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Allelopathy Journal 29 (1): 107-124 (2012) International Allelopathy Foundation 2012
Tables: 4, Figs : 7
Screening of Mediterranean wild plant species for
allelopathic activity and their use as bio-herbicides

F. ARANITI, A. SORGON, A. LUPINI and M.R. ABENAVOLI
*


Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale
(BIOMAA), Universit Mediterranea di Reggio Calabria, Facolt di Agraria - Salita
Melissari, I-89124 Reggio Calabria RC, Italy
E. Mail: mrabenavoli@unirc.it

(Received in revised form : October 13, 2011)

ABSTRACT

Seventeen wild plant species from the Mediterranean area (Calabria,
Southern Italy) were assayed for their allelopathic activity and as potential source of
new natural herbicides for weed control. The inhibitory effects of aqueous extracts of
17 Mediterranean plant spp. were studied on seed germination and root elongation of
Lactuca sativa L. of these 4-species [Calamintha nepeta (L.) Savi, Hypericum
hircinum L. ssp. Majus (Aiton) Robson, Artemisia arborescens L. and Euphorbia
rigida Bieb] proved most inhibitory to weeds and lettuce seedlings. Root elongation
proved more sensitive than seed germination. The phytotoxicity of aqueous extracts of
most phytotoxic plant species persisted till 112 days. The aqueous extracts inhibited
the seed germination and root growth of Chenopodium album, Sinapis alba,
Echinochloa crus-galli weeds. C. album and S. alba weeds were most sensitive to all
aqueous extracts, while E. crus-galli was most tolerant. In pot experiments, plant
residues caused stronger inhibition in shoot than in roots. The phytotoxicity followed
the order: A. arborescens > E. rigida > C. nepeta H. hircinum. These results might
help in developing the natural Mediterranean plant extracts for weeds control.

Keywords: Allelopathy, dose-response curve, extracts, pot experiments, root growth,
seed germination, shoot growth, weeds.

INTRODUCTION

Weeds are major constraints to crop production as they reduce both crops yield
and quality through their competition for light, moisture and nutrients (9,46,53). Presently
weed management, is done with herbicides (17,38), which adversely affect the
environment and human health (1,16) and weeds develop resistance to them (26,28).
Therefore, allelopathy phenomenon may provide alternative biological weed control (8).
Indeed, these natural compounds/allelochemicals with their phytotoxic activity, novel
molecular structures, new sites of action and rapid biodegradation could provide tools for
new herbicidal templates. Some allelochemicals from fungi, lichens, plants or other living
organisms have already been claimed as natural herbicides (15), antibiotics (48),
fungicides (47) or insecticides (42). In plant allelochemicals, cinmethylin (a monoterpene
derivative of 1,4-cineole), major component of plant essential oils, different kinds of
triketone, structurally derived from the natural compound leptospermone, benzoxazinoids
*
Correspondence author
Araniti et al 108
isolated from many species of Poaceae family, sorgoleone, a benzoquinone exuded from
sorghum roots, have potential uses in agriculture as weed control agents (15,36). Hence
presently, the search of plant phytotoxins among crops, weeds and invasive plants,
toxicologically and environmentally beneficial, is major challenge in agrochemical
research.
The Mediterranean area is an important pool of global biodiversity (37)
apparently due to the specific climatic conditions, the origin of flora, the habitat
heterogeneity and the geological, paleogeographical and historical factors. In particular,
due to the semiarid and arid climate in summer season and the volatilization plays a major
role in plant-plant interactions. Several aromatic shrubs possess the phytotoxic activity.
Essential oils from Mediterranean Labiatae inhibited the germination and root growth of
three test seeds and a positive correlation was found between their total monoterpene
content and the inhibitory activity (4). Beside the volatile compounds, water soluble from
aromatic shrubs of Mediterranean-type ecosystems inhibited the germination and growth
of some species (18). Finally, Brassica fruticulosa, Chenopodium album and Malva
silvestris, showed a strong phytotoxic activity (14).
In this study, 17-wild plant species (Table-1) from Mediterranean flora were
screened for their allelopathic potential on Lactuca sativa L. (41) and their potential as
natural herbicides for weed management. The aqueous extracts of shoots were assessed for
their effects on the seed germination and root growth of lettuce. Furthermore, to
understand whether the most phytotoxic species could be a source of molecules for weed
management, three different experiments were done to: (i) Determine the persistence of
their phytotoxicity during storage period, (ii) Determine the phytotoxic potential of their
decaying residues in pots against test species and (iii) Evaluate their effects against on
Chenopodium album, Sinapis alba and Echinochloa crus-galli weeds.

