Partial replacement of Fe(o,o-EDDHA) by humic substances for Fe nutrition and fruit quality of citrus Mar Cerdán 1 , Antonio Sánchez-Sánchez 1 *, Margarita Juárez 1 , Juan J. Sánchez-Andreu 1 , Juana D. Jordá 1 , and Dolores Bermúdez 1 1 Agrochemistry and Biochemistry Department, Faculty of Sciences, University of Alicante, 03080 San Vicente del Raspeig, Alicante, Spain Accepted May 23, 2007 Summary The most widely used Iron (Fe) fertilizer in calcareous soils is the synthetic chelate Fe(o,o-EDDHA). However, humic sub- stances are occasionally combined with Fe chelates in drip irrigation systems in order to lower costs. We investigated the effect of various mixtures of Fe(o,o-EDDHA) and a commer- cially available humic substance on Fe availability in a calcar- eous soil from Murcia, Spain (in vitro experiment) and on leaf Fe content and fruit-quality attributes of Citrus macrophylla (field experiment). In the in vitro experiment, a calcareous soil was incubated for 15 d with solutions of sole Fe(o,o- EDDHA) and humic substance and of a mixture of humic substance and Fe(o,o-EDDHA) to determine the dynamics of available Fe. While the mixture did not significantly increase the available soil Fe, it did decrease the rate of Fe retention in the surface soil compared to sole Fe(o,o-EDDHA). In the field experiment, the substitution in the application solu- tion of 67% of Fe(o,o-EDDHA) by commercial humic sub- stance increased leaf P in lemon trees from 0.19% with sole Fe(o,o-EDDHA) to 0.30% and leaf Fe from 94 mg kg –1 to 115 mg kg –1 . Some quality parameters like vitamin C content and peel thickness were also improved with a partial substitu- tion of Fe(o,o-EDDHA) by humic substances. We conclude that a partial substitution of commercial Fe chelates by humic substance can improve crop Fe uptake and may thus be eco- nomically attractive. The underlying physiological mechan- isms and ecological implications require further studies. Key words: calcareous soil / Citrus macrophylla / Fe(o,o-EDDHA) / iron deficiency  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1436-8730/07/0408-474 474 DOI: 10.1002/jpln.200520594 J. Plant Nutr. Soil Sci. 2007, 170, 474–478 1 Introduction Iron chlorosis in citrus is frequently associated with cultivation on calcareous soils. In these soils, the Fe-mobilizing activity of citrus, being a strategy I plant, is neutralized by large amounts of bicarbonate, usually requiring an external appli- cation of Fe (Marschner , 1995). Fe(o,o-EDDHA) is one of the most effective Fe fertilizers in calcareous soils. However, commercial Fe(o,o-EDDHA) che- lates are costly and show different degrees of effectiveness under field conditions, since they contain large amounts of soluble Fe bonded as (o,p-EDDHA) (Gómez-Gallego et al., 2002), which is less effective than (o,o-EDDHA) and can result in a 50% decline in synthetic-chelate efficacy (Goos and Tarrida, 2006). On the other hand, humic substances are relatively cheap and able to complex many metals, including Fe, in addition to being sources of N, P, or S (Varanini and Pinton, 2001). The application of humic substances can increase the concentra- tion of Fe, Mn, Cu, and Zn in calcareous soil solution (Olmos et al., 1998). Moreover, they improve the growth of roots and root hairs (Pinton et al., 1999), increasing the root surface and promoting the uptake of K, P, or Fe (Cesco et al., 2002). Humic substances can also stimulate the reduction of Fe(III) to Fe(II) in a reaction mediated by microorganisms (Scott et al., 1998), and there is evidence of a positive effect of humic substances on the activity of plasma membrane H + ATPase (Varanini and Pinton, 2001). Consequently, a combined application of Fe chelates with humic substances in citrus fertigation systems appears to be a promising and economically attractive approach to counter- act Fe deficiency and enhance the efficiency of synthetic che- lates. A preliminary study (Sánchez-Sánchez et al., 2002) showed that the application of a 1:1 (w/w) mixture of commer- cially available Fe(o,o-EDDHA) and humic substances can improve Fe uptake by lemon trees in calcareous soils. How- ever, the causes of this improvement are currently not well understood. In this paper, we studied the reaction of various mixtures of Fe(o,o-EDDHA) and a commercially available humic substance on the Fe availability in a calcareous soil, crop Fe uptake, and fruit-quality attributes of citrus. 2 Materials and methods 2.1 Fertilizer substrates and soil characteristics The commercial Fe(o,o-EDDHA) fertilizer used in this experi- ment was analyzed for its total Fe, Cu, Mn, and Zn contents by atomic-absorption spectrometry (AAS) according to the method described by Commission Directive (1993). The Fe(o,o-EDDHA) quantification in the fertilizer was done fol- lowing the ion-pair chromatography method described by Lucena et al. (1996). The product contained 5.6% soluble Fe of which 3.2% were chelated by (o,o-EDDHA), 0.02% soluble Cu, 0.08% soluble Mn, and 0.04% soluble Zn. The difference * Correspondence: A. Sánchez-Sánchez; e-mail: antonio.sanchez@ua.es