wild oat (Avena fatua

Agrochimica, Vol. LVI - N. 3May-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, 71122 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: [email protected]
The present contribute was presented at the XXIX National Congress of the Italian Society of Agricultural
Chemistry, September 21-23, 2011, Foggia (Italy).
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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, 15°34’ 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 H2O), 15 mg/kg available P (Olsen method), 800 mg/
kg exchangeable K (NH4Ac), 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/m2/s PAR was used. A dose of 60 kg/ha of 18/46
fertiliser (18% elemental N, 46% P2O5, by weight) and 90 kg/ha NH4NO3 (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
Allelochemicals from wild oat roots
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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
100 × 2.1 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, 24-30 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:
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A. Iannucci et al.
Table 1. – Bio-agronomic traits of the wild oat plants at the four growth stages.
Stage
Tillering
Stem extension
Heading
Milky ripe
LSD(0.05)
Aerial fresh weight
(g/plant)
Root fresh weight
(g/plant)
Height
(cm)
L/S†
10.9c
24.4ab
30.1a
19.0bc
8.5
5.1c
12.2a
8.4b
6.7bc
2.9
52.6b
54.9b
112.2a
123.6a
16.3
10.9a
1.6b
0.3c
0.2c
1.2
Values within a column not followed by the same letter are significantly different at p ≤ 0.05. † 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
0.306 µg/kg to 0.563 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 (Rt) of standards of phenolic compounds.
Compound
Standard Rt (min)
4-Hydroxybenzoic acid
Vanillic acid
Syringic acid
Vanillin
p-Cumaric acid
Syringaldehyde
Ferulic acid
6:59
9:33
10:92
11:69
12:24
13:39
14:26
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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
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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
4-Hydroxybenzoic acid
Vanillic acid
Syringic acid
Vanillin
p-Cumaric acid
Syringaldehyde
Ferulic acid
Mean
LSD(0.05)
Concentration (µg/kg soil dry matter)
Tillering
Stem
extension
Heading
Milky ripe
Means
0.0736b
0.0147e
0.0224de
0.0850a
0.0217de
0.0628c
0.0257d
0.0437
0.0090
0.1115b
0.0219d
0.2027a
0.1182b
0.0324d
0.0491c
0.0261d
0.0803
0.0136
0.1171c
0.0241f
0.1772a
0.1286b
0.0340e
0.0483d
0.0253ef
0.0793
0.0088
0.0363d
0.0145e
0.1485a
0.0573b
0.0073f
0.0466c
0.0172e
0.0468
0.0050
0.0846c
0.0188f
0.1377a
0.0973b
0.0239e
0.0517d
0.0236e
0.0045
Values within a column not followed by the same letter are significantly different at p ≤ 0.05.
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.
Allelochemicals from wild oat roots
191
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Summary. – We explored the allelopathic potential of rhizospheric soil of wild
oat (Avena fatua L.) during plant growth. Concentrations of phenolic compounds
were determined in rhizosphere soil of plants grown under controlled conditions and
harvested at four developmental stages (tillering, stem extension, heading, milky ripe).
HPLC analysis showed production of seven phenolic compounds in rhizospheric soil
of wild oat: 4-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, p-cumaric
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acid, syringaldehyde, ferulic acid. Higher total concentrations for these phenols in the
rhizospheric soil were recorded at stem extension and heading (mean, 0.558 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.