Soybean nodulation and chlorophyll concentration (SPAD value) affected by some of micronutrients

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1 Available online at Annals of Biological Research, 2011, 2 (2) : ( ISSN CODEN (USA): ABRNBW Soybean nodulation and chlorophyll concentration ( value) affected by some of micronutrients Soheil Kobraee*, Keyvan Shamsi and Siros Ekhtiari, Department of Agronomy and Plant Breeding, Islamic Azad University, Kermanshah Branch, Kermanshah, Iran _ ABSTRACT There were relationships between Chlorophyll concentration, nodulation and quality and quantity traits in soybean and micronutrients have effects on these relationships. In order to study some of these effects, the pot experiment was done at the research field of the Islamic Azad University of Kermanshah province, Iran in The experimental design was a factorial experiment based on Randomized Complete Block with three replicates. The experiment consist of 27 treatments included of three rates of Zinc (0, 4, and 8 mg Zn kg -1 as ZnSo 4.7H 2 o), three rates of iron (0, 4, and 8 mg Fe kg -1 as FeSo 4 ), and three rates of manganese (0, 15, and 30 mg Mn kg -1 as MnSo 4.4H 2 o). At 48 days after sowing and before harvesting, The -502 meter reading were performed on 5 leaves per experimental pot and then plants were harvested. Number of nodule per plant and fresh weight were determined, after drying, mass nodules and shoot dry weight were measured. The results shown that, there were significant differences in the number, fresh & dry weight of nodules per plant different levels of Zinc, Iron and manganese application. plant height was not affected by Zn and interaction effects of Zn and Fe. The maximum number of nodules per plant was obtained from 4, 8 and 30mgZn, Fe and Mn per kg soil, respectively. Maximum plant height, total dry weight and value were obtained in Zn 4 Fe 8 Mn 30 with 46.8(cm), 3.37(gr), and 31.9, respectively. Key word: chlorophyll, micronutrient, nodulation, soybean, value. INTRODUCTION Among environmental factors, nutrients availability within plants can be critical limitations on plant production. Micronutrients, known as trace elements that are required in small quantities by plants. Micronutrients deficiency such as Zinc, Iron and Manganese were limited growth [11, 12, 14], symbiosis and nodulation [1, 5, 14, 35, 38, 42], photosynthesis [1, 3, 22, 38], chlorophyll concentration [2, 16, 28, 31, 32, 40], dry matter accumulation [2, 5, 14, 16], and plant nutrient imbalanced [9, 10, 24]. Soybean is one of the non-conventional oilseed legume crops of the 414

