerance, propagation via underground rhizomes and seeds that mature in large numbers, etc. Additionally, the success of Canadian goldenrod’s invasion is determined by its allelochemicals that affect seed germination, root formation and whole growth of nearby plants. Allelopathy of various extracts and essential oils (EOs) of S. canadensis on seed germination and growth of lettuce (Lactuca sativa L.) and garden pepper cress (Lepidium sativum L.) was evaluated and compared with other Solidago species (S. virgaurea, S. × niederederi) collected from the same growing locality in Lithuania. Soil characteristics (conductivity, pH and major elements) of the collecting site were determined. Aqueous flower extracts of all studied Solidago species showed the highest inhibitory effect on model plants. Canadian goldenrod leaf water/diethyl ether extract showed highest inhibitory effect in all relative concentrations (1.0; 0.1; 0.01) suppressing growth of L. sativa (from 0 to 2.3 mm compared with 22.7 mm for control samples) and L. sativum (from 0.5 to 16.8 mm compared with 35.3 mm in control). It was noticed that garden pepper cress was more susceptible to Solidago spp. inhibitory effects than lettuce. S. canadensis root EOs comprised mainly of limonene (35.0%) and β-pinene (26.2%) and inflorescence oils containing α-pinene (21.6%), germacrene D (15.1%), limonene (10.2%) and lupenyl acetate (9.8%) exhibited the highest inhibitory effect on lettuce and garden pepper cress growth. Relative germination and vigor index of model plants was conducted. Chemical composition of extracts and EOs was determined by HPLC/DAD/TOF and GC/MS techniques.


Introduction
There are about 140 species of Solidago L. (Asteraceae) in the world, of which 115 are native to North America, around 8 species originate from Mexico, and about 13 species are indigenous to South America, the Azores, Europe and Asia [1]. All Solidago species found in local flora or cultivated as ornamentals are herbaceous and rhizomatous perennials (up to 2.5 m in height) with yellow ray florets.

Chemical Composition of Solidago canadensis Methanol/Water Extracts
Twenty-two compounds were identified tentatively in the leaf, inflorescence and root methanol/water (70:30 v/v) extracts of Canadian goldenrod (Table 3). All constituents were detected by DAD and TOF in positive or negative ionization mode. Some compounds provided m/z ions by both (positive and negative) ionizations.

Chemical Composition of Volatile Organic Compounds (VOCs) in Solidago canadensis Water/Diethyl Ether Extracts
The main compositional data of chemical analysis performed by GC/FID and GC/MS of S. canadensis extracts of different acidity obtained from various plant organs are presented in Table 4.

Chemical Composition of Solidago canadensis Essential Oils (EOs)
A gas chromatography (GC) analysis equipped with FID and GC/MS techniques (respectively for quantitative and qualitative purposes) was applied for chemical analysis of S. canadensis EOs obtained from various plant parts such as inflorescences, leaves and roots. The principal compositional data are presented in Table 5. In total, up to 98 compounds were identified in the EOs, comprising up to 95.4%.

Allelopathic Effects of Solidago canadensis Water/Diethyl Ether Extracts
The allelopathic effect of leaf, inflorescence and root etheric extracts of S. canadensis on seed germination and growth of Lactuca sativa and Lepidium sativum is presented in Figure 1.   (one-way ANOVA test, df = 9, p < 0.05). Data are presented as means of treatments (n = 60) ± SD (bars); asterix (*) indicates statistically significant difference compared to the control; letters (a, b and c) denote statistically significant difference between fractions (Tukey's test, df = 2, p < 0.05).

Relative Germination (RG) and
Vigor Index (VI) of Lettuce (Lactuca sativa L.) and Garden Pepper Cress (Lepidium sativum L.) Affected by S. canadensis, S. virgaurea and S. × niederederi Extracts Extracts of S. canadensis, S. virgaurea and S. × niederederi Khek (hybrid between S. canadensis and S. virgaurea) significantly affected seed germination and growth of tested lettuce (Lactuca sativa) ( Figure 2) and garden pepper cress (Lepidium sativum) (Figure 3). The root extracts of S. virgaurea showed the highest effect on lettuce and pepper cress's relative germination (RG) and vigor index (VI).

