A comparison of phytotoxic potential among the crude extracts from Parthenium hysterophorus L. extracted with solvents of increasing polarity

There is a worldwide search for the safe, effective and eco-friendly compounds of plant origin to combat the weed species and other pests which are responsible for the great impact on the growth and productivity of agricultural crops. In this present study, a comparison was made to evaluate the phytotoxicity potential of sequentially extracted solvent (hexane, ethyl-acetate, methanol) extracts of Parthenium hysterophorus L. (aerial parts) in vitro through bench-top seed germination assay ( Vigna radiata L.). One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) were done for statistical analysis of the data. The study reveals that germination, growth and vigour was significantly (P<0.05) reduced by ethyl-acetate and methanol extracts. The present study concluded that phytotoxicity of ethylacetate and methanolic crude extracts of Parthenium hysterophorus could be exploited as potential bioherbicide for future weed management programme and the development of bioherbicide for commercial use.


INTRODUCTION
Synthetic chemicals improved crop yields quantitatively and qualitatively but has created undesirable impacts on health, environmental quality and development of resistance and cross resistance.Due to these problems, there is a serious efforts to find alternatives to these chemicals.Among those alternatives, phytochemicals shows a potential approach for the management of noxious and other pests in a proper manner.Besides pest management they are environment friendly, because they are biodegradable, rarely contain halogenated atoms, and possess novel target sites that are different from synthetic chemicals.They also exhibit a large degree of novelty and structural diversity and are highly sought after for the discovery of new agrochemicals.The discovery of novel modes of action would lead to the development of new weed management tools.
There are many weed species that are phytotoxic in nature.Among the weeds Parthenium hysterophorus L., is an aggressive weed native to Southern North America, Central America, the West Indies and Central South America [1], having allelopathic effect and drastically retards the growth of many species [2].Parthenium is an invasive exotic weed commonly known as Bitter weed, False ragweed, Fever few, Ragweed, Carrot weed, White top, Chatak chandani, Congress grass & Star weed.Its botanical name is Parthenium hysterophorous L. and belongs to the Class: Magnoliopsida, Family: Asteraceae (Compositae).It is a herbaceous plant, and a native of Tropical America.It is an annual herb and has a deep taproot and erect stem, which becomes woody with age.Height varies of Parthenium weed between 50-150 cm, leaves are deeply lobed.It is pale green in colour and has soft hair.Parthenium weed flower is creamy white in color.The weed has a large number of highly branched stems.It has small (1-2mm long) black seeds with white scales.They are not visible to the naked eye.The countries where it has been reported with the presence of this weed are South Africa, Mozambique, Madagascar in Africa, China, India, Vietnam, Nepal in Asia, Guatemala, Honduras, Belie etc. in Central America, Mexico, in South America and in United States [3].
The successful spread of Parthenium in so many parts of the world including India has mainly been attributed to its allelopathic properties, which enables it to compete effectively with crops and pasture species [4].It can play an important roles in the determination of plant diversity [5], dominance, succession, and climax of natural vegetation and in the plant productivity of agro ecosystems.Apart from its competitive ability, the invasiveness of the noxious weed P. hysterophorus L., is thought to be due to an ability to displace other species by means of allelopathy.The allelochemicals released from Parthenium affecting many plant species are sesquiterpene lactones and phenolics [6].Parthenin is the major sesquiterpene lactone whereas caffeic, vanillic, ferulic, chlorogenic and anisic acids are the major phenolics [7,8].These two synergistically acting groups of allelochemicals signifi-cantly decrease the seed germination and subsequent growth in many crops [8].

Plant material for extraction
The plant sample (Parthenium hysterophorous L) was collected from local vegetation of Baruipur, 24-Paraganas (south) [22 0 21 ' 29 '' N Latitude and 88 0 26 ' 37 '' E Longitude and altitude of 42 feet] , West Bengal, India.The samples were cut into small pieces, air dried at room temperature (25±3 0 C), then oven dried for 2 days at 40 0 C and powdered by mechanical grinder to store in air tight packets which are now ready for experiment.

Seed material for bioassay
Vigna radiata L. Seeds were used in this experiment as target organism for bioassay.The seed materials were procured from the Department of Agronomy, BCKV, Mohanpur, Kalyani, Nadia (W.B.).

Extraction
The crude extracts were prepared by macerating 250 gm. of powdered Parthenium with 1 L. solvents of choice with increasing polarity (hexane, ethyl-acetate and methanol) following exhaustive sequential extraction.Extraction was done in multiple set and pooled together.The crude extracts were filtered through Whatman No.1 filter paper and were evaporated to a gummy mass, which were then diluted with minimum solvents & remaining

Bioassay
10%, 25%, 50%, 75%, 100% (v/v) test extracts thus obtained from hexane, ethyl acetate and methanol crude extracts were used for bioassay along with a treatment as control with distilled water.A commercial herbicide Pendiguard (pendimethylene 50%EC) at FR dose was used as standard check control.Physiological Experiments of the investigation were carried out with viable surface sterilized seeds of Vigna radiata, pretreated with the test extracts of different concentrations for 2 hrs, and placed in Petri dishes containing moistened filter paper.
Germination data were recorded every 12 hr.intervals up to 120 hr of seed soaking following the International Rules for Testing [9].Seven germination indices, germination percentage (GP), germination index (GI), germination energy (GE), speed of emergence (SE), mean germination time (MGT), seedling vigour index (SVI), and coefficient of the rate/velocity of germination (CRG) were obtained & then calculated from the same data by using the equations [10] described below (Table -1).Morphological parameters like root length, shoot length and seedling height were measured in fresh samples after 120 hrs.Phytoxicity, inhibition and relative root elongation were calculated following the equations [11] described.One-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) were done for statistical analysis of the data.

