Allelopathy Journal 33 (2): 267-276 (2014) Table: -, Figs : 4 0971-4693/94 US $ 5.00 International Allelopathy Foundation 2014 Phytotoxicity and accumulation of secondary metabolites in Heracleum mantegazzianum (Apiaceae) L. BALEZENTIENE* and M. RENCO1 Aleksandras Stulginskis University, Studentu 11, Akademija, LT-53361 Kaunas, Lithuania E. Mail: ligitaba@gmail.com (Received in revised form: January 2, 2014) ABSTRACT The phytotoxic effects and total phenols content (TPC) of H. mantegazzianum was tested on perennial ryegrass (Ryegrass perenne) and winter rapeseed (Brassica napus) seed germination to find their response ex situ. H. mantegazzianum leachates inhibited the germination. The seeds of oil rapeseed germination was 82% in lowest concentration (0.02% w/v) and 0% in most concentrated leachates (0.2% w/v). Nonetheless, the 0.02% leachates of leaf and seed stimulated the ryegrass germination by 17 and 2% over the control, respectively. The TPC varied depending on the hogweed parts and leachate concentration. At 0.2% leachate concentration, the highest content of phenolic compounds accumulated in leaf (89.16 mg mL-1) and seed (67.54 mg mL-1), possibly due to intensive conversion of synthesized materials in these hogweed parts. Key words: Allelopathy, Brassica napus, germination, H. mantegazzianum, phenolics, rapeseed, ryegrass, Ryegrass perenne INTRODUCTION A tall forb giant hogweed [Heracleum mantegazzianum Somm. et Lev., syn. Heracleum speciosum Weinmann, Spondylium pubescens Hoffmann and Pastinaca pubescens (Hoffmann) Calestani, etc.)] [family Apiaceae (Magnoliophyta)] originated from the Western Greater Caucasus (Russia, Georgia) and is now most dangerous invasive species throughout Europe (33-35). The Heracleum mantegazzianum Somm. et Lev. is one of the three Heracleum spp. found in most Central Europe countries with phototoxic effects due to photoactive furanocoumarins injurious to human and animal health, as well as suppresses the local biodiversity. Recently it has become widely naturalized and invasive throughout Western Europe, North America, Australia, New Zealand, etc. with a continuing increase in its distribution (31,36,37). H. mantegazzianum is dangerous invader in about two-thirds of German districts, (34). In Czech Republic, the front of populations of H. mantegazzianum are advancing at 10 m per year (27,28, 30). H. mantegazzianum has also invaded the Slovakia from Ukraine by seed. Its individual plants over 12 years old have been reported from extremely dry localities. Though mature plant is strictly monocarpic and dies after flowering, but there are some reports of polyc*arpy. At maturity stage the plant has accumulated enough resources for reproduction. It develops *Correspondece author, Aleksandras Stulginskis University, Studentu 11, Akademija, LT-53361 Kaunas, Lithuania 268 Balezentiene and Renco generative stem upto 5 m in height and 10 cm in diameter at the base and each plant annually produces 10,000 -20,000 fruits. It forms pure stands, resulting in change in ecosystems diversity and landscape (31). The species has phototoxic effects by means of photoactive furanocoumarins, which could be injurious for human and animal health (16). This species is enlisted as invasive alien plants (23) and is strongly recommended to take measures to prevent its introduction and spread to new areas, or to manage unwanted populations. One of the mechanisms of invasive species success is the production and release of allelopathic compounds by invader that are harmful to plant neighbours in the introduced range (12). Moreover, plant biochemistry may also influence the invasions either directly (i.e. production of phytotoxins) or indirectly (i.e. by making a plant less payable to insects). Consequently, allelopathy is likely to play an important role in shaping the community structure of successive invaders after the successful establishing on the restored sites (17,32). Among the secondary metabolites, phenolic compounds influenced the interactions in communities thus playing an important role in plant-plant interference (3,19). These compounds also play important role in seed germination, plant development, growth, xylogenesis and flowering (8,13). Considering all above, we attempted to investigate into the allelopathic activity of H. mantegazzianum. This study aimed to assess the secondary metabolites namely total phenols content (TPC) and phytotoxic effects of H. mantegazzianum on perennial ryegrass (Ryegrass perenne) and winter rapeseed (Brassica