Relative Germination (RG) and Vigor Index (VI) of Lettuce (Lactuca sativa L.) and Garden Pepper Cress (Lepidium sativum L.) Affected by S. canadensis, S. virgaurea and S. × niederederi Extracts
Extracts of S. canadensis, S. virgaurea and S. × niederederi Khek (hybrid between S. canadensis and S. virgaurea) significantly affected seed germination and growth of tested lettuce (Lactuca sativa) ( Figure 2) and garden pepper cress (Lepidium sativum) ( Figure 3). The root extracts of S. virgaurea showed the highest effect on lettuce and pepper cress's relative germination (RG) and vigor index (VI).
Data of inhibitory effects of root, leaf and inflorescence aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi on germination rate (GR), relative germination (RG) and vigor index (VI) of lettuce and garden pepper cress seeds are presented in Tables S2-S4.

Allelopathic Effects of Water Extracts of Various Solidago Species: S. canadensis, S. virgaurea and S. × niederederi
The allelopathic effect of leaf, inflorescence and root aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi on the seed germination and growth of Lactuca sativa and Lepidium sativum is presented in Figure 4. Data of inhibitory effects of root, leaf and inflorescence aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi on germination rate (GR), relative germination (RG) and vigor index (VI) of lettuce and garden pepper cress seeds are presented in Tables S2-S4.

Allelopathic Effects of Water Extracts of Various Solidago Species: S. canadensis, S. virgaurea and S. × niederederi
The allelopathic effect of leaf, inflorescence and root aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi on the seed germination and growth of Lactuca sativa and Lepidium sativum is presented in Figure 4.   Data are presented as mean of treatments (n = 60) ± SD (bars); asterix (*) indicates statistically significant differences compared to the control; letters (a, b and c) denote statistically significant differences between various species (Tukey's test, p < 0.05).

Inhibitory Effect of Solidago canadensis EOs on Seed Germination and Seedling Growth of Lettuce (Lactuca sativa) and Garden Pepper Cress (Lepidium sativum)
The essential oils of S. canadensis significantly affected seed germination and growth of tested lettuce and garden pepper cress ( Figure 5). The EOs of Canadian goldenrod roots and inflorescences showed the highest effect on lettuce and garden pepper cress growth ( Figure 5A,C).

Inhibitory Effect of Solidago canadensis EOs on Seed Germination and Seedling Growth of Lettuce (Lactuca sativa) and Garden Pepper Cress (Lepidium sativum)
The essential oils of S. canadensis significantly affected seed germination and growth of tested lettuce and garden pepper cress ( Figure 5). The EOs of Canadian goldenrod roots and inflorescences showed the highest effect on lettuce and garden pepper cress growth ( Figure 5A,C).  , leaves-(B) and inflorescences-(C)) on germination and growth of lettuce and garden pepper cress (one-way ANOVA test, df = 3, p < 0.05). Data are presented as the mean of treatments (n = 60) ± SD (bars); asterisk (*) indicates a statistically significant difference compared to the control.

