Flora - Morphology, Distribution, Functional Ecology of Plants

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Seasonal dynamics of allelopathically significant phenolic compounds in globally successful invader Conyza
canadensis L. plants and associated sandy soil
Lola Djurdjevi Gordana Gaji, Olga Kosti, Sneana Jari, Marija Pavlovi, Miroslava Mitrovi, Pavle Pavlovi
Department of Ecology, Institute for Biological Research Sinia Stankovi, University of Belgrade, Bulevar Despota Stefana 142, 11060, Belgrade, Serbia http://dx.doi.org/10.1016/j.flora.2012.09.006 , How to Cite or Link Using DOI Permissions & Reprints
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Abstract
The seasonal dynamics of total phenolics and phenolic acids in the stems of the global invader Conyza canadensis, from March (young plants in the form of rosettes) to September (fruit abscission and the beginning of plant decline), and in sandy soil were monitored monthly in non-native areas. The highest amount of total free phenolics was found in its tissues (31,000 g g1) during the flowering and fruiting time (August). Bound phenolics peaked (up to 8443 g g1) during shoot elongation and intensive plant growth (MayJune) and in September. In the stems, bound phenolic acids (p-coumaric, ferulic, p-hydroxybenzoic, vanillic and syringic) have a maximum twice, in May and in August, with ferulic acid predominating (up to 951.6 g g1). Free phenolic acids in the plant's tissue

peaked in May (plant elongation). In the soil under C. canadensis, the amount of bound phenolics decreased between March and June, before increasing up to the full bloom phase of the plants (August). The amount of bound phenolic acids was several times greater than that of free ones, with maximum values in August. C. canadensis is a highly important source of phenolics in the ruderal phytocoenosis in new areas. In order to better explain the mechanisms of the spread and domination of invasive plants in non-native areas, in which allelopathy plays a decisive role, it is necessary to measure the production of allelochemicals in tissue and their accumulation in soil at the shortest possible intervals and link this with the phases of plant development.

Keywords
Allelopathy;

Conyza canadensis domination;

Degraded habitat; Globally successful invader; Phenolic acids; Phenolics

Introduction
Allelopathy has been explored as a potential driver of plant invasion in the context of the novel weapon hypothesis (NWH), part of which emphasizes that some invasive plants may have stronger allelopathic impacts on other species in introduced areas than in native areas because the former may not be as adapted to the specific allelochemcials of invaders as plants in the native range (Callaway and Ridenour, 2004). There is increasing evidence that many plant invaders interfere with native plants through allelopathy. This allelopathic interference may be a key mechanism of plant invasiveness ( [Bousquet-Mlou et al., 2005],[Dorning and Cipollini, 2006], [Callaway and Maron, 2006], [Inderjit et al., 2008], [Thorpe et al., 2009], [Pisula and Meiners, 2010], [Murrell et al., 2010], [McEwan et al., 2010] and [Kim and Lee, 2011]). Many results suggest that the inherently stronger competitive and allelopathic effects of invasive plants on plants in non-native areas than on plants from its native range may contribute to its invasive success. Invasive plants excel in their new ranges because they produce novel metabolites to which native species possess little resistance ( [Ni et al., 2010], [Thorpe et al., 2009], [Barto et al., 2010], [Orr et al., 2005] and [Hierro and Callaway, 2003]). Rice divided all the inhibitory compounds that are important in allelopathy (phytoncides and kolines), which are secondary metabolites in higher plants, into 15 groups of compounds (Rice, 1974). The group of allelochemicals that has been studied most are phenolic compounds, which are universally distributed in all plants, in all plant organs, as free and bound forms of which several thousand exist. They are part of the composition of lignin, the most abundant polymer in plants or are bound to cell

