Evaluation of Application Methods Efficiency of Zinc and Iron for Canola ( Brassica napus L . )

In order to evaluation of application method efficiency of zinc and iron microelements in canola, an experiment was conducted in the Agricultural Research Station of Eastern Azerbaijan province in 2008. The experimental design was a RCBD with eight treatments (F1: control, F2: iron, F3: zinc, F4: iron + zinc in the form of soil utility, F5: iron, F6: zinc, F7: iron+ zinc in the form of solution foliar application, and F8: iron + zinc in the form of soil utility and foliar application). Analysis of variance showed that there were significant differences among treatments on given traits, antioxidant enzymes activity, fatty acids percentage, plant height, seed weight to capitulum weight ratio, protein percentage, oil percentage, oil yield, 1000 seed weight, seed yield, nitrogen, phosphorous and potassium percentage of leaves, zinc and iron content of leaves and capitulum diameters. The highest seed yield, oil yield, oil percentage, 1000 seed weight, seed weight to capitulum weight ratio and protein percentage were obtained from the soil and foliar application of iron + zinc treatments (F8). Also, the highest amounts of nitrogen, phosphorous and potassium concentration in leaves were achieved from control treatment which was an indication of non-efficiency of iron and zinc on the absorption rate of these substances in the leaves. The correlation between effective traits on the seed yield, such as, capitalism diameter, number of seed rows in capitulum, seed weight to capitulum weight ratio and 1000 seed weight were positively significant. In general, foliar and soil application of zinc and iron had the highest efficiency in aspect of seed production. The comparison of the various methods of fertilization showed that foliar application was more effective than soil application. Also, micronutrient foliar application increased concentration of elements, especially zinc and iron. Antioxidant enzymes activity was different in response to treatments also the highest palmitoleic, oleic and myristic acid were observed in F6 and F7 treatments.


Introduction
Canola (Brassica napus L.) is grown in different agroclimatic zones of the world, differing in soil nutrient status.The use of foliar fertilizing in agriculture has been a popular practice with farmers since the 1950s, when it was learned that foliar fertilization was effective and economic.Recent research has shown that a small amount of nutrients, particularly Zn, Fe and Mn applied by foliar spraying increases significantly the yield of crops (Sarkar et al., 2007;Wissuwa et al., 2008).Also, foliar nutrition is an option when nutrient deficiencies cannot be corrected by applications of nutrients to the soil (Crabtree, 1999;Sarkar et al., 2007;Cakmak, 2008).It is likely therefore, in open-field conditions, where the factors that influence the uptake of the nutrients are very changeable, foliar fertilization can get considerable importance.Among the micronutrients, Zn and Fe nutrition can affect the susceptibility of plants to drought stress (Sultana et al., 2001;Khan et al., 2003;Cakmak, 2008).physical and chemical properties.The research field had a loamy soil.Details of soil properties are shown in Tab. 1.

Field preparation and applying the treatments
After plough in fall and two disks in spring, the land was flatted by leveller and then plots were prepared.The experimental design was laid out in a Randomized Complete Block Design with four replications.The plots had 4 m length and 3 m width consisted of five rows, 0.6 m apart.Between all plots, 1 m distance was kept to eliminate all influence of lateral water movement.According to results of soil analysis 50 k.ha - super phosphates triple and 300 kg.ha -1 urea was used.All of super phosphates triple and one third of urea were distributed in plots and mixed with surface soil before seed sowing.Rest of urea was used when plants have 6 or 8 leaves and at flowering stage as topdress.Different treatments of micronutrient soil application which were including: 25 k.ha -1 iron from secostrine and 40 kg.ha -1 from zinc sulphate were distributed in plots and mixed with surface soil before seed sowing.

Seed sowing and irrigation
The canola seeds (Brassica napus L. C.V. 'SLM 046 ') were sown on 15 July.The between row distance and within distance were 60 cm and 25 cm, respectively.The flooding Irrigation was carried out as similar in all of plots during study.Two week after sowing weed control was efficiently performed by hand.Micronutrient foliar application was done at two times, one time when plants had 6 or 8 leaves and another when they have 10 or 12 leaves (early of flowering stage).The iron and zinc were sprayed on plants with concentrate of 2 parts per thousand and 3 part per thousand, respectively.In control treatments plants were sprayed by water.Description of all treatments is shown in Tab. 2.