Germination parameters Equation
Germination percent (GP) GP=[(number of germinated seeds at final count)/total number of seeds sets for bioassay] ×100 International Letters of Natural Sciences Vol. 33

RESULTS
The hexane, ethyl acetate and methanol crude extracts of Parthenium were used to study the germination process of V. radiata seeds upto 120 hrs.The Germination Kinetics was studied for the three crude extracts (Figure -1).It has been revealed that there are nonsignificant changes in germination kinetics (Figure -1A) in hexane crude dilutions where as there is a significant changes in germination kinetics among the treatments of ethyl-acetate (Figure -1B) and methanol (Figure -1C) crudes.The ethyl-acetate treatments show a 50% reduction of germination in comparison to control (100% germination).Table -2 -3) have significant (P < 0.05) effect on all calculated germination indices except MGT.The GP, GI, CRG, SE, GE, SVI showed a reduction trend with increase in concentration.The 100% ethyl-acetate crude shows least germination followed by 75% treatment which shows maximum reduction as evident by GP(98%) , GI(98.7%) , CRG(83.3%),SE(88.9%),GE(99.3%) and SVI(100%).The lowest concentration (10%) of ethyl-acetate crude treatments also show a significant reduction on all germination indices GP(59.3%),GI(73.4%),CRG(40.93%),SE(60.33%),GE(85.33%) and SVI(97.7%).Almost similar trend of changes were observed in Methanol treatments.At any dilutions (Table-4) significant (P < 0.05) effect on all calculated germination indices except MGT were observed.The 75% methanolic treatment shows maximum reduction of germination indices, as evident by GP(78%) , GI(81.5%) , CRG(27.7%),SE(31.3%),GE(84.3%) and SVI(99.4%)as compared to control.On the contrary there is no significant changes for was noted for the H, EA and M solvents as untreated control.The increasing trend of MGT compared to the decreasing trend of GP, GI, CRG, SE, GE, and SVI indicated a significant inhibition or delay of germination of the test species caused by the Parthenium crude extracts.The overall result show maximum reduction in ethyl-acetate treatments followed by methanol treatments than the hexane treatments.Parthenium plant extracts have significant effect on seedling growth of V. radiata (Fig- 2).

A
Treatments Length (mm)

78
International Letters of Natural Sciences Vol. 33 All the ethyl-acetate treatments (Figure -2B) shows inhibitory effect on the root and shoot growth of the test species followed by methanolic treatments (Figure -2C).There is a decreasing trend in root, shoot and seedling growth as the concentration increases except in the hexane treatments (Figure -2A).At 100% concentration root, shoot and seedling growth was inhibited by 36, 39, and 37% in respect of control respectively for hexane extract; 100, 100 and 100% of control for ethyl-acetate extract; 95, 100 and 96% of control for methanolic extract.The inhibitory effect was greater on the growth of roots than shoot length.The lowest (10%) concentration of ethylacetate and methanol crude extracts also shows a similar trend.The gross toxicity potential of Parthenium extracts was corroborated by phytotoxicity and inhibition (Figure -3).At 100% concentration of hexane extract (Figure -3A) maximum phytotoxicity and inhibition was observed 37 and 15% respectively.The ethyl-acetate extracts (Figure -3B) and International Letters of Natural Sciences Vol. 33 methanolic extracts (Figure -3C) of all concentration shows significant (P<0.05)toxicity on growth of test species.In both the treatments phytotoxicity and inhibition follows a concentration dependent trend as noted earlier for germination studies.At 100% concentration, phytotoxicity and inhibition was noted by 100 and 100% respectively for ethylacetate extract; 94 and 65 % for methanolic extract, followed by 75, 50, 25, 10% concentrations in both treatments.

DISCUSSION
The allelopathic effect of different concentration of solvent extracts of Parthenium hysterophorus have been found inhibitory to all parameters viz.Seed germination to metabolism of Vigna radiata (mung bean) seeds.Maximum inhibition of germination measured through 'germination indices" and growth of Vigna seeds were observed for ethylacetate and methanol crude extracts over hexane extracts.Overall the extract at different concentration reduces germinability and slows down germination kinetics which is considered to be the important visible and reliable indices for the evaluation of Allelopathic Effect.GP index indicated the total germination percent of a seed lot after certain period of time when germination became constant.As it is measured by total germination relative to total number of seeds set for germination, GP cannot explain the delayed germination.In contrast, GI is a measure of both percentage and speed of germination and assigns maximum arithmetic weight to seeds that germinate during first count and less weight to those that germinate later.The higher the GI, GE, SE, SVI, and CRG values compared to control, the lower the inhibition, and vice versa, except for MGT [10].The delay or inhibition of germination caused by phytotoxic plant extracts or substances was also reported by Anjum [12] and Hussain [13] as also corroborated by Tefera [2], Regina et al, [14].

CONCLUSION
Therefore in conclusion, ethylacetate and methanolic crudes Parthenium extracts has a strong inhibitory effect on seed germination.However the effect is more prominent in ethylacetate crude extracts over methanol extracts.Hence it could be a potential source of bioherbicide with proper management.Since Parthenium has become a major invasive in different parts of the country.Therefore all efforts should be made to manage its use in bioherbicide formulation which may be beneficial to farmers as well as environment friendly.

Table 2 .
Effect of hexane crude extract dilutions of Parthenium on germination.

Table 3 .
Effect of ethylacetate crude extract dilutions of Parthenium on germination.

Table 4 .
Effect of methanol crude extract dilutions of Parthenium on germination.

International Letters of Natural Sciences Vol. 33
It has been found that all calculated germination indices except MGT, has significantly changed in hexane extract dilutions (Table-2).Ethyl-Acetate treatments at any dilutions (Table