Effects of Copper Slag and Organic Matters on Physiological Indices of Iron Deficiency in Sorghum (Sorghum bicolor)

Document Type : Research Article


1 Ph.D. Graduate Faculty of Water and Soil Engineering, Gorgan University of Agricultural Sciences and Natural Resources

2 Associate Professors and Assistant Professor, Faculty of Water and Soil Engineering, Gorgan University of Agricultural Sciences and Natural Resources

3 Associate Professors Faculty of Water and Soil Engineering, Gorgan University of Agricultural Sciences and Natural Resources

4 Assistant Professor, Faculty of Water and Soil Engineering, Gorgan University of Agricultural Sciences and Natural Resources

5 Assistant Professor, Department of Civil Engineering, Sirjan University of Technology, Sirjan


Introduction: Iron deficiency is one of the most common nutritional problems of plants in arid and semi-arid soils especially in calcareous soils. Iron is essential to many cellular activities, required for optimum growth and development, however it is insoluble in aerated soils at neutral or basic pH, therefore, iron deficiency is common in these soils. The problem is usually solved by using iron synthetic chelates which is a very expensive option. There is, therefore, a need for cheaper and more effective alternatives to traditional Fe fertilizers. Several reports have shown that application of Fe factory by-product to soil tends to raise the availability of Fe and reduce Fe deficiency in plants. Application of organic compounds to soil may improve the solubility of the minerals containing micronutrients and correct their deficiencies in alkaline and calcareous soils. A large amount of slag is produced annually at the Sarcheshmeh Copper Complex, Kerman Province, Iran. So far, the copper slag, however, has not been tested as an Fe fertilizer in calcareous soils. Since about 53.8% of slag obtained from copper concentrate melting is composed of iron oxides, we, thus, examined the effect of copper slag along with organic compounds on the level of upper leaf iron, photosynthetic pigments, SPAD index, the activity of plant enzymes and the level of active iron in the upper leaves of sorghum by performing a factorial experiment in a completely randomized design.
Materials and Methods: In order to study the effect of copper slag (one of by-products of melting copper concentrate in Sarcheshmeh Copper Complex, Kerman Province) and organic compounds (cow manure and pistachio skin) on total leaf iron content, photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll), antioxidant enzymes activity (Guiacol Peroxidase, Glutathione peroxidase) and concentration of active iron of young leaves developed of sorghum, a pot experiment was conducted in the greenhouse with three replicates per treatment. We applied experimental treatments including 5 levels of organic matter (pistachio skin, cow manure at 2 and 4 wt. % and control sample), and 11 levels of iron (copper slag, copper slag with sulfur, copper slag with sulfur and thiobacillus, acidic slag (each 2 levels each), sequesterine, foliar application of EDTA, and control sample) to a soil sample with low iron content. At the end of the incubation period, sorghum bicolor was cultured in the above treatments. Ten seeds were sown in each pot. Seedlings were thinned to 4 when they were about 10 cm high. During the growth period, pots were irrigated with distilled water as needed. Before harvesting, SPAD, the concentration of photosynthetic pigments, level of active iron and activity of plant enzymes were also measured in fresh plant samples. Furthermore, the concentration of Fe in the leaves was measured. Analysis of variance was performed using software SAS and significant differences were determined based on LSD (Least Significant Difference Test) at p < 0.05 level.
Results and Discussion: The interaction between slag treatments and organic compounds showed that treatments of 4 wt. % of cow manure with slag of 4 times of recommended soil test value (C4S4,  and 4 wt. % of cow manure with slag of 4 times of recommended soil test value with sulfur and thiobacillus (C4S4S°T), had significant effects on increasing photosynthetic pigments pigments (chlorophyll a, chlorophyll b, total chlorophyll), SPAD index, activity of plant enzymes (Guiacol peroxidase, Glutathione peroxidase) and active iron. The highest active iron level in the young leaves developed (54.06 mg / kg) was observed in (C4S4S°T) treatment which showed a significant increase compared to the control treatment (17.14). Increased concentration of active iron was also observed due to application of treatments (slag, organic compounds and the interaction between treatments) in sorghum young leaves. The photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll), SPAD index and activity of plant enzymes (Guiacol peroxidase, Glutathione peroxidase) were more correlated with active iron in young leaves developed compared to total iron concentration in these leaves. This indicates that active iron can be used as an index to detect iron deficiency.
Conclusion: The higher level of active iron in the young leaves developed was more associated with physiological indices of sorghum as compared with total Fe concentration in these leaves. Therefore, this parameter can be used as an index to detect iron deficiency. In this study, increasing the level of slag consumed and consequently increasing Fe concentration in the leaf resulted in a significant increase in chlorophyll a, chlorophyll b, total chlorophyll, carotenoids and activation of the plant enzymes.


