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International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.9, No.08 pp 01-11, 2016
The Influence of Lithovit Fertilizer on the Chemical Constituents and Yield Characteristics of Cotton Plant under Drought Stress 1Magdy A. Shallan, 1Hazem M.M. Hassan, 2Alia A.M. Namich and *2Alshaimaa A. Ibrahim 1Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, Egypt 2Cotton Research Institute, Agricultural Research Center, Giza, Egypt Abstract : Nano-fertilizers are used recently as an alternative to conventional fertilizers for slow release and efficient use by plants. This study was carried out to evaluate the effects of Lithovit (nano-CaCO3) fertilizer on chemical constituents and yield characteristics of cotton plant under drought stress. The cotton plants pre-treated with four concentrations of nano-CaCO3 (3000, 6000, 9000 and 11000 ppm) then exposed to drought stress. The obtained results showed that pretreatment of cotton plants under drought stress with nano-CaCO3 caused increase of pigments content, total soluble sugars, total phenolics, total soluble proteins, total free amino acids, proline content, total reducing power, total antioxidant capacity and antioxidant enzyme activities and enhancement of yield characteristics. The optimum concentration of nano-CaCO3 to alleviate the drought stress in cotton plant was 11000 ppm. Finally, it can be concluded that foliar application of nano-CaCO3 can reduce the adverse effects of drought on cotton plants. Key words: Drought stress – Cotton – Lithovit – Nanoparticles - Chemical constituents – Yield. Introduction Cotton is a white fibrous agricultural product that has a wide variety of uses, from textile production, to creating paper, to producing oil and food products1. Cotton, Gossypium barbadense L., is the major fiber and oil crop in Egypt and it has been playing for along time, an important role in Egyptian national economy. It is the most important cash crop and considered as the main source of foreign exchange for the country. Water deficit is the most important factor limiting crop yield worldwide. Plant growth, including biochemical and physiological processes, is affected by water deficit stress2. The results of water deficit stress depend on the severity and duration of drought as well as the growth stage and genotype of the plant. The effect of drought on growth, yield and yield component and quality characters are very different and serious. In cotton plants, the sensitivity to drought stress during flowering and boll development has been well established. Lint yield is reduced by decrease in boll production due to reduction in flowering sites and increased boll abscission when the plant is exposed to extreme drought during reproductive development3. The turgor decrease is the first effect of drought stress that influences cell growth rate and its final volume. The phenomenon probably is the most sensitive drought-related process, resulting in decreasing the development rate, stem growth, leaf growth and also decreasing the stomatal diameter. The drought stress affect directly or indirectly photosynthesis via affecting the carbohydrate metabolism. Due to drought stress the photosynthesis decreases, flower and bud fall increases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsincreases and competition between vegetative and reproductive for obtaining carbohydrates increases. Leaf area development in cotton in the response to drought stress is more sensitive than stomatal photosynthesis and any changes in terms of decrease or increase of carbon uptake by change in photosynthesis rate, is resulted in the decrease of boll maintenance on plantsDevelopment and application of new types of fertilizers using innovative nanotechnology are one of the potentially effective options of significantly enhancing the global agricultural productions needed to meet the future demands of the growing population. Indeed, the review of available literature indicates that some engineered nanomaterials can enhance plant growth in certain concentration ranges and could be used as nanofertilizers in agriculture to increase agronomic yields of crops and/or minimize environmental pollution9. Lithovit is a natural calcium carbonate (nano-CaCO3) foliar fertilizer supplemented with calcium micronutrient which delivers fine particles (<10 µm) that can easily be adsorbed directly through the stomata of plant leaves. The micronutrients supplied with Lithovit influence plant metabolism and cell wall formation (Ca), resulting in a product that has potential to increase and sustain improved plant metabolism. Inside the leaf