Impaginato 503 Adv. Hort. Sci., 2018 32(4): 503-510 DOI: 10.13128/ahs-22083 Allelopathic activities of celery extract and its fractions against Corchorus olitorius, Echinochloa crusgalli and Portulaca oleracea weeds R. El-Mergawi (*), E.R. El-Desoki Botany Department, National Research Centre, 12622 Cairo, Egypt. Key words: Apium graveolens, germination, natural herbicide, phenolic acids. Abstract: Aqueous extract of celery (2.5-20 g l-1, w/v) was evaluated for its phy- totoxicity against three weed species, under laboratory and greenhouse condi- tions. Celery extract had a strong inhibitory effect on germination and seedling growth of Corchorus olitorius, Echinochloa crusgalli and Portulaca oleracea seeds. From dose response curves of tested seeds, LC50 were calculated to be in the range from 6.3 to 8.3 g l-1 for germination percent, from 7.2 to 8.0 g l-1 for shoot length and from 1.7 to 3.6 g l-1 for root length. Completely inhibition of root growth was exhibited C. olitorius and P. oleracea at 7.5 g l-1 corresponded with 15 g l-1 for E. crusgalli seed. Total phenolics in celery extract at 20 g l-1 con- stituted 201 mg l-1. Ten phenolic acids were identified in extract by HPLC, among of them p-coumaric acid and p-hydroxybenzoic acid were presented in high amounts. Aqueous extract was partitioning between three solvents, hexa- ne, methylene chloride, ethyl acetate. Generally, water residue after partition- ing aqueous extract with the three solvents had the most phytotoxic effect on seedling growth of target seeds. In greenhouse trial, foliar spray of aqueous extract of celery (30, 60 and 90 g l-1) and its fractions did not produce any signif- icant effect on growth of two-weeks-old C. olitorius, or E. crusgalli or P. oler- acea weeds. 1. Introduction Worldwide, weeds caused about 34% yield losses among the major crops. Herbicide application is the most reliable weed control methods. Negative impacts of herbicides on environmental human health and her- bicide resistant weeds were considered the two problems faced in weed management with herbicides (Jabran et al., 2015). Thus, manipulating the allelopathy can help to improve weed control in agriculture and increase the acceptance of agricultural products in today´s demanding consumer markets (Trezzi et al., 2016). Allelopathy has been used as the basis for identifying plant species which may contain phytotoxic chemicals. These natural compounds can (*) Corresponding author: ragabelmergawi05@hotmail.com Citation: EL-MERGAWI R., EL-DESOKI E.R., 2018 - Allelopathic activities of celery extract and its fractions against Corchorus olitorius, Echinochloa crusgalli and Portulaca oleracea weeds. - Adv. Hort. Sci., 32(4): 503-510 Copyright: © 2018 El-Mergawi R., El-Desoki E.R. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 22 November 2017 Accepted for publication 28 May 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(4): 503-510 504 offer excellent potential for new herbicidal solutions, or lead compounds for new natural herbicides (Duke et al., 2000; Vyvyan, 2002). The main purposes of research on allelopathy include the application of the allelopathic effects to agricultural production, reduc- tion of the input of chemical pesticides and conse- quent environmental pollution, and provision of effective methods for the sustainable development of agricultural production and ecological systems (Han et al., 2013; Jabran et al., 2015). Knowledge concerning allelopathy can also be a key component in supporting organic farming, for which weed con- trol is a major problem (Trezzi et al., 2016) Organic farming can involve reduced weed infestation by using plant extracts or intercropping plant species with an allelopathic potential (Bajwa et al., 2015). Plant extracts are the way of using allelochemicals for weed control in agroecosystems, as they have been already used as post-emergence natural herbi- cides in some countries. In Pakistan, for example, an aqueous extract deriving from