wall polysaccharides ([Harborne, 1980], [Bate-Smith, 1969], [Saunders and McClure, 1976], [Kgel, 1986], [Whitmore, 1976],[Djurdjevi et al., 1998], [Djurdjevi et al., 2004], [Djurdjevi et al., 2005a], [Djurdjevi et al., 2005b] and [Djurdjevi et al., 2011]). Phenolic compounds enter soil through being released from aboveground plant parts by precipitation, through glandular trichomes, through active leaching via roots, and through the microbial degradation of dead plant remains, mostly due to the degradation of lignin ( [Alexander, 1965], [Tang and Young, 1982], [Whitehead et al., 1983], [Sterling et al., 1987], [Grodzinsky et al., 1987],[Carballeira and Reigosa, 1999], [Reigosa et al., 1999a], [Kgel-Knabner, 2002], [Hyder et al., 2002a], [Hyder et al., 2002b], [Djurdjevi, 1991], [Djurdjevi and Oberan, 1998], [Djurdjevi et al., 1997], [Djurdjevi et al., 1999], [Djurdjevi et al., 2000] and [Djurdjevi et al., 2003]). In terms of their abundance and primary productivity, the dominant species in plant communities are of great importance as the main source of phenolics, and therefore have a significant effect on plant littersoil interactions. Soil fertility is influenced by phenolics from dominant plant or microbial sources not only because they are important precursors of stable soil humic substances, but also for their effects on soil nutrient dynamics (N, P, K, Mn, Fe, Cu, etc.), pH, ion-uptake, the rate of litter decomposition, the composition and activity of soil microbial communities, soil quality, soil C and N cycling, macro- and micronutrient availability, soil aggregation, plantwater relations and soil respiration (Djurdjevi et al., 2009). Phenolic toxins originating from dominant plants and accumulating in soil can lead to the inhibition of seed germination, as well as of root and seedling growth, photosynthesis, water and ion uptake of target plants, glyoxylic enzyme activities, etc. As a consequence of the negative effects of phenolics originating from the dominant species, there is a reduction in the abundance of the other species in a phytocoenosis or their complete disappearance, hence disturbing the existing biodiversity ( [Muller, 1966], [Lodhi and Rice, 1971],[Rice, 1974], [Rice, 1979], [Lodhi, 1975], [Mersie and Singh, 1988], [Lyu and Blum, 1990], [Li et al., 1992],[Aliotta et al., 1993], [Barkosky and Einhellig, 1993], [Souto et al., 1995], [Reigosa et al., 1999b], [Barkosky et al., 2000], [Muscolo and Sidari, 2006], [Hussain and Reigosa, 2011], [Hussain et al., 2011], [Timsina et al., 2011], [Djurdjevi et al., 2004], [Djurdjevi et al., 2005a], [Djurdjevi et al., 2008] and [Djurdjevi et al., 2011]). One of the globally successful invaders is Conyza canadensis L. (horseweed), originating from North America. Now it is widespread as a neophyte across almost the whole world (a cosmopolite). It was introduced into Europe in the 17th century and was first noted in Serbia in 1874 and in the vicinity of Belgrade in 1878 around houses, in gardens, along roadsides, in sand and fields ( [Pani, 1874] and [Pani, 1878]). In Serbia, it is a naturalized species, which spreads from ruderal to arable land ( [Gaji, 1975], [Jovanovi, 1994], [Vrbnianin et al., 2004], [Vrbnianin et al., 2008] and [Jari, 2009]). The introduced C. canadensiscovers large areas of degraded habitats on the sandy levees next to the Danube and Tami rivers, dominating the other species in the phytocoenosis with its aboveground mass and abundance. As it plays a significant role in pioneer communities in degraded habitats ( [Djurdjevi et al., 2011] and [Zaplata et al., 2011]), we monitored the seasonal dynamics during the vegetative season (MarchSeptember) and measured the amount of phenolic acids and

total phenolics at monthly intervals in the plant itself and in the soil under it, from the appearance of rosettes to the end of the generative phase, fruiting and plant decline. This is the first work to study the seasonal dynamics of phenolics in the tissue of the invasive dominant species C. canadensis and the soil under it. It is believed that through taking samples at monthly intervals throughout the development of C. canadensis from the rosette phase to that of seed abscission and plant decline, we will have a complete insight into the dynamics of phenolic compounds formed in the plant tissue and their translocation into the soil under it, as opposed to taking a sample once a year or on just few occasions during the vegetative season. This is of crucial importance for the study of the allelopathic capacity of this invasive species, and its effect on the other species in the phytocoenosis, and its survival in newly-invaded areas away from its native one.

Materials and methods

Investigated plant community and collection of plant material and soil samples
Conyza canadensis, a cosmopolitan agricultural and ruderal weed, occupies large areas of the sandy
levees at the confluence of the Tami and Danube rivers as a pioneer species, creating dense microcomplexes and dominating the other herbaceous species in the ruderal meadow phytocoenosis due to its aboveground mass and abundance. This species is among the first to colonize the bare sandy terrain and plays an important role in vegetation succession on the sands and in increasing biodiversity. The studied ruderal phytocoenosis with meadow elements dominated by C. canadensis is located on the upper ridge of a sandy levee alongside the River Tami, close to its confluence with the Danube, not far from Panevo (12 km north-east of Belgrade, Serbia). In addition to the dominant C.