MATERIALS AND METHODS

Plant material and aqueous extract preparation
Based on their abundance and stronger growth than other plants and their affinity
with other potentially allelopathic species (5,21,23,27,39,45,9), 17 wild plant species, were
collected from June to October 2008 during various phenological stages (Table 1) from
Aspromonte, Southern Italy (Calabria). As preliminary screening, the aboveground parts
of plants (leaves and green stems), responsible for volatilization were collected and oven
dried at 40 C until a constant weight, then grounded and stored at room temperature.
Ten g powder of each species were soaked in 200 ml distilled water in a
continued orbital shaker for 48 h. The mixture was filtered through four layers of
cheesecloth and then centrifuged at 3000 rpm for 60 min. The supernatant was filtered
again through Whatman no. 2 filter paper and sterilized by 0.22 m Millipore filter. The
aqueous extracts were stored at -20C until bioassay tests were conducted. The extraction
was done at room temperature, in dark conditions (to avoid photo degradation). Each
aqueous extract was diluted appropriately with distilled water to give the following final
concentrations 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 75 and 100%, Where, 0: control
with no extract and 100 % : undiluted extract.

Screening of Mediterranean wild plant species for use as bio-herbicides

109
Table 1. Wild Mediterranean plant species studied

English name Botanical name Family

Sampling Stage I. Heading
Purple mistress Moricandia arvensis (L.) DC Brassicaceae
Stinking Tutsan Hypericum hircinum L. ssp. Majus Clusiaceae
Tree Spurge Euphorbia dendroides L. Euphorbiaceae

Sampling Stage II. Flowering ( Main Shoot)
Curry plant Helichrysum italicum (Roth) Don Asteraceae
Montpelier Cistus monspeliensis L. Cistaceae
Rockrose Cistus salvifolius L Cistaceae
Rockrose Cistus incanus L Cistaceae
St John's wort Hypericum perforatum L. ssp. veronense Clusiaceae
Golden wreath wattle Acacia cyanophylla Lindley Fabaceae
Spiny Broom Calicotome infesta (Presl) Guss Fabaceae
Lesser Calament Calamintha nepeta (L.) Savi Lamiaceae
Fernleaf lavender Lavandula multifida L. Lamiaceae

Sampling Stage III. Development of fruit
Woormwood Artemisia arborescens L. Asteraceae

Sampling Stage IV. Ripening or maturity of fruit and seed
Mastic tree Pistacia lentiscus L. Anacardiaceae
Upright Myrtle Spurge Euphorbia rigida Bieb. Euphorbiaceae
Jerusalem Sage Phlomis fruticosa L. Lamiaceae
Albardine, Lygeum Lygeum spartum L. Poaceae

Bioassays
Seed Germination : Seeds of Lactuca sativa L. (variety Parris Island Cos USA) were
surface sterilized with 15% (v/v) NaClO solution for 15 min and then rinsed thoroughly
with distilled water. Ten seeds were evenly distributed into a Petri dish (6 cm dia) between
a double moistened filter paper with 2 ml of different concentrations of aqueous extracts as
per treatments. Petri dishes were then placed in dark in growth chamber [25 1 C and
70% relative humidity]. Seeds showing 1 mm long extrusion of radical after 24 and 48 h
were considered to have germinated. Total Germination (G
T
), was calculated as under:



Where, N
T
: Number of germinated seeds and N: Total number of seeds sown (10).