sorghum shoots with a 10% concentration was left to ferment for several weeks and was subsequently sprayed post emer- gence for weed control. This fermented water extract, known as “Sorgaab”, reduced weed density a n d w e e d d r y w e i g h t u p t o 5 0 % i n f i e l d t r i a l s (Cheema and Khaliq, 2000; Cheema et al., 2002). Celery (Apium graveolens L.; Apiaceae) has been cultivated for the last 3000 years, notably in Egypt, and was known in China in the fifth century BC. It has been used as a popular aromatic herb and spice (Chevallier, 1998). Earlier studies of A. graveolens led t o i s o l a t i o n o f s o m e p h e n o l i c c o m p o u n d s a s phthalides (Tang et al., 1990; Momin and Nair, 2001) and furocoumarins (Garg et al., 1979). These com- pounds are reported for their insecticidal, nematici- dal, antifungal and phytotoxic activities (Kato et al., 1977; Momin and Nair, 2001; Pavela and Vrchotová, 2013). Recently, Sbai et al. (2017) reported that the aqueous extract (10-50 g/L) of A. graveolens had great inhibitory effect on root growth of germinated seeds of lettuce (in the range between 80% and 90%). They isolated six compounds which included three phthalides [senkyunolide A, (3S)-butylphthalide and sedanolide], two furanocoumarins (bergapten and scopoletin) and one phenyl propanoid (p-hydrox- yphenethyltrans ferulate). Senkyunolide A compound was the most toxic on lettuce germination and shoot growth, however, p-hydroxyphenethyl trans-ferulate was the most toxic on root growth. However, in spite of the wide range of biological activity of celery extract, but information concerning the herbicidal activity of this extract is rarely avail- able. Therefore, the main objective of this study was to evaluate the herbicidal activity of the aqueous cel- ery extract and its fractions against Corchorus olito- rius, Echinochloa crusgalli and Portulaca oleracea weeds, with the goal of developing an effective plant derived herbicide. Moreover, the phenolic acids that considered the main source of all bioactive phenolic substances were identified in celery extract via HPLC. 2. Materials and Methods Plant material Plants of celery (A. graveolens L. var. dulce) were purchased from a local market in Cairo, Egypt. Identification of celery based on morphological traits that extensive observation of mature plants. The leaves located at the top of the leaf stalks were col- lected and dried in hot-air oven at 50°C for 72 h, powdered and used for extraction. Preparation of aqueous extract Different concentrations (w/v) of extracts were prepared by soaking known weight of dried leaves in known volume from distilled water at room tempera- ture, and shaken for 24 h. The extracts were filtered through a Whatman No. 1 filter paper and kept at 4°C in the dark until use. Germination bioassay Mature seeds of Corchorus olitorius, Echinochloa crusgalli and Portulaca oleracea were collected from plants growing in fields of the experimental station of t h e N a t i o n a l R e s e a r c h C e n t r e ( R e s e a r c h a n d P r o d u c t i o n S t a t i o n , N u b a r i a r e g i o n , B e h a i r a Governorate, Egypt. Uniform healthy seeds were selected. Seeds were surface-sterilized with sodium hypochlorite (0.1%, w/v) for 2 min, washed under running tap water for 5 min followed by distilled water for 2 min, and stored for further use. Twenty seeds were placed in a 9-cm plastic Petri dish lined with a single Whatman No. 1 filter paper, and then 4 mL of each extract concentration (2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 g l-1) was added onto filter paper. Distilled water (4 mL) was applied to a Petri dish to serve as a control. Four replicates of Petri dishes of each treatment were placed, in a complete- ly randomized manner, in a growth chamber at 25°C. After 5 days, germination percent, shoot length and root length of germinated seeds were determined. El-Mergawi and El-Desoki - Allelopathich activies of celery extract as