Main Subjects

1- Aebi H. 1984. Catalase in vitro. Methods in Enzymology 105: 121–126.
2- Allison L.E., and Modie C.D. 1965. Carbonate. p. 1379-1396. In C.A. Black (ed.), Method of Soil Analysis Part 2. Soil Science Society of America and American Society of Agronomy, Madison, WI, USA.
3- Amal A.M., and Aly A.A. 2008. Alteration of some secondary metabolites and enzymes activity by using exogenous antioxidant compound in onion plants growth under seawater salt stress. American Journal of Scientific Research 3: 139-146.
4- Banuls J.A., Quinones B., Martin E., Primo-Millo E., and Legaz  F. 2003. Effects of frequency of iron chelate supply by fertigation on chlorosis in citrus. Journal of Plant Nutrition 26: 1985-1996.
5- Benton J., Jones J.R., and Case V.W. 1990. Sampling, handling and analyzing plant tissue sample. p. 389-429. In: R.L. Westerman (ed.), Soil Testing and Plant Analysis. Soil Science Society of America and American Society of Agronomy, Madison, WI, USA.
6- Bremner J.M. 1996. Nitrogen-total. p. 1-89. In: D.L. Sparks, Methods of Soil Analysis, Part 3. Soil Science Society of America and American Society of Agronomy, Madison, WI, USA.
7- Chance B., and Maehly A.C. 1955. Assay of catalases and peroxidases. Methods in Enzymology 11: 764-755.
8- Chen Y., and Avnimelech Y. 1986. The role of organic mater in modern agriculture. Martinus Nijhoff Publishers, Dordrecht Netherland.
9- Chocano C., Garcia C., Gonzalez D., de Aguilar J.M., and Hernandez T. 2016. Organic plum cultivation in the Mediterranean region: The medium-term effect of five different organic soil management practices on crop production and microbiological soil quality. Agriculture, Ecosystems & Environment 221: 60-70.
10- Daived F., and Ksander G. 1989. Influence of external iron concentration on active iron for four species of aquatic macrophytes. Journal of Aquatic Plant Management 27: 65-69.
11- De La Guardia M.D., and Alcantara E. 2002. A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince, pear and olive genotypes under iron deficiency stress. Plant Soil 241: 49-56.
12- Ediga A., Ramgopal M., Narasimha G., and Balaji M. 2010. Effect of Pig Iron Slag Particles on Soil Microbial and Enzyme Activities. European Journal of Applied Sciences 2(3): 122-126.
13- Fabrizio P., Iori V., Beone T., Mirabile D., and Zacchini M. 2017. Effects of a ladle furnace slag added to soil on morpho-physiological and biochemical parameters of Amaranthus paniculatus L. plants. Journal of Hazardous Materials 329: 339-347.
14- Gee G.W., and Bauder J.W. 1986. Particle-size analysis. p. 383-411. In A. Klute (ed.), Methods of Soil Analysis Part 1: Physical and Mineralogical Methods. Soil Science Society of America and American Society of Agronomy, Madison, WI, USA.
15- Havlin J., Beaton J.D., Tisdale S.L., and Nelson W.L. 2005. Soil fertility and fertilizers, 7th edition. Pearson Prentice Hall.
16- Hesse P.R. 1971. A text book of soil chemical analysis. John Murray. London
17- Hirai M.K. Higuchi K., Sasaki H., Suzuki T., Maruyama T., and Tadano T. 2007. Contribution of iron associated with high molecular weight substances to the maintenance of the SPAD value of young leaves of barley under iron deficient conditions. Journal of Soil Science and Plant Nutrition 53: 612-620.