Root : Five pre-germinated lettuce seeds (24 h), selected for uniformity in root length,
were placed in sterile Petri dishes (6 cm dia) and moistened with 2 ml of each aqueous
extract at different concentrations in the same conditions previously described. The Petri
dishes were then placed in a growth chamber [25 1 C and 70% relative humidity]. At 24
Araniti et al 110
and 48 h, an image of lettuce roots for each treatment and species was captured by scanner
(Epson Expression 800, Regent Instruments, Quebec, Canada) and the Total Root Length
(TRL) was measured using the WinRhizo Pro System v. 2002a software (Instruments
Rgent Inc., Quebec, Canada).

Extracts Stability: A stability test on the most phytotoxic extracts (A. arborescens, C.
nepeta, E. rigida, H. hircinum) was done with minor modifications (24). Briefly, 500 ml
of 100% aqueous extract of each species were stored in glass reagent bottles at 4 C in
dark for 112 days. The pH and Electrical Conductivity (EC) were monitored over time.
During the same experimental period, the persistence of the extract phytotoxicity during
storage was investigated based on lettuce seed germination and root elongation. The ED
50

values of both processes, previously estimated, were used as concentrations. The bioassay
procedure was already described above.

Pot experiment
Plastic pots (7 cm dia) were filled with 120 g silver sand/commercial potting
mixture (9:1 v/v). The potting mixture was sieved through 2 mm sieve before mixing. The
mixture was added at 5 and 10% (w/w) of A. arborescens, C. nepeta, E. rigida, H.
hircinum plant residues (leaves and stems). Pots without plant residues (0%) were used as
a control. The pots were then placed in a growth chamber with a temperature [251 C
with a 16-h photoperiod, a photon flux rate of 120 mol m
2
s
1
and a relative humidity of
70%] for 7 days. Then, lettuce seedlings were transplanted and sub-irrigated daily with 10
ml deionised water. The plants were harvested 16 days after transplanting; fresh and dry
weight of the root and shoot, root length and leaf area were measured.


Bioassays on weeds
Seed germination and root elongation of 3-weeds (Chenopodium album, Sinapis
alba, Echinochloa crus-galli) were assayed to evaluate the inhibitory effect of most
phytotoxic extracts (Artemisia arborescens, Calamintha nepeta, Euphorbia rigida,
Hypericum hircinum). The procedure was similar to that described above, with two
modifications: 15 seeds were used for germination (G
T
%) and 10 pre-germinated seeds for
root elongation (TRL). The measurements were recorded 72 h after treatment.

Experimental design and statistical analysis
A completely random design with 4 replications was used to evaluate the effects
of aqueous extracts on lettuce germination and root elongation processes.
To compare the effects of aqueous extracts of different species, seed germination
and root elongation data were fitted with a non-linear regression model using the following
log-logistic response equation (6,50), extensively applied for investigating the phytotoxic
effect or toxic efficacy of several allelochemicals (43) and herbicides (40):




( ) [ ]
50
/ ln
1
ED x B
e
C D
C y
+

+ =
Screening of Mediterranean wild plant species for use as bio-herbicides

111
where, C : lower response at indefinitely large doses, D: mean response of control, B:
slope around the ED
50
and ED
50
: dose causing 50% of total response (considered as the
index of phytotoxic capability of aqueous extracts).
By comparing the ED
50
values in the extracts, it was possible to determine the
phytotoxicity of different plant species. The comparison of phytotoxicity of aqueous
extracts among the plant species was done by one-way ANOVA using the ED
50
as a
variable and plant species as the main factor. The ED
50
data were first checked for
deviations from normality (Kolmogorov-Smirnov test) and tested for homogeneity (Leven
Median test). Tukeys test comparison was used to compare the mean values of ED
50