weeds control 505 Extract concentrations required to cause 50% inhibi- tion of different germination criteria (germination percentage, root length and shoot length) were cal- culated by Probit analysis. Determination of total phenolics in aqueous extract Extract of celery at 20 g l-1 was subjected to deter- m i n e t o t a l c o n t e n t s o f p h e n o l i c s u s i n g F o l i n - Ciocalteu reagent (Singleton and Rossi, 1965). Analysis of phenolic acids in aqueous extract by High- Performance Liquid Chromatography (HPLC) Phenolic acids in aqueous celery extract at a con- centration 20 g l-1 were hydrolyzed with sodium hydroxide (McKeehen et al., 1999) and subjected to HPLC analysis. Approximately 15 ml of aqueous extract was added to 15 ml of 8 N NaOH in a 50 ml Pyrex centrifuge tube, purged with nitrogen, shaken for 2 h in the dark with a shaker and acidified with ice-cold 6 N HCl to reduce pH to 2. Sample was cen- trifuged at 3000g, and the supernatant was decanted into separatory funnel. The supernatant was extract- ed with ethyl acetate (3 x 50 ml) with shaking for 10 s, and the mixture was allowed to settle for 5 min between extractions. The phenolic acids rich ethyl acetate fraction was dried by addition of anhydrous sodium sulfate and concentrated using rotary evapo- rator at 40°C to dryness. The residue was re-solubi- lized in 2.5 ml of methanol and filtered through a 0.2 μm PTFE filter prior to analysis. HPLC analysis was performed using equipment from Shimadzu (Japan): a Shimadazu LC-2010A liquid chromatograph, a Shimadazu SPDM10A Diode Array Detector and a Shimadzu Class-vp V6.12 SP4 offline processing sys- tem. Phenolics were analyzed using a Luna RP-C18 (2) column (250×4.6 mm i.d, 5 μm, Phenomenx). The mobile phase consisted of a mixture of acetate buffer: acetonitrile (9:1, v/v). Acetate buffer was pre- pared by dissolving 6.35 g sodium acetate in one-liter H2O and 20 ml acetic acid. The detecting wavelength was 260 nm. Standard phenolic acids (gallic, proto- catechuic, p-hydroxybenzoic, syringic, ferulic and p- coumaric) were purchased from Sigma Aldrich and vanillic, caffeic, salicylic and cinnamic were pur- chased from Fluka. Greenhouse bioassay Another experiment was performed to study the effect of the aqueous. celery extract on the growth of two-weeks-old C. olitorius, E. crusgalli and P. oler- acea weeds grown under controlled conditions in an experimental greenhouse (25±3°C, 12 h photoperi- od). Aqueous extracts at four concentrations (0, 30, 60 and 90 g l-1) were prepared as previously men- tioned and used in a greenhouse bioassay. Plants were raised from the collected seeds in 12-cm diame- ter earthenware pots. The pots were filled with 750 g soil (sand:peat moss = 3:1, w/w) and 20 seeds of each plant species were sown per pot. Two weeks after emergence, plants were sprayed with plant extracts at 0, 30, 60 and 90 g l-1 concentrations. Extract solutions were applied to shoots of tested weeds using a Epoca sprayer (Italy). The solution was sprayed evenly over the entire surface of the plant, including the adaxial and abaxial surface of leaves. A total of 48 pots were maintained (i.e. 4 extract con- centrations × 3 weeds × 4 replicates) in a completely randomized design. Seven days after extract spray- ing, the plants were examined for visible injury levels and the percent of chlorotic and necrotic areas were recorded. Fractionation of extract One hundred milliliter of high extract concentra- tion that used either in germination trial (20 g l-1, w/v) or in greenhouse trial (90 g l-1, w/v) was subse- quently partitioned with organic solvents with increasing polarity. The extract was partitioned three times with 200 mL aliquots of each solvent: hexane, methylene chloride (MeCl2) and ethyl acetate (EtOAc) using separatory funnel. Solvents were dried by addi- tion of anhydrous, sodium sulfate, filtered and evap- orated using Buchi Rotary Evaporator at 40°C to dry- ness. The residues of hexane, MeCl2 and EtOAc