Plant sampling, harvesting and scrutiny
At early flowering, number of leaves and leaf area were counted.Leaf area was estimated by multiplying of length and width of leaves in 0.68 coefficients (Rao and Saran, 1991).Also, fresh leaves samples were taken of similar leaves.The samples were frozen in liquid nitrogen and stored at -80°C until biochemical analysis.
After that, at flowering stage four upper leaves were taken for determine of N, P, K, Mn, Fe and Zn content of leaves.The samples were washed by distilled water and dried in oven at 70°C during 48 h.Total N, P, K, Mn, Fe and Zn content were determined through atomic absorp-tion of plant-available Fe +2 to unavailable Fe +3 .Plant yield on many soils is, therefore, limited by poor Fe availability, rather than a low Fe content in the soil.Also Fe leaching is the main pathway for Fe loss in coarse-textured soil with high pH, while excessive Fe uptake was the main pathway for Fe loss in clay-textured and acid soil.Application of Zn or Fe has been reported significant positive effects, in most cases, on growth measurements and chemical composition of safflower (Lewis and McFarlane, 1986), lupine ( Brennan et al., 2001), cumin (El-Sawi andMohamed, 2002), soybean (Gadallah, 2000;Heitholt et al., 2002), barley (Genc et al., 2004;Hebbern et al., 2005), wheat (Lu et al., 2004), sunflower (Mirzapour and Khoshgoftar, 2006), mustard (Chatterjee andKhurana, 2007), common bean (Fernandes et al., 2007) and rice (Wissuwa et al., 2008).Even though iron is one of the most abundant elements in soils, the low solubility product of iron minerals makes the inorganic form of iron unavailable to plants and forms the most common widespread nutritional disorder world over (Welch et al., 1991).Plants subjected to iron deficiency excess respond in different ways (Abadia, 1992).It is believed that under conditions of iron stress, some plants can increase their absorption capacity for iron.The mechanism affecting the acquisition of this essential microelement is often present in aerobic soils in the form of Fe (Marschner, 1995).
However, to the best of our knowledge, information regarding application method efficiency of zinc and iron on the growth and development of canola is not available.Therefore, the purpose of this study is to understand whether application of micronutrients as foliar application is better or soil application.

Materials and methods
The experiment was conducted on the research farm of Agricultural Research Station of Eastern Azerbaijan province, Azerbaijan, Iran (37º58´ N latitude, 46º3´ E longitude and altitudes of 1320 m) during 2008 growing seasons.Site of study has cold winter and warm summer.The yearly average precipitation (30-years long term period) which is mostly occurred during the spring months is 273.1 mm.The mean annual temperature was 11°C.

Soil sampling and analysis
Prior to the beginning of experiment, soil samples were taken in order to determine the physical and chemical properties.A composite soil samples were collected at a depth of 0-30 cm.It was air dried, crushed, and tested for The fatty acid compositions of the canola seed oils were determined by gas chromatography (GC) (Metcalf et al., 1966).The contents of palmitoleic, linolenic, oleic and myristic acids were determined using a computing integrator.The effects of the independent variables on oil content and palmitoleic, Linolenic, oleic and myristic acid concentrations of the oil were analyzed on a percentage basis.
Oil percentage and protein percentage were measured by soxhlet and Kjeltec method, respectively.Oil yield was calculated via product seed yield in percentage oil.

Catalase
Catalase activity was estimated by the meth od of Cakmak and Horst (1991).The reaction mixture contained 100 μL crude enzyme extract, 500 μL 10 mm H 2 O 2 and 1400 μL 25 mm phosphate buffer.The decrease in the absorbance at 240 nm was recorded for 1 min.

Glutathione reductase
Glutathione reductase activity was determined by the method of Halliwell and Foyer (Halliwell and Foyer, 1978).Its activity was assayed in a 1 mL reaction mixture containing 0.25 mL of 100 mM potassium phosphate buffer (pH 7.0), 0.05 mL of 10 mM oxidized glutathione, 0.12 mL of 1mM NADPH, 0.48 mL of distilled water, and 0.1 mL of enzyme extract.The resultant decrease in NADPH was observed at 340 nm.