among the plant species during their exposure time (P < 0.05). Clusters analysis was also
done to identify discrete groups of plant species with similar toxic ED
50
of both
germination and root elongation of lettuce 24 and 48 h after exposure. Clustering was
performed using a centroid hierarchical approach based on a minimum variance linking
method with Euclidean distance as the similarity measure (25).
The germination and root elongation responses of weed species at different
concentrations of A. arborescens, C. nepeta, E. rigida, H. hircinum aqueous extracts were
statistically evaluated as described above. The same completely random design with 4
replications was also used to evaluate the persistence of the phytotoxic effect of A.
arborescens, C. nepeta, E. rigida, H. hircinum aqueous extracts. For each observation, the
means were compared using the t-Student test (P < 0.05).
Finally, a completely random design with three replications was used in pot
experiments. Root length, leaf area, fresh and dry weight of lettuce shoot and root were
evaluated for normality (Kolmogorov-Smirnov test) and tested for homogeneity (Leven
Median test). Means were compared by Tukeys test (P < 0.05). All statistical analyses
were conducted using SPSS ver. 6.1 software (Insightful Corporation, USA).

RESULTS AND DISCUSSIONS

Petri plate Lactuca sativa L. bioassays

Seed germination bioassay: Seed germination responses of lettuce to aqueous extracts
differed significantly depending on the plant species, extract concentration and time of
exposure. After 24 h of contact, the aqueous extracts of H. hircinum, C. nepeta and
A. arborescens, at 25% of concentration, totally inhibited the lettuce seed germination,
while 50% or 75% extract concentrations were needed to cause the same inhibitory effect
on other plant species (Fig. 1). On the other hand, the extracts of P. lentiscus and C.
salvifolius did not influence the lettuce seed germination (Fig. 1). After 48 h of exposure,
while the aqueous extract of E. rigida maintained the same phytotoxic effect on lettuce
seed germination, the magnitude of inhibition of H. hircinum, C. nepeta, E. dendroides
and M. arvensis extracts decreased. In addition, the extracts of remaining species showed
some inhibitory effects and delayed the lettuce seed germination (Fig. 1). Hence, the
aqueous extracts derived from spontaneous Mediterranean plant species on seed
germination caused three effects: delay, inhibition or null effect.
The non-linear regression fits of the aqueous extract curves of all plant species
were characterized by a high statistical significance (P < 0.001) for lettuce seed
Araniti et al 112
germination (Fig. 1). The comparison of ED
50
values for lettuce seed germination (48 vs
24 h) showed that this parameter increased 48 h after exposure to all aqueous extracts
confirming a decrease in their magnitude of inhibition. The inhibitory effects of extracts of
C. nepeta (30%), H. hircinum (43%), E. rigida (50%) and H. perforatum (50%) decreased
slightly, but were markedly reduced in A. arborscens (63%), E. deindroides (78%),
M. arvensis (67%) extracts. Furthermore, the extracts of A. cyanophylla, C. infesta,
L. spartum, C. monspeliensis and H. italicum only delayed germination (ED
50
values at 48
h not detected) and P. lentiscus and C. salvifolius did not inhibit germination (Table 2).



Figure 1. Dose-response curves of Total Germination Index (G
T
, %) of Lactuca sativa L. seeds
exposed to aqueous extracts of different plant species for 24 () and 48 h (). All dose-
response curves were significance at P < 0.001.

Root Elongation Bioassay: All aqueous extracts inhibited the root elongation of lettuce
seedlings and the degree of inhibition increased with the increase in extract concentration
(51). After 24 h of exposure, the low concentrations of all extracts inhibited the root
elongation (51 to 84%) than control and at 48 h, the inhibition was 58-85% (Fig. 2).
Furthermore, the highest concentration of C. nepeta, C. infesta and H. perforatum extracts
caused brownish colour and turgor loss in lettuce roots (data not shown).
The aqueous extract curves of all plant species fitted by non-linear regression
showed high statistical significance (P < 0.001) for root elongation (Fig. 2). At 24 h after
exposure, the ED
50
values ranged from 3.4 % to 62.8 %, (Table 3). The aqueous extract
curves of all plant species fitted by non-linear regression showed high statistical
significance (P < 0.001) for root elongation (Fig. 2). At 24 h after exposure, the ED
50

values ranged widely from 3.4 % to 62.8 %, (Table 3). At 48 h after exposure, in contrast