frac- tions were dissolved in 100 mL of DMSO-water solu- tion (0.1%, v/v). These three fractions and water residue (H2O fraction) were subjected to bioassay as previously mentioned. Statistical analysis All data was subjected to ANOVA analysis using costat software to evaluate the effect of extract rates upon germination and growth parameters of tested weeds. After ANOVA, the parameters that were sta- tistically significant (A. graveolens extracts with P≤0.05), were subjected to Pro-bit analysis using LdP line. Pro-bit curves were derived by plotting the extract concentration (on the x-axis) and inhibition % (on the y-axis). With the curves obtained, LC50 value was calculated for each parameter. This value repre- sents the extract concentration at which 50% inhibi- Adv. Hort. Sci., 2018 32(4): 503-510 506 tion in different germination parameter occurs. 3. Results Effect of celery extract on germination percentage of seeds As shown in Table 1, aqueous extract of celery had a strong inhibitory effect on germination per- centage of the three tested seeds and the inhibition increased with concentration. Increasing extract con- centration above 5 g l-1 produced gradual decrease in germination percentage of C. olitorius. The highest reduction effect was produced by extract at highest concentrations, 17.5 and 20 g l-1 (89% and 93%, respectively). Also, germination percentage of E. crusgalli and P. oleracea was reduced by the extract at all tested concentrations. The reduction percent- age varied from 37% and 9%, respectively at the low- est concentration to 96% and 73%, respectively at the highest ones. Effect of celery extract on Seedling growth of seeds Aqueous extract of celery exhibited a great inhibitory effect on shoot length of the three germi- nated seeds (Table 2). The extract improved the shoot growth of E. crusgalli and P. oleracea at the lowest concentrations. The progressive increase in extract concentration followed by progressive reduc- tion in shoot length of all tested seeds. More than 90% reduction in shoot length of three germinated seeds was obtained by soaking seeds in celery extract at 17.5 and 20 g l-1. An inhibition of root growth was observed in pres- ence of all extract concentrations and roots more sensitive than shoots (Table 2). Root length of three target seeds varied in their response to different extract concentrations and E. crusgalli was less effec- tive one. Soaking seeds in celery extract at the lowest concentration produced great reduction in root elon- gation of all treated seeds ranged between 36% and 71%, relative to control. Using extract at 7.5 g l-1 and above completely inhibited root growth of both P. oleracea and C. olitorius, whereas complete inhibi- tion of E. crusgalli roots was obtained at 15 g l-1. Extract concentrations required to cause 50% inhibi- tion (LC50) Inhibition percent and levels of LC50 of all germina- tion parameters of tested weeds were calculated and dose-response curves were illustrated in figure 1. The three tested weeds varied in their inhibition percent- ages as affected with celery extract. Among tested seeds, germination percent of P. oleracea was con- Concentration (g l-1) Germination % C. olitorius E. crusgalli P. oleracea Control 100±0 a 100±0 a 100±0 a 2.5 95±5 a 63±7 b 91±9 b 5 93±2 a 67±4 b 84±4 b 7.5 49±5 b 52±2 c 73±3 c 10 41±4 c 43±5 c 74±7 c 12.5 23±3 d 39±7 c 60±2 d 15 11±5 e 25±4 d 40±2 e 17.5 11±5 e 14±5 d 37±2 e 20 7±2 e 4±1 e 27±3 f Table 1 - Effect of aqueous celery extract on germination per- centage of three weed species Values are given as means of three replicates ± standard error. Means with the same letters in a column are not significantly dif- ferent at P<0.05. Table 2 - Effect of aqueous celery extract on shoot and root lengths of three weed species Values are given as means of three replicates ± standard error. Means with the same letters in a column are not significantly different at P<0.05. Rate (g l-1) Shoot length (cm) Root length (cm) C. olitorius E. crusgalli P. oleracea C. olitorius E. crusgalli P. oleracea Control 2.56±0.14 a 3.35±0.35 a 2.17±0.13 b 2.41±0.08 a 1.83 ±0.26 a 1.95±0.23 a 2.5 2.74±0.25 a 2.91±0.34 b 2.50±0.23 a 0.81±0.17 b 1.17 ±0.12 b 0.57±0.07 b 5 2.21±0.19 b 2.43±0.19 c 2.19±0.60 b 0.51±0.11 c 0.65 ±0.20 c 0.29±0.06 c 7.5 1.38±0.40 c 1.73±0.33 d 0.58±0.40 c 0.00 d 0.51 ±0.17 c 0.00 d 10 0.76±0.28 d 0.79±0.20 e 0.60±0.06 c 0.00 d 0.25 ±0.06 d 0.00 d 12.5 0.36±0.06 e 0.77±0.20 e 0.56±0.31 c 0.00 d 0.22 ±0.01 d 0.00 d 15 0.10±0.02 e 0.53±0.31 ef 0.15±0.08 d 0.00 d 0.00 e 0.00 d 17.5 0.05±0.03 e 0.27±0.14 ef 0.14±0.14 d 0.00 d 0.00 e 0.00 d 20 0.00 f 0.06±0.03 f 0.11±0.06 d 0.00 d 0.00 e 0.00 d El-Mergawi and El-Desoki - Allelopathich activies of celery extract as weeds control 507 sidered a less sensitive to celery extract. Since, value of LC50 of P. oleracea constituted 13.4 g l-1 corre- sponded with 8.3 and 6.3 g l-1 for C. olitorius and E. crusgalli, respectively. Whereas, inhibition percent of shoot length did not produce a great variation among tested seeds and LC50 calculated to be 7.8, 7.2 and 8 g l-1 for C. olitorius, E. crusgalli and P. oleracea seeds, respectively. As shown in figure 1, root length exhib- ited the maximum inhibition effect as affected by cel- ery extract when compared with germination per- cent and shoot length. Moreover, root length of test- ed seeds varied in their inhibition percent as affected with celery extract. Depending on LC50 levels, root length of P. oleracea (1.65 g l-1) was considered the most sensitive, followed by C. olitorius (1.8 g l-1) and E. crusgalli (3.6 g l-1). Total phenolics and phenolic acids content in aqueous extract In this study, we determine total phenolics for cel- ery extract at a concentration 20 g l-1 and found that the extract contained high amounts of phenolics con- Fig. 1 - Dose-response curves showing the effect of celery extract (inhibition % and LC50) on percent germination and seedling growth of three tested seeds. 508 Adv. Hort. Sci., 2018 32(4): 503-510 stituted 201 mg l-1, as gallic acid. Using HPLC, ten p h en o l i c a c i d s w ere d et ec t ed i n t h i s a q u eo u s . extract, of which six compounds consisted of benzoic acid derivatives and four of cinnamic acid derivatives (Table 3). Among the identified phenolic acids, p- hydroxybenzoic acid, p-coumaric acid and ferulic acid were presented in high amounts (18.72, 30.32 and 11.12 mg l-1, respectively). Except the minor quantity of cinnamic acid, moderate concentrations from other phenolic acids were determined in celery sam- ple. Effect of fractions on germination of weeds As shown in Table 4, fractions obtained from the aqueous extract of celery varied in their effects on germination criteria of target seeds. As for germina- tion percentage, the significant reduction effect was recorded only for MeCl2 fraction on P. oleracea (15%) as well as for H2O residue on both C. olitorius (30%) and P. oleracea (36%). But, all examined fractions did not produce any significant effect on germination percentage of E. crusgalli seeds. Whereas, H2O frac- tion induced a great reduction on shoot elongation of C. olitorius, E. crusgalli and P. oleracea seeds, reached 64%, 47% and 74%, respectively relative to control. With less extent, hexane and MeCl2 fractions reduced shoot length of E. crusgalli. With few excep- tions, all fractions reduced root elongation of target seeds. Water fraction was the most toxic, inducing an inhibition of 99%, 94% and 50% in root length of P. oleracea, C. olitorius and E. crusgalli, respectively. Whereas, the other fractions, hexane, MeCl2 and EtOAc fractions reduced root length of E. crusgalli and P. oleracea between 17% and 43%, relative to control. Effect of celery extract and its fraction on growth of weeds Data presented Table 5 revealed that spraying aqueous celery extract and its fractions did not pro- duce any significant effect on growth of two-weeks old of either C. olitorius, or E. crusgalli or