intercellular spaces Lithovit particles break down and release gaseous CO2 enhancing the CO2 concentration at the photo-synthetically active area within the plant leaves. The normal concentration of CO2 in the atmosphere is approximately 0.04 vol. %, which means that most cultivated plants fail to achieve the optimum level of photosynthetic rate, which is achieved at near to 0.1 vol. % CO2. The Lithovit particles that enter the intracellular "compartment" dock, with its negative surface of the cell membrane, where they produce a negative potential. Lithovit’s mode of action is to increase CO2 levels within the plant leaf structure and by implication enhance photosynthetic efficiency. The additional supply of micronutrients from the Lithovit complex provides a source of key plant available elements required to aid photosynthetic activity10. Maswada and Abd El-Rahman11 reported that Lithovit treatments significantly increased total chlorophyll, total carotenoids, total soluble sugar and proline concentrations under stress conditions compared to control wheat plants. Exposure to elevated CO2 significantly affects activity of antioxidative enzymes in plants. It decreased SOD activities in spruce, pine and oak12,13 and reduced CAT activity in spruce and tobacco12,14. Pritchard et al.15 investigated the effects of elevated compared to current atmospheric CO2 concentration (720 and 365 Ml/l, respectively) on antioxidative enzymatic activities of two soybean genotypes. They found that elevated CO2 significantly decreased activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione reductase (GR) in both soybean genotypes. Lithovit has a very significant influence on the growth it had significant positive influence on stem height, crown diameter, and leaf number plants under salt stress of maize16, Koelreuteria paniculata17, wheat11, Hayward kiwifruit10 and tomato18. The aim of this study was to evaluate the effects of Lithovit (nano-CaCO3) on chemical constituents and yield characteristics of cotton plant under drought stress. Materials and Methods Materials Plant material: Cotton (Gossypium barbadense L. cv Giza 94) seeds were obtained from the Plant Physiology Department, Cotton Research Institute, Agricultural Research Center, Giza, Egypt. Chemicals: Lithovit was purchased from Agrolink Co., Egypt. Folin reagent, Pyrogallol and trichloroacetic acid were purchased from Acmatic Co., Egypt. All other chemicals were of analytical reagent grade. Methods Experimental design and treatments: The experiment was conducted in two summer seasons 2014 and 2015 at Sakha Research Station of Plant Physiology Department, Cotton Research Institute, Agricultural Research Center, Kafr El-Sheikh, Egypt. This experiment was carried out to study the effects of spraying cotton plants with different concentrations of nano-CaCO3 (3000, 6000, 9000 and 11000 ppm) on chemical constituents and yield characteristics under drought conditions. Seeds of cultivar Giza 94 were sown in clay loam soils on 24th of April 2014 in the first season and on the 28th April 2015 in the second one. The experimental plot consisted of rows, 3.5 m long and 0.6 m width (plot area = 14.70 m2) of the Agricultural Experimental Sakha Station Farm of the Agriculture Research Center, Kafr El-Sheikh, Egypt. All plots were fertilized at a rate of 60 kg N/fed in the form of urea (46.5% N) in two equal doses, the first dose was added after thinning (before the first irrigation), while the second dose was applied before the second irrigation. All plots received an adequate amount of fertilizer in order to produce healthy plants. Fertilization was carried out according to recommendation of Cotton Research Institute, phosphorus fertilizer was applied during soil preparation in form of calcium superphosphate (15.5% P2O5) at a rate of 15.5 kg P2O5/fed. Potassium fertilizer was applied after thinning at a rate of 24 kg K2O/fed in the form of the potassium sulphate (48% kg K2O). Irrigation was carried out regularly at the plant needs using tap water until the start of flowering stage, then the plots preventing water supply for 24 days till the appearance of sing of permanent wilting (drought stress) to take samples and back to irrigate plants. Plants were sprayed with Lithovit (nano-CaCO3) at start of flowering stage and the untreated plots (control) were irrigated with tap water continuously. Plant samples: Plant samples (whole plant and leaves) were taken at flowering stage (74 days from sowing) during the experimental period. In this stage, 6 plants were taken from each treatment (3 plots). The soil particles were washed off the roots by a stream of tap water. At harvest