canadensis, whose abundance and cover was 100%, a further 28 herbaceous plant species were also
found, but their abundance and cover were significantly less ( Djurdjevi et al., 2011). Plant material and soil samples were collected along the upper ridge of the levee from an area 1 km in length, 5 m in height and with a base width of 20 m. Five sampling plots (10 m 20 m) were selected from this transect along the levee ridge. At each plot 30 plants of the same height and growth stage of C. canadensiswere randomly sampled together with 5 samples of soil. Samples of plant material (aboveground plant parts) and soil were taken once a month in order to monitor the seasonal dynamics of phenolics, as follows: March (young plants in the form of rosettes, 12 cm in diameter), April (young plants in the form of rosettes, 510 cm in diameter), May (beginning of elongation, young plants, 1015 cm long), June (phase of intensive plant growth, plants up to 1.5 m in height), July (beginning of flowering), August (in full flower and beginning of fruiting, plants up to 2 m in height), September (fruit abscission, the beginning of plant decline). In terms of time, the development phases of the American species C. canadensis that plants go through in native areas (Weaver, 2001) coincide completely with the phases plants go through in introduced areas. The plant material was air-dried, ground and sifted through a sieve with 2 mm diameter holes. A surface layer of sandy soil (010 cm) was taken from those microcomplexes with complete domination of C. canadensisand with an absence of all other herbaceous plants. After the removal of visible plant

remains, the soil was air-dried at room temperature, ground and sifted through a sieve with 2 mm diameter holes.

Preparation of phenolics-containing extracts from Conyza canadensis

Both phenolic acids and total phenolics were extracted from plant material (5 2 g) with distilled water over a 24 h period. Water extracts were evaporated and the residues were dissolved in water, the pH adjusted to 2.0 with 2N HCl, and the mixture was transferred to ethyl acetate. This was then evaporated to dryness and the residue dissolved in 4 ml of 80% (v/v) MeOH (free phenolics) and used for HPLC analysis or stored at 20 C until use. Bound phenolics were prepared by boiling the residue in 2N HCl for 60 min before transferring to ethyl acetate.

Extraction of phenolics from soil samples

Free forms of phenolics were extracted from 5 30 g of dried sandy soil with distilled water over a 24 h period. The extracts were evaporated and the residues were dissolved in water, the pH adjusted to 2.0 with 2N HCl, and the mixture was transferred to ethyl acetate. This was then evaporated to dryness and the residue dissolved in 4 ml of 80% (v/v) MeOH (free phenolics). The residual soil was treated with 15 ml of 2N NaOH and, after boiling for 24 h, the mixture was acidified with concentrated HCl to pH 2.0. Phenolic compounds were transferred to ethyl acetate (3 50 ml) and were evaporated to dryness. The dry residue was dissolved in 4 ml 80% MeOH (bound phenolics), all procedures following Hennequin and Juste (1967), [Katase, 1981a] and [Katase, 1981b], and [Djurdjevi et al., 2007a] and [Djurdjevi et al., 2007b].

Determination of total phenolics

Total phenolics (free and bound) were measured by spectrophotometry (Shimadzu UV 160 spectrophotometer) using the Folin-Ciocalteu reagent (Feldman and Hanks, 1968). A standard curve was constructed using different concentrations of ferulic acid. Units of total phenolics were expressed in micrograms of ferulic acid equivalent per gram dry weight.

Determination of phenolic acids by HPLC analysis

Phenolic acids were detected between 210 and 360 nm using a Hewlett Packard diode array detector (HP 1100 HPLC system). Separation was achieved with a Nucleosil 100-5C18 column; 5 m; 4.0 mm 250 mm (Agilent Technologies, USA). A step-gradient of acetonitrile in water was used: 15% acetonitrile (5 min, gradient), 30% acetonitrile (20 min, gradient), 40% acetonitrile (25 min, gradient), 60% acetonitrile (30 min, gradient), 60% acetonitrile (35 min, gradient), and 100% acetonitrile (45 min, isocratic). In order to avoid tailing of the phenolic acids, 0.05% orthophosphoric acid was added to the solvents. The flow rate was 1 ml min1 and the injection volume was 5 l. The identity of the phenolic acids was determined by comparing the retention times and absorption maxima of known peaks with pure standards. p-Hydroxybenzoic and syringic acids (Acros organics, USA), and ferulic, vanillic and p-coumaric acids (Serva, Germany) were used as phenolic standards. Units of phenolic acids

were expressed in micrograms per gram dry weight. All analyses were performed in triplicates ( [Djurdjevi et al., 2007a] and [Djurdjevi et al., 2007b]).

Statistical analyses
The statistical evaluation of the differences in the total content of phenolics and the composition of phenolic acids in plant material and soil samples was performed with two-sided ANOVA tests. The data were processed using the Statistica 6.0 for Windows statistical package.

