Dynamics of release of allelochemical compounds from roots of wild oat (Avena fatua L.)

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1 Agrochimica, Vol. LVI - N. 3 may-june 2012 Dynamics of release of allelochemical compounds from roots of wild oat (Avena fatua L.) A. Iannucci *, M. Fragasso, C. Platani, A. Narducci, V. Miullo, R. Papa Centro di Ricerca per la Cerealicoltura (CRA), S.S. 16, km 675, Foggia, Italy Received November 14, 2011 Received in revised form April 26, 2012 Accepted May 3, 2012 Keywords: Avena fatua L., phenolic compounds, plant growing stage, rhizospheric soil Introduction. Wild oat (Avena fatua L.) is considered to be among the world s worst crop weeds (Hassan and Khan, 2003). It is the most prevalent annual grass in durum wheat (Triticum durum Desf.) fields in the environments of southern Italy. Due to its competitive nature, wild oat results in reductions in the availability of nutrients for crops, thereby reducing productivity both quantitatively as well as qualitatively. The negative interactions between weeds and crops might be due to a combination of competition and allelopathic factors (Narwal et al., 2005). As reported by Schumacher et al. (1983), wild oat is involved in interactions with wheat plants through the production and release of phytotoxic substances known as allelochemicals. Allelopathy is defined as any direct or indirect effect of one plant on another that is mediated through the production of chemical compounds that are released into the environment (Rice, 1984). Plant roots actively release allelochemicals that have pivotal roles in this phenomenon. A number of phytotoxic compounds have been identified in plant root exudates (Narwal et al., 2005). In particular, phenolic compounds are one of the main groups of substances involved in the allelopathy of wild oat roots (Pérez and Ormeno-Nuñez, 1991). As reported by Schumacher et al., (1983), wild oat can release scopoletin and phenolic acids such as p-coumaric acid, vanillic acid and ferulic acid, as well as other phenolic compounds. However, the isolation and identification of chemicals from donor plants with biological activity do not demonstrate that these compounds interfere in nature through allelopathy (Inderjit and Weston, 2000). Indeed, retention, transformation and transport * Corresponding author: anna.iannucci@entecra.it The present contribute was presented at the XXIX National Congress of the Italian Society of Agricultural Chemistry, September 21-23, 2011, Foggia (Italy).

2 186 A. Iannucci et al. of allelopathic chemicals in soil and physicochemical and biological components of the soil can influence the fate of allelopathic chemicals, and thus of allelopathy, in soil (Inderjit, 2001; Inderjit et al., 2010). For these reasons, it is important to isolate, identify and characterize phenolic compounds from the soil. At present, the study of phytotoxins released by intact roots of living crop plants is considered one of the most promising approaches to exploit allelopathy in annual crops (Duke et al., 2005; Macas et al., 2007). Furthermore, the extent of allelopathy by a plant can vary with age, part and type of cultivar being used (Batish et al., 2001). Therefore, to define the activities of the phytotoxic compounds of wild oat and to promote successful strategies for the control of this weed, there is the need for improved knowledge of the seasonal dynamics of their release, and of the accumulation of allelochemicals in the rhizospheric soil. The objectives of this study were: (i) to identify the phenolic compounds that might have important roles in the phytotoxic potential of wild oat rhizosphere soil; and (ii) to determine whether their concentration varies during plant growth. Material and methods. Plant growth and soil sampling. Seeds from wildtype oat populations were collected from agricultural fields at the Cereal Research Centre farm located in Foggia (Italy) (41 28 N, E, 76 m a.s.l.) in 2011 and stored at 4 C. These wild oat seeds were surface sterlized for 10 min in a 1:10 (v/v) dilution of commercial hypochlorite bleach, and then rinsed several times with distilled water. Five sterilised seeds were placed in each pot (diameter, 18 cm; height, 14 cm), which contained 2 kg of a soil mixture (soil:sand:peat, 60:30:10; v/v). The soil was an unsterilized loam soil (USDA classification system) with the following characteristics: 21% clay, 43% silt, 36% sand, ph 8 (in H 2 O), 15 mg/kg available P (Olsen method), 800 mg/ kg exchangeable K (NH 4 Ac), and 21 g/kg organic matter (Walkey Black method). Silica sand with a grain size that ranged from 0.4 to 0.1 mm was used. A commercial product containing peat-moss was used (Terravera Universal mould). After emergence, the seedlings were thinned to one per pot. The pots were placed in a growth chamber with daily light/dark and temperature cycles that were changed as a function of the plant growth. Thus the growth conditions were regulated according to the mean values of the photoperiod and temperature of the plain environments of southern Italy. A light of 1000 µmol photons/m 2 /s PAR was used. A dose of 60 kg/ha of 18/46 fertiliser (18% elemental N, 46% P 2 O 5, by weight) and 90 kg/ha NH 4 NO 3 (26% elemental N) were applied at sowing and at plant tillering, respectively. No further nutrient solutions were used. The plants were regularly watered to 70% of the field capacity. The control treatment consisted of two pots without plants, stored under the same conditions. The plants and soils were sampled four times during the growing season, according to the defined growth stages of tillering, stem extension, heading and milky ripe. At each