Discussion
It is known that S. canadensis L., being an expansive perennial weed, forms persistent, species-poor plant communities. According to the European habitat classification system (EUNIS), the growing sites (Lithuania, Vilnius, Trakai municipality, Lentvaris) of S. canadensis, S. virgaurea and hybrid S. × niederederi plants can be attributed to the category of anthropogenic herb stands with dry perennial anthropogenic herbaceous vegetation (EU-NIS2020 code V38 and E5.1) [62]. Diagnostic species of the growing locality was mugwort (Artemisia vulgaris L.); and characteristic species are the following: couch grass (Elytrigia repens (L.) Gould), mugwort (Artemisia vulgaris L.), common yarrow (Achillea millefolium L.), field bindweed (Convolvulus arvensis L. Around 50% of this territory was occupied by S. canadensis plants, distributed almost evenly over the area ( Figure S1 in Supplementary Materials). The rapid spread of the herb is due to several reasons. Canadian goldenrod plant has broad climatic tolerance; also it is tolerant to a wide range of soil nature and fertility, texture conditions and soil pH [63][64][65]. Another important aspect is plant lifecycle and dispersion. S. canadensis is a perennial rhizomatous hemicryptophyte and spreads both via seeds and underground rhizomes. The plant has a relatively long flowering time (from late July to mid-September or October). Individual clones are long-lived (can reach up to 100 years) and spread fast with each year. One sprout of Canadian goldenrod can mature up to 20 000 seeds per season. Seeds are responsible for long-distance dispersal and the colonization of unoccupied sites. However, rhizomatous growth is significant for the expansion of S. canadensis populations.
The soil of the growing habitat was investigated and characterized by some main parameters. Soil pH values ranged from 6.15 ± 0.07 to 6.65 ± 0.27 and conductivity was from 92.33 ± 0.55 to 125.63 ± 6.04 µS/cm (Table 1). According to the soil map of the National Atlas of Lithuania (the new FAO classification of Lithuanian soils (LTDK-99) has been adopted), typical alluvial soils have been found in this area. The elemental composition of the soil was typical ( Table 2) and concentrations of hazardous and heavy metals were below limitary values according to the Lithuanian health regulations (V-114 HN60:2004) [66].
The success of the Canadian goldenrod invasion may be explained by its allelopathic compounds. It is known that allelochemicals can be obtained from different parts of the plant (leaves, stems, flowers, buds, fruits and roots) and can affect seed germination and root formation on neighboring plants, as well as the growth of the whole plant. The allelopathic effect of S. canadensis on other plants has not been studied very widely. Thus, it is important to identify allelopathic compounds and their role in the spread of invasive species. Mainly polyphenolic acids and flavonoids were identified in the methanol/water extracts from S. canadensis inflorescences, leaves and roots ( Table 3). The identity of some compounds was confirmed by matching of chromatographic data with corresponding reference compounds. Most of the determined constituents were identified formerly in Canadian goldenrod extracts [32,35,36,[46][47][48][49]. The retention times of compounds varied slightly for each sample. Three compounds with a molecular mass of 354 were identified by both positive and negative ionization and by DAD as neochlorogenic, chlorogenic and 4-Ocaffeoylquinic acid in Canadian goldenrod leaf and inflorescence extracts. Feruloylquinic acid and narcissin were found only in the leaf extract, while caffeoylshikimic acid and its glucoside, cinnamic and ferulic acids were determined purely in the root extract. In addition, two saponins and cinnamtannin A2 were identified in the Canadian goldenrod root extract. No one constituent was determined in extracts from all plant parts. It should be mentioned that some common compounds such as afzelin, astragalin, kaempferol, nicotiflorin, quercetin, quercitrin and some others previously identified in S. canadensis extracts [32,35,36,46,48,49] were determined in the present study in small amounts or even under detection limits.
The inhibitory effects of S. canadensis leaf, inflorescence and root exudates on seed germination and growth of lettuce (Lactuca sativa) and garden pepper cress (Lepidium sativum) differed slightly. Canadian goldenrod leaf ( Figure 1B) and inflorescence ( Figure 1C) extracts were found to be the most active and seed germination of lettuce and garden pepper cress was inhibited up to 100% at a relative concentration of 1.0. However, the neutral leaf extract showed the highest inhibitory effect in all relative concentrations (1.0; 0.1; 0.01) suppressing growth of Lactuca sativa (from 0 to 2.3 mm compared with 22.7 mm for control samples) and Lepidium sativum (from 0.5 to 16.8 mm compared with 35.3 mm in the control group) (Figure 1).