Results
Seasonal dynamics of total phenolics and phenolic acids in the tissue of the aboveground parts of Conyza canadensis
Fig. 1A presents the results of the seasonal measurements of the amount of total phenolics (free and bound forms) in the vegetative parts of C. canadensis from March, when the plants where in the rosette phase, to September, when the plants were in the phase of fruit abscission and the beginning of plant decline. The amount of total free phenolics gradually increased (from 1898 to 4973 g g1 between the rosette phase and the phase of intensive plant growth (March June). At the beginning of flowering and during the phase of full flowering and fruiting (JulyAugust), there is a sudden and multiple increase in the amount of total free phenolics in comparison to the previous phases (up to 31,000 g g1). In the C. canadensis stem, the amount of bound phenolics is several times lower than free forms and they are at their peak during the initial phase of elongation (May) and intensive plant growth (June), and also in September, during the phase of fruit abscission and the beginning of plant decline.
Fig. 1. Seasonal dynamics of total phenolics and phenolic acids (free and bound forms) in C.

canadensis plants (A total phenolics; B free phenolic acids; C bound phenolic acids; D percentage of
phenolic acids in relation to the total phenols). Notice the different y-axis scales. Figure options

In the tissue of this species, we detected 5 phenolic acids in their free forms ( p-coumaric, ferulic, phydroxybenzoic, vanillic and syringic), reaching their highest levels in May (the beginning of plant elongation). Ferulic and p-coumaric acids, which are derivatives of cinnamic acid, predominate, while p-hydroxybenzoic, vanillic and syringic acids (derivatives of benzoic acid) are present in lower quantities for the majority of the vegetation season (Fig. 1B). Bound forms of phenolic acids (Fig. 1C) peak twice, in May and August, when ferulic acid predominates (up to 952 g g1). The amount of bound phenolic acids in the tissue is several times higher than free forms, particularly at the beginning of plant elongation (May) right up to the phase of fruit abscission and plant decline (September). If one considers the percentage ratio of free and bound forms of phenolic acids to the total free and bound phenolics during vegetation (Fig. 1D), it can be seen that free acids reach their highest concentrations (16.7%) in May (the beginning of elongation), after which their amount

decreases sharply until July (the beginning of flowering); thereafter it increases gradually to 4.6% by the end of vegetation. On the other hand, the percentage of bound phenolic acids increases markedly between April (young plants in the form of rosettes, 510 cm in diameter) and August, during the phase of full flowering and the beginning of fruiting (34.7%). In summary, the trend of the seasonal dynamics of bound phenolic acids is opposite to that of free ones with their percentage in the aboveground parts of C. canadensis increasing up to the end of vegetation, while free acids decrease.

Seasonal dynamics of total phenolics and phenolic acids in sandy soil under Conyza canadensis
The results of the seasonal measurements of the amount of total phenolics (free and bound) in the soil underC. canadensis are given in Fig. 2A. Bound phenolics can be seen to have clear seasonal dynamics. Their amount falls rapidly from March (179 g g1), when the plants are in the rosette phase, to June (109 g g1), when the levels begin to increase again. On the other hand, changes in the amount of free phenolics are not so marked and they are at their lowest levels from May to July (the beginning of elongation and the beginning of the flowering of plants). In the soil, the amount of bound phenolics is several times higher than free forms, which is the case for the whole period of vegetation.
Fig. 2. Seasonal dynamics of total phenolics and phenolic acids (free and bound forms) in sandy soil under C.

canadensis (A total phenolics; B free phenolic acids; C bound phenolic acids; D percentage of phenolic
acids in relation to the total phenols). Notice the different y-axis scales. Figure options

Only three free forms of phenolic acids in the soil under C. canadensis have clear seasonal dynamics (p-coumaric, ferulic and syringic acids), i.e. their amounts increase from March, when the plants are in the rosette phase, to August or the phase of full flowering and the beginning of fruiting (Fig. 2B). Their amounts vary from 2 to 2.83 g g1 at the beginning of vegetation up to 5.24 g g1 during August. The amounts of p-hydroxybenzoic and vanillic acid do not change significantly during the period of vegetation and range from 0.57 to 0.99 g g1. The amount of bound phenolic acids in the soil falls insignificantly between March and April, after which there is a gradual rise until July (the beginning of flowering), with all five phenolic acids reaching their highest levels in August during the phase of full flowering and the beginning of fruiting (Fig. 2C). Ferulic (30.0 g g1), p-hydroxybenzoic (19.56 g g1) and vanillic acids (15.92 g g1) predominate. The percentage of free and bound phenolic acids in terms of the total free and bound forms of phenolics in the soil under C. canadensis during the vegetative period is shown in Fig. 2D. It can be seen that the level of free acids as a percentage of the total free phenolics increases dramatically between March, when the plants are in the form of rosettes, 1 2 cm in diameter, and July (the beginning of flowering), when free phenolic acids account for as much as 73. 7% of the total free phenolics in the soil. The percentage of bound phenolic acids gradually increases between April, when the plants are in the form of rosettes, 510 cm in diameter, and August, when they reach their maximum level of 43.5%. During the phase of fruit abscission and the beginning of plant senescence

(September), the percentages of free and bound phenolic acids in the soil are almost equal (35.1% and 30.6%, respectively).

