Study Of Parthenium hysterophorus L. Extracts (First clean-up Fractions) On Seed Germination Behaviour In Search Of Bioactive Fractions for Preparation of Bioherbicide Formulations.

: Increase in productivity is directly related with increase pesticide consumption. So there is a strong desire to use “greener” chemistry to produce more toxicologically and environmentally benign pesticides and natural products. In this present study, a comparison was made to evaluate the phytotoxicity potential of first cleanup fractions obtained from sequentially extracted solvent (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 fractions. A significant changes in soluble and insoluble sugar content, protein, amino acid content and cellular metabolic activity was aslo observed by 1F3, 1F4, 1F6, and 1F9 fraction s. The present study concluded that phytotoxicity of four fractions ( 1F3, 1F4, 1F6, and 1F9 ) from 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
With the advancement of civilization, mankind has two primary goal in mind -(i) achieving food security and (ii) improving the quality of life. To achieve the earlier point we always ignore our traditional resources and relied on the synthetic agrochemicals for productivity increase and pest management. Today, more than 60% of our population is involved in agriculture sector, which dominates the economic scenario of our country. The increasing pattern of productivity is directly related with increasing trend of pesticide consumption Due to favourable growth and multiplication criteria offered by a tropical country like India pests (insects, fungal pathogens, nematodes, weeds, rodents, birds, mollusc etc.) assume a serious proportions throughout the year. Year round cropping system also encourage these pest populations. So, losses due to pests are far greater than the temperate countries. Crop reduction due to pests accounts for 25-35%. This has been a reason for intensive use of pesticides. Natural product especially plant product is used as the one of main sources of biocides as pharmaceuticals, crop protection chemicals and many more since time immemorial. Many highly effective chemicals have so far been isolated from plant sources especially as pest control agents viz., nicotine, pyrethrins, cinerins, rotenone, ryanodine etc. [1]. In recent times number of breahthrough work has been done in respect of biopesticides with particular reference to botanicals, microbial pesticides, pheromones, IGR, plant growth regulators, and new more promising crop protection molecules. A detailed review of naturally occurring bioactive crop protecting chemicals has been discussed by Vyvyan [2] and Dyan [3,4]. However, in today's perspective, there is a strong desire to use "greener" chemistry to produce more toxicologically and environmentally benign pesticides and natural products. Natural products, especially phytochemicals have been the potential source of many pesticides, either used directly as Isolation for cleanup: Isolation for clean-up of the crude extracts (Ethyl acetate and Methanol crude extract) was then subjected to liquid column chromatography on silica gel G (Merck 60-100 mesh) as adsorbent following solvent of increasing polarity. The elution was done with solvents wef 100% hexane to 100% methanol as eluent of increasing polarity with two gradients. Elutes, as fractions (200ml each) were collected and marked accordingly (Table-1). The fractions were then concentrated on rotary vacuum evaporator (Buchi type) and were subjected to phytochemical analysis and bioassay.

Dark green coloration
Salkowski test 5 ml extract was added with 2 ml of chloroform and 3 ml of concentrated sulphuric acid H 2 SO 4 .

Reddish brown color of interface Tannins
Braemer's test 10% alcoholic ferric chloride was added to 2-3ml of methanolic extract (1:1) Dark blue or greenish grey coloration of the solution Dilute HNO 3 On addition of dil. HNO 3 solution to the ethanolic extract Reddish color

Pink or red coloration
Bioassay: The first cleanup fractions 1F1, 1F2, 1F3, 1F4, 1F5, 1F6, 1F7, 1F8, 1F9, and 1F10 from ethyl acetate and methanol crude were used for bioassay with distilled water as control set. Physiological and biochemical tests were then performed with the pre-treated seeds.

ANALYSIS OF PHYSIOLOGICAL PARAMETERS
Germination behavior: Germination data were recorded every 12 h. intervals up to 120 h. of seed soaking following the International Rules for seed 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 described below (Table-3) .

Germination parameters Equation Reference
Germination percent (GP) GP = [(number of germinated seeds at final count)/total number of seeds sets for bioassay] ×100 Global method N i = number of newly germinated seeds at time T i .
Mavi et al. [11] Seedling vigour index (SVI) SVI= (seedling length × germination percent)/100 Islam et al. [12] Speed of emergence (SE) SE= (number of germinated seeds at the starting day of germination/number of germinated seeds at the final day of measurements) ×100 Modified from Islam et al. [12] Germination energy (GE) GE= (percent of germinated seeds at the day of germination/total number of seeds set for bioassay) ×100 Modified from Ruan et al. [13] Coefficient of the rate of germination (CRG) where, N 1 =number of germinated seeds at time T 1 ,N 2 =number of germinated seeds at time T 2 ,N n =number of germinated seeds at time T n.
Relative root Elongation R = (L/Lr) ×100, where R is the relative root elongation, L is the mean root length in treatments, Lr is mean root length the of control.
Growth Study: Morphological parameters like root length, shoot length and seedling height were measured in fresh samples after 120 hrs. TTC (2, 3, 5-Triphenyl Tetrazolium Chloride) stain ability was done according to the method of Halder [18]. TTC stain ability, were analysed by differential staining pattern, 3 separate categories of seeds (viz., fully stained, partly stained, and not stained) were visually made.