2 world. Worldwide, it is estimated that leguminous plants are fixing 80 million tones of nitrogen annually from the vast free supply in the air. In contrast fertilizer manufactures produce at high cost only million tones of nitrogen annually [13]. [26] Stated that soybean plants fix more than 276 hg of nitrogen/ha in the aboveground plant parts. Nitrogen accumulation rates average between grams/ha/day during reproductive development. Research showed that inoculation of seed increased the plant height, photosynthetic rate, plant biomass, number, dry and fresh weight of nodules per plant and yield components of soybean as compared with noninoculated control [4, 7, 18, 21, 33]. Leaf total chlorophyll concentration, decreased with Zn, Mn and Fe deficiency [2]. Metals such as Zn, Fe and Mn are required in the biosynthetic pathway of chlorophyll. These metals are involved directly in electron transport reactions and are essential for the synthesis of of chlorophyll [2, 28, 31]. [16] Reported that chlorophyll ( 502) values were greater when Mn and Zn were added to the soil than the 0 ppm treatment. Research on the effects of Zn, Fe, Mn and interaction it's on soybean nodulation and chlorophyll ( 502) values is limited. [5] Reported that soybean nodulation unaffected by Zinc application that research was shown that the highest number of nodules obtained to 20kgZnha -1, while dry matter and shoot biomass were significantly affected by Zinc application. Zinc application increased nodules formation in soybean [42]. [1] Stated that in nodules of legumes, Fe is an essential for leghaemoglobin biosynthesis that transported oxygen within cells. Results of [20] was shown that Fe and Mn concentration up to 50 µm in solution were not affected on root, shoot and nodule mass. In that experiments, chlorophyll concentration in soybean leaves with added of Mn was increased. Based on [41] studies, using from 502 for the estimation of chlorophyll content in leaves was more useful method as compared to the extraction method. Research on the effects of Zn, Fe, Mn and interaction effects on soybean nodulation and chlorophyll concentration are limited. Therefore, the objective of this study was to evaluation the effects of Zn, Fe and Mn applied on the number and weight of nodules in roots, shoot biomass and chlorophyll concentration ( value) in soybean in climatic conditions of Kermanshah, Iran. MATERIALS AND METHODS The experiment was done at the research field of the Islamic Azad University of Kermanshah province, Iran ( ' N, ' E; 1351 m elevation) in The experimental design was a factorial experiment based on Randomized Complete Block with three replicates. Surface soil was collected from an agricultural field and passed through a 2-mm mesh screen. The texture of the soil based on silty clay with ph 7.6, total organic matter 1.8%, electrical conductivity (EC e ) 0.46 dsm -1, total nitrogen 0.09%, available phosphorus 7.4 mg kg -1, available potassium 435 mg kg -1, zinc, iron and manganese 0.56, 5.1 and 3.2 mg kg -1, respectively. The experiment consist of 27 treatments included of three rates of Zinc (0, 4, and 8 mg Zn kg -1 as ZnSo 4.7H 2 o), three rates of iron (0, 4, and 8 mg Fe kg -1 as FeSo 4 ), and three rates of manganese (0, 15, and 30 mg Mn kg -1 as MnSo 4.4H 2 o). All pots were fertilized with 20 mg N kg -1 as NH 4 NO 3, 40 mg P kg -1 as H 2 PO 4.2H 2 O. Six seeds of soybean (cv. Williams) inoculated with BradyRhizobium japonicum and were sown directly in plastic pots containing 4 kg of the soil. After 48 days, plants were harvested and samples were washed in deionized water. For measure of dry weight, samples were dried oven at 70 0C and 48 hours. Nodules are removed from the roots and number of nodule per plant and fresh weight were determined, after drying, mass nodules were measured. At 48 days after sowing and before harvesting, The -502 meter reading were performed on 5 leaves (from the uppermost leaf of youngest fully developed trifoliate leaf) per experimental pot for all replicates. All of data obtained from the measurements and analyses were evaluated statistically by the analysis of variance and F-test means comparison conducted by least significant difference (LSD) test. Correlation coefficient significance was evaluated by student s t-test. 415

3 RESULTS AND DISCUSSION The results of analysis of variance were shown in Table1. There were significant differences in the number, fresh & dry weight of nodules per plant different levels of Zinc, Iron and manganese application. In addition, effects of Fe and Mn on plant height was significant, likewise, plant height was not affected by Zn and interaction effects of Zn and Fe (Table1). [40] Stated that plant height of soybean increased when that Fe was applied. The simple effect of these elements was shown in Figure1. The maximum number of nodules per plant was obtained from 4, 8 and 30mgZn, Fe and Mn per kg soil, respectively. The results was shown that with application of zinc up to 4 mgkg -1 soil fresh and dry weight of nodules per plant were increased and then decreased (Fig1). [5, 42] Stated that zinc application increased nodules formation and shoot biomass in soybean. Shoot dry weight was not affected by zinc application but with application of Zn up to 8mgkg -1 soil shoot dry weight was decreased (Table1). Research of [17] indicated that Mn and Zn application were increased biomass accumulation, but were not affected number of nodules and nodule mass per plant in soybean. In nodules of legumes, iron is an essential for leghemoglobin biosynthesis, that transported oxygen within cells. Nodule formation in soybean is very sensitive process to Fe deficiency [19]. value affected by zinc, iron and manganese application and there was significant differences between different levels of these micronutrients application (P<0.01). Zn, Fe, and Mn increased chlorophyll concentration, as measured with a -502 meter. In the zinc study, chlorophyll content at 4mgZnkg -1 soil increased, but with excess decreased. [16] Found that chlorophyll values (- 502) were greater when Mn and Zn were added to the soil. With application of iron up to 8 mg per kg soil, values were increased. The highest values belonged to 30mgkg -1 soil 1 Mn (Fig1). The interaction effects of these elements were shown in Table1. Except of plant height and shoot dry weight, interaction effects of these elements on fresh & dry weight of nodules per plant (P<0.01) and value (P<0.05) were significant. The highest number, fresh and dry weight of nodules per plant were found in Zn 0 Fe 4 Mn 30 mgkg -1 soil with 27.3, 1.70(gr), and 0.41(gr), respectively. Maximum plant height, total dry weight and value were obtained in Zn 4 Fe 8 Mn 30 with 46.8(cm), 3.37(gr), and 31.9, respectively. [2] Reported that in zinc, iron and manganese deficiency conditions, Leaf chlorophyll concentration was decreased. Metal such as Zn, Fe and Mn are required in the biosynthetic pathway and essential for the synthesis of chlorophyll [28, 31, 40]. The lowest value was obtained at Zn 0 Fe 0 Mn 0 treatment. Based on Table 3, there were highly significant positive correlation between number of nodules per plant and shoot dry weight (r=0.525 ** ) and value (r=0.661 ** ). There is a significant positive correlation between shoot dry weight and value (r=0.731 ** ). 416