Superoxide dismutase
The activity of SOD was measured according to the method of Giannopolities and Ries (1977).The assay medium contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 mM p-nitro blue tetrazolium chloride, 2 mM riboflavin, 0.1 mM EDTA and 5 mL enzyme extract.Glass test tubes containing the assay medium were illuminated with a fluorescent lamp (120 W); identical tubes that were not illuminated served as blanks.After illumination for 15 min, the absorbance was measured at 560 nm.One unit of enzyme activity was determined as the amount of the enzyme to reach an inhibition of 50% nitro blue tetrazolium reduction rate by monitoring the absorbance at 560 nm.

Reduced glutathione
The reduced glutathione content was assayed as described by Griffith and Meister (1979).200 mg of fresh material was ground with 2ml of 2% metaphosphoric acid and centrifuged at 17,000 rpm for 10 min.Adding 0.6 ml 10% sodium citrate neutralized the supernatant.One milliliter of assay mixture was prepared by adding 100 μl extract, 100 μl distilled water, 100 μl of 6mM 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and 700 μl of 0.3mM NADPH.The mixture was stabilized at 25 ºC for 3-4 min.Then 10 μl of glutathione reductase was added and read the absorbance at 412 nm in spectrophotometer.

Statistical analysis of data
All data were analyzed from analysis of variance (ANO-VA) using the MSTAT-C.Least significant difference test was used to measure statistical differences between treatment methods and controls.Also the charts were drawn by Excel, Microsoft Office 2003.

Results and discussion
The results of analysis of variance showed that, effect of micronutrients was significant on all traits except number of leaves in plant, leaf area, number of seed row in capitulum, seed number in capitulum, kernel to seed percentage and Mn content (Tab.3 and 4).

Antioxidant enzyme activity
The highest POD activity was observed in F 6 and F 8 treatment while the lowest activity was related to F 2 treatment (Tab.5).There was not significant difference between F 1 and F 3 and between F 5 and F 7 treatments (Tab.5).
It seems that Zn foliar application causes increase of POD activity because soil application had not additive effect on activity of this enzyme in contrast Fe application decreased POD activity.Our findings were in agreement with the results reported by Jiang and Huang (2001) and Habibi et al. (2004).The simultaneous increase in the activity of these enzymes contributes to a decrease of the deleterious effects of H 2 O 2 under stress.
Control treatment had the highest CAT activity (Tab.5).The lowest CAT activity was achieved from F 4 , F 6 and F 8 treatments, as among these treatments were not significant difference (Tab.5).Similarly, there was no difference between F 3 and F 5 application method.Fe and Zn application decreased CAT activity, due to scavenging of reactive oxygen species by other antioxidant enzymes.These results were agreement with the results reported by Bailly et al. (2000).
Glutathione reductase was affected by fertilizers too.As F 2 and F 3 treatments had the highest enzyme activity while the lowest was observed from F 8 treatment (Tab.5).There was not significant difference between F 4 and F 6 treatments too (Tab.5).The highest and the lowest GSH activity were related to F 1 , F 2 , F 3 and F 6 , F 7 , F 8 treatments, respectively (Tab.5).
The results showed that, F 7 and F 8 treatments increased significantly SOD activity.The lowest activity was observed in F 1 , F 3 and F 5 treatments (Tab.5).Cakmak (2000) reported that Zn deficiency may inhibit the activities of a number of antioxidant enzyme.
It has been demonstrated that environmental stress induces oxidative stress in plant tissues.Exposes chloroplasts to excessive excitation energy, may increase generation of reactive oxygen species and induce oxidative stress.To overcome the effects of oxidative stress, plants make use of a complex antioxidant system.Relatively higher activities of reactive oxygen species scavenger enzymes have been reported in many stressed plants, which suggest that the antioxidant system plays an important role in plants against environmental stresses (Habibi et al., 2004).
Superoxide dismutase may function as a reactive oxygen species scavenger, by converting O 2 -to H 2 O 2 (Baily et al. 2000).Hydrogen peroxide is converted to oxygen and water by CAT and POX .Even though high SOD activity protects plants against superoxide radicals, it can not be considered solely responsible for membrane protection against peroxidation.In general, micronutrient application had different effects on antioxidant enzymes activity, in some cases increase and in some cases decrease was observed.

