JURNAL HERBISIDA 1

0971-4693/94 US $ 5.00 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. SORGONÀ, A. LUPINI and M.R. ABENAVOLI*

Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale (BIOMAA), Università Mediterranea di Reggio Calabria, Facoltà di Agraria - Salita

Melissari, I-89124 Reggio Calabria RC, Italy E. Mail: [email protected]

(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

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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 µm Millipore filter. The aqueous extracts were stored at -20°C 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

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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 (GT), was calculated as under:

Where, NT: 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

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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 Règent 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 ED50 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 [25±1 °C with a 16-h photoperiod, a photon flux rate of 120 µmol 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 (GT %) 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/ln1 EDxBe

CDCy

+

−+=


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where, C : lower response at indefinitely large doses, D: mean response of control, B: slope around the ED50 and ED50 : dose causing 50% of total response (considered as the index of phytotoxic capability of aqueous extracts). By comparing the ED50 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 ED50 as a variable and plant species as the main factor. The ED50 data were first checked for deviations from normality (Kolmogorov-Smirnov test) and tested for homogeneity (Leven Median test). Tukey’s test comparison was used to compare the mean values of ED50 among the plant species during their exposure time (P < 0.05). Cluster’s analysis was also done to identify discrete groups of plant species with similar toxic ED50 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 Tukey’s 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

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germination (Fig. 1). The comparison of ED50 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 (ED50 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 (GT, %) 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 ED50 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 ED50 values ranged widely from 3.4 % to 62.8 %, (Table 3). At 48 h after exposure, in contrast


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Table 2. ED50 (%) 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 (Tukey’s 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.

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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. ED50 (%) 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 (Tukey’s test). The values within the brackets indicated the standard error (N=3). Cluster analysis: A cluster analysis of ED50 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 ED50 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


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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 ED50 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

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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 (Tukey’s test at P < 0.05, N= 4).


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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 %) (Tukey’s 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

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(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 (GT, %) 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 (ED50) 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 ED50 clearly indicated that


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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.

ED50 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).

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Table 4. ED50 (%) response of seed germination (GT) 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

GT TRL GT TRL GT TRL GT 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 (Tukey’s 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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