3 Allelochemicals from wild oat roots 187 sampling, following the measurement of their height, the plants were collected by pulling them from the soil in the pots. The roots were shaken gently to remove and collect their root-zone, or rhizosphere, soil. The plants were then cleaned and weighted as the shoots (separated in leaves and stems) and the roots. The leaf/stem (L/S) ratios were calculated from the fresh weights. The rhizospheric soil samples were dried at 30 C under vacuum. Phenolic compounds. Samples of the rhizosphere soil from the wild oat plants were mixed thoroughly and sieved (2 mm mesh) to remove root tissue. One hundred grams of oven dried soil was extracted with 300 ml methanol (agitation, 48 h at 25 C; centrifugation, 1,200 g for 30 min). According to Kong et al. (2006) 100% methanol was used because this allows maximum extraction of the hydrophilic compounds. The extracts were concentrated under vacuum at 40 C, and the residues were dissolved in methanol (6 ml) and filtered through 0.45 µm filters prior to injection of 2 µl into the HPLC system (HP 1200). The HPLC was equipped with a reverse-phase Zorbax SB-C18 column (Eclipse mm, 1.8 µm) with a diode array detector. The temperature of the column oven was set to 35 C. For the analysis, linear gradient elution was used, with the mobile phases of acetonitrile (solution A) and aqueous 1% acetic acid (solution B), as follows: 100% solvent B at 0 min; 85% solvent B at 12 min; 50% solvent B at 20 min; 0% solvent B at 22 min; 100% solvent B at 24 min; isocratic elution of 100% B, min. The flow rate was 0.4 ml/min, with detection at 280 nm. The phenolic compounds were identified through their retention times and their UV spectra, as compared to those of standards. The analyses of the allelopathic compounds were repeated three times, with three extracts in each sample. In this study we reported the net phenolic compound concentrations, obtained by substracting the values found in the control soil. Statistical analysis. The experiments were carried out using a completely randomised design, with three replicates. Two pots with no seeds were included as the controls. The data were subjected to analysis of variance using the Statistica software (StatSoft version 7.1 StatSoft, Inc., Tulsa, Oklahoma, USA), and the means were examined by least significant difference (LSD) at the 0.05 probability level. Results and discussion. The bio-agronomic traits recorded for these wild oat plants at the four plant growth stages are reported in Table 1. The greatest fresh weights of the aerial and root parts of the plants were seen at the heading and stem extension stages, respectively. The L/S ratio was the greatest at tillering, while the plant height was greatest at the milky ripe stage. As reported by Anderson (1985), it is important to determine the growth and development of such grass plants because they are strong indicators of the growing conditions. Furthermore, knowledge of wild oat development is important to determine any relationships between plant growth and changes in the chemical compounds in the rhizosphere soil. The analysis of the root exudates led to the identification of seven phenolic compounds in the rhizospheric soil from the wild oat roots:

4 188 A. Iannucci et al. Table 1. Bio-agronomic traits of the wild oat plants at the four growth stages. Stage Aerial fresh weight Root fresh weight Height L/S (g/plant) (g/plant) (cm) Tillering 10.9 c 5.1 c 52.6 b 10.9 a Stem extension 24.4 ab 12.2 a 54.9 b 1.6 b Heading 30.1 a 8.4 b a 0.3 c Milky ripe 19.0 bc 6.7 bc a 0.2 c LSD (0.05) Values within a column not followed by the same letter are significantly different at p L/S: leaf/stem ratio. 4-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, p-cumaric acid, syringaldehyde and ferulic acid. The retention times of their peaks were compared with those of authentic phenolic acid standards (Tab. 2). Many of these phenolic compounds have been identified as allelochemicals in other cereal species (Wu et al., 2001; He et al., 2005). Through the growth stages of the wild oat plants, the total concentrations of these combined phenolic compounds in the rhizospheric soil varied from µg/kg to µg/kg soil dry matter (Fig. 1). The total concentrations of the phenolic compounds increased from tillering to stem extension and heading, and then decreased during the seed filling period (to the milky ripe stage). Kong et al. (2006) found several phenolic acids in soil-grown rice until heading, and their concentrations also varied with growth stages. Here, the peak concentration of the total phenols in the rhizospheric soil samples corresponded to when the wild oat plants showed the highest fresh weights. This is probably because at the stem extension and heading stages, the plants are actively assimilating Table 2. HPLC retention times (R t ) of standards of phenolic compounds. Compound Standard R t (min) 4-Hydroxybenzoic acid 6:59 Vanillic acid 9:33 Syringic acid 10:92 Vanillin 11:69 p-cumaric acid 12:24 Syringaldehyde 13:39 Ferulic acid 14:26