For comparison, different extracts of various Solidago species were tested in order to evaluate allelopathic properties. Solidago canadensis, S. virgaurea and S. × niederederi extracts significantly affected seed germination and growth of tested lettuce ( Figure 2) and garden pepper cress (Figure 3). Solidago virgaurea root extracts have shown the highest effect on lettuce and pepper cress relative germination (RG) and vigor index (VI). At a relative concentration of 0.5, the RG of lettuce reached 53.57% ( Figure 2A); pepper cress (65.52%) ( Figure 3A) (G test, p < 0.05) and VI were 390.83 and 2829.79, respectively. At the highest tested concentration (1.0), germination RG was 0% for lettuce and 8.62% for pepper cress. Aqueous root extracts of S. × niederederi significantly affected only garden pepper cress RG at 1.0 concentration and seed germination was totally suppressed (Figure 3, A). Leaf extracts from S. × niederederi had an effect only on pepper cress's RG and were 67.86% and 73.21% at 0.5 and 1.0 relative concentrations (G test, p < 0.05) ( Figure 3B). Solidago canadensis and S. × niederederi inflorescence extracts affected lettuce ( Figure 2C) and pepper cress's RG and VI ( Figure 3C). However, S. × niederederi inflorescence extract (at relative concentrations 0.5 and 1.0) suppressed garden pepper cress seed RG completely ( Figure 3C) (G test, p < 0.05).
For comparative purposes, aqueous extracts of native and invasive Solidago species growing at the same locality were tested under laboratory conditions (Figure 4). Solidago canadensis, S. virgaurea and S. × niederederi extracts significantly affected the seed germination and growth of lettuce (Lactuca sativa) and garden pepper cress (Lepidium sativum). It is evident that Solidago's inhibitory effects differed on species. In the case of S. virgaurea, root and inflorescence extracts indicated the highest allelopathic activity, while for S. canadensis and S. × niederederi both leaf and flower extracts demonstrated similar inhibitory effects on model plants (Figure 4). The strongest inhibitory effects were caused by aqueous extracts of inflorescences of all tested Solidago species at 1.0 and 0.5 of relative concentration. In this case, seed germination and growth was reduced from 81.2 to 94.8% for lettuce (L. sativa); and 100% for garden pepper cress (L. sativum). The allelopathic effects of the plant extracts are implemented usually by using the whole plant or leaves only [49][50][51][52][53][54][55][56][57][58]. Contrary to this, our research confirmed the importance of studying individual morphological parts (leaves, flowering tops and roots) and the effects of their aqueous/etheric extracts. To the best of our knowledge, allelopathy of Solidago spp. root extracts were investigated for the first time. Aqueous extract of S. canadensis, S. virgaurea and S. × niederederi inflorescences showed the highest inhibitory effect on seed germination and growth of model plants (Lactuca sativa and Lepidium sativum). In comparison to parental plant species hybrid (S. × niederederi) also effectively suppressed germination and growth of the tested plants. Comparing model plants, it was noticed that garden pepper cress (Lepidium sativum) was more susceptible to Solidago sp. inhibitory effects than lettuce (Lactuca sativa).
A comparison of our results on allelopathy of Solidago spp. extracts with previously published data [49][50][51][52][53][54][55][56][57][58] is complicated for several reasons: different preparation of extracts, their chemical composition is not always known and various model plant species have been used for the tests.
The experimental data evaluated that S. canadensis EOs significantly affected seed germination and growth of lettuce (Lactuca sativa) and garden pepper cress (Lepidium sativum) ( Figure 5). Root EOs, comprised mainly of limonene (35.0%) and β-pinene (26.2%) and inflorescence oils, containing main components α-pinene (21.6%), germacrene D (15.1%), limonene (10.2%) and lupenyl acetate (9.8%) exhibited the highest inhibitory effect on lettuce and garden pepper cress growth ( Figure 5A). Root EOs at the highest concentrations (1.0 and 10 ppm) suppressed lettuce seed germination completely. Garden pepper cress seed growth was affected at all concentrations and plant lengths were from 24.85 ± 5.36 mm (at an EO concentration 0.1 ppm) to 21.63 ± 6.98 mm (at EO 1.0 ppm), and 0 mm (at EO 10 ppm) in comparison to 56.33 ± 8.08 mm for the control samples. Inflorescence EOs at the highest concentrations (1.0 and 10 ppm) suppressed lettuce seed germination and growth almost completely (plant lengths from 0.85 ± 0.81 mm to 0 mm, respectively) ( Figure 5C). Garden pepper cress seeds growth was affected at all concentrations and plant lengths were from 22.97 ± 8.20 mm (at EO concentration 0.1 ppm) to 15.98 ± 7.35 mm (at EO 1.0 ppm), and 0.15 mm ± 0.13 mm (at EO 10 ppm) in comparison to 56.33 ± 8.08 mm for the control samples. From this experiment, we can state that root and inflorescence EOs showed the highest inhibitory effects on model plant seed germination and growth. It is noticed that lettuce seeds were more susceptible to S. canadensis EO's inhibitory effects than garden pepper cress seeds. Our data are in correlation with allelopathy of EOs of aerial parts of S. canadensis collected from natural populations in Slovakia [24] and Poland [25]. The oils of different chemical composition (containing α-or β-pinene, germacrene D, limonene, thymol, epi-bicyclosesquiphellandrene, β-cadinene, γ-cadinene, δ-cadinene, α-or γ-muurolene, α-cubebene or β-elemene as main components) significantly inhibited seed germination and growth of garden pepper cress and other model plant species [24,25].

