Phytotoxic effect of plant extracts on physiology of cotton ( Gossypium hirsutum L.) plants

Background: Plant-derived compounds are alternatives of synthetic insecticides in sustainable agriculture. Objective: This study investigated the phytotoxic effect of higher concentrations (2, 4, 8 and 16%) of four plants extracts (Azadirachta indica, Mentha arvensis, D. stramonium and Citrus limonium) on cotton plants. Methods: Each concentration was replicated four times to check the phytotoxic effect (CO2-in, CO2-out, H2O-in, H2O-out and photosynthesis absorption rate (PAR) in randomized complete block design. Data was recorded after 12, 24, 48 and 72 hours of spray with the help of Photosynthetic CL 340 meter. Results: The results showed that CO2-in was more affected by the D. stramonium (131.65±0.38) at 8% concentration. The overall progress showed that C. limonium was more affected the CO2-in of cotton crop. CO2-out was less affected by the C. limonium (117.83±1.46) at 4% concentration than M. arvensis (116.99±1.25) at 8% concentration and D. stramonium (115.77±0.74) at 16% concentration, but was more affected by the A. indica (118.15±0.71) at 4%. H2O-in was more affected by the C. limonium (0.39±0.05) than D. stramonium, A. indica and M. arvensis at 16% concentration. H2O-out of cotton was least affected by the D. stramonium (7.63±0.01) at 2% and more affected by the C. limonium (1.56±0.15) at 16% concentration. PAR was more affected by the A. indica (931.47±8.39) at 4% concentration and least affected by the M. arvensis (1499.7±9.94) at 8% concentration. Conclusions: Different dosages of various botanicals influenced the opening and closing of stomata and photosynthesis of cotton plants.


INTRODUCTION
Cotton (Gossypium hirsutum L.) is known as world most important cash crop. It is utilized as a part of various items like lint in textile; cottonseed is utilized as vegetable oil and feeds of animal. Cottonseed cake is a rich source of value protein (Sarwar et al., 2013). Cotton assumes a key part in monetary improvement of both developed and developing nations. Cotton has been known as crude material for industrialization, riches and advancements of a nation, which give wage to various segments like well being, education and transportation.
Cotton is attacked by different insect pests in various stages, which cause lessening in the yield specifically or by implication. Roughly 160 species attack on the cotton at various stages like borers, sapsuckers and defoliators and cause around 60% yield misfortunes annually (Halbert and Manjunath, 2004). By feeding different insect pests reduce the quantity and decrease the quality by transmitting distinctive diseases (Manjunath, 2004). Numerous manufactured sprays are utilized to control insect pest and increase the fruit setting in the cotton crop (Dayan et al., 2009). Synthetic insecticides have undesirable impact on other non-target species, their residues remain in the food which is a reason of environmental pollution and ecosystem.
Synthetic insecticides must be replaced with those items which are friendly to environment. For a long time botanicals are being utilized option of manufactured insecticides for pests administration in light of the fact that these are more secure for condition and human wellbeing. Around 46 families of plants are utilized as botanicals which have insecticidal value (Isman and Machial, 2006) Some farmers utilized high concentrations of insecticides to control the insect pests, yet there was no change in yield even diminishment. Development and yield of products lessened when OPI were utilized as a part of higher than suggested concentrations (Shehata and El-Khawas, 2003). It is observed that the broadly higher concentration of pesticide application caused negative effect on the physiology of the crop, generally on the photosynthesis of the crop. The development and yield of the crop additionally exasperates when the photosynthesis rate of product aggravate. The utilization of contact fungicide copper impact on the chloroplasts, photosynthesis and chlorophyll biosynthes (Petit et al., 2012).
Normally happening monoterpenes demonstrated phytotoxicity against maize plants, they impact on roots and leaves of maize crop. Carvone was most phytotoxic against maize than monoterpenes (Shehata and El-Khawas, 2003). Strawberry seedling is very influenced when treated with over 3% concentration of limonene (Ibrahim et al., 2001). The seedling of carrot and cabbage were exceptionally influenced when treated with the 9% concentration of limonene (Ibrahim et al., 2004). Citrus photosynthetic was changed by the ramifications of pesticides (Jones et al., 1983). The present research was conducted to evaluatephytotoxic effects (CO2-in, CO2-out, H2O-in, H2O-out and photosynthesis absorption rate PAR) in cotton due to higher concentrations of different botanicals. In present research harmful effects of higher doses of botanicals were studied. The main objective was the evaluation of phytotoxic effects (CO2-in, CO2-out, H2O-in, H2Oout and photosynthesis absorption rate PAR) in cotton due to higher doses of different botanicals.