P. oleracea weeds. Table 3 - Phenolic acids in aqueous extract of celery at concen- tration 20 g l-1 Means with the same letters in a raw are not significantly different at P<0.05. Table 4 - Effect of fractions obtained from aqueous extract of celery on germination of three weeds (% of control) Acid derivatives Concentration (g l-1) Benzoic acid derivatives p-Hydroxybenzoic acid 18.72 Gallic acid 8.56 Vanillic acid 8.8 Syringic acid 6.4 Protocatechuic acid 9.28 Salicylic acid 7.84 Cinnamic acid derivatives Cinnamic acid 0.64 Caffeic acid 9.52 p-Coumaric acid 3.032 Ferulic acid 11.12 Table 5 - Effect of aqueous celery extract and its fractions on the growth of two-weeks-old of three weeds Criteria Weed species Fractions Control Hexane MeCl2 EtOAc H2O Germination (%) C. olitorius 100 a 104 a 96 a 102 a 70 b E. crusgalli 100 a 98 a 104 a 102 a 95 a P. oleracea 100 a 107 a 85 b 109 a 64 c Shoot length C. olitorius 100 a 97 a 102 a 102 a 36 b E. crusgalli 100 a 86 b 83 b 95 a 53 c P. oleracea 100 a 95 a 100 a 87 a 26 b Root length C. olitorius 100 a 101 a 74 b 103 a 6 c E. crusgalli 100 a 57 c 83 b 79 b 50 c P. oleracea 100 a 61 b 76 b 63 b 1 c Treatments Injuries ( % of control) C. olitorius E. crusgalli P. oleracea 30 g l-1 2±0 a 0±0 a 1±0 a 60 g l-1 3±1 a 2±5 a 3±2 a 90 g l-1 3±2 a 3±1 a 3±1 a Hexane fraction 2±1 a 3±2 a 4±2 a MeCl 2 fraction 4±2 a 1±0 a 1±0 a EtOAc fraction 3±1 a 2±1 a 3±1 a H 2 O residue 4±1 a 1±0 a 1±0 a Values are given as means of three replicates± standard error. Means with the same letters in a column are not significantly dif- ferent at P<0.05. 509 El-Mergawi and El-Desoki - Allelopathich activies of celery extract as weeds control 4. Discussions and Conclusions Aqueous extract of celery was evaluated for its phytotoxicity against three plant species typically present as weeds in summer crops, under laboratory and greenhouse conditions. The extract displayed a great inhibition on germination percentage and seedling growth of target seeds. In line of these results, Sbai et al. (2017) reported that lettuce germi- nation was completely inhibited by celery extract at concentration above 20 g l-1. They extracted and identified the allelochemicals compounds that responsible of toxicity namely phthalides, among of them senkyunolide A was the most toxic in lettuce g e r m i n a t i o n . C u r r e n t s t u d y r e v e a l e d t h a t t h e inhibitory effect of celery extract on germination per- cent varied between tested seeds, and P. oleracea possessed the least sensitivity. Variation between dif- ferent plant species in their sensitivity to plant extracts was previously observed by many investiga- tors (Al-Humid and El-Mergawi, 2014; Han et al., 2008). This study showed that root growth was the most sensitive to extract than shoot growth. Since, LC50 of shoot length of three target seeds ranged between 7.2 g l-1 and 8 g l-1 whereas, values of LC50 for root growth are calculated to be in the range of 1.7 g l-1 and 3.6 g l-1. These results are in agreement with Sbai et al. (2017), who reported that celery extract had more pronounced effects on roots of let- tuce, rather than shoots. Generally, in germination bioassay, water extract of allelopathic plants have more pronounced effect on root rather than shoot growth (Inderjit and Dakshini, 1995; Muhammad et al., 2011). This may be attributed to the fact that roots are the first to absorb the phytotoxic com- pounds (Turk and Tawaha, 2002). In order to identify and distribute the chemical groups of toxic allelochemical constituents, celery aqueous. extract was partitioning between three organic solvents varied in their polarities. Generally, the most phytotoxic compounds were represented water residue (H2O fraction). It can be suggested that the high toxicity compounds in celery extract may be related to presence of more polar compounds. These results are in general agreement with the results obtained by Sbai et al. (2017). Who observed that extraction of celery with