Screening of Mediterranean wild plant species for use as bio-herbicides

113
Table 2. ED
50
(%) response of germination of lettuce seeds to aqueous extract of different plant
species at 24 and 48 h after exposure (Estimated by the log-logistic equations)

Plant species Exposure time (h)
24 h 48 h

Lavandula multifida 43.8 (1.5)
cd
97.6 (4.3)
a

Phlomis fruticosa 59.9 (0.8)
b
81.4 (4.7)
b

Artemisia arborescens 14.4 (0.05)
g
62.8 (0.5)
d

Euphorbia rigida 36.4 (2.2)
de
49.8 (0.08)
e

Cistus monspeliensis 76.1 (0.3)
a
103 (0.3)
a

Helychrisum italicum 75.5 (0.4)
a
104 (0.2)
a

Calamintha nepeta 17.6 (1.5)
fg
30.1 (3.2)
f

Euphorbia dendroides 25.4 (0.07)
ef
78.0 (1.6)
bc

Acacia cyanophylla 77.7 (1.7)
a
N.D.
Calicotome infesta 50.4 (0.05)
c
N.D.
Lygeum spartum 26.5 (0.04)
f
N.D.
Moricandia arvensis 26.4 (0.5)
f
66.6 (5.5)
cd

Hypericum hircinum 15.1 (0.9)
g
43.4 (2.8)
e

Hypericum perforatum 31.5 (3.7)
ef
50.2 (0.03)
e

Cistus incanus 61.1 (1.7)
b
78.4 (1.0)
bc

Pistacia lentiscus N.D. N.D.
Cistus salvifolius N.D. N.D.
Different letters along the columns indicated significant differences at P<0.05 (Tukeys test). N.D.:
Not detectable. The values within the brackets indicated the standard error (N=3).




Figure 2. Dose-response curves of Total Root Length (TRL, cm) of Lactuca sativa L. seeds exposed
to aqueous extracts of different plant species for 24 () and 48 h (). All the dose-
response curves pointed out a significance level of P < 0.001.
Araniti et al 114

to seed germination, all plant species aqueous extracts showed lower toxicity in
L. multifida (2.8%), C. infesta (3.1%), P. lentiscus (4.4%), H. hircinum (5.3%),
A. arborescens (5.4%) than in H. italicum (16.9%), L. spartum (14.1%), A. cyanophylla
(13.6%) and C. monspeliensis (11.7%) (Table 3). Thus root elongation is best indicator of
allelopathic effects of plant extracts, being more sensitive to allelochemicals (7,11,35).

Table 3. ED
50
(%) response of root length of lettuce to aqueous extract of different plant species at
24 and 48 h after exposure (values estimated by the log-logistic equations)
Plant species Exposure time (h)
24 h 48 h

Lavandula multifida 39.5 (0.2)
abc
2.8 (1.2)
bc

Phlomis fruticosa 25.4 (7.6)
abc
7.3 (1.2)
abc

Artemisia arborescens 3.4 (0.3)
c
5.4 (1.5)
bc

Euphorbia rigida 4.5 (0.6)
c
6.4 (0.3)
abc

Cistus monspeliensis 15.8 (3.9)
abc
11.7 (3.1)
abc

Helychrisum italicum 62.8 (16.5)
a
16.9 (3.7)
a

Calamintha nepeta 23.7 (1.2)
abc
8.8 (2.8)
abc

Euphorbia dendroides 27.8 (7.1)
abc
7.1 (2.3)
abc

Acacia cyanophylla 49.6 (9.4)
abc
13.6 (1.7)
abc

Calicotome infesta 11.1 (3.5)
bc
3.1 (0.7)
c

Lygeum spartum 22.9 (13.8)
abc
14.1 (1.5)
abc

Moricandia arvensis 45.9 (0.8)
abc
7.3 (0.3)
abc

Hypericum hircinum 8.9 (3.9)
bc
5.3 (2.3)
bc

Hypericum perforatum 25.4 (6.5)
abc
6.5 (2.7)
abc

Cistus incanus 12.3 (3.7)
bc
7.1 (2.3)
abc

Pistacia lentiscus 7.6 (1.1)
c
4.4 (0.7)
bc

Cistus salvifolius 62.3 (26.2)
ab
7.8 (0.5)
abc

Different letters along the columns indicated a significant difference at P<0.05 (Tukeys test). The
values within the brackets indicated the standard error (N=3).