stage (180 days after sowing), samples (ten plants) from each plot were taken. Chemical analysis: Cotton leaves were taken randomly after flowering stage to carry out the chemical analysis as follows: Determination of pigments content: The chlorophyll a, b and total chlorophyll contents were determined according to the method19 and carotenoids content was determined according to method20. Determination of total soluble sugars: Total soluble sugars were determined in ethanol extract of cotton leaves by the phenol-sulfuric acid method according21. Determination of reducing sugars: Reducing sugars were determined colormetrically according to Folin and Wu method22. Determination of Non-Reducing Sugars: Non-reducing sugars were calculated by the difference between total soluble sugars and total reducing sugars. Total phenolics content: Total phenolics were determined in ethanol extract of cotton leaves using Folin-Ciocalteau method23. Determination of total soluble protein: Total soluble proteins were extracted from cotton leaves according to24 and determined by the method25 of Lowry-Folin. Determination of total free amino acids: Total free amino acids were determined in ethanol extract of cotton leaves by ninhydrin method26. Determination of proline content: Proline content of cotton leaves was determined according to method27. Assay of antioxidant enzymes activities Extraction of antioxidant enzymes: Crude enzyme extract was prepared for assay of catalase (CAT), peroxidase (POX), superoxide dismutase (SOD) and glutathione reductase (GR) activities according to24. Assay of catalase activity: Catalase (EC 1.11.1.6) activity was measured according to the method28 as follows: The assay mixture contained 2.6 ml of potassium phosphate buffer solution (50 mM, pH 7.0), 0.4 ml of H2O2 solution (15 mM) and 0.04 ml of enzyme extract. The decomposition of H2O2 was followed by the decline in absorbance at 240 nm. The enzyme activity was expressed in U/mg protein (U = 1 mM of H2O2 reduction/min/mg protein). Assay of peroxidase activity: Peroxidase (EC 1.11.1.7) activity was assayed according to the method29 as follows: The assay mixture of POX contained 2 ml of phosphate buffer solution (0.1 M, pH 6.8), 1 ml of pyrogallol solution (0.01 M), 1 ml of H2O2 solution (0.005 M) and 0.5 ml of enzyme extract. The solution was incubated for 5 min at 25°C, after which the reaction was terminated by adding 1 ml of H2SO4 solution (1.25 M). The amount of purpurogallin formed was determined by measuring the absorbance at 420 nm against a blank prepared by adding the extract after the addition of H2SO4 solution at zero time. The activity was expressed in U/mg protein. One U is defined as the change in the absorbance by 0.1 min/mg protein. Assay of superoxide dismutase activity: Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed according to the method30 as follows: The reaction mixture contained 2.35 ml of phosphate buffer (50 mM, pH 7.8), 0.30 ml of methionine solution (10 mM), 0.10 ml of Nitroblue tetrazolium solution (1 mM), 0.20 ml of EDTA solution (0.01M), 0.20 ml of enzyme extract and 0.05 ml of riboflavin solution (0.2 mM). The absorbance of reaction mixture was measured at 560 nm. The increase in absorbance in the absence of enzyme was taken as 100 and 50% initial was taken an equivalent to 1 unit of SOD activity. Assay of glutathione reductase activity: Glutathione reductase (GR; E.C. 1.6.4.2) activity was assayed according to the method31 as follow: The assay mixture was composed of 1.20 ml of phosphate buffer solution (50 mM, pH 7.8), 0.10 ml of extract and 0.05 ml of NADPH (83 μM in 0.1% NaHCO3). After incubation at 25°C for 10 min, 0.15 ml of GSSG solution (1 mM) was added, and the decrease of NADPH absorption was monitored for 3 min at 340 nm using a UV/Visible Spectrophotometer. The NADPH concentration change [μmol NADPH (ml extract)-1min-1] was calculated. Determination of total antioxidant capacity: Total antioxidant capacity was determined in ethanol extract of cotton leaves using the phosphomolybdenum method32 as described33 as follows: A known volume (0.01 ml) of extract was added to test tube then completed to a constant volume (0.3 ml) with DW. 3.0 ml of reagent solution (0.6 M sulfuric acid, 28.0 mM sodium phosphate and 4.0 mM ammonium molybdate) were added to each tube and mixed well then incubated at 95°C for 90 min. Blank was prepared by the same procedure without extract. After cooling to room, the absorbance of the solution was measured at 695 nm using spectrophotometer against blank. Increased absorbance of the reaction mixture indicated increased total antioxidant capacity. Determination of total