18- Hopkins J., and Tudhope G.R. 1973. Glutathione peroxidase in human red cells in health and disease. British Journal of Haematology 25: 563–575.
19- Iglesias I.R., Dalmau X., Marce M.C., Delcampillo V., and Torrent J. 2003. Fertilization with iron(II)-phosphate effectively prevents iron clorosis in pear trees pyrus commonis. Acta Horticulturae 511:65-72.
20- Kaplan M., and Orman S. 1998. Effect of elemental sulfur and sulfur containing waste in a calcareous soil in Turkey. Journal of Plant Nutrition 21:1655-1665.
21- Karla Y.P. 1998. Hanbook of Reference Methods for Plant Analysis. CRC Press, Washington D.C. USA.
22- Katyal J.C., and Sharma B.D. 1984. Some modification in the assay of Fe2+ in 1-10, o-phenanthroline extracts of fresh plant tissues. Plant Soil 79: 449-450.
23- Knudsen D., Peterson G.A., and Pratt P.F. 1982. Lithium, Sodium and Potasium. p. 225-246. In: A.L. Page et al. (ed.). Methods of soil analysis (part II), Chemical and microbiological properties, American Society of Agronomy, Madison, WI, USA.
24- Lindsay W.L., and Norvell W.A. 1978. Development of a DTPA soil test for zinc, iron, manganese and copper. Soil Science Society of America Journal 42: 421-428.
25- Lombardi L., Sebastiani L., and Vitagliano C. 2003. Physiological, Biochemical and Molecular Effects of in vitro induced iron deficiency in peach rootstock. Journal of Plant Nutrition 26: 2149-216.
26- Majidi A. 2013. Assessing the nutritional status of apple orchards in western Azerbaijan, and guidelines to improve the quantity and quality of product. The First Scientific Conference on Agricultural and Rural Development with a Focus on National Production, Piranshahr, Iran. (In Persian)
27- Marshner H. 1995. Mineral nutrition of higher plants. Academic press. New York.
28- McKersie B.D., Murnaghan J., Jones K.S., and Bowley S.R. 2000. Iron superoxidase dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance. Plant Physiology 122: 1427-1437.
29- Miller R.H., and Keeney D.R. 1982. Methods of Soil Analysis Part 2: Chemical and Microbiological prpperties.  Soil Science Society of America and American Society of Agronomy, Madison, WI, USA.
30- Minami M., and Yoshikawa H. 1979. A simplified assay method of superoxide dismutase activity for clinical use. Clinica Chimica Acta 92: 337–342.
31- Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7: 405-410
32- Mohammad M.J., Najim H., and Khresat S. 1998. Nitric acid-and O-Phenanthrolineextractable iron for diagnosis of iron chlorosis in citrus lemon trees. Communications in Soil Science and Plant Analysis 29: 1035-1043.
33- Mokhtari S., Hodaji M., and Kalbasi M. 2014. The effect of steel converter slag application along with sewage sludge in iron nutrition and corn plant yield. Journal of Life Sciences Biotechnology and Pharma Research 3: 96-104.
34- Nelson D.W., and Sommers L.P. 1986. Total carbon, organic carbon and organic matter. p. 539-579. In A.L. Page etal. (ed.), Methods of Soil Analysis Part 2. Soil Science Society of America and American Society of Agronomy, Madison, WI, USA.
35- Nogami R., Tam L.T., Anh H.T.L., and Quynh H.T.H. 2016. Growth promotion effect of steelmaking slag on Spirulina platensis. Journal of Physics. Conference Series 704: 12-19.