MATERIALS AND METHODS
The present study was carried out at College of Agriculture, University of Sargodha to check phytotoxic effect of high concentrations of four botanicals, Neem (Azadirachta indica), (Mentha arvensis), Datura (Datura stramonium), Lemonene (Citrus limonium), on cotton. The cotton variety MNH-886 was sowed with Chopa method on 30-May-2014. The row to row distance was 30 inches and plant to plant was 12 inches.
From the field of College of Agriculture, University of Sargodha fresh leaves of D. stramonium were collected. A. indica, M. arvensis and C. limonium leaves were collected from Bhakkar. The plant materials were washed with distilled water separately. For drying leaves were put at room temperature (25 o C) for two weeks. It ensured that adequate air was streaming to evade damping. After shade drying leaves were granulated with electric processor for 45 seconds for making concentrated powder. At that point took leaves powder 40 grams put in conical flask and included ethyl liquor 320 mL in it. The mixture of powder and ethyl liquor was put on mechanical shaker for legitimate mixing for 72 hours. After blending the stock arrangement was put for 48 hours. The blend of stock solution was sifted with channel paper (What man channel paper No.1). Now the plant extract was prepared. A similar methodology was drilled for all plant leaves, respectively (Fiaz et al., 2012 cotton. Data of CO2-in, CO2-out, H2Oin, H2O-out and Photosynthetic absorption rate (PAR) of cotton crop was recorded by Photosynthesis meter Cl 340. M. Stat C 8.1 version was used for analysis of variance (ANOVA).

Phytotoxic impact of various concentrations of various botanicals against CO2-in on cotton crop
The results (

Various botanicals phytotoxic impact at various concentrations against CO2-out of cotton crop
The results ( The results ( Table 2) also represented that after 72 hours at 2% concentration of C. Limonium (145.641.17), CO2-out word movement of cotton crop was less influenced which differ significantly from all other treatments. The 4% concentration of D. stramonium (122.951.14) was more phytotoxic against CO2-out of cotton crop having significant difference from all other treatments. The results revealed that 8% concentrations of A. indica, M. arvensis, D. stramonium and C. limonium have almost similar phytoxicity against CO2-out of cotton crop. These finding indicated that the 16% concentration of D. stramonium (129.642) following 72 hours of spray was more phytotoxic against CO2out of cotton crop.

Phytotoxic impact of various concentrations of various botanicals against H2O-in of cotton crop
The results (Table 3) demonstrated that the concentration of 2% all treatments, 12 hours after spray, did not differ significantly from each other. The results revealed that cotton crop at 4% concentration of A. indica (4.630.11) and D. stramonium (4.73 0.10) were less phytotoxic and are statistically at par. The 8% concentration of D. stramonium (2.610.05) and M. arvenis (3.640.18) did not differ significantly from each other and were more phytotoxic than A. indica (3.740.16). The results also revealed that the 16% concentration of A. indica (2.420.03) was less phytotoxic and showed significant difference from all other treatments.  The results (Table 3) showed that the 2% concentration of D. stramonium, M. arvensis and limonene were statistically at PAR and more toxic against H2O-in of cotton crop than the A. indica (5.370.09). The outcomes demonstrated that M. arvensis (3.450.13) after 24 hours of spray at 4% concentration was more phytotoxic against H2O-in of cotton crop than the A. indica (4.630.14) but less toxic than the limonene (2.510.07) and D. stramonium (2.540.16). The 8% concentration of D. stramonium (1.390.12) differs significantlyfrom all other treatments. The 16% concentration of D. stramonium (1.530.12) and M. arvensis (1.56 0.17) were statistically at PAR and the phytotoxicity of these two botanicals were more as compared to the limonene (2.480.14) and A. indica (2.770.09).