reducing power: The total reducing power was determined in ethanol extract of cotton leaves according to the method34 as described by35 as follows: A known volume (1 ml) of ethanol extract was mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%). The mixture was incubated at 50oC for 20 min. Then, 2.5 ml of trichloroacetic acid (10%) was added to mixture, which was then centrifuged for 10 min at 3000 rpm. The upper layer of solution (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl3 (0.1%). The absorbance was measured at 700 nm against a blank using UV-Vis spectrophotometer. Increased absorbance of the reaction mixture indicates increase in reducing power. Yield and its components: Yield and its components, including plant height (cm), number of fruiting branches/plant, number of open boll/plant, boll weight (g), lint percentage, seed index (g) and yield (k/f) were recorded. Relative water content: Relative water content was determined according to the method36. Statistical analysis: The results were analysed by an analysis of variance (P>0.05) and the means separated by Duncan’s multiple range test. The results were processed by CoStat computer program (1986). Results and Discussion Foliar application of Lithovit on cotton plant under drought conditions Chemical constituents of cotton leaves: Cotton leaves obtained from this experiment were employed to determine their contents of chlorophyll a, b, total chlorophyll, carotenoids, total soluble sugars, reducing sugars, non-reducing sugars, total phenols, total soluble proteins, total free amino acids and proline in addition to determine the antioxidant enzyme activities (catalase, peroxidase, superoxide dismutase and glutathione reductase), total antioxidant capacity and total reducing power. The obtained results are presented in Tables 1, 2, 3, 4 and 5. Pigments content: Data presented in Table 1 showed that under drought conditions the contents of chlorophyll a, b, total chlorophyll, carotenoids of stressed cotton plants were decreased in comparison with control plants under normal conditions. Foliar application of cotton plants with different concentrations of Lithovit (3000, 6000, 9000 and 11000 ppm) under drought conditions significantly increased chlorophyll a, b, total chlorophyll and carotenoid contents of cotton plants to be more than control plants. This increasing in pigments content of cotton plants as a result of application of Lithovit with the different concentration (especially 11000 ppm) may be due to those Lithovit particles can enhance the growth and productivity of crops by means of increasing the natural photosynthesis. These results are in agreement with11,37,38. Carmen et al.39 suggested Lithovit fertilizer particles, finely sprayed on the leaf surface are absorbed and transformed in CO2. Therefore, Lithovit fertilizer can significantly enhance photosynthesis because the external factor that limiting photosynthesis is the natural content of CO2 in the air. Table 1: Effect of foliar application of Lithovit (nano-CaCO3) on Chlorophyll (Chl) a, b, total chlorophyll and carotenoids contents in leaves of cotton plant under drought stress
Carotenoids (mg/g DW)
Chlorophyll pigments (mg/g DW)
Concentration (ppm)
Treatment
Total Chl
Chl b
Chl a
0.62c±0.036
5.10c±0.016
2.46b±0.031
2.64d±0.029
Control (under normal conditions)
0.46d±0.047
3.65f±0.035
1.43f±0.034
2.22e±0.021
Drought stress conditions
0.65c±0.026
3.95e±0.024
1.71e±0.018
2.24e±0.022
3000
nano-CaCO3
Drought stress conditions
0.70b±0.017
4.56d±0.018
1.79d±0.049
2.76c±0.017
6000
0.87a±0.012
5.66b±0.022
1.94c±0.029
3.72b±0.033
9000
0.90a±0.024
8.24 a±0.023
3.64 a±0.018
4.61a±0.017
11000
0.0435
0.0359
0.0393
0.0349
L.S.D
-Values are means of three replicates ± SE. Numbers in the same column followed by the same letter are not significantly different at P<0.05. DW: dry weight. Total soluble sugars content: Table (2) shows the effect of foliar application of Lithovit on total soluble sugar, reducing and non-reducing sugar contents in leaves of cotton plant under drought stress. The obtained results revealed that the contents of total soluble sugars, reducing sugars and non-reducing sugars of cotton leaves were increased with increasing the concentration of Lithovit. Lithovit was more effective at concentration 11000 ppm. According to Ahmed et al.40 the total soluble and non-reducing sugars in cotton leaves were decreased at bolling stage under drought conditions. Lithovit application improves photosynthesis process, that due to may be related to its high content of carbonates, as source of CO2, and calcium. However, it is found that elevated CO2 and exogenous calcium application have positive effects on photosynthesis improvement. The effect of Lithovit, as a CO2 reservoir, on total soluble sugar accumulation can be explained by its stimulatory effect on carbon assimilation11,41. Table 2: Effect of foliar application of Lithovit (nano-CaCO3) on total soluble sugar, reducing and non-reducing sugar contents in leaves of cotton plant under drought stress