36- Olsen R.A., Brown J.C., Bennett J. H., and Blutne D. 1982. Reduction of Fe3+ as it relates to Fe chlorosis. Journal of Plant Nutrition 5: 433-445.
37- Olsen S.R., Close V., Watnebe F.S., and Dean L.A. 1954. Estimation of available phosphorous in soil by extraction with sodium bicarbonate. United States Department of Agriculture 939: 1-19.
38- Orman S., and Kaplan M. 2007. Effects of elemental sulphur and organic manure on sulphur, zinc, and total chlorophyll contents of tomato in a calcareous sandy loam soil. Soil Science Society of America Journal 55: 85-90.
39- Pestana M., Varennes A., and Faria E.A. 2003. Diagnosis and correction of iron chlorosis in fruit trees. Food, Agriculture and Environment 1: 46-51.
40- Peyvandi M., Kamali Jamakani Z., and Mirza M. 2011. Comparison of nano Fe chelat with Fe chelate effect on growth parameters and antioxidant enzymes activity of Satureja hortensis. New Cellular and Molecular Biotechnology Journal 2(5): 25-32.
41- Pierson E., and Clark R.B. 1984a. Chelanting agent differences in ferrous iron determination. Journal of Plant Nutrition 7(1-5): 91-106 .
42- Pierson E., and Clark R.B. 1984b. Ferrous Iron determination in plant tissue. Journal of Plant Nutrition 7(1-5): 107-116.
43- Porra R.J., Thompson W.A., and Kriedemann P.E. 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biophysica Acta 975: 384–94.
44- Rahmani A., Mirza M., and Tabaei Aghdai S.R. 2013. Effects of different fertilizers (macro and micro element) on quantity and quality of essential oil and other byproducts of Rosa damascena Mill. Iranian Journal of Medical Sciences 29(4): 747-759.
45- Rahimizadeh M., Habibi D., Madani H., Mohammadi G.N., Mehraban A., and Sabet A.M. 2007. The effect of micronutrients on antioxidant enzymes metabolism in sunflower (Helianthus annus L.) under drought stress. Helia 30: 167-174.
46- Ranieri A., Castagna A., Baldan B., and Soldatini G.F. 2001. Iron deficiency differently affects peroxidase isoforms in sunflower. Journal of Experimental Botany 52: 25-35.
47- Rao A.C.S. 1993. Diagnosis of nutrient deficiencies of citrus orange orchards in Jiroft Valley of Iran. Agrochimica 37(l-2): 41-54.
48- Razmjoo S. 1997. Manual of analysis of fruit and vegetable products. 9th. ed. Tata MC Grow Hill, New Delhi.
49- Reddappa Reddy M. 2006. Effect of calcium, sulphur and boron on the yield andcomposition of corn (Zea mays L.) under water deficit stress. Plant Growth Regulation 54: 205–209.
50- Rombola A.D., Bruggemann A.F., LopezMillan M., Tagliavini J., Abadia B., Marangoni B., and Moog. P.R. 2002. Bioshimical responses to iron deficiency in kiwifruit (Actinidia deliciosa). Tree Physiology 22: 869-875.
51- Roosta H.R., and Karimi H.R. 2012. Effects of alkali-stress on ungrafted and grafted cucumber plants: using two types of local squash as rootstock. Journal of Plant Nutrition 35(12): 1843-1852.
52- Rosado R., del Campillo M.C., Martinez M.A., Barron V., and Torrent J. 2002. Longterm effectiveness of vivianite in reduce iron chlorosis in olive trees. Plant Soil 241: 139-144.
53- Samar S.M., and Shahabian M. 2003. Effect of organic manure enrichment with sulfur and sulfate on increasing availibility of iron in a calcareous soil. National Seminar of production and application of sulfur in the country. Mashhad, Iran. (In Persian)