Correlation between total phenolics and phenolic acids in the tissue of Conyza
canadensis and the soil substrate
Data on the correlation between total phenolics (free and bound forms) and phenolic acids (free and bound forms) in the stem of C. canadensis and the soil under it is given in Table 1. There is a positive correlation between total free phenolics in the plant tissue and the total bound phenolics and bound phenolic acids in the soil (p < 0.01). There is also a positive correlation between the total bound phenolics in the tissue of C. canadensis, on the one hand, and free and bound phenolic acids in the tissue of this species, on the other (p < 0.05). There is a significant negative correlation between total bound phenolics in the tissue of C. canadensis and total free phenolics in the soil (p < 0.001). The same is true for the correlation between the amount of bound phenolic acids in the tissue of the plant and the total free phenolics in the soil. As the amount of total free phenolics in the soil increases, so too does the amount of bound forms in it (p < 0.001). There is also a positive correlation between free phenolic acids in the plant, on the one hand, and bound phenolic acids in the plant and free phenolic acids in the soil, on the other (p < 0.05).
Table 1. Correlation coefficients between total phenolics and phenolic acids in C. canadensis tissues and associated sandy soil. CFPh CFPh CBPh SFPh SBPh CFPAs CBPAs SFPAs SBPAs 1.00 CBPh 0.10 1.00 SFPh 0.15 0.75*** 1.00 SBPh 0.50** 0.25 0.65*** 1.00 CFPAs 0.25 0.41* 0.28 0.35 1.00 CBPAs 0.15 0.41* 0.33* 0.05 0.34* 1.00 SFPAs 0.21 0.85 0.21 0.09 0.38* 0.02 1.00 SBPAs 0.51** 0.04 0.05 0.39* 0.19 0.26 0.27 1.00

*, **, ***Significantly different at 0.05, 0.01 and 0.001 level, respectively, by t-test (n = 5). CFPh, C.

canadensis free total phenolics; CBPh, C. canadensis bound total phenolics; SFPh, soil free total phenolics;
SBPh, soil bound total phenolics; CFPAs, C. canadensis free phenolic acids; CBPAs, C. canadensis bound phenolic acids; SFPAs, soil free phenolic acids; SBPAs, soil bound phenolic acids. Table options

Discussion
Seasonal dynamics of total phenolics and phenolic acids in the tissue of Conyza canadensis
In terms of allelopathy, Conyza canadensis is a species that has not been studied enough either in native areas or introduced areas. As a pioneer weed species, it dominates during the first few years and is of great importance in vegetation succession in abandoned fields and in anthropogenically disturbed landscapes ([Booth, 1941], [Keever, 1950], [Rice, 1974], [Rice, 1979], [Kapustka and Moleski, 1976], [Shaukat et al., 2003], [Gao et al., 2009], [Djurdjevi et al., 2011] and [Zaplata et al.,