napus) seed germination in Petri dishes and to record their response ex situ. MATERIALS AND METHODS Site of plant sampling and leachate preparation Invasive hogweed samples were collected from Slovakia for preparing the aqueous leachates for bioassay in laboratory of Aleksandras Stulginskis University, Lithuania. In 2013, the Heracleum mantegazzianum was collected in village Lekárovce, Slovakia (5 km from Ukraine border). The site of plant collection (48°36´29-58´´N, 22°08´15-24´´E, 106 m.a.s.l., soil was sandy-loamy, pH: 6.8) is adjacent meadow of Uh River. Plant leachates Plant parts were chopped into 0.5 cm long pieces and air dried. Ten g portion of each air dried plant part was immersed in 50 ml distilled water (0.2% w/v solution) in 15 x 20 x 5 cm plastic tray and kept at 25°C in an incubator. After 12 h, the aqueous leachates were filtered through Whatman No 1 filter paper. The aqueous leachate was diluted to concentrations of 0.2, 0.1, 0.05 and 0.02 % (w/v). These diluted leachates and a distil water control (0%) were used for germination assays. Germination bioassay Allelopathic activity of H. mantegazzianum plant parts was determined based on seed germination and recalculated to conventional coumarine units (CCU). The plants were sampled in summer to prepare aqueous leachates for bio-screening (Table 1). The Phytotoxicity of secondary metabolites in Heracleum mantegazzianum 269 biochemical (allelopathic) characteristics of H. mantegazzianum aqueous leachates were examined at maturity (end July). Principal (0-9) and secondary (0-9) growth stages were defined as per the universal BBCH (Biologische Bundesanstalt, Bundessortenamt and Chemical industry) Scale and coded using uniform two-digit code of phenologically similar growth stages of all monocot and dicot plant species (20). The plant samplings were done at 50% maturity of plants. Germination was recorded at 50% seeds germination (G50) in distilled water (control). The Control G50 rate was equated to 100%. This method enables to assess both inhibitory and stimulatory effects of leachates. Owing to quick and high germination and phytotoxins, oil rapeseed (Brassica napus L., Dicot) cv. Kasimir (NPZ / Saaten-Uninio, Germany) and perennial ryegrass (Lolium perenne L., Monocot) cv. Sodrė were chosen as acceptor plants (7,39). These species are ideal to assess the allelochemicals impact in bioassays (5). Fifty seeds were placed on filter paper in each 6-cm dia Petri dish and 5.0 ml aqueous plant leachate (0,0.2,0.1,0.05 and 0.02% concentration, w/v) was added as per treatment. Distilled water served as control. Treatments were replicated four times. Petri dishes were kept at 26°C for 16 h in incubator. Germination was considered when radicle emerged from the seed coat. Seed germination rate was used to calculate the allelopathic potential of aqueous leachates in conventional coumarine units (CCU) (10) and then universal index of allelochemicals activity - CCU, was evaluated by nomogram. Phenolic compounds Total phenolics content (TPC) in leachates were determined by Singleton and Rossi’s method (29). To determine the TPC, standard curve with chlorogenic acid (C16H18O9, C3878, Sigma, Aldrich, Germany) was prepared. One ml of 0.2-0.02% leachate solution was mixed with 45 ml distilled water. One ml of Folin-Ciocalteu reagent (Merck, Darmstadt, Germany) was added and mixed thoroughly. After 3 min, 3 ml of Na2CO3 was added and then the mixture was kept for 2 h. The absorbance was measured at 760 nm. Samples were analyzed in two replications. Identification and quantification of individual target polyphenolic compounds was done by UV-Vis spectrophotometry (Bechman DU40, Germany). To evaluate the effects of selected chemicals as a standard equivalent on total phenolics in H. mantegazzianum, the content was calculated on the basis of standard curve of chlorogenic acid. Equivalent value was calculated by multiplication of absorbance of each sample by a single value of equivalent chemical weight per absorbance unit determined under the same condition. In crude leachates, each concentration TPC of H. mantegazzianum was expressed on fresh weight basis as milligram per g chlorogenic acid equivalent (CAE). Statistical analysis The confidence limits of the data were based on Student theoretical criterion. Standard errors (SE) were calculated at statistical significance of p < 0.05. Correlations between germination, TPC and CCU were calculated. The results of allelopathic effects were statistically evaluated by using the statistical package STATISTICA of Stat Soft for Windows standards. Results of germination, phenols concentration and CCU are presented as a mean ± SE of 4 independent analyses (n=4) at the ≤0.05 probability level. 