Cluster analysis: A cluster analysis of ED
50
values estimated at 24 and 48 h after
exposure on both seed germination and root elongation was done to assess, which plant
species showed the strongest allelopathic potential. P. lentiscus, C. salvifolius, A.
cyanophylla, C. infesta, L. spartum, C. monspeliensis and H. italicum were excluded from
this analysis, which caused a delay or null effect on lettuce seed germination (see Table 2).
Three discrete groups were obtained as under:

(i). Cluster I : C. nepeta, H. hircinum, A. arborescens and E. rigida;
(ii). Cluster II : H. perforatum, M. arvensis and E. dendroides and
(iii). Cluster III : P. fruticosa, C. incanus and L. multifida.

To compare the different clusters in terms of phytotoxicity, they were plotted on
an average of ED
50
values (24 and 48 h) for seed germination and root elongation,
respectively. The aqueous extracts of C. nepeta, H. hircinum, A. arborescens and E.rigida
(cluster I) showed the highest and most wide-spectrum phytotoxicity; whereas, the
aqueous extracts of H. perforatum, M. arvensis and E. dendroides (cluster II) showed
Screening of Mediterranean wild plant species for use as bio-herbicides

115
toxicity on root elongation, by contrast, the aqueous extracts of P. fruticosa, C. incanus
and L. multifida (cluster III) were more phytotoxic to seed germination (Fig. 3). Thus,
subsequent experiments were done with the aqueous extracts of most phytotoxic species:
C. nepeta, H. hircinum, A. arborescens and E. rigida.


Figure 3. Cluster analysis on the mean ED
50
values estimated at 24 and 48 h on both seed
germination and root elongation of lettuce treated with the aqueous extracts of 10 wild
plant species [A. arborescens (AA), E. rigida (ER), H. hircinum (HH), C. nepeta (CN),
H. perforatum (HP), M. arvensis (MA), E. dendroides (ED), L. multifida (LM), P.
fruticosa (PF), C. incanus (Cin).] of Mediterranean area.

Persistence of Phytotoxicity: All aqueous extracts did not change in phytotoxicity during
the storage period, causing a marked reduction in lettuce root elongation at both 24 and 48
h after exposure (Fig 4). In particular, the aqueous extract of A. arborescens showed the
highest phytotoxicity, causing over time (112 days) an average inhibition of root
elongation of 70% and 84%, after 24 and 48 h, respectively, compared to control. Even as
C. nepeta, H. hircinum and E. rigida aqueous extracts caused 60 and 80 % inhibition in
root elongation (Fig. 4). These results showed their potential as future natural herbicides.
The pH and EC of all aqueous extracts ranged from 4.89 to 5.87 and 2.88 to 4.98
dS/m. After 28 days of storage period, some extracts exhibited little increase in both pH
and EC values, which was not correlated with their phytotoxic activity (data not shown).
Thus, this variation in both parameters may be due to the biodegradation products
associated with allelochemicals (36) which, did not affect the phytotoxicity of extracts.