4 Soheil Kobraee et al Annals of Biological Research, 2011, 2 (2): Number of nodule per plant Number of nodule per plant Manganese fertilizer(mgkg -1 soil) Fersh weight of nodule Dry weight of nodule Dry weight of nodule Fersh weight of nodule Number of nodule per plant Fresh weight of nodule Dry weight of nodule Height of plant (cm 417

5 Soheil Kobraee et al Annals of Biological Research, 2011, 2 (2): Height of plant (cm Shoot dry weight Shoot dry weight Reading Shoot dry weight Height of plant (cm Reading Reading Figure1. Effect of Zinc, Iron and manganese on number of nodule per plant, fresh and dry weight of nodule per plant, plant height, shoot dry weight and reading in soybean. 418

6 Source of variation Block Zn Fe Zn Fe Mn Zn Mn Fe Mn Zn Fe Mn Error Coefficient of variation (%) Table1. Analysis of variance of soybean evaluated traits d.f NNP ** ** ** ** ** ** ** FWN ** 0.81** 0.16** 0.39** 0.07 ** 0.11 ** 0.09 ** MS DWN ** 0.037** 0.017** 0.011** 0.007* 0.014** 0.005** H ns ** ns ** 9.56 ns ns ns SDW 0.16 ns ** 0.32* 3.49** 0.21 ns 0.94** 0.18 ns ** ** 18.36** 87.45** 2.07 ns 1.55 ns 4.86* ns, * and ** : Non significant, significant at 5 and 1% levels of probability, respectively -NNP: Number of nodule per plant; FWN: Fresh weight of nodule per plant; DWN: Dry weight of nodule per plant; H: Plant height; SDW: Shoot dry weight, and : value. Table2: Mean comparison Number of nodule per plant; Fresh weight of nodule per plant; Dry weight of nodule per plant; Plant height; Shoot dry weight, and value in soybean according to LSD test in %5 level Rate of fertilizer Means (kg/ha) Zn Fe Mn NNP FWN DWN H SDW j 0.72 g 0.16 hij 32.6 hi 1.45 i 20.3 m j 0.70 g 0.13 ij 32.8 hi 1.60 ghi 21.7 lm ij 0.75 g 0.11 j 31.7 i 1.52 hi 22.5 klm gh 1.07cdef 0.21 defgh 33.5 fghi 1.49 hi 21.0 lm hi 1.01def 0.22cdefgh 38.5bcdefgh 1.72 fghi 23.2 jkl a 1.74 a 0.41 a 40.1 bcde 2.21 de 27.2 def de 1.08cdef 0.24bcdefg 37.6cdefghi 2.12 def 25.4 fghij ab 1.37 b 0.28 bcd 39.4 bcdefg 2.71 bc 27.8 cde efg 1.25 bc 0.29 bc 39.6 bcdef 2.26 cd 28.4 bcd fgh 1.18bcde 0.29 bc 33.0 ghi 1.75 efghi 21.7 lm 4 0\ de 1.03def 0.28 bcd 33.5 fghi 1.87defghi 24.3 ijk abc 1.11cdef 0.23cdefgh 33.7 efghi 1.52 hi 24.1 ijk fgh 0.98ef 0.19 fghi 33.0 ghi 1.68 fghi 25.6 efghi de 1.09cdef 0.27 bcde 42.4 abc 3.07 ab 29.7 abc ab 1.35 b 0.29 bc 40.5 abcd 2.97 ab 30.2 ab cde 1.12cdef 0.27 bcde 38.5bcdefgh 2.28 cd 27.0 defg ab 1.19bcd 0.25bcdefg 44.3 ab 3.15 ab 30.1 abc ab 1.72 a 0.31 b 46.8 a 3.37 a 31.9 a h 0.98ef 0.19 fghi 35.2 defghi 2.03 defg 21.7 lm hi 0.68 g 0.13 ij 35.1 defghi 2.09 defg 24.4 hijk de 1.02def 0.19 fghi 36.7cdefghi 1.97 defgh 26.7 defgh bcd 1.10cdef 0.22cdefgh 35.2 defghi 1.71 fghi 24.8 ghijk def 0.96 f 0.19 fghi 39.6 bcdef 2.99 ab 25.1 fghij ab 1.17bcde 0.26bcdef 42.8 abc 3.10 ab 26.1defghi efg 1.18bcde 0.29 bc 38.8bcdefgh 2.01 defg 24.3 ijk def 1.17bcde 0.18 ghij 40.4 abcd 3.03 ab 27.8 cde de 1.09cdef 0.20 efghi 41.6 abcd 3.08 ab 26.2defghi -Similar letters in each column shows non-significant difference according to LSD test in %5 level. -NNP: Number of nodule per plant; FWN: Fresh weight of nodule per plant ; DWN: Dry weight of nodule per plant ; H: Plant height (cm); SDW: Shoot dry weight ; and : value. 419