54- Sandra R., Crnojevic H., and Sandev D. 2013. Effect of electric arc furnace slag on growth and physiology of maize (Zea mays L.).  Acta Biologica Hungarica 64(4): 490–499.
55- Schenkeveld W.D.C., Dijcker R., Reichwein A.M., Temminghoff E.J.M. and Riemsdijk W.H. 2008. The effectiveness of soil- applied FeEDDHA treatments in preventing iron chlorosis in soybean as a function of the o, o-FeEDDHA content. Plant and Soil 303: 161-176
56- Shah B., Sulaimana S., Jamal P., and Alam M.S. 2014. Production of heteregenous catalysts for biodiesel synthesis. Environmental Chemical Engineering 5(2): 73-75.
57- Shariatmadari H., Rezainejad Y., Abdi A., Mahmoudabadi A., and Karam M. 2009. Effect of Converter Sludge and Slag of Isfahan Iron Melting Factory on Corn Yield and Some Heavy Metal Uptake in a Calcareous Soil. Journal of Water and Soil Science 12(46): 667-680. (In Persian with English abstract)
58- Sharma P.K., and Hall D.O. 1991. Interaction of salt stress and photoinhibition on photosynthesis in barley and sorghum. Journal of Plant Physiology 138(5): 614-619.
59- Sikka R., Kansal B.D. 1994. Effect of fly-ash application on yield and nutrient composition of rice, wheat and on pH and available nutrition status of soils. Bioresource Technology 51: 199-203.
60- Sposito G., Lund L.J., and Chang A.C. 1982. Trace metal chemistry in arid zone field soils amended with sewage sludge, I.: Fractionation of Ni, Cu, Zn, Cd, and Pb in solid phases. Soil Science Society of America Journal 46: 260-264.
61- Sudahono D., Byrne H., and Rouse R.E. 1994. Greenhouse screening of citrus rootstocks for tolerance to bicarbonate induced iron chlorosis. Horticultural Science 29: 113-116.
62- Sun B., Jing Y., Chen K., Song L., Chen F., and Zhang L. 2007. Protective effect of nitric oxide on iron deficiency-induced oxidative stress in maize (Zea mays). Journal of Plant Physiology 164: 536-543.
63- Terry N. 1980. Limiting factors in photosynthesis. I. Use of iron stress to control photochemical capacity in vivo. Plant Physiology 65: 114-120.
64- Therios I., Chouliaras V., Bizas D., Boukouvalas S., Palioura E., Hatzidimitriou E., Basioukas D., Gioldasis V. and Stavrou N. 2005. Changes in leaf biochemical and physiological indices due to iron deficiency in citrus. AgroThesis 1: 18-25.
65- Tsipouridis C., Almaliotis D., Thomidis T., and Isaakidis A. 2006. Effects of different sources of iron, hormones and Agrobacterium tumefaciencs on chlorophyll and iron concentration in the leaves of peach trees. Horticultural Science 33(4): 140-147.
66- Wiersma J.V. 2005. High rates of Fe-EDDHA and seed iron concentration suggest partial solutions to iron deficiency in soybean. Agronomy Journal 97: 924-934.
67- Wonge J.W.C., Lik L., Zhoul X., and Selvam A. 2007. The sorption of Cd and Zn by different soils in the presence of dissolved organic matter from sludge. Geoderma 137: 310-317.
68- Youfa L., Yonggui W., Rongrong X., and Cong Y. 2018. Effects of plant litter decomposition on chemical and microbiological characteristics of artisanal zinc smelting slag using indigenous methods. Journal of Geochemical Exploration 292-301.
Volume 35, Issue 6 - Serial Number 80
January and February 2022
Pages 841-823
  • Receive Date: 26 November 2019
  • Revise Date: 18 November 2021
  • Accept Date: 29 November 2021
  • First Publish Date: 29 November 2021