2011]). As far as we know, this is the first work to study the seasonal dynamics of phenolic compounds which are of high allelopathic efficiency in the tissue of C. canadensis, during the plant's development phases and in the soil under it, in non-native areas. As a result of taking samples of plant material at monthly intervals, we were able to measure precisely the amount of total phenolics and phenolic acids in the aboveground parts of C. canadensis from the beginning of growth, when the plants were in rosette form, to fruit abscission and plant senescence. Our data on the seasonal dynamics of phenolics in the tissue of C. canadensis represent a step forward in the study of natural concentrations of phenolics as important allelochemicals of this in Europe and many other regions of the world alien species. According to literature surveys, allelochemicals in plants have usually been analyzed and measured only annually or three times a year in line with the seasons spring, summer and autumn (e.g., [Rice, 1974], [Rice and Pancholy, 1973], [Rice and Pancholy, 1974], [Gonzlez et al., 1995], [Hyder et al., 2002a], [Hyder et al., 2002b], [Gallet and Lebreton, 1995], [Gallet and Pellissier, 1997], [Gallet et al., 1999], [Reigosa et al., 1999a], [Djurdjevi et al., 1997], [Djurdjevi et al., 1998], [Djurdjevi et al., 2008] and [Wardle et al., 1998]). There are copious data which support the novel weapon hypothesis for the dominance of invasive plants, according to which many plant invaders interfere with native plants through allelopathy. Phenolic compounds [( )-catechin, apigenin and luteolin, chlorogenic gallic, caffeic, vanillic, p-coumaric, syringic and ferulic acids] synthesized in the tissue of an invasive species play a main role in this process ( [Cipollini et al., 2008], [Dorning and Cipollini, 2006], [Batish et al., 2006], [Thorpe et al., 2009], [He et al., 2009], [Hierro and Callaway, 2003], [Callaway and Ridenour, 2004] and [Callaway and Maron, 2006]). In the tissue of C. canadensis we found 5 phenolic acids in their free forms (p-coumaric, ferulic, p-hydroxybenzoic, vanillic and syringic), with highest concentrations in May (the beginning of plant elongation). Ferulic and p-coumaric acids, which are derivatives of cinnamic acid, predominated, while p-hydroxybenzoic, vanillic and syringic acid (derivatives of benzoic acid) were present in lower amounts for the majority of the vegetation season. The amount of free phenolic acids was several times lower in the tissue of C. canadensis than bound forms. Free phenolic acids can have a direct allelopathic effect on surrounding plants after having been released into sandy soil, while bound forms can only do so after the microbial degradation of lignin from dead plant parts. In the tissue of C. canadensis, up to 31 mg g1 of total free phenolics were measured during the plant's elongation phase and the phase of fruit abscission and plant senescence, and 5 phenolic acids, which accounted for 7.4% of the total phenolics. According to data from literature, the following phenolics are found in the stems: gallic acid, vanillic acid, syringic acid and catechol (Shaukat et al., 2003), eugenyl -Psd, scutellarin, luteolin-7-O--D-glucuronide, quercetin, quercetin-3-O--D-glucopyranosid and luteolin (Liu et al., 2011). Inflorescences of C. canadensis contain many more polyphenols, flavonoids (as apigenin) and some organic acids than leaves and shoots. Condensed polyphenols predominate in shoots (Vinogradova, 2007). We suppose that in our case, due to the higher amount of phenolics in C. canadensis inflorescences than in leaves and stems, there is an increase in the amount of free phenolics at the beginning of flowering and in the phase of full flowering and beginning of fruiting (21.3 and 31 mg g1, respectively) during July and August.

The trend of the seasonal dynamics of bound phenolic acids is opposite to that of free ones as their percentage in the aboveground parts of C. canadensis increased by the end of the vegetation season, while free acids decreased, which occurred due to incorporation into the structure of lignin, the most abundant polymer of woody and also slightly lignified herbaceous plants, like C. canadensis. Ferulic and p-coumaric acids are the main phenolic acids present in the cell wall of monocotyledons and especially of Poaceae. It seems likely that dimerization reactions occur in the cell walls when graminaceous plants are growing. Such a mechanism could be involved in cross-linking adjacent pcoumaroyl and feruloyl groups in arabinoxylans, leading to increased cell wall rigidity and decreased biodegradability of the wall polysaccharides (Hartley et al., 1990). Theodoru et al. (1987) showed that cell walls of barley stems contain p-coumaric (6.8 mg g1), ferulic (3.4 mg g1) and p-hydroxybenzoic acid (0.1 mg g1). These acids become incorporated into cell wall lignin, as demonstrated by Lowry et al. (1993). Phenolic constituents (lignins and phenolic acids) and carbohydrates are assembled in a tight architecture, which differs according to the plant species. Ferulic and diferulic acid are esterified with the polysaccharides of the cell walls ( [Scalbert et al., 1985], [Kamisaka et al., 1990], [Lewis and Yamamoto, 1990], [Besle et al., 1994], [Kondo et al., 1994] and [Provan et al., 1994]). Information about these and general biosynthetic pathways in the plant's metabolism of phenolic substances is provided by Httenschwiler and Vitousek (2000).