Pot experiments
All residues of 4-spontaneous plant species drastically inhibited the lettuce growth (5 and
10 % w/w), than control (Fig. 5). In particular, 5 and 10% (w/w) residues of A.
arborescens showed complete inhibition of lettuce plant growth. Similar behavior was
Araniti et al 116


Figure 4. Changes in phytotoxicity of C. nepeta (A, B), H. hircinum (C, D), A. arborescens (E, F), E.
rigida (G, H) aqueous extracts, on root elongation of Lactuca sativa L. exposed for 24 and
48 h, stored at 4 C in dark condition. Statistical significance of differences between treated
and untreated (0%) plants: *p < 0.05; **p < 0.01; ***p < 0.001 (Tukeys test at P < 0.05,
N= 4).
Screening of Mediterranean wild plant species for use as bio-herbicides

117
also observed with 10% (w/w) residue of E. rigida (Fig. 5). The lowest concentration of
residues (5%) of E. rigida, C. nepeta and H. hircinum caused a strong inhibition (96%,
89% and 85%, respectively) in lettuce root length, which was also observed with 10%
concentration of C. nepeta (98%) and E. rigida (91%) (Fig. 5). The 5% residue
incorporation of H. hircinum, C. nepeta, and E. rigida, inhibited the leaf area (LA) by 93,
92 and 90 % respectively. This effect increased at higher residue concentrations i.e was
concentration dependent (Fig. 5).



Figure 5. Effects of residue incorporation of A. arborescens (AA), C. nepeta (CN), H. hircinum (HH)
and E. rigida (ER) on root length (A), leaf area (B), root fresh weight (C), shoot fresh
weight (D), root dry weight (E) and shoot dry weight (F) of lettuce seedlings. Different
letters indicates a significant difference versus control (Ctr) (0 %) (Tukeys test at P < 0.05,
N= 4).

The residues incorporation of all species at both concentrations (5 and 10%)
caused significant reduction in the fresh and dry weight of shoot than the root system (29)
(Fig. 5). The sensitivity of shoot growth in Italian ryegrass to allelochemicals is known
Araniti et al 118
(20). The reduction in dry shoot biomass may be explained by the reduction of LA which
is associated with the plant capacity to capture the light and produce photosynthesis (34).
These observations suggested a common strategy in plant species in terms of elongation
and biomass allocation in roots in response to allelochemicals. Although the responses
varied depending on the test plant species (30,31), but they could be attributed to the
specific allelopathic compounds, where action was enhanced when the residues were
added in the soil mixture before planting (12).

Bioassay on weeds

Seed germination: The aqueous extract of A. arborescens, at 25% concentration,
completely inhibited the seed germination in both C. album and S. alba, while higher
concentrations of C. nepeta and H. hircinum (50 %) and E. rigida (75 %) extracts were
needed to cause the same inhibitory effect (Fig. 6).



Figure 6. Dose-response curves of Total Germination Index (G
T
, %) of C. album L., S. alba L. and
E. crus-galli L. seeds exposed to aqueous extracts of C. nepeta (A), A. arborescens (B),
E. rigida (C) and H. hircinum (D). N=4. All the dose-response curves pointed out a
significance level of P < 0.001.

The data were fitted by a non-linear regression and the curves of all species were
characterized by high statistical significance (P < 0.001) for both seed germination and
root elongation bioassays. In seed germination of C. album and S. alba, the aqueous
extracts of four species caused 50% inhibition (ED
50
) at following concentratios: 7.3 and
12% (A. arborescens), 14.14 and 19.25% (C. nepeta), 6.92 and 49.71% (E. rigida) and
29.8 and 82.7% (H. hircinum). The comparison of ED
50
clearly indicated that
Screening of Mediterranean wild plant species for use as bio-herbicides

119
A. arborescens extract was most phytotoxic. In addition, the aqueous extract of
A. arborescens inhibited the E. crus-galli (58%) (Table 4) which was tolerant to C. nepeta,
H. hircinum or E. rigida and never reached 50% of inhibition (data indicated as ND)
(Table 4). These results confirmed the resistance ability of E. crus-galli to chemical
compounds as previously reported (32).

Root Elongation: Root elongation was more sensitive to allelopathic compounds than
seed germination, hence the root elongation of weeds was strongly reduced by all aqueous
extracts (Fig. 7).