Treatment
Concentration (ppm)
Carbohydrate (mg/g FW)
Total Soluble Sugars
Reducing Sugars
Non- Reducing Sugars
Control (under normal conditions)
32.08c±0.046
21.34c±0.018
10.64c±0.008
Drought stress conditions
27.57f±0.024
19.73f±0.040
7.83f±0.024
Drought stress conditions
nano-CaCO3
3000
28.80e±0.028
20.16e±0.025
8.64e±0.029
6000
30.54d±0.016
20.36d±0.022
10.18d±0.029
9000
36.24b±0.018
23.07b±0.028
13.17b±0.014
11000
41.43a±0.027
26.38a±0.027
15.05a±0.011
L.S.D
0.0426
0.0411
0.0317
-Values are means of three replicates ± SE. Numbers in the same column followed by the same letter are not significantly different at P<0.05. FW: fresh weight. The phenolics content: The accumulation of polyphenols, in adverse climate conditions and increasing the CO2 concentration, are probably examples of adaptive protection strategies. Polyphenols constitute a large group of diverse molecules, implemented in ROS detoxification and in the protection of the photosynthetic apparatus. Polyphenols can also retard microbial and enzymatic decomposition by forming resistant compounds or by inhibiting microbial activity. Data presented in Table 3 indicated that the drought conditions increased the phenolics content as compared with control plants. The results revealed that the spraying of cotton plants with Lithovit (nano-CaCO3) under drought conditions increased the contents total phenolics content in comparison with control plants especially at concentration 11000 ppm. The results showed improved antioxidant status indicating formation of antioxidants in all treatments. These results are in harmony with the results of Abdallah42 mentioned that drought conditions tended to increase total phenols of cotton leaves at all stages of growth in Giza 70 (all stages), Dandara (seedling) and Giza 69 (squaring). While, Ibrahim5 revealed that water stress tended to reduce total phenols content significantly in wilted cotton leaves (Giza 90) as compared to turgid. This variation in results may be due to differences in plant varieties and cultivars, experimental conditions and drought stress period. The enrichment of CO2 supply has been shown to significantly affect secondary metabolites formation including phenolics and flavonoids in various plant or cell cultures43,44,45. Total soluble proteins: The results in Table 3 revealed that the total soluble proteins were increased significantly in cotton leaves under drought stress as compare with plant under normal condition. The results in Table 3 indicated that the foliar application of Lithovit under drought conditions significantly increased total soluble proteins especially with concentration 11000 ppm of nano-CaCO3. The increase in total soluble proteins during drought stress was due to the expression of new stress proteins5,46. The increase in Lithovit treatments may be due to any effects of elevated CO2 on crop protein content could be ameliorated by increased use of N fertilizer. When plants grew under elevated CO2 conditions, the rate of photosynthesis was higher, as were the nitrate reductase and glutamine synthetase activities; thus, drought had less effect on the protein content. This outcome could also result from the fact that under elevated CO2 conditions, nitrogen uptake was higher than under ambient conditions47,48. Table 3: Effect of foliar application of Lithovit (nano-CaCO3) on total phenol, total soluble protein, total free amino acids and proline contents in leaves of cotton plant under drought stress
Treatment
Concentration (ppm)
Total Phenols (mg/g FW)
Total soluble proteins (mg/g FW)
Total free amino acids (mg/g FW)
Proline (µmol/g FW)
Control (under normal conditions)
11.17f±0.024
8.0d±0.589
12.35f±0.029
3.25f±0.029
Drought stress conditions
16.17e±0.029
22.8c±0.283
18.85e±0.028
73.32e±0.021
Drought stress conditions
nano-CaCO3
3000
19.42d±0.041
23.0bc±0.432
20.55d±0.023
142.79d±0.018
6000
26.18c±0.032
24.0b±0.632
22.75c±0.018
182.64c±0.022
9000
28.95b±0.024
30.0a±0.432
25.06b±0.025
208.55b±0.040
11000
32.12a±0.045
30.8a±0.365
28.79a±0.038
225.55a±0.014
L.S.D
0.0377
0.0552
0.0414
0.0384
