JOURNAL OF MARINE SCIENCES & ENVIRONMENTAL TECHNOLOGIES Vol. 3, Issue No. 2 (December-2017)

This study aims to investigate the allelopathic effects of Artemisia herba-alba Asso. aqueous extracts on the germination of Ceratonia siliqua L. seeds and seedlings. Three concentrations 100%, 50 %, 25%, and control (distill water) were used for testing. Seeds and seedlings of Ceratonia siliqua were exposed to above-mentioned concentrations. One-way ANOVA test was adopted to examine the effect of these treatments on germination rate and root length for the seeds of Ceratonia siliqua. Our results showed no significant differences between the concentrations on germination rate as the F-Value and P-Value were 2.88 and 0.08 respectively. However, high significance occurred after testing concentrations against root length with F-value reaching (139.89). Tukey HSD (Honestly Significant Difference) test showed that highest significance in means was imposed by both (100 % and 50 %) levels of concentration on root length. It also shows a strong inverse relation between root length and concentrations levels reflected in R-squared value of (0.93). The treatments affected Ceratonia siliqua roots by inhibiting their length significantly (F-Value: 21.1). In conclusion, the increase of concentration levels result in the inhibition of mainly root length. That may explain the law survival of Ceratonia siliqua seedlings in regions where Artemisia herba-alba grow. However, further studies are required to investigate other factors.


Introduction
One of the most fundamental driving forces in forest ecology, and eventually in forest dynamics and biodiversity, is plant-to-plant relationships (Dunnett & Grime, 1999;Escudero et al., 2000;and Brooker, 2006).Allelopathy in plants can be defined as the effects of one plant on other plant through the release of chemical compounds in the environment (Rice, 1984;Hussain & Khan, 1988;and Mallik, 2008).Therefore, the natural regeneration of certain species in forest ecosystems maybe affected by allelopathy.Several studies have reported the inhibitive behavior of different plants on the germination of their seeds or on the seeds of other species, which results in the alternation of seed bank viability in forests as it discussed in the current context.In arid and semi-arid regions of the world, it is important to consider all factors that may affect the plant community succession, composition and dynamics since the competition between plants is very high due to the limited water and nutrition resources in such parts of the planet (Escudero et al., 2000).Chemical microenvironment constitutes determinant factors that promote or inhibit seed germination are proved to be produced by a number of species in arid and semi-arid regions (Long et al., 2015).
Artemisia herba-alba Asso (white wormwood) is a perennial shrub occurs generally on the dry steppes of the Mediterranean regions in Northern Africa (Morocco, Tunisia, Algeria, and Libya), Western Asia (Arabian Peninsula) and Southwestern Europe (www.theplantlist.org).It is a micro-chamaephyte, perennial plant in which the living parts of aerial shoots become very reduced in summer, 20-40 cm, aromatic, tomentose, greyish, easily uprooted owing to its superficially ramified roots.Heads sessile, oblong, 3-4 mm, 2-4 flowered (IUCN, 2005).The species belongs to Asteraceae family and has other Synonyms namely: A. aethiopica L., A. aragonensis Lam., A. lippii Jan ex Besser, A. ontina Dufour, and Seriphidium herba-alba Soják.It is used as an antiseptic and antispasmodic in herbal medicine (Yashphe et al., 1987).In terms of its chemical composition, A. herba-alba has five main substances: α-thujone; campholene aldehyde; 2, 4-Hexadiene, 2, 3-dimethyl-; Artemisia triene and Sabinene (Sbayou et al., 2014).A very recent research conducted by Arroyo et al. (2016) cited most important studies that investigated the allelopathic properties of A. herbaalba and concluded that it is a remarkably allelopathic species, especially with the increase of concentration.According to Arroyo et al. (2016), the application of A. herba-alba aqueous extract reduced seedling emergence from the seed bank by 50%.Other authors reported similar results regarding the exhibition of allelopathy by many herbaceous, aromatic shrubs and trees species in regions that have similar environmental conditions to the Mediterranean The Allelopathic Effects of Artemisia herba-alba Asso.Aqueous ………………………… Faculty of Marine Resources, Al-Asmarya Islamic University, Libya.