Soil Analysis
Preparation of soil samples for elemental analysis, conductivity and pH measurements was done in such way: all samples of the soil were dried at room temperature, later ground and sieved through a bolt (2.0-2.5 mm of perforation).
A mixture of 20 mL of sifted soil and 40 mL of deionized water was rested for 1 h in an ultrasonic bath; later the mixture was filtered and the conductivity of aliquots was measured using the conductivity and temperature meter AD3000 EC/TDC (Adwa, Szeged, Hungary).
Sample preparation for pH measurements was as follows: a mixture of 5 mL of soil and 25 mL of deionized water was shaken for 1 h in an ultrasonic bath, stored for 2 h and filtered; measurements of the aliquots were performed by pH-meter Orion 3 Star (Thermo Fisher Scientific, Waltham, MA, USA), calibrated using buffer solutions of pH 4.01, 7.00 and 10.04.
Elemental analyses were undertaken with the inductively coupled plasma-optical emission spectroscopy (ICP-OES) method. Procedure of sample preparation: 5 g of soil and 50 mL of 1 M HCl was stirred for 30 s, and then rested for 24 h. The mixtures were filtered and analysis was performed using the Optima 700 DV spectrometer (Perkin Elmer, Waltham, MA, USA).

Plant Material
Solidago canadensis, S. virgaurea and S. × niederederi plants (up to 2.5-3.0 kg) were collected at full flowering stage (in August 2020 and 2021) in a derelict field (Eastern Lithuania, Vilnius, Trakai municipality, Lentvaris, 54 • 38 53.5 • N 25 • 07 37.9 • E). The habitat is depicted on the geographic information system map ( Figure 6). The area of the investigated populations was up to 150 m 2 . Plants were collected randomly from four different sampling sites at full flowering stage. Raw material (above-and below-ground plant parts) was taken immediately to the laboratory and dried at room temperature (20-25 • C) under shade conditions for 2 weeks. Leaves, inflorescences and roots were separated before drying. and 50 mL of 1 M HCl was stirred for 30 s, and then rested for 24 h. The mixtures were filtered and analysis was performed using the Optima 700 DV spectrometer (Perkin Elmer, Waltham, MA, USA).

Plant Material
Solidago canadensis, S. virgaurea and S. × niederederi plants (up to 2.5-3.0 kg) were collected at full flowering stage (in August 2020 and 2021) in a derelict field (Eastern Lithuania, Vilnius, Trakai municipality, Lentvaris, 54°38′53.5° N 25°07′37.9° E). The habitat is depicted on the geographic information system map ( Figure 6). The area of the investigated populations was up to 150 m 2 . Plants were collected randomly from four different sampling sites at full flowering stage. Raw material (above-and below-ground plant parts) was taken immediately to the laboratory and dried at room temperature (20-25 °C) under shade conditions for 2 weeks. Leaves, inflorescences and roots were separated before drying.
Plant material was identified by Dr. M. Rasimavičius, and voucher specimens of S. canadensis, S. virgaurea and S. × niederederi were deposited at the Vilnius University Herbarium (WI, Vilnius, Lithuania) with code numbers P33666, P33663 and P33664, respectively.
Lactuca sativa and Lepidium sativum seeds were selected as the model plants. Seeds were bought from a local vegetable market.

Essential Oil Isolation
The essential oils from S. canadensis were isolated by hydrodistillation of dried material (up to 100 g each) in a Clevenger-type apparatus for 2 h according to the European Pharmacopoeia. The ratio of plant material to water was 1:20. A yellow-gray, greasy mass Plant material was identified by Dr. M. Rasimavičius, and voucher specimens of S. canadensis, S. virgaurea and S. × niederederi were deposited at the Vilnius University Herbarium (WI, Vilnius, Lithuania) with code numbers P33666, P33663 and P33664, respectively.
Lactuca sativa and Lepidium sativum seeds were selected as the model plants. Seeds were bought from a local vegetable market.

