**2.3 Pot trial studies**

90 Soybean Physiology and Biochemistry

2007; Rojas, et al., 2008; and mobilization of Fe from Fe oxides by EDDHA ligands has been

Still, the question of how much individual FeEDDHA components actually contribute to supplying soil-grown plants with Fe had remained unaddressed up until recently. An understanding of this issue is however particularly relevant for agricultural practice, since nowadays the composition of FeEDDHA products in terms of FeEDDHA components varies greatly. An efficient use of FeEDDHA fertilizer, implying maximizing the benefits in terms of crop yield and Fe uptake by plants, while minimizing the applied FeEDDHA dosage, is desirable both for the applier in view of cost efficiency, and from an environmental perspective to minimize the input of synthetic chemicals into the environment. In practical terms efficient FeEDDHA application translates into applying the right fertilizer (right composition) at the right moment in the right quantity. This requires a profound understanding of the effectiveness of individual FeEDDHA components in soil application. This chapter aims to inform on recent advances made in understanding the performance of FeEDDHA components in soil application (Schenkeveld et al. 2008; 2010a; 2010b). In a series of pot trial studies with soybean, FeEDDHA-facilitated Fe uptake was examined in relation to 1) the composition of the FeEDDHA treatments, 2) the soil solution concentrations of the FeEDDHA components as a function of time, and 3) the moment of FeEDDHA application.

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

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.

studied by Perez-Sanz and Lucena, 1995.

**2. Materials and methods** 

Schenkeveld et al., 2010a.

**2.2 FeEDDHA solutions** 

**2.1 Soil** 

The effectiveness of FeEDDHA components in providing soybean plants from the chlorosis susceptible cultivar Mycogen 5072 with Fe was examined in three pot trial studies.

#### *Effect of FeEDDHA treatment composition on Fe uptake– pot trial 1*

In pot trial 1, soybean plants were given FeEDDHA treatments similar in Fe dose (≈ 7 mg l-l Fe in the pore water; 0.13 mM), but differing in FeEDDHA component composition. Four FeEDDHA treatments (16