-Values are means of three replicates ± SE. Numbers in the same column followed by the same letter are not significantly different at P<0.05. FW: fresh weight. Total free amino acid and proline contents: The data presented in Table 3 showed that total free amino acid and proline contents of cotton plants under drought stress were increased significantly as comparison with control plants. Lithovit treatments on cotton plant under drought stress increased significantly total free amino acid and proline contents as comparison with plants untreated under normal and drought conditions. By decreasing water potentials, proline accumulation involved in osmoregulation appeared to allow additional water to be taken up from the environment, thus counteracting the influence of drought stress on the plant tissues. Accumulation of proline caused proline synthesis or decrease proline degradation or High proline concentration measured in sludge-treated nodules could also contribute to a protective role as scavenger of ROS49. The large amount of accumulated free proline might also be a source of energy and N for the plants used when stress is relative50. Proline would be a good storage form of nitrogen because of its metabolic proximity and ready conversion to glutamic acid which is considered a key compound in nitrogen metabolism. These results are in agreement with Ibrahim5 in cotton, Hammad and Ali8 in wheat, Lum et al.51 in barley plants and Shinde and Thakur52 in pea. Total antioxidant capacity and reducing power: The results of total antioxidant capacity and total reducing power in cotton leaves under drought stress are presented in Table 4. The obtained results showed that the spraying of cotton plants by Lithovit (nano-CaCO3) at concentration 11000 ppm increased total antioxidant capacity (1.096±0.005) and at concentration 9000 ppm increased total reducing power (1.228±0.007) in cotton plants under drought conditions. This may be related to the CO2 concentration has a substantial influence on the stress sensitivity of plants via changes in antioxidant enzyme activity. When oxidative stresses are present, elevated CO2 helps to increase the synthesis and activities of antioxidants that tend to alleviate the various problems caused by the stresses. In addition, the possibility exists that the greater concentrations of antioxidant compounds in plant tissues caused by the historical rise in the air's CO2 content53. Table 4: Effect of foliar application of Lithovit (nano-CaCO3) on the activities of total antioxidant capacity and total reducing power in leaves of cotton plant under drought stress
Treatment
Concentration (ppm)
Total antioxidant capacity (O.D 695 nm)
Total reducing power (O.D700 nm)
Control (under normal conditions)
0.683f±0.003
0.481e±0.006
Drought stress conditions
0.793e±0.002
0.882d±0.009
Drought stress conditions
nano-CaCO3
3000
0.846d±0.004
0.940b±0.003
6000
0.872c±0.003
0.944b±0.005
9000
1.013b±0.002
1.228a±0.007
11000
1.096a±0.005
0.913c±0.006
L.S.D
0.0059
0.0100
-Values are means of three replicates ± SE. Numbers in the same column followed by the same letter are not significantly different at P<0.05. Antioxidant enzymes: Upon stress, plants have developed several antioxidation strategies to demolish the harmful effects of ROS. Several enzymatic and nonenzymatic antioxidative systems such as SOD, CAT, POD, GR and phenolic compounds, etc., are responsible for combating ROS mediated damage. The results obtained in Table 5 showed that the catalase activity was significantly increased by drought treatment in as compared to turgid leaves. The foliar application of Lithovit to cotton plants under drought conditions increased the activities of catalase, peroxidase, superoxide dismutase and glutathione reductase in comparison with control and untreated stressed plants. Ali et al.53 reported that CAT and POD are also the major H2O2 detoxifying enzymes in plants. According to our result both CAT and POD activity continued to increase throughout the culture period. The combined action of CAT and POD is critical in mitigating the effects of oxidative stress, since both act on H2O2 converting it in to water and oxygen. The increased GR activity was accompanied by an increase in total glutathione content, suggesting that a predominant GSH synthesis took place. A significant increase in glutathione content was found under different stress conditions and playing an important role for ROS scavenging54. Frenandez-Trujillo et al.55 and Ali et al.53 inducted that increases in the atmosphere's CO2 concentration may increase various plant antioxidant enzymes and thereby reduce the negative effects of various abiotic stresses. Table 5: Effect of foliar application of Lithovit (nano-CaCO3) on the activities of catalase and peroxidase, superoxide dismutase and glutathione reductase in leaves of cotton plant under drought stress.