Essential Oil Isolation
The essential oils from S. canadensis were isolated by hydrodistillation of dried material (up to 100 g each) in a Clevenger-type apparatus for 2 h according to the European Pharmacopoeia. The ratio of plant material to water was 1:20. A yellow-gray, greasy mass with a sweet characteristic odor was obtained. Hydrodistillation yielded 1.25, 1.11 and 0.95% (v/w, on a dry weight basis) of EO from S. canadensis inflorescences, leaves and roots, respectively. Yields of the EOs slightly ranged according to the collection time (2020 and 2021). The obtained oils were dried over anhydrous sodium sulfate, kept in closed dark vials and stored in a refrigerator; the samples were diluted with a mixture of pentane and diethyl ether (1:1) before analysis.

Preparation of S. canadensis, S. virgaurea, S. × niederederi Extracts for Allelopathic Tests
Thirty g of crushed herbal material (separately: flowers, leaves and roots) and 500 mL of distilled water were kept at 40 • C for 24 h, then filtered and three portions of 25 mL were prepared. In order to obtain acidic (pH ca. 3) and alkaline (pH ca. 11) solutions, 0.1 M HCl and 0.5 M KOH were applied. An amount of 1.5 mL of the solution used for allelopathic tests was considered as 1.0 of relative concentration.
Then, each portion was extracted with 5 mL of diethyl ether for 5 min. Diethyl ether was evaporated. The dry residues were dissolved in deionized water before germination assay.

Preparation of S. canadensis Extracts for HPLC-DAD-TOF Analysis
Samples of air-dried flowers, leaves and roots of S. canadensis were ground into a homogenous powder and protected from light and humidity until analysis. Up to 1 g of crushed herbal material and 15 mL of solvent (mixture of methanol and water (70:30, v/v)) were used for extraction. The extraction procedure was performed in an ultrasonic bath at room temperature for 50 min. The mixture was filtered through a filter paper for qualitative analysis (pore size 11 µm) using nylon syringe filters (0.22 mm).

GC (Flame-Ionization Detector FID) Analysis
Quantitative analyses of the EOs and extracts were carried out on HP 5890II chromatograph equipped with an FID (Hewlett Packard, Palo Alto, CA, USA), using DB-5 ((5%-phenyl)-methylpolysiloxane; 50 m × 0.32 mm × 0.25 µm) and HP-FFAP (polyethylene glycol 30 m × 0.25 mm i.d., film thickness 0.25 µm) capillary columns (Agilent, J&W Scientific, Santa Clara, CA, USA). The GC oven temperature was programmed as follows: from 50 • C (isothermal for 1 min) increased to 160 • C (isothermal for 2 min) at a rate of 5 • C/min, then increased to 250 • C at a rate of 10 • C/min, and the final temperature was kept for 4 min. The temperature of the injector and detector was maintained at 250 • C. The flow rate of carrier gas (hydrogen) was 1 mL/min. At least 3 replicates per analysis were performed.
The conditions of chromatographic separation were the same as for GC (FID) analysis. The temperature of the injector and detector was 250 • C. The flow rate of carrier gas (helium) was 1 mL/min, split 1:20. At least 2 replicates per analysis were performed. The temperature of ion source was 220 • C. Mass spectra in electron mode were generated at 70 eV, 0.97 scans/second, mass range 33-400 m/z.

Identification of Individual Components
The percentage composition of the EOs and VOCs was computed from GC peak areas without correction factors. Qualitative analysis was based on comparison of retention indexes on both columns (polar and nonpolar), co-injection of some reference terpenoids (α-, β-pinene, 1,8-cineole, linalool, camphor, β-caryophyllene, α-humulene and caryophyllene oxide) and C 8 -C 28 n-alkane series; and mass spectra with corresponding data in the literature [61] and computer mass spectra libraries (Flavors and Fragrance of Natural and Synthetic Compounds 2 (FFNSC 2), Wiley and NIST). Identification was approved when computer matching with the mass spectral libraries showed a probability above 90%. The relative proportions of the oils and extracts constituents were expressed as percentages obtained by peak area normalization, all relative-response factors being taken as one.
The MS (TOF) acquisition parameters were as follows: mass range 100-1700 m/z, rate 1.42 spectra/s, time 704.2 ms/spectrum. Ionization source conditions were drying gas temperature 300 • C, drying gas flow rate 3 L/min, nebulizer 15

Allelopathy of Solidago canadensis EOs
Allelopathic activity of S. canadensis EOs (from roots, leaves and inflorescences, separately) was tested in vivo, using model plant seeds (Lactuca sativa and Lepidium sativum). Different concentrations of S. canadensis EOs dissolved in dimethyl sulfoxide (DMSO) were added. Control tests were performed with DMSO in deionized water (1 µL/mL).