The results (Table 3) demonstrated that the
A. indica (6.340.14) at 2% concentration following 48 hours of spray had significant difference and least toxicity against H2O-in of cotton crop while the other extracts were phytotoxic and more phytotoxic was D. stramonium (3.560.04). The results revealed that 4% concentration of M. arvensis differ significantly and had morephytotoxicitythan all the other plant extracts. The results also showed that the D. stramonium at 8% concentration was more phytotoxic against H2O-in of cotton crop. While limonene (3.570.20) at 16% concentration phytotoxicity differ significantly and was less as compared to all the other plant extracts which were statistically at par.
The results (Table 3) also demonstrated that the limonene (6.390.14) at 2% concentration following 72 hours of spray had significant difference and was least toxic against H2O-in of cotton crop. The results revealed that C. limonium and D. stramonium had similar phytotoxicity at 4% concentration against H2O-in of cotton crop. The phytotoxicity of 8% concentration of different botanicals differ significantly and was in order as limonene > A. indica > D. stramonium > M. arvensis which were 5.740.01, 3.500.12, 2.640.05 and 2.600.02 respectively as compared to the average of control 7.640.02. The phytotoxicity at 16% concentration of M. arvensis (2.630.02) differs significantly and was more as compared to all other plant extracts.

Phytotoxic impact of various concentrations of various botanicals against H2O-out of cotton
The results (Table 4) demonstrated that the limonene at 2% concentration after 12 hours of spray had significant difference and was less phytotoxic against H2O-out of cotton crop. The phytotoxic affects of M. arvensis (3.770.04) and limonene (3.660.00) at 4% concentration was statistically at PAR and were less than the D. stramonium (1.640.11) and The result (Table 4) revealed that at 2% concentration phytoxicity of C. limonium (4.760) against H2O-out of cotton crop differ significantly and was more than all the other treatments. Similar results of C. limonium were also recorded at 4%, 8%and 16% concentrations.

Phytotoxic impact of various concentrations of various botanicals against PAR of cotton
The result ( M. arvensis (1022.5329.6) had more phytotoxicity against PAR of cotton crop and both were statistically at par. The PAR of cotton crop was more influenced by the D. stramonium (1016.0162.2) at 16% concentration.
The present finding showed that at 2% and 8% concentration, neem and mint affected the CO2-in of cotton crop. CO2-out was affected by mint and neem at 16% and 4% concentrations, respectively. Both neem and mint at 16% concentration affected the H2O-in of cotton crop. PAR of cotton crop was more affected by the neem at 4% concentration. The reason of such outcomes was that different dosages of different botanicals in various way influenced the opening and shutting of stomata and photosynthesis color of cotton crop. The dosages which cause higher phytotoxicity of cotton crop, have more influence the stomata (influence the in word and out word development of CO2 and H2O) and photosynthesis color (impact the PAR).

CONCLUSIONS
The results of present research coincide with the finding of (Nijënstein and Ester, 1998), and (Ibrahim et al., 2004) who discovered that the concentrations of limonene 90 and 120 mL were more phytotoxic. The present investigation showed that the limonene 8% and 16% were more phytotoxic against CO2-in of cotton crop following 12 long periods of shower, at 4% affected the CO2-out, H2O-in was more influenced at 16% and H2O-out of cotton was more influenced at concentration of 16%.

CONTRIBUTIONS
MNZ: designed and performed the experiments, analyzed the data, interpreted the results, and drafted the manuscript. NM, JI, and MFS: did the project adminstration, revised the manuscript and helped in data analysis. TI, HAB, MSB, and AU: helped in data collection, investigation and visulization.

ACKNOWLEDGEMENTS
Sincere thanks to College of Agriculture, University of Sargodh providing research facilities.