Fatty acids
Analysis of fatty acids by GC showed that, the application of Zn and Fe increased the fatty acids compared to the control (Tab.5).In case of palmitic, palmitoleic and myristic acid foliar application was better than soil application while regarding linolenic acid soil application had better effect.Between foliar application and soil application was not difference on oleic acid content but applica- For a given means within each column followed by the same letter are not significantly differences (p < 0.05) Fig. 10.Changes of oil yield due to different methods of iron and zinc application.F 1 = control, F 2 = Iron (soil application), F 3 = Zinc (soil application), F 4 = Iron + Zinc (soil application), F 5 = Iron (foliar application), F 6 = Zinc (foliar application), F 7 = Iron + Zinc (foliar application), F 8 = Iron + Zinc (soil and foliar application).For a given means within each column followed by the same letter are not significantly differences (p < 0.05)

seed weight
The highest 1000 seed weight was observed in F 8 treatment and other foliar application treatments.Zinc is required for the biosynthesis of the plant growth regulator such as IAA and for carbohydrate and N metabolism which leads to high yield and yield components.This may be due to provision of macro and micro nutrients at latter stages which might have enhanced accumulation of assimilate in seeds and thus resulting in heavier seeds.

Seed yield
The highest seed yield was produced from F 8 treatment.Masoni et al. (1996) showed that, zinc increases seed yield.Heitholt et al. (2002) reported that seed yield of soybean increased while Cu, Mn, Zn, and Fe applied individually.The increase in yield with soil applied Fe might have been the result of increase in the amount of available Fe in soil.The increase in seed yield with foliar application of Fe could be attributed to the direct absorption of the element by the foliar sprayed with Fe.
In proceed to the previous studies (Cakmak, 1999), all methods of Zn application for plants significantly increased grain yield.Micronutrients increases photosynthesis rate and improves leaf area duration thus seed yield will be increased.Zinc plays important role in tryptophan biosynthesis, later is precursor of auxine also zinc is founded in phosphoenolpyruvate carboxylase structure.Another element that is iron is necessary to chlorophyll synthesis and its critical element in electron transport chain in photosystems.Iron deficiency leads to many disorders in chloroplasts.Ferredoxin is an important ironcontaining protein involved in electron-transfer.Singh et al. (1975) observed that, zinc increases canola significantly seed yield.The lowest seed yield was obtained from control treatment.

Leaf nitrogen content
The results showed that, micronutrient application decreased leaf nitrogen content.The highest nitrogen content was related to control treatment while the lowest content was obtained from F 8 treatment.Foliar application had more effect on nitrogen content reduction.It seems that, decrease in nitrogen content is due to competitive effect between elements such as zinc and nitrogen.

Leaf phosphorous content
Similar upon results, micronutrient fertilizing decreased phosphorous content in leaves.These results were inconsistent with that obtained from Movchan and Sobornikova (1972).The highest phosphorous content was related to control treatment.Zinc decreases phosphorous content because there is competitive effect between phosphorous and zinc in uptake of ions.Sahota and Arora (1981) reported that there was no significant effect of Zn on P content of leaves.tion of these elements increased significantly oleic content than control treatment (Tab.5).Zakaria et al. (2001) concluded that Zn foliar application significantly increased the total unsaturated fatty acids in cotton.
The highest palmitoleic acid was related to F 3 , F 5 , F 6 , F 7 and F 8 treatments also, the highest and the lowest linolenic acid percentage were achieved from F 3 , F 4 , F 5 and control treatment, respectively (Tab.5).
The highest myristic percentage was achieved from F 6 and F 7 treatments (Tab.5).It was observed that control treatment had the lowest myristic percentage (Tab.5).
Canola oil consists of different types of saturated and unsaturated fatty acids (palmitic acid, stearic acid, oleic acid, linoleic acid etc.).The palmatic acid and stearic acid are the major saturated fatty acids, whereas oleic and linoleic acids are unsaturated.Fatty acid composition of canola in particular and other oil seed crops in general, are influenced by fertilizing management.The findings of present study are shows that fatty acid composition is affected by iron and zinc application, for example, Kheir et al. (1991) found that the higher N-rate increased the percentage of unsaturated fatty acids and decreased saturated fatty acids of flax oil.