Figure 7. Dose-response curves of Total Root Length (TRL, cm) of C. album L., S. alba L. and
E. crus-galli L. seedlings exposed to aqueous extracts of C. nepeta (A), A. arborescens (B),
E. rigida (C) and H. hircinum (D). N=4. All the dose-response curves pointed out a
significance level of P < 0.001.

ED
50
values for root elongation were highly variable ranging from 1.48 % in S.
alba treated with C. nepeta to 46.1 % in E. crus-galli treated with E. rigida aqueous
extracts (Table 4), making it impossible to establish a hierarchy of phytotoxicity. In
particular, E. rigida (4.6%) was more toxic than H. hircinum (7.07%), A. arborescens
(7.9%) and C. nepeta (17%) to inhibit C. album (Table 4). Whereas, in S. alba the
strongest effect was caused by C. nepeta (1.48%) followed by A. arborescens (8.7%), E.
rigida (11.63%) and H. hircinum (13.7%) extracts (Table 4). Finally, C. nepeta (4.4%),
H. hircinum (7.2%) and E. rigida (46.1%) extracts reduced E. crus-galli root elongation, while
A. arborescens did show any effect (data indicated as ND) (Table 4).


Araniti et al 120
Table 4. ED
50
(%) response of seed germination (G
T
) and root elongation (TRL) of weeds to aqueous
extracts of different plant species (estimated by the log-logistic equations)
Aqueous extracts
A. arborescens C. nepeta E. rigida H. hircinum
Weed species
G
T
TRL G
T
TRL G
T
TRL G
T
TRL
C. album 7(0.3)
a
8(1)
a
14(0.7)
a
17(4)
a
30(2.5)
a
5(0.8)
a
30(2.9)
a
7(1.3)
a

S. alba 13(0.5)
a
9(0.5)
a
19(0.8)
b
2(0.1)
b
50(7.3)
b
12(1.6)
a
83(9.7)
b
14(1.7)
b

E.crusgalli 58(4.8)
b
ND ND 4(0.7)
b
ND 46(5.4)
b
ND 7(0.6)
a

Different letters along the columns indicated significant differences at P<0.05 (Tukeys test). N.D.:
Not Detectable. The values within the brackets indicated the standard error (N=4).

Several hypotheses could be advanced to explain the weed responses to aqueous
extracts in both physiological processes: a selective activity of allelochemicals for target species
(16); a different level of weed species tolerance to such allelochemicals. This latter hypothesis
could be due to versatile detoxification systems of weeds to counter the phytotoxicity of
chemicals, by reducing their uptake, and/or partitioning the chemical from the target site and/or
modifying the molecules (13). Furthermore, different seed size, the coat permeability of weeds
may also cause the varying responses to allelochemicals (52).

CONCLUSIONS

Among 17-wild plant species, aqueous extracts of A. arborescens, E. rigida,
C. nepeta and H. hircinum drastically inhibited the seed germination and root growth of
L. sativa. These also inhibited the seed germination and root elongation of common weed
species (Chenopodium album, Sinapis alba) and with less inhibition in Echinochloa crus-galli.
Hence these are promising source of bio-herbicides.
Conversely, the remaining plant species strongly inhibited the root elongation, but
delayed the lettuce seed germination. Their allelopathic potential and their efficacy as source of
natural compounds was also confirmed in pot culture by adding the residues of A. arborescens,
E. rigida, C. nepeta and H. hircinum to soil mixture (simulating the field conditions) reduced
the shoot growth less than root growth of lettuce. The phytotoxicity of these plant species
followed the order: A. arborescens > E. rigida > C. nepeta H. hircinum. Their extracts
phytotoxicity to these plant species had never been tested on the plant growth.
The Mediterranean area is important source of potential natural herbicides in
sustainable agriculture. Before the original hypothesis of allelopathic effects and their possible
use as herbicidal templates can be confirmed additional research is required: (i) To distinguish
the allelopathic activity of separate plant organs (leaves versus stems), (ii) To investigate the
allelopathic effects of root exudates of these species and (iii) To isolate, identify, and
characterize the allelochemicals responsible for phytotoxic effects through a bioassay-guided
fractionation.

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