Treatment
Concentration (ppm)
Catalase activity (U/mg protein)
Peroxidase activity (U/mg protein)
Superoxide dismutase (U/mg protein)
Glutathione reductase (U/mg protein)
Control (under normal conditions)
0.073f±0.004
0.316f±0.002
0.401e±0.002
0.09d±0.018
Drought stress conditions
0.132e±0.007
0.420e±0.007
0.619d±0.004
0.05e±0.029
Drought stress conditions
nano-CaCO3
3000
0.163d±0.006
0.486d±0.009
0.637c±0.007
0.15c±0.024
6000
0.177c±0.009
0.496c±0.004
0.660fb±0.007
0.12bc±0.018
9000 Lint %
0.196b±0.004 Seed index
(g)
0.520b±0.003
0.667b±0.006
0.20b±0.041
11000
0.217a±0.003
0.721a±0.004
0.690a±0.003
0.24a±0.022
L.S.D
0.0093
0.0084
0.0080
0.0371
-Values are means of three replicates ± SE. Numbers in the same column followed by the same letter are not significantly different at P<0.05. Yield characteristics: The obtained results in Table (6) showed that all yield characteristics i.e. plant height, number of fruiting branches, number of boll, boll weight (g), seed index (g), lint%, yield K/F and leaf relative water content of cotton plants were decreased under drought stress conditions in comparison with control plants of Giza 94 cultivar. CO2 fixation is reduced and photosynthetic rate decreases, decrease in photosynthetic pigments, carbohydrates accumulation and nitrogenous compounds as showed in Tables (1 and 2). The decrease in yield and yield components in cotton crop under drought conditions has also been reported by5,56-60. The present study showed that pre-treatment of cotton plants under drought stress with Lithovit (nano-CaCO3) decreased adverse effects of drought stress and the nano-CaCO3 at 11000 ppm concentration was actively involved in the regulation of plant growth. Lithovit acts as a long term reservoir supplying plants with CO2; thus it can enhance growth and productivity of plants, particularly C3-plants as wheat and cotton by increasing Pn (photosynthesis net) because higher CO2 can suppress ribulose-1,5-bisphosphate (RuBP) oxygenase activity; decrease photorespiration; and increase carbon assimilates for plant growth and development. When elevated CO2 concentrations generally increase plant growth through increased carbon assimilation, biomass and leaf area of plants11,61. All exogenous treatments, particularly Lithovit application, significantly enhanced grain and straw yields of plants. This effect could be related to the influence of Lithovit, as a source of calcium and CO2 reservoir62,63,64. Nassef and Nabeel65 revealed that the foliar application of Lithovit enhanced significantly plant height, main head length, main head diameter, main head weight and total head yield (ton/feddan) in favor of 0.05% concentration in the two growing seasons. The highest broccoli yield was obtained from Calabrese cultivar planted with direct seeds and subjected to 0.05% Lithovit as foliar application. Also, transplanting the same cultivar without using Lithovit fertilizer gave better growth and higher yield. Sala et al.66 found that Lithovit foliar application on wheat seedlings influenced the synthesis and accumulation of fresh matter and dry matter in wheat shoots and roots. Rebalancing of the shoot: root ratio was recorded for fresh weight and dry weight. Table 6. Effect of foliar application of nano-CaCO3 on yield characteristics of cotton plant (cv Giza 94) under drought stress conditions season 2015
Relative water content (%)
Yield K/F
Boll weight (g)
No. of open boll/plant
No. of fruiting branch/plant
Plant height (cm)
Concentration (ppm)
Treatment
60.74 a±1.82
6.67a±0.12
40.70d±0.21
12.12ab±0.21
3.21a±0.14
20.52 a±1.29
18.25 b±0.95
148.51c±1.50
Control (under normal conditions)
49.42 c±1.29
3.80e±0.24
40.45e±0.11
12.03b±0.26
3.18a±0.18
19.00a±0.82
18.06b±0.13
137.53e±0.58
Drought stress conditions
52.15 bc±1.09
4.51d±0.11
40.88cd±0.15
12.16ab±0.22
3.23a±0.14
19.25a±0.96
18.25b±0.50
144.02d±0.50
3000
nano-CaCO 3
Drought stress conditions
52.93 bc±1.63
5.76c±0.09
41.32b±0.09
12.23ab±0.21
3.25a±0.11
19.51a±1.29
18.54b±1.29
145.01c±0.82
6000
54.78 b±1.49
5.96b±0.14
41.70a±0.16
12.31ab±0.18
3.31a±0.11
19.75a±0.51
18.51b±0.50
146.54b±0.58
9000
55.23 b±1.81
6.02b±0.14
41.96c±0.15
12.44a±0.16
3.34a±0.14
20.75a±1.50
19.75a±0.51
148.75a±0.50
11000
4.313
0.168
0.219
0.315
0.208
1.652
1.110
1.237
L.S.D
- Values are means of three replicates ± SE. Numbers in the same column followed by the same letter are not significantly different at P<0.05. References
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