Seasonal dynamics of total phenolics and phenolic acids in the sandy soil of a ruderal meadow community dominated by Conyza canadensis
The accumulation of total bound phenolics in the soil under C. canadensis occurs between June, when the plants are in the phase of intensive growth (up to 1.5 m in height), and August, when the plants are in the phase of full flowering and fruiting. At the same time, in the period from June to August, plant tissues contain the highest concentration of total free phenols which can be easily released into the soil by precipitation during the vegetation period and after plant decay due to the only low lignification of Conyza cell walls. However, the amount of free and bound phenolic acids as a percentage of the total free and bound phenolics in the soil increases from the first rosette phases in plant development (MarchApril) right up to the phase of full flowering and fruiting (JulyAugust). During that period, the release of allelochemicals into the soil occurs via leachates and volatiles from live or dead plant roots, leaves, stems, branches and the inflorescence, which coincides with the findings of many authors on many other dominant species in such phytocoenoses ([Koeppe et al., 1976], [Tang and Young, 1982], [Rice, 1984], [Inderjit and Dakshini, 1994], [Inderjit and Dakshini, 1995], [Inderjit, 1996], [Batish et al., 2009] and [Lorenzo et al., 2011]). Roots exude a variety of lowmolecular weight organic compounds. Some of them, especially the phenolics (catechin, phydroxybenzoic, caffeic, p-coumaric, ferulic, gallic, gentisic, protocatechuic, salicylic, sinapic, and syringic acid), influence the growth and development of surrounding plants and soil microorganisms ( [Bertin et al., 2003], [Thorpe et al., 2009], [Batish et al., 2006] and [Batish et al., 2009]), in addition to influences upon organismic interactions (directing by allelopathy the competition between plants, supporting beneficial symbioses, influencing resistances against pests, etc.)

The whole C. canadensis plant (stem, branches and leaves) is covered with hairs and glandular trichomes (Weaver, 2001). They are sources for the release of phenolic compounds, which are secreted and washed away by precipitation into the soil under the plant. C. canadensis is characterized in its growing places by a large number of individuals (in May, 1000 young plants, 10 15 cm in height, per m2 were counted) and a large aboveground mass, as the plants reach a height of up to 2 m in summer. Hence, phenolics released via the surface of the plant account for a significant proportion of their total amount. The release of phenolic compounds via the hairs and glandular trichomes of various plant species has been documented by the research of many authors ( [Av and Tingey, 1986], [Sterling et al., 1987], [Wagner, 1991] and [Salminen et al., 2004]). The investigated soil at the levee in the pioneer meadow community can be categorized as young sandy soil in the first phases of pedogenesis, in which the dominant species C. canadensis has an important role to play, due to the decomposition of its dead organic remains and/or the compounds released during vegetation by its roots and aboveground parts. Although the dominant C.

canadensis creates high levels of free phenols (31 mg g1), only an insignificant percentage (0.6%) are
bound to sand particles because of the low absorbent capacity of the latter, as compared to soil particles. As consequence, quite an amount of the phenolics released from the plants are washed away by precipitation into deeper layers and hence become inaccessible to plants. The significantly higher percentage of free phenolic acids for the majority of the growing season (maximum 73.7% in July) when compared to bound forms (maximum 43.5% in August) highlights this, too. Their presence in the sandy soil under C. canadensis, although in smaller amounts than in natural soils, indicates the initiation of pedogenetic processes, processes of organic matter transformation, and humus formation. On the other hand, the presence of phenolics as secondary plant metabolites points to the influence of these invasive dominant plants in the overbuilding of substrate, such as sand deposits ( Djurdjevi et al., 2011). Gordon (1998) hypothesizes that many invasive non-indigenous species do alter ecosystem properties at several levels. Out of 31 species studied in Florida, 12 20 (3964%) potentially alter ecosystem properties and of geomorphology, hydrology, biogeochemistry, invasive and disturbance. alter the Also, Weidenhamer Callaway (2010) emphasize that species can

biogeochemistry of ecosystems, as secondary metabolites released by invasive species may play important roles in soil chemistry, as well as plantplant and plantmicrobe interactions. As opposed to our monitoring of the dynamics of phenolics in soil at monthly intervals during the vegetation period of the dominant invasive C. canadensis, data from other such cases are available only seasonally (spring, summer, autumn) and not directly related to the plant ontogenetic stages. Lodhi (1975) isolated ferulic, caffeic, and p-coumaric acids from soils under hackberry ( Celtis

laevigata) trees. There, the phytotoxicity of each phenolic acid was highest in January and April and
lowest in September. However, phenolic concentrations in soils collected in August were not as high as those collected in January and April. The highest concentration of caffeic acid in the humus of bilberry (Empetrum hermaphroditum) in northern Sweden was recovered during snowmelt in May (Gallet and Lebreton, 1995). Muscolo and Sidari (2006)examined seasonal variations in the content of water-soluble phenolics extracted from litter and the Ah horizon of forest soil under Pinus laricio Poiret trees. Similar to our findings, the results indicated a seasonal variation of phenolic substances