5 Allelochemicals from wild oat roots 189 Fig. 1. Dynamics of release of the total phenolic compounds into the rhizosphere soil during the growth stages of wild oat. Data within each column are LSD (0.05) values. metabolites. After these periods, the nutrient removal for the filling of the seeds becomes the most important biological process, which results in a decrease in the concentrations of these phenolic compounds in the shoots at the later growth stage (El-Shatnawi, 2004). The concentrations of the seven phenolic compounds identified in the rhizospheric soil of wild oat are shown in Table 3. The concentration of each individual compound varied considerably in relation to the plant growth stage. The highest maximum concentration was recorded for syringic acid at the stem extension stage (0.203 µg/kg soil dry matter), while the lowest maximum concentration was for p-cumaric acid at the milky ripe stage (0.073 µg/kg soil dry matter). Most of these phenolic compounds in this experimental study have been recorded in root exudates of seedlings of wild oat (Pérez and Ormeno-Nuñez, 1991). Phenolic compounds derived from the shikimic and acetic acid (polyketide) metabolic pathways in plants are among the main category of allelochemicals in nature (Blum et al., 1991; Turk and Tawaha, 2003; Chon et al., 2005; Li et al., 2010). Phenolic compounds inhibit seed germination and plant growth and influence other physiological processes (Djurdjevic et al., 2004). For instance, ferulic acid and p-coumaric acid inhibit hydraulic conductivity and nutrient uptake of plant roots, which results in growth inhibition (Blum et al., 1991). Furthermore, Putnam and Tang (1986) noted that in all cases of allelopathy that had been studied, this phenomenon appeared to involve a

6 190 A. Iannucci et al. Table 3. Levels of the seven identified phenolic compounds in the rhizosphere soil of wild oat at the four developmental stages of the plant growth. Compound Concentration (µg/kg soil dry matter) Tillering Stem Heading Milky ripe Means extension 4-Hydroxybenzoic acid b b c d c Vanillic acid e d f e f Syringic acid de a a a a Vanillin a b b b b p-cumaric acid de d e f e Syringaldehyde c c d c d Ferulic acid d d ef e e Mean LSD (0.05) Values within a column not followed by the same letter are significantly different at p complex of chemicals; thus no single phytotoxin has been shown to be solely responsible for, or produced as a result of, interference from a neighbouring plant. Also of note, according to Wu et al. (2000), plant roots can regulate the exudation of allelochemicals into the growth environment to promote seedling allelopathy during the growing season. In conclusion, as exudation of allelochemicals from living roots into the rhizospheric soil is an active metabolic process, their presence in the rhizospheric soil here demonstrates their directed release by the plant into the growth environment. Furthermore, the present study demonstrates that wild oat roots can exude varied amounts of phenolic acids into their growth soil, and that the quantities exuded depended on the developmental stage of the plant growth. This evidence is important for the management of crop weed interactions in field crops, because the seriousness of the potential damage is also a function of the growth stage of the weed. However, to understand the allelopathic mechanisms of phenolics more clearly, further studies on their production, roles, and fates in soil environments are necessary.

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8 192 A. Iannucci et al. acid, syringaldehyde, ferulic acid. Higher total concentrations for these phenols in the rhizospheric soil were recorded at stem extension and heading (mean, µg/kg soil dry matter). Syringic acid and vanillic acid showed the highest (0.138 µg/kg soil dry matter) and lowest (0.019 µg/kg soil dry matter) mean values, respectively, over all of the four developmental stages of plant growth. These data suggest that wild oat exudes allelopathic compounds and levels of phenolics into the rhizospheric soil vary according to plant maturity.