Phytochemical screening and allelopathic evaluation of aqueous and methanolic leaf extracts of Populus nigra L

Allelopathic compounds are secondary metabolites which are produced during different metabolic pathways and apparently they have no prominent role in plants’ growth and development. However, these compounds are supposed to play an important role in defense and interactions of producers with receivers. In this study, aqueous and methanolic leaf extracts of Populus nigra were screened for the presence of secondary metabolites and tested for the allelopathic effects on seed germination of four wheat cultivars (Ghazanvi, Siran, Atta Habib and Janbaz). Phytochemical screening revealed the presence of alkaloids, flavonoids, steroids, terpenoids, saponins, tannins, phlobatanins, and glycoside, reducing sugars, triterpenes, phytosterols and proteins in leaf extracts. Allelopathic bioassay demonstrated that leaf extracts had detrimental influence on the seed germination of four cultivars of wheat, which depended on the amount of powder dissolved in extracts (10, 20, 40 and 80 g). Methanolic extracts exhibited greater degree of phytotoxicity than aqueous leaf extracts. Among the tested cultivars, Janbaz and Ghaznavi were more sensitive to aqueous and methanolic extracts particularly at the highest dose of concentration (80 g). The study suggests the presence of wide range of bioactive compounds in P. nigra leaves with phytotoxic potentials on wheat germination.


Introduction
Allelopathy is an important biological phenomenon which is directed by the releases of biological molecules from different plant parts in the form of rootexudates, leachates from above ground parts, volatilization or decomposed residues to interact with other plants, microbes and other organisms for either beneficial or negative association [1,2]. The process is intricate in nature which may be due to the interaction of various groups of chemical compounds like alkaloids, flavonoids, phenolics, glycosides, lactones, quinines, volatile terpenes, organic acids compounds and other secondary metabolites (allelochemicals) -which are produced during secondary metabolic pathways and apparently have no active role in growth and development of plants but are concerned with defense and interaction with other organisms [3,4]. Usually, the allelopathic effect of one plant on the other is perceived to be due to the synergetic or antagonistic effects of different compounds in order to make the environment conducive for allelopathic plants [5]. In many countries including Pakistan, wheat is grown close to poplar (Populus nigra) -a multipurpose tree of significant economic importance. However, the leaves of these trees might release toxic compounds when they fall and undergo decay process in the soil. This is a common problem but the underline mechanism is not fully understood. A number of researchers assessed the allelopathic effects of different Poplar species on agricultural crops which include Populus deltoides [6-8], Populus tomentosa [9], and Populus euphratica [10]. However, research is lacking in review on phytochemical screening and allelopathic effects of P. nigra on wheat. Thus understanding of the allelopathic nature of Populus leaves, their phytochemical screening and effects on wheat are crucially needed for possible crop modification pattern in an agro-farming system where populous trees are cultivated. The aim of the present work was therefore to carryout phytochemical screening of aqueous and methanolic leaf extracts of P. nigra and to test them on germination of wheat.

Materials and methods Extraction and fractionation
Leaves of P. nigra were collected from District Charsadda in October 2014; washed with running tap water for removing the surface contaminants and dust, then dried at room temperature for two weeks in shade, and was crushed using electric blender. The leaf powder (800 g) was extracted by soaking it in fresh water and methanol at room temperature separately. The extracts were then filtered and concentrated by Rotavapor under reduced pressure at temperature below 50°C. The final residue obtained was weighed (15.2 g) in fresh water and (21.1g) in methanol. Each of the extract was screened for the presence of phytochemicals as per the standard procedures [11]. The aqueous extract (210 g) was subjected to fractionation and was partitioned successively with nhexane, ethyl acetate, chloroform and methanol. Each extract was evaporated to dryness under reduced pressure that yielded ethyl acetate (7 g), chloroform (5 g) and methanol fraction (20 g) respectively. However, no n-hexane fraction was obtained. Phytochemical Screening Crude extracts of P. nigra including hot water and tap water and methanolas well as their sub-fractions were screened for qualitative determination of various secondary metabolites according to standard analytical methods [12].

Test for reducing sugars
To the extracts in separate test tubes 3-4 drops Fehling's solution was added and heated. The appearance of red color revealed reducing sugar was present. Steroids 2 ml of acetic anhydride and 2 ml concentrated H2SO4 were added to 5 ml of the extracts in separate test tubes. Change of color from violet to blue confirmed the presence of steroids. Glycosides 2 ml of glacial acetic acid containing 1 drop of ferric chloride solution and 1 ml of concentrated H2SO4 were added to 4 ml of extracts. Appearance of a brown ring indicated the presence of glycosides.

Tannins test
Crude extracts (0.5 g) were suspended in water and heated on water bath and filtered, followed by adding few drops of ferric chloride to the filtrates. The appearance of green color indicated the presence of tannins.

Alkaloids test
Crude extracts (0.5 g) were warmed with 5 ml of 2% H2SO4 for 2 minutes, filtered and 2-3 drops of Dragendrof's reagent was added to each filtrate and observed for red precipitation.

