**2. Materials and methods**

#### **2.1 Soil**

The calcareous soil used for the pot experiments was collected at a site located in Santomera (Murcia, Spain), from the top soil layer (0 – 20 cm). Plants grown on Santomera soil became chlorotic under field conditions. Pre-treatment of the soil consisted of air drying and sieving (1 cm). Santomera soil is a clay soil with a lutum fraction of 260 g kg-1 and a CaCO3 content of 520 g kg-1. The soil has a high pH: 8.0 (pH-CaCl2) and a low soil organic carbon (SOC) content: 5 g kg-1. The dissolved organic carbon (DOC) concentration amounts 30 mg l-1 (0.01 M CaCl2), and Fe availability parameters are low: oxalate extractable Fe content: 0.30 g kg-1 Fe, and diethylene triamine penta acetic acid (DTPA) extractable Fe content: 3.5 mg kg-1 Fe. A more complete overview of soil characteristics of Santomera soil is presented in Schenkeveld et al., 2010a.

#### **2.2 FeEDDHA solutions**

Depending on the pot trial experiment, FeEDDHA solutions were prepared from EDDHA stock solutions varying in EDDHA component composition, from a solid o,o-H4EDDHA mixture (purity: 99%), or from separated solid racemic o,o-EDDHA (purity: 100%), meso o,o-EDDHA (purity: 99.5%) and o,p-EDDHA (purity: 90%). Racemic and meso o,o-H4EDDHA were obtained by separation of the o,o-H4EDDHA mixture, as described in Bannochie and Martell (1989) and Bailey et al. (1981). Solid H4EDDHA was first dissolved in sufficient 1 M NaOH solution. An FeCl3 solution was added to the EDDHA solution in a 2- 5% excess, based on a 1:1 stoichiometry between Fe and ethylene diamine (incorporated in the EDDHA ligands). pH was raised to 7 ± 1, and the solution was stored overnight in the dark to allow excess Fe to precipitate as Fe(hydr)oxides. Subsequently the FeEDDHA solutions were filtered over a 0.45 μm nitro cellulose filter (Schleicher & Schuell, refno: 10401114) and further diluted for application in the pot trial. The composition of FeEDDHA solutions was examined at t=0 and at the end of the experiment by ICP and HPLC analysis.