contents in soil, with the highest concentrations of water-soluble phenolic acids in autumn and the lowest in summer. According to our previous research, the seasonal dynamics of phenolics in soil depends on the type of phytocoenoses and on their dominant species. Among forests in Serbia, in a sessile oak forest the highest amount of total phenolics occurs in summer ( Djurdjevi and Oberan, 1998), the amount falls between spring and autumn in a Scots pine and European black pine forest ( Djurdjevi et al., 1997), it falls in spring, but increases during summer and autumn in a forest of black ash and Virgilius oak (Djurdjevi et al., 1998), and it falls between March and May in the rhizosphere soil of the vernal ephemeroid Arum maculatum in a beech and linden forest (Djurdjevi et al., 2008). Kuiters and Denneman (1987) studied monthly fluctuations in soil phenolics content (free and bound forms) under stands with Fagus sylvatica, on strongly podzolized sandy soil. A distinct seasonal pattern could be found in the soil phenolic content at this site. The Ca(OH) 2-extractable concentration, including water-soluble as well as reversibly bound phenolics, rose to 139% during and after the leaffall period and reached the highest concentration in late winter (January). Thereafter, from January to March, the concentration fell to its former level and was stable until leaf-fall in autumn. The watersoluble concentration showed a seasonal pattern as well, but the extent of these fluctuations was much smaller. The authors argued that the fall in phenolic acids in spring was the result of increased microbial activity due to increased soil temperatures and to the soil's adsorption of organic matter during humification. In the soil under the invasive C. canadensisanalyzed in present investigation, the concentration of free phenolics also changed insignificantly during the growing period, as opposed to bound phenolics, where the changes were more marked and significant. The decrease in the amount of bound phenolics between March and June can probably also be attributed to increased microbial activity due to high soil moisture and temperature in early summer at this open ruderal meadow community on a sandy levee, and to the soil's adsorption of organic matter during humification. A substantial amount of phenolic compounds may have been transported also into the deeper layers of the sandy soil, which is not able to adsorb them due to the coarseness of its particles. Some of the phenols are further degraded also by many organisms which used them for energy gain and syntheses in cell metabolism ( [Felbeck, 1971], [Haworth, 1971] and [Martin and Haider, 1971]). The phenolic compounds originating from C. canadensis tissue, in addition to the phenolics which occur through microbial degradation of organic material, mainly lignin, and those phenolics which are produced by soil microorganisms, enter the soil and become part of the humus fraction. Humic acid, the most abundant constituent of humus, and the similar type fulvic acid, is a complex polymer of various phenolic units with linked peptides, amino acids and some polysaccharides ( [Martin and Haider, 1969], [Martin and Haider, 1971], [Haider et al., 1975], [Flaig, 1971], [Felbeck, 1971] and [Haworth, 1971]). Phenolic compounds make up an important component of the humus layer of different soil types ( [Guenzi and McCalla, 1966],[Hennequin and Juste, 1967], [Katase, 1981a], [Katase, 1981b], [Kgel, 1986], [Kuiters and Denneman, 1987], [Djurdjevi et al., 2000], [Djurdjevi et al., 2003], [Djurdjevi et al., 2004], [Djurdjevi et al., 2005a],[Djurdjevi et al., 2006], [Djurdjevi et al., 2009] and [Banach-Szot, 2007]).

Conclusions
The dynamics of allelopathically significant phenolic compounds in the globally successful invader C.

canadensis L. and the sandy soil where the plants were growing, have a marked seasonal character
and coincide with certain phases in plant development. The highest amount of phenolic phytotoxins in the tissue was measured during the phase of plant elongation and intensive growth, and again during the phase of fruit abscission and plant decline. It can be seen that the level of free acids in the soil under C. canadensis, as a percentage of the total free phenolics, increases dramatically between March, when the plants are in the form of rosettes, 12 cm in diameter, and July, the beginning of flowering. In these months free phenolic acids account for as much as 73.7% of the total free phenolics in the soil. The percentage of bound phenolic acids gradually increases between April, when the plants are in the form of larger rosettes, 510 cm in diameter, and August, when their maximum level (43.5%) is reached. Phenolic acids can serve as reliable indicators of phenolics metabolism and turnover, starting from their biosynthesis in plant tissues to their incorporation into stable fractions of soil humus. The results presented in this work on the seasonal dynamics of phenolics in the aboveground parts of the dominant alien plant C. canadensis and the soil under it, as well as on the allelopathic effects of the invader upon native species (Djurdjevi et al., 2011), support the novel weapon hypothesis (Callaway and Ridenour, 2004). An important role in the spread of this invasive species into new areas and its domination over native species is played by phenolic compounds as the most abundant group of allelochemicals. These exert regulatory roles in many processes, not only in plants themselves, but as well in the ecosystem as a whole.

Acknowledgements
This work was supported by the Ministry of Education, Science and Technological Development of Serbia (Grant No. 173018). The authors would like to thank Dr. Anka Dini, a retired Senior Scientist, for her extensive help during field research into the domination of C. canadensis and vegetation succession on sandy levees. We thank Jonathan Pendlebury, lector at Belgrade University's Faculty of Philology, for proofreading this paper.

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