Saponins test
Crude extracts (0.5 g) were shaken with 5 ml of distilled water and was heated to boiling. Formation of froth with persistence time of 5 minutes or more represent saponins. Triterpenes and phytosterols test To 5 ml extracts 2 ml chloroform was added and filtered. To this filtrate 3 ml concentrated H2SO4 was added, shaken and allowed to stand and observed for golden yellow color that's indicate triterpenes, while red color in the lower layer represent phytosterols.

Flavonoids test
Crude extracts (0.5 g) were dissolved in 1ml diluted NaOH and 3-4 drops HCl was added. Yellow color that turns colorless indicates the presence of flavonoids.

Anthraquinones test
Crude extracts (0.5 g) were boiled with 5 ml 10 % HCl for few minutes in water bath, filtered and allowed to cool. To the filtrate 3 ml CHCl3 was added. 2-3 drops of 10% ammonia was added to the mixtures and heated. Formation of rose-pink color indicated the presence of anthraquinones.

Phlobatanins test
Crude extracts (0.5 g) were dissolved in 5 ml distilled water and filtered. The filtrate was boiled with 1ml of 2% HCl solution. Formation of precipitation shows the presence of phlobatanins.

Terpenoids test
Crude extracts (0.5 g) were mixed with 2 ml of chloroform and concentrated H2SO4 (3 ml) was carefully added to form a layer. The formation of a reddish brown coloration at the interface indicated positive results for the presence of terpenoids.

Seed bioassay
Allelopathic bioassay for seed germination of four wheat cultivars was performed at Botany Department. 10 seeds of each wheat cultivar were placed on filter paper in sterilized petri dishes. 5 ml of each aqueous and methanolic extracts at 10, 20, 40 and 80 g were applied to petri dishes which were arranged in a completely randomized design. Distilled water was used as control treatment. For each treatment, five replicates of petri dishes were used. Germination % was calculated in each treatment at 10 th day of the onset of germination bioassay. Least significant difference test was used to record the effect of extracts on germination % of wheat cultivars.

Results
Phytochemical evaluation of the aqueous leaf extracts of P. nigra revealed that secondary metabolites such as phenols, alkaloids, flavonoids, steroids, terpenoids, saponins, tannins, glycoside, reducing sugars, triterpenes and phytosterols were present in the samples. In methanolic extracts glycoside, reducing sugars and phytosterols were not detected while other compounds were present (Table 1). Furthermore, the organic fractions such as chloroform, ethyl acetate and methanol showed differential indication for the tested phytochemicals ( Table 2). Germination bioassay demonstrated that four varieties of wheat differed in their response to the applied extracts; showing decline at increasing concentration of extracts (Fig. 1). In aqueous extract treatments, lowest germination percentage (28%) in cultivars Siran and Atahabib respectively at 80 g extract concentration was observed, which was significantly lower than control i.e., 68 and 70% respectively. In cultivar Ghaznavi, 34% germination was observed at 80 g extract against 64% in control. Cultivar Janbaz revealed 31% germination at 80 g when compared to control where it was 64%. All the extract concentrations showed inhibitory effects on germination of four wheat varieties; however, highest concentration (80 g) was more phytotoxic.  Effect of methanolic extracts on seed germination of four cultivars of wheat is presented in (Fig. 2). Results revealed that all the extract concentrations were strongly phytotoxic which significantly lowered germination parentage in all the cultivars (or varieties?); however, unlike to aqueous extracts, 40 g extract concentration was more drastic in effects than other concentrations in methanolic extract. In cultivar Ghaznavi, lowest germination (12%) was observed at 40 g when compared to control (64%).
Germination was 18% at 80 g extracts. Similar tendency was recorded in cultivar Janbaz where germination was significantly lowered to 14% by 40 g extract concentration. Cultivar Siran was found most susceptible to the allelopathic stress at 40 g where only 10% seeds germinated while in Atahabib germination percentage was 20%.
In general, methanolic extracts were found to be more inhibitory than aqueous extracts towards seed germination of wheat.

Concentration of extract (g)
Ghaznavi Siran Atta Habib Janbaz activities of respiratory enzymes in seed germination thus causing inhibitory effect on its germination. Enzymes that mostly affected by phenolic compounds are aldolase and glucosephosphate isomerase, involved in glycolysis and glucose 6-phosphate dehydrogenase, an enzyme involved in the first step of oxidative pentose phosphate pathway [17]. Our results generally agree with previous studies conducted on the allelopathic activities of several plants on wheat germination and growth [18,19].

Conclusion
The present study suggests that P. nigra is an allelopathic plant, which is capable of suppressing the germination of tested wheat cultivars. P. nigra leaves crude extracts contain different bioactive compounds e.g., alkaloids, flavonoids, steroids, terpenoids, saponins, tannins, phlobatanins, and glycoside, reducing sugars, triterpenes, phytosterols and proteins which might be responsible for its allelopathy effects.