7 Table3. Pearson correlation coefficients among evaluated traits in soybean NNP NNP FWN DWN H SDW FWN ** DWN ** ** H * * * TDW ** * ns ** ** ** * ** ** -ns, * and **: Non significant, significant at 5 and 1% levels of probability, respectively -NNP: Number of nodule per plant; FWN: Fresh weight of nodule per plant ; DWN: Dry weight of nodule per plant ; H: Plant height (cm); SDW: Shoot dry weight ; and : value. Acknowledgments The authors wish to thank from The Islamic Azad University for supporting projects. This research was supported by Islamic Azad University, Kermanshah Branch, Kermanshah, Iran. CONCLUSION The legume-rhizobia symbiosis is particularly sensitive to nutrients deficiency. Rhizobium japonicum is one of important species of genus Rhizobium which infect the root of soybean plant. Research showed that inoculated of seed soybean with this bacteria increased the plant height, photosynthetic rate, plant biomass and dry weight of nodules [4, 7, 18], Whereas, other researcher such as [29, 37] Reported that seed inoculation had no effect on nodule formation, nodule number and plant biomass. The main product of symbiosis nitrogen fixation in soybean root nodules is ureides [36]. Soybean transports of this ureides to the plant shoot, manganese is a cofactor required in the catabolism of the ureides in the leaves [34]. In nodules of soybean, Iron is an essential for leghaemoglobin biosynthesis that transported oxygen within cells [1]. Zinc is an important role as a functional, structural and regulatory cofactor of a large number of enzymes [23]. Also leaf chlorophyll content in plants were adversely affected by Zinc deficiency. In this experiment was used from the 502 for the measurement of the chlorophyll content in soybean leaves that were used as a diagnostic tool for the evaluation of plant nutrients, such as iron, manganese, nitrogen, magnesium and other micronutrients related to chloroplast pigment contents. Many researches were conducted on the usefulness of 502 as a nondestructive analysis method for determination of chloroplast pigments [6, 8, 15, 25, 27, 30, 39]. In this experiment, using Zinc, Iron and Manganese increased the evaluated traits the highest value of which obtained by application 4, 8 and 30 mg per kg soil Zinc, Iron and Manganese. REFERENCES [1]-Abdelmajid, K., and Chedly, A. Journal of Plant Nutrition and Soil Science : [2]-Adams, M. L., Norvell, W. A., Philpot, W. D., and Peverly, J. H.. Agron J : [3]-Ahlbrink, R., Semin B. K., Mulkidjanian. A. Y. & Junge, W. Biochimica et Biophysica Acta :

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