270 Balezentiene and Renco RESULTS AND DISCUSSION The leachates of H. mantegazzianum inhibited the seed germination of oil rapeseed and perennial ryegrass (Fig. 1,2). Though leachates phytotoxicity differed on tested acceptor-species, yet the common trend was observed. The germination of both acceptor species depended on H. mantegazzianum plant parts and their leachates concentration. Oil rapeseed: H. mantegazzianum leachates inhibited the rapeseed germination (Fig. 1). The most variability in germination (0-82%) occurred in seed leachates. It ranged between 82% in lowest concentration (0.02% w/v) and 0% in most concentrated leachates (0.2% w/v), possibly due to the increased total phenolics concentration (Fig. 5). Complete inhibition of seed germination occurred in 0.2% leaf leachates. Figure 1. Impact of H. mantegazzianum leachates on oil rapeseed germination Except 0.05 and 0.1% root leachates, all tested leachates inhibited the seed germination over Control (Fig. 1). Leachates of root (38-94%), stem (46-90%) leaf (66100%) and seed (18-100%) were inhibitory. These findings correspond with inhibitory effects of other invasive hogweed species, i.e. H. sosnovskyi (2). Ryegrass: Unlike dicot rapeseed, monocot ryegrass was less sensitive to hogweed leachates possibly due to less permeability of seed coat and the faster germination (Fig. 2). Although, leachates had variables effects on ryegrass germination. Most leachates inhibited the ryegrass germination, but 0.02% leaf and seed leachates stimulated the germination than control. The leaf and seed leachates did not cause complete inhibition of ryegrass’ germination, unlike complete inhibition of rapeseed. The different germination response may be due to seed size, seed structure and differences in permeability of seed coat (18). Seed germination and seedling emergence are biological events initiated by Phytotoxicity of secondary metabolites in Heracleum mantegazzianum 271 water imbibition followed by enzymatic metabolism of stored nutrients. The environment and quality of seed affects these events. The quick germinating rapeseed need shorter period for G50, whereas ryegrass needs longer G50. The seed coat (Testa) structure responds to scarification for improving the germination (21,38). The thick, lignified seed coat of ryegrass was more impermeable to leachates and reduced the phenols availability to embryo than rapeseed. Hence germination was lower in ryegrass than rapeseed in tested leachates. Figure 2. Impact of giant hogweed leachates on germination of perennial ryegrass (mean ±SE) The stem and root leachates drastically inhibited the germination (24-44% and 2494 %, respectively) than control. The leaf leachates inhibited the germination (96%) as well as stimulated it (17%), possibly due to wide variations in leaf TPC (Fig. 3). 0.05-0.2 % seed leachates caused lowest inhibition (2-24%), however, 0.02% seed leachates stimulated the germination (2%). Stimulatory effects occurs at lower concentrations (14). Such stimulatory effects in 0.02% leaf and seed leachates of hogweed may be due to lower concentrations of allelochemicals and their different compositions. The functional activity of allelochemicals depends both on its concentrations and exposure time to acceptors-species (i). These results suggested that the invasive H. mantegazzianum may acquire spreading advantage in new territories through inhibitory effects on germination due to accumulated allelochemicals. Moreover, results ex-situ may not represent the natural field environment, hence, the more detailed bioassay in field could follow this ex-situ study to minimize the gap between lab and natural conditions. Total phenolics content and conventional coumarin units (CCU) Phenolics compounds play major role in ecosystems functionality and are involved in many plants interactions with their biotic and abiotic environment (9). They 272 Balezentiene and Renco can accumulate in different plant parts, tissues and cells during ontogenesis and under the influence of various environmental stimuli (13). TPC in hogweed parts was variable (8.86 and 87.98 mg mL-1) and exhibited different effects on germination of test species (Fig. 3). Strong correlations were found between TPC and germination of acceptor species, namely rapeseed (r= -0.8) and ryegrass (r= -0.7) due to impact of TPC on germination. The leachates TPC drastically inhibited the dicot rapeseed germination than monocot ryegrass germination, hence, germination was lower in rapeseed (30%) than in ryegrass (64%) in tested leachates. The highest accumulation of phenolic compounds was in leaf (89.16 mg mL-1) and seed (67.54 mg mL-1), possibly due to most intensive conversion of synthesized materials in these hogweed parts (Fig. 3). However the content of phenolics compounds are similar in two invasive allelopathic Heracleum species, [H. mantegazzianum and H. sosnovskyi (2)]. Figure 3. Total phenolics content in leachates of different plant parts of Heracleum mantegazzianum at maturity stage (mean ±SE) TPC levels were similar in root (10.04-50.37 mg mL-1) and stem (7.10-55.14 mg mL ) leachates. These findings of H. mantegazzianum correspond with lower TPC accumulation in all root leachates than in shoot leachates of 2-year old H. sosnowskyi plants during the vegetative growth (2). The 0.02% leaf leachates of lowest TPC (11.81mg ml-1) stimulated the germination of slow germinating ryegrass. Stimulation (ryegrass) or inhibition (rapeseed) in germination in same TPC leachates possibly depended on leachates composition (1), seed anatomy, germination duration and nature of test spp. (18,38). The thick, lignified seed coats of perennial ryegrass were more impermeable, which reduced the phenols availability to embryo than rapeseed. Consequently, lower TPC in root of H. mantegazzianum caused less inhibitory effects on dicots neighbour plants than leaf and seed. More specifically, higher phenolics content were in H. mantegazzianum leaf and seed, which drastically suppressed the dicots neighbouring species, than monocots grasses and thus help in spread of this invasive species. The higher content of phytotoxins in leaf -1 Phytotoxicity of secondary metabolites in Heracleum mantegazzianum 273 and seed of H. mantegazzianum caused maximum inhibition in rapeseed germination. These findings are in agreement with other findings (4,14). The phenolic compounds are striking example of metabolic plasticity enabling plants to adapt to changing biotic and abiotic environments (6). Moreover, phenolic compounds play major role in seed development and germination and thus may protect plants against various stresses. Consequently universal expression of TPC, induced by different phenolic compounds namely conventional coumarine units (CCU), might be used to compare the biochemical activity of various plant species (10). We have found that content of CCU varied with TPC. CCU content of H. mantegazzianum decreased from maximum 1364 in 0.2% leaf and seed leachates with the drastic inhibitory ffects on acceptor-rapeseed decreased to 4.3 in 0.02% leaf leachate with the highest stimulatory effects on acceptor-ryegrass (Fig. 4). Such variable response of test-species to CCU content may be possibly related to differences in anatomy and permeability of their seed coats. Figure 4. CCU content in giant hogweed leachates in response to test-species (mean ±SE) Among the various reasons of invasive species success in new environment may be its chemical interaction with recipient community due to the absence of tolerance of resident flora to new chemicals produced by the invader, e.g. H. mantegazzianum. The hypothesis, that allelopathy may be an important mechanism in the plant invasion may encourage development of general research models of invasive susceptibility in ecosystems. Invasive species H. mantegazzianum exhibited high biochemical activity due to the accumulation of phenolics compounds. For acceptor-rapeseed, the strongest phytotoxicity was observed from the 0.2% leachates of leaves and seeds at the flowering stage. The complete inhibition (0% germination) was induced by highest contents of phenolics (89.16 mg mL-1 and 67.54 mg mL-1 respectively) and CCU (1364). This significant relationship between acceptor rapeseed and ryegrass germination and CCU was confirmed by strong negative correlation (r=-0.8). 274 Balezentiene and Renco CONCLUSIONS It is not only giant size of H. mantegazzianum, vigorous uptake of nutrients and reproduction of high seed yield (22,26,33), but also biochemical activity that substantially contributes to its success. The results suggested that invasive plant species may induce ecological risk and acquire spreading advantage in new land by using ‘novel weapons’ to inhibit germination of neighbour plant species. 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