ANALYSIS OF BIOCHEMICAL PARAMETERS
In the phytotoxicity experiment soluble, insoluble carbohydrate, total free amino acid, protein and cellular respiratory activity were recorded. Soluble and insoluble carbohydrate from seed Kernels was determined following method described by McCready et al, [19]. Quantification of total free amino acid in seed kernel was followed as per the method of Moore and Stein [20] modified by Bhattacharjee [21]. Protein in seed kernels was estimated following the method by Bradford [22] using BSA as standard. The respiratory activity of intact seeds was analysed by the reaction of tetrazolium chloride (TTC) according to the method of Rudrapal and Basu [23].
Statistical Analysis: One-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) were done for statistical analysis of the data at 5% level using IBM SPSS v.20.

Phytochemical characterization of P. hysterophorus extracts (first cleanup fractions) obtained from ethylacetate and methanolic crude extracts.
The first cleanup fractions (1F1 to 1F10) were tested for qualitative chemical tests for various phytochemical constituents and they confirmed the presence of triterpenes, steroids, alkaloids and flavonoids (Table-4).

Effect of P. hysterophorus extracts (first cleanup fractions) on germination and growth of V. radiata
There is a significant change in germination parameters of ethyl acetate and methanol fractions ( Figure-1

Methanol crudes
Hex elute - The methanol fractions (Table-6) also have significant (P < 0.05) effect on all calculated germination indices. The GP, GI, CRG, SE, GE, SVI decreased in 1F6, 1F9, followed by 1F8, 1F7, and 1F10 except MGT, which is increased.  Ethylacetate fractions, 1F3, 1F4 and 1F5 shows maximum inhibitory effect on the root and shoot growth of the test species followed by 1F1 and 1F2 ( Figure-2A). The maximum root, shoot and seedling growth was inhibited by 1F3 fraction; while the minimum inhibition was noted for 1F2 fraction. Methanol fractions 1F7 shows maximum inhibitory effect on root and shoot growth of germinating Vigna seeds (Figure-2B). The toxicity potential of ethylacetate and methanol extracts was also supported by phytotoxicity, inhibition and relative root elongation (Figure-3A,B). The cleanup fractions 1F3, 1F4, 1F6, 1F9 and 1F7 shows significant (P<0.05) toxicity on growth of test species. The strong phytotoxicity, inhibitiory activity and least root elongation was noted for 1F3, 1F4, 1F6, 1F9 fraction; whereas minimal toxicity potential were observed in 1F5, 1F8, 1F10. TTC staining pattern and TTC stainability (Table-7

Effect of ethyl acetate and methanolic fractions on biochemical parameters of germinating Vigna seeds:
The Biochemical component (Table-9-10) shows high soluble carbohydrate in 1F3 and 1F6 treatments (11.46 mg g -1 and 9.76 mg g -1 fresh weight respectively) followed by 1F2, 1F9. On the contrary, insoluble carbohydrate at highest quantity was noted in 1F4, 1F3 and 1F6 treatments (260.14, 245.77 and 248 mg g -1 fresh weight respectively). Total free aminoacids follows the same pattern as soluble carbohydrate. Cellular respiration measured through formazan percent were drastically reduced in 1F3, 1F6, 1F9, and 1F4 treatments.

DISCUSSION
Presence of terpenoides, streroides, flavonoids, tannins and alkaloids in the ethylacetate and methanol elutes are in agreement with the findings of [24][25][26][27][28][29][30][31][32][33][34][35]. Gross allelopathic potential of P. hysterophorus, revealed from the seed germination bioassay. Among the ethylacetate and methanol extracts, 1F3, 1F4 elutes from ethylacetate and 1F6, 1F9 elutes from methanol fraction indicates a significant inhibitory response on seed germination as also explained from the germination indices. Overall the extract (1F3, 1F4, 1F6, 1F9) reduces germinability and slows down germination kinetics which is considered to be the important visible and reliable indices for the evaluation of allelopathic Effect. Germination percent (GP) index indicated the total germination percent of a seed lot after certain period of time (120 h.) when germination became constant. As it is measured by total germination relative to total number of seeds set for germination, GP alone 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 [36]. On the other hand, mean germination time (MGT) was calculated as the weighted mean of germination time. The number of seeds germinated in the interval of time is used as weight. Here the use of weighted mean is extremely important as it considers that a different number of seeds germinate in the interval of time [c.f. 37]. Hence, MGT is proportional to reduced and poor germinability. The delay or inhibition of germination caused by phytotoxic plant extracts or substances was also reported by Anjum et al [38] and Hussain et al. [39] as also corroborated by Tefera [5], Regina et al. [40], Rashid et al, [41], Dhole and Dhole [42]. Similar trends of germination retardation were also reported by Marwat et al [43] in Avena fatua and Lepidium pinnatifidum. The data thus indicating a strong allelopathic effect. Respiratory ability were also reduced in these treatments over other treatments. These results are in strong agreement with Batish et. al. [44]. Abrahim [45] reported that interference of monoterpenes with respiratory ability can lead to germination and growth retardation. Cellular metabolic activity in terms of sugar, total protein, total free amino acids were also impared in the four treatments (1F3, 1F4, 1F6, and 1F9). Similar type of observation were also recorded by Kiran et al. [46], Padhy et al. [47].

CONCLUSION
The present study concluded that phytotoxicity of four fractions (1F3, 1F4, 1F6, and 1F9) from 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.