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ISSN: 2413-5267 ecosystems (Thompson, 2005;and Scognamiglio et al., 2013).It seems that most species of the genus Artemisia have general positive tendency towards allelopathy, yet with different extents between its species.Examples of this tendency are documented by several recent studies.Elshatshat (2010) reported a proportional relationship between seven concentration levels of Artemisia herba-alba aqueous extract and the inhibition level on Tomato and Wheat seed germination.Salhi et al. (2011) found that the aqueous root extract of Artemisia herbaalba has no significant effect on the germination of Avena fatua L. and Polygonum convolvulus L, whereas the aerial parts aqueous extract showed higher significance effect on same measured parameters.Abd El-Fattah et al. (2011) examined the allelopathy of Artemisia princeps extracts (3.75 and 5% w/v) and they were phytotoxic to germination of wheat.The reduction in the germination percentage reached 15 to 30% by treatment with A. princeps after 8 days of treatment.Bataw and Hughes (2013) investigated the effect of Artemisia herba-alba on common bean and concluded that the 4% aqueous extract of shoots contain allelopathic chemicals and phytotoxic compounds that may lead to an increase in unsaturated and saturated fatty acids in germinated common bean seedlings.Other results obtained by Tilaki et al. (2013) showed that increase in essential oil concentration reduced root lengths, seed germination percentage and vigor index of Agropyron desertorum and Agropyron cristatum.
Understanding the role of allelopathy exhibited by plants in forest ecosystems may represent a potential tool for management (Cummings et al., 2012).Weed management, seed bank and seedling establishment and growth can be controlled by inducing or, alternatively, reducing naturally produced chemical compounds derived from local spices (Caboun & Jhon, 2015).
To make a brief summary of what we discussed above, it is important indeed to understand the role of allelopathy phenomenon.Such importance should be a priority in regions where natural vegetation is degraded like what we have in Libya.Studies about the role of allelopathy in forest ecosystems development are, in fact, rare (Elshatshat, 2010).For these reasons, we present this study to examine the effect of Artemisia herba-alba aqueous extract on a vital forestry species Ceratonia siliqua L.
C. siliqua from Caesalpiniaceae family It is one of the most important species.It is used locally for the production of honey and the Carob syrup.It is also a good source of food for wild animals and demotic livestock.It faces a decline in the production of seedlings despite the massive amount of seeds produced by mother trees in natural habitats.Over-grazing could be a major reason for the loss of fresh seedlings but in some inaccessible locations, it is hardly to notice any natural regeneration.Hence, comes the present study to observe the possible role of allelopathy on this species and to, further, understand about the action of Artemisia herba-alba in nature.

Preparing the aqueous extract of Artemisia herba-alba
After the collection of the plants, they were dried in a shady place at room temperature for ten days.At the forestry department laboratory of Omar Al-Mukhtar University, the dried areal parts (leaves and stems) were ground into powdered form.Then 50 g of the powder were diluted into 500 ml of distilled water (Bajalan et al., 2013).Next to that, this mixture was left on a Shaker for 24 hrs in room temperature at speed of 120 rpm (Ghorbani et al., 2008).A four-folded cotton fabric was used as a filter to separate rough solid particles from solution, and then it was centrifuged with the speed of 2000 rpm for 15 minutes (Bajalan et al., 2013).Three concentrations of solutions were prepared based on volume/volume percent (v/v.%) except for the basic solution 100 % which was a weight/ volume percent (w/v.%) (Elshatshat, 2010).Three concentrations were prepaid (25, 50, and 100%) in addition to the distilled water as a control.

Preparing Germination and Seedling Experiments
The seeds of C. siliqua were cleaned and then floating test was implemented.To break seeds dormancy and deviate its effect on germination, a chemical treatment was applied.According to (Bostan, and Kilic, 2014), treating C. siliqua seeds with Sulphuric acid (95 %) for 20 The Allelopathic Effects of Artemisia herba-alba Asso.Aqueous ………………………… Faculty of Marine Resources, Al-Asmarya Islamic University, Libya.