non-polar solvents, petrole- um ether or chloroform had no significant effect on lettuce germination, contrarily to the high toxicity effect of methanol extract (polar solvent). The high toxicity of celery extracts may be attributed to the present water soluble compounds as saponins, glyco- sides, hormones or enzyme which could affect growth directly or by altering the mobilization of storage compounds during germination (Chaves and Escudero, 1997; El-Khatib, 1997). For the greatest inhibition effect of aqueous cel- ery extract, we analyzed phenolics and phenolic acids in the extract at a concentration 20 g l-1. The results indicated the presence of high amount of phenolic compounds (201 mg l-1 as gallic acid) may explain the greatest effect of celery extract. In line of these results, Jung et al. (2011) found high levels of pheno- lic in celery (51.09 mg g-1 dw, as gallic acid). Phenolic acids are the precursor of all phenolic constituents in plants as well as the bioactive constituents in celery extract. Identification and determination the concen- tration of the phenolic acids in the aqueous extract was conducted by using HPLC. Ten phenolic acids were identified in celery extract, among of them p- hydroxybenzoic acid, p-coumaric acid and ferulic acid were presented in relatively high amounts. In line of these results, Yang et al. (2010) identified caffeic acid, p-coumaric acid, and ferulic acid in celery extract. Presence of high concentration of total phe- nolic and phenolic acids in areal parts of celery was previously reported by many investigators (Yang et al., 2010; Sbai et al., 2017). In greenhouse trial, we evaluated the effects of foliar s pray of aqueous extract of celery and its frac- tions, hexane, MeCl2, EtOAc and H2O-residue on growth of two-weeks-old C. olitorius, or E. crusgalli or P. oleracea weeds. Celery extract at 30, 60 and 90 g l-1 as well as the obtained fractions did not produce any significant effect on growth of three examined weeds. In general, the growth of two-weeks-old weeds tended to be less sensitive to the test frac- tions than the weed germination process. These results are in agreement of those obtained by Inderjit and Weston (2000), they found that greenhouse bioassays do not adequately predict the responses observed in laboratory bioassay. Hence, weed germi- nation might be the most sensitive index with which to judge allelopathy of plant extracts and its fractions under natural conditions (Corrêa et al., 2008). In conclusion, in this study aqueous extract of cel- ery was evaluated for its phytotoxicity against three weed species under laboratory and greenhouse con- ditions. The extract displayed a great inhibition on germination percentage and seedling growth of tar- get seeds. Root growth of C. olitorius and P. oleracea was completely inhibited by extract at 7.5 g l-1, corre- sponded with 15 g l-1 for E. crusgalli. Celery aqueous extract was fractionated by using three less polar sol- 510 Adv. Hort. Sci., 2018 32(4): 503-510 v e n t s ; h o w e v e r , w a t e r e x t r a c t d i s p l a y e d t h e strongest inhibition effect on germination of three target seeds. Celery extract had a relatively high amount of phenolics and phenolic acids. High con- centrations of water celery extract 30, 60, 90 g l-1) and the obtained less polar fractions did not produce any significant effect on growth of two weeks-old of tested weeds. Water extract of celery may be a use- ful source for the future development of pre-emer- gence bio- herbicide. References AL-HUMAID A., EL-MERGAWI R., 2014 - Herbicidal activi- ties of seven native plants on the germination and growth of Phalaris minor, Echinochloa crusgalli, Portulaca oleracea and Lactuca sativa. - J. Agric. Sci. Tech. A, 4: 843-852. B A J W A A . A . , M A H A J A N G . , C H A U H A N B . S . , 2 0 1 5 - Nonconventional weed management strategies for modern agriculture. - Weed Sci., 63(4): 723-747. 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