Bioassay for Seed Germination
Lactuca sativa and Lepidium sativum seeds were surface sterilized (by 70% ethanol for approximately 2 min) and then thoroughly washed thrice with deionized water. The seeds (20 per dish) were maintained in the dark at 25 • C in glass Petri dishes (Ø 9 cm) containing a sheet of Whatman filter paper and 1.5 mL of test or control (DMSO 1 µL/mL in deionized water) solution for 5 days. Three replicates were performed for each concentration. The number of germinated seeds was counted after 5 days of incubation time, and each seed was considered to have germinated when the radicle emerged ( Figure S2  Twenty seedlings per Petri dish were measured after 5 days of incubation. Seedling root length (RL) and seedling height (H) were measured and are presented as plant length (PL). Germination rate (GR), relative germination (RG) and vigor index (VI) of L. sativa and L. sativum were determined according to the literature [67] (Table 6). Table 6. Calculation of germination rate (GR), relative germination (RG) and vigor index (VI) of L. sativa and L. sativum.

Statistical Data Analysis
The obtained results were statistically processed by calculating the Pearson correlation coefficient (r); the results were expressed as mean values, range intervals, and standard deviation (SD) values, using XLSTAT (trial version, Addinsoft 2014, Paris, France).
The PAST version 4.03 was applied for data statistical analysis, to determine reliability and significant differences. The comparison of independent groups was performed by one-way ANOVA test. Differences between the control and treatment groups were compared using Tukey test, applying a significance level of α = 0.05. The G test was used to determine statistically significant differences in the assessment of germination rate and relative germination. Statistically significant differences were set at p values equal to or lower than 0.05.

Conclusions
The ecosystem of invasive Solidago canadensis native to Lithuania and Solidago virgaurea and their hybrid S. × niederederi was investigated. Soil parameters and population characteristics of investigated area where three Solidago species grow collectively are defined in detail.
Allelopathic effects of the plant aqueous extract are usually implemented by using the whole plant or leaves only. However, our research confirmed the importance of studying individual morphological parts and the effects of their extracts and EOs. Aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi flowers showed the highest inhibitory effect on model plants (Lactuca sativa and Lepidium sativum) seed germination and growth. In comparison to parental plant species, the hybrid (S. × niederederi) also effectively suppressed tested plants' germination and growth. When comparing model plants it was noticed that garden pepper cress was more susceptible to Solidago inhibitory effects than lettuce.
Root and inflorescence EOs of S. canadensis showed the highest inhibitory effects on model plant seed germination and growth. It was noticed that lettuce seeds were more susceptible to Canadian goldenrod EO inhibitory effects than garden pepper cress seeds. Allelopathic properties of S. canadensis are related with the chemical composition of various extracts and EOs.
This study revealed that it is important to investigate allelopathic mechanisms and to improve our understanding about its role in the spread of invasive species. Furthermore, we recommend to control non-native goldenrods before the flowering period and not leave removed biomass during the restoration process of territories which are colonized by these plants. Obtained data could be important for further regulation and monitoring of the spread of invasive Solidago species.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12071421/s1. Table S1. Minor constituents (<3.0%) identified in inflorescence, leaf and root EOs of S. canadensis. Table S2. The effect of root aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi on germination rate (GR), relative germination (RG) and vigor index (VI) of lettuce (Lactuca sativa L.) and garden pepper cress (Lepidium sativum L.) seeds. Table S3. The effect of leaf aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi on germination rate (GR), relative germination (RG) and vigor index (VI) of lettuce and garden pepper cress seeds. Table S4. The effect of inflorescence aqueous extracts of S. canadensis, S. virgaurea and S. × niederederi on germination rate (GR), relative germination (RG) and vigor index (VI) of lettuce and garden pepper cress seeds. Figure S1. Sampling site of plant material of Solidago species (S. canadensis, S. virgaurea and S. × niederederi) in Eastern Lithuania (Vilnius, Trakai municipality, Lentvaris). Figure S2. Control samples of Lactuca sativa L. seeds (A) and L. sativa seeds after treatment of aqueous S. canadensis root extract (B). Control samples of Lepidium sativum L. seeds (C) and L. sativum seeds after treatment of aqueous S. canadensis root extract (D).