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ISSN: 2413-5267 minutes would result in high germination percentage reaching (95-99 %).A test was conducted to confirm these results to be used in this experiment.Results of the current study was very similar and, hence, used in this study.Completely Randomized Design CRD was implemented.80 treated seeds were randomly collected and randomly distributed on 16 Petri dishes (90 mm diameter), five seeds per dish.Each treatment concentration was applied on four Petri dishes (Four replicates).The parameter of interest in this part of the study was the germination rate after the application of (distilled water, 25, 50, and 100% of Artemisia herba-alba).All dishes were placed in a low temperature incubator at 21 o C for eight days (Abd El-Fattah et al., 2011;Al-Watban & Salama, 2012;and Bataw & Hughes, 2013).Percentage of germination for each dish was calculated and the root length for germinated seeds was measured.For the evaluation of growth parameters (total length, root length, and number of leaves), 12 early young seedlings of C. siliqua were randomly measured.Each three seedlings were irrigated by the same concentration level.Root length, stem length and number of leaves were recorded.One-way ANOVA (P < 0.05) was carried out using Minitab 18 (Trail Licensed Version, 2017) to compare the germination among treatments.

Seed Germination
The germination started in the control treatment in 48 hours from the beginning of the trial reaching 100% in all four dishes.Observations were recorded every 48 hrs and the germination was tested in eight days (Abd El-Fattah et al., 2011;and Al-Watban & Salama 2012).The analysis of variance showed that there are no significant differences between the effects of A. herba-alba aqueous extracts on the germination of C. siliqua seeds after eight days of the experiment.Similar findings on the germination of C. siliqua has been reported by (Bataw and Hughes, 2013;and Tilaki et al., 2013).

Root Length of Germinated Seeds
The measurements taken by the end of the germination trials showed a very clear significan as shown in Table (1).Tukey HSD (Honestly Significant Difference) test revealed that all treatments exhibited high significance between all pair-wised comparisons (Arroyo et al., 2016).As it appears from Table (1), subjecting seeds to higher concentration results in the inhibition of root length.These findings confirm what has been demonstrated by (Elshatshat, 2010;Bataw & Hughes, 2013;and Tilaki et al., 2013).

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ISSN: 2413-5267 total length and number of leaves in the case of C. siliqua.Significance appeared when roots length of C. siliqua seedlings were tested against concentration.
Table 2. ANOVA for the differences of means between stem total length, root length, number of seeds α = 0.05 Based on results of Table (2), Tukey HSD test was conducted to compare between the effects of the treatments on root length of C. siliqua seedlings.As it explained in Table (3), comparing the effect of treatments on root length for C. siliqua seedlings revealed that all treatments have significant effect compared to control.However, no significant differences between both levels 25 % and 50 % nor between 50 % and 100%.Apparently, the later level of concentration had the highest significance in inhibiting roots to reach only 1.19 cm in average.Thus, it may be explanatory to study the regression analysis and understand the relation between tested concentrations and both root length and number of leaves of examined species.

Conclusion
The findings of this study, in general, go along with all conclusions cited in literature reviewed.It is noticed clearly that despite testing the Artemisia herba-alba aqueous extracts on different species (forestry, weeds and crops), similar results were obtained and reported.An extrapolation of these findings may be the key to the begging of adopting new management tools in the field of forest ecology.
Effects of Artemisia herba-alba aqueous extracts on germination & root length of C. siliqua.α = 0.05 Means within columns followed by different letters are significantly different according to Tukey (HSD) P<0.05.Noticeably, there was a systematic and proportional pattern relates the decline in root length of C. Siliqua and the increase in the concentration of Artemisia herba-alba aqueous extracts.The function between both variables is statistically illustrated in Figure (2).The coefficient of determination value (R 2 ) reached to 0.93 and the model of regression can be expressed by bellow given equation: RL = 12.57-14.44 C + 5.967 C 2 ………………... (1) Whereas: RL is root length and C is concentration value.

Figure 2 .
Figure 2. Analysis of regression between root lengths of C. Siliqua seedlings and concentrations of Artemisia herba-alba aqueous extracts

Figure 3 .
Figure 3. Regression between concentration root length for C. siliqua along with the associated coefficient of determination value.

Table 3 .
Tukey HSD test for the effects of treatments on the root length of C. siliqua seedlings Figure (3) reveals fair regression between the only dependent variable in our experiment that is concentration and explanatory variable (Root length for C. siliqua).The coefficient of determination value (R 2 ) was 0.73 for root length and the model of regression can be expressed by bellow given equation: RL is root length and C is concentration value Faculty of Marine Resources, Alasmarya Islamic University, Libya.E-8ISSN: 2413-5267