Phytotoxic effects of Thuja orientalis L. and Thevetia peruviana (Pers.) schum crude extracts on wheat seed germination

The present studies focus on the phytotoxic effects of four crude extracts viz-viz n-hexane, ethyl acetate, methanol and butanol of two plants Thuja orientalis and Thevetia peruviana at two different concentrations i.e, 10 mg/ml and 1 mg/ml against wheat grains germination. Wheat grains (Triticum aestivum) germination percentage, germination velocity, root inhibition percentage, root shoot length, seedlings fresh dry weights and moisture content was determined. Maximum inhibition of germination was exhibited by n-hexane and butanol concentrations of Thuja orientalis and ethyl acetate of Thevetia peruviana. Germination velocity was reduced at maximum level by n-hexane and butanol extracts of Thuja orientalis and Thevetia peruviana respectively. Butanol and methanol extracts of Thuja orientalis and Thevetia peruviana were highly effective in reducing the shoot length while maximum reduction in root length was exhibited by ethyl acetate and n-hexane extracts of the two plants respectively. Ethyl acetate extract of both the plants was most effective in root inhibition against wheat grains. Maximum reduction in seedling moisture content was recorded in seedlings treated with methanolic extract at the same concentration of Thuja orientalis and Thevetia peruviana. Over all the phytotoxic effects of both the plants for all the parameters were more pronounced at 10 mg/ml concentration.


Introduction
A number of phytotoxic or growth promoting substances or allelochemicals have been found in different plant species and in different tissues of plants and soils [1].Allelochemicals are usually the plant secondary metabolites that have a great potential as natural pesticides and as growth promoters [2].Many plant species, including some important crops, can produce and release allelochemicals into the environment to suppress the growth of other plants [3].
Release and activity of allelochemicals may be affected by environmental factors such as soil moisture, soil pH, soil organic matter content or soil salinity [4].Secondary metabolites may be induced in plants as a response to environmental stress like drought, nutrient deficiency or salinity [5].Induction of secondary metabolites or allelochemicals may be important for the defense of plants in situations where several threats or stress factors act simultaneously [6].It is now well established that release of allelochemicals has many advantages for many plants especially under environmental stressful conditions such as drought, nutrient deficiency etc [7].Allelochemical compounds had injurious effect on the growth and development of crop plants.Allelochemicals released either from leaves, stem, bark of living or dead trees or plants can be classified into terpenes, glucosides, coumarins, aldehyde and phenolic compounds.The leaves of Eucalyptus are main releasing source of toxic compounds.The volatile compounds are also considered a threat to the environment [8].It is difficult to separate resource competition from allelopathy under field conditions.To overcome this problem, various laboratory screening techniques have been developed to measure allelopathy without the interference of resource competition [9, 10].Allelopathy can be used in weed management in two ways: by selecting an appropriate crop variety or incorporating an allelopathic character into a desired crop variety, and by applying residues and straw as mulches to grow an allelopathic variety in a rotational sequence that allows residues to remain in the field [11].Thuja orientalis L. belongs to Cupressaceae and is commonly known as Morphenkhi.The genus has five species, two native to North America and three to Eastern Asia.They are commonly known as arbovitea.It has been widely cultivated as an ornamental timber tree for its source of wood, shade in different kind of soils.It can also be grown as dense hedge [12].Thuja orientalis L. plants are an evergreen tree growing to 15m at a slow rate.The seeds ripen from September to October.The flowers are monoecious (individual flowers are either male or female, but both sexes can be found on the same plant) and are pollinated by wind.

Preparation of extracts
Extracts were prepared by simple maceration technique.Plant material was shade dried at room temperature, ground to powder form and were placed in a sealed plastic bags separately duly labelled.60g of each plant material was soaked in 600 ml nhexan, ethyl acetate, butanol and methanol for 12 days at room temperature.The mixture was mixed daily with mechanical shaker.After 12 days the extract was filtered by means of filter paper (Whatman filter paper #1).The filtrates were rotary evaporated in order to separate the gummy extract from solvents.Each gummy extract was put in a beaker duly labelled.The recovered solvent was put again in residue for further extraction and the process was repeated thrice.The four extracts were allowed to evaporate till complete dryness.Each extract was weighed.Thuja orientalis n-hexan, ethyl acetate, butanol and methanol extracts weighed 4gm, 4.9gm, 4.9gm and 14.7gm respectively whereas Thevetia peruviana nhexane, ethyl acetate, butanol and methanol extract weighed 3gm, 3.4gm, 3.3gm and 17.9gm respectively.

Preparation of stock solution and dilution
Two solutions were made for each extract.500 mg of the extract was dissolved in 50 ml of pure methanol to prepare stock solution (10 mg/ml).50 mg of extract was dissolved in pure methanol to make a dilute solution (1mg/ml) as given in Table 1.
Where N1, N2, N3--------Nn = proportion of seeds which germinated on day 1, 2, 3-----n following set up of the experiment.'S' varies from 100 (if all the seeds germinated on the first day following set up) to 0 (if no seeds are germinated by the end of the experiment).This has advantage over percentage germination, because it is usually more sensitive ''S'' indicator of allelopathic [26].Percentage of moisture content was calculated by the following formula % of moisture content =Fresh weightdry weight × 100 Fresh weight

Statistical analysis
Statistical analysis was conducted using statistics 9 software for the analysis of data for standard deviation (SD).