Protein percentage
The highest and the lowest protein percentage were observed in F 8 treatment and control, respectively.Kaya and Higgs (2002) pointed that the application of Zn fertilisers significantly increased the protein content of tomato.But it was in consistent with results of Singh, S. (2000) which suggest that protein content does not affected by fertilizers treatment and is related to genotypes.Cakmak et al. (1989) stated that under condition of zinc deficiency protein content decreases.Iron and zinc are two important elements in enzymes structure involved in amino acid biosynthesis and thus amino acids are the base of protein synthesis, protein content increases in use of these micronutrients.Our results are agreed with results of Zakaria et al (2001) on cotton.

Oil yield and percentage
The F2, F4 and F8 treatments produced the highest oil percentage .The lowest oil percentage was obtained from foliar application treatment and control treatment.It seems that, soil application of micronutrients is more benefit to oil biosynthesis.Singh and Sinha (2005) reported the decrease in oil concentration may be due to oxidation of some polyunsaturated fatty acids.The highest and the lowest oil yield were achieved from F8 and control treatments.In general, there was not significant different between micronutrient application method.Because of direct relation between seed yield and oil yield, increase in seed yield causes in higher oil yield (Singh, S. 2000).sorption.Sometimes response of the plants is different to application methods of fertilizers, for example in calcareous soil Fe and Zn are not available for plants, in this times, foliar application is a useful method for nourish of the plants.The results of this study demonstrated that, Fe and Zn had positive effect on yield and quality of canola oil.In addition to this, we also suggest that Ca and Fe should be sprayed on plants to reach the best quality and quantity in canola production.

Leaf potassium content
There was significant decrease in potassium content of plants which were treated with iron or zinc than control plants.The lowest potassium content was observed in F 8 treatment.It shows that, iron and zinc decreases potassium content in leaves of canola.Stoyanova and Doncheva (2002) remarked that the K content of the stems and leaves was not significantly affected by different concentrations of Zn.

Leaf iron content
The highest iron contents were observed in four treatments, F 8 , F 6 , F 5 and F 4 , respectively .The application of Fe through soil as well as foliar application caused a marked increase in the total content of Fe in the fenugreek plants.Whereas in foliar application method iron absorption is faster and easier that soil application the highest iron content was observed in these treatments.In soil application due to organic matters banding with chemical fertilizers that have high ability to absorb and hold nutrients, and positioning these substances near hairy roots, results in better availability to plant and thus causing in higher iron content.Studying previous researches show that best results achieved in using iron sulphate (Kalbasi et al. 1998).And may be the use of zinc sulphate causes better absorption in iron.Iron deficiency leads to chlorophyll degradation and chlorosis.It's reported that, iron is an essential element in protein synthesis and iron deficit decreases plant growth (Agarwala et al., 1965).Also, iron is involved electron transport in photosystems.Already decrease of chlorophyll content due to iron deficit was reported by Masoni et al. (1996).

Leaf zinc content
The highest zinc content was observed in F 6 , F 7 and F 8 treatments, respectively.Foliar application of zinc increased zinc content more than soil application.Zinc uptake is easier form leaves in compare soil application.Zinc uptake is lower in lime soils.Grawel and Graham (1999) have been reported that, zinc application increases zinc concentration in seed, root and leaves.Increase in Zn concentration reported in soil application of S and Fe.These results were in agreement with those reported by El-Gazzar et al. (1979) and Foregoni et al. (1974).Zinc is essential element in enzymatic system such as superoxide dismutase enzyme.Zinc plays important role in auxine and protein synthesis and it is essential for seed setting.

Conclusions
Iron and zinc uptake are controlled by the two major factors, availability of these elements in the soil and the ability of plants to acquire them.Application methods of micronutrients are very important to attain the best ab-