Results and discussion
Phytotoxicity of tested medicinal plant species was checked on wheat.The germination velocity of wheat was decreased by n-hexane extract of Thuja orientalis.However their effect was more at 10 mg/ml concentration.Maximum decrease in germination velocity was exhibited by the same extract as compared to respective control or other extracts of the same plant.
The ranking of extracts of Thuja orientalis for their phytotoxic effects on germination velocity of wheat was as follows: n-hexane< butanol<ethyl acetate < methanol (Table 2).However the germination velocity of butanol extract at both the concentrations was the same.Maximum decrease in germination velocity was exhibited by the butanol extract as compared to respective control or other extracts of the Thevetia peruviana plant at 10 mg/ml concentration.The ranking of extracts of Thevetia peruviana for their phytotoxic effects on germination velocity of wheat was as follows: butanol<ethyl acetate<n-hexane < methanol (Table 3).The results showed maximum inhibition of wheat germination was caused by n-hexane and butanol extracts as compared to control of the plant Thuja orientalis.On the basis of inhibitory effect of extracts at 10mg/ml concentration on seed germination of wheat, the extracts were ranked as n-hexane =butanol<ethyl acetate < methanol (Table 2).
For the inhibitory effect of extracts of Thevetia peruvianaat10mg/ml concentration on seed germination of wheat, the extracts were ranked as ethyl acetate <butanol <methanol < n-hexane (Table 3).
The results presented in Table 2 showed that at higher concentration of 10 mg/ml, average shoot length was decreased by application of extract as compared to control.It was found that butanol extract of plant Thuja orientalis was highly effective in decreasing the shoot length of wheat as compared to control and other extracts and are ranked as butanol< ethyl acetate< nhexane < methanol (Table 2).
It was found that methanol extract of plant Thevetia peruviana at concentration 10 mg/ml was highly effective in decreasing the shoot length of wheat as compared to control and other extracts and are ranked as methanol< ethyl acetate=n-hexane < butanol (Table 3).It is further stated that the results were pronounced at higher concentration as compared to low concentration of 1 mg/ml.The results presented in Table 2 showed that at higher concentration of 10 mg/ml, average root length was decreased by application of extract as compared to control.It was found that ethyl acetate extract of plant Thuja orientalis was highly effective in decreasing the root length of wheat as compared to control and other extracts and are ranked as ethyl acetate< butanol< n-hexane < methanol (Table 2).It is further noticed that results were more pronounced at this concentration as compared to that of 1 mg/ml.It was found that n-hexane extract of plant Thevetia peruviana at concentration 10 mg/ml was highly effective in decreasing the root length of wheat as compared to control and other extracts and are ranked as n-hexane< ethyl acetate< methanol< butanol (Table 3).Like seedling fresh weight, the dry weight of the wheat was also decreased by the application of different plant extracts of the two selected medicinal plants under investigation.More pronounced phytotoxic effects on shoot dry weight of wheat was exhibited by methanol extract of Thuja orientalis.Maximum phytotoxic effects were exhibited on shoot dry weight of wheat by methanol extract of Thuja orientalis as compared to control and other plant extracts.
The other extracts reduced the seedling fresh weight to maximum level.The moisture content of wheat seedling was affected to its maximum level with respect to their fresh and dry weights at 10 mg/ ml concentration of the methanol extract followed by extracts butanol, ethyl acetate and n-hexane respectively and the results were quite pronounced at this higher concentration (Table 2).The ranking of the extracts for their phytotoxic effects on wheat seedling dry weight at 10 mg/ml concentration for Thevetia peruviana was as follows: methanol< ethyl acetate< butanol< n-hexane (Table 3).
The results also showed that maximum wheat root inhibition was also exhibited at 10mg/ml as compared to 1 mg/ml concentration by all the four extracts of Thuja orientalis.The ranking for this parameter was as follows: ethyl acetate>butanol>n-hexane> methanol (Table 2).The same results were also shown by all the tested extracts of Thevetia peruviana and were ranked as follows: ethyl acetate= n-hexane> methanol>butanol (Table 3).The results highlighted that phytotoxic activity of selected medicinal plants extracts on wheat germination was dose dependent.It was found that at 1 mg/ml of extract concentration, butanol extract of Thuja orientalis exhibited maximum decrease in germination velocity of wheat.The sequence of different extracts for their phytotoxic effects on germination velocity of wheat at 1 mg/ml concentration was as follows: butanol<n-hexane<ethyl acetate=methanol (Table 4).The results established that maximum inhibition of seed germination in wheat was caused by ethyl acetate extract of Thevetia peruviana at both the concentrations followed by methanol at higher concentration only.The results further revealed that both concentrations of butanol extract of Thevetia peruviana exhibited decrease in germination velocity of wheat but only the higher concentration was effective in inhibiting the germination.The root inhibition % of wheat was less decreased at 1 mg/ ml concentration for all the four extracts as compared to 10 mg/ml for both the plants and their ranking is as follows.For Thuja orientalis it was ethyl acetate> butanol> n-hexane> methanol where as for Thevetia peruviana it was ethyl acetate> methanol>n-hexane > butanol.
Ethyl acetate extracts at 1 mg/ml concentration for both the plants was most effective in wheat root inhibition.(Table 4  and 5).The moisture content of wheat seedling with respect to their fresh and dry weights at 1 mg/ml was lower in wheat seedling treated with methanol extract of Thuja orientalis as compared to control and other extracts of the same plant,however the intensity of decrease was lower than that at 10 mg/ml concentration.Maximum reduction in seedling moisture content was recorded in seedlings treated with methanolic extract at the same concentration of Thuja orientalis and Thevetia peruviana (Table 4 and 5

Table 5 . Thevetia peruviana phytotoxicity against wheat grains germination (1 mg/ml)
ConclusionFrom the present studies it is concluded that both the plants exhibit phytotoxic effects but more pronounced effects are seen at higher concentration against wheat.Hence it is recommended that further research is required to isolate active allelopathic principles in order to destroy unwanted weeds especially the grasses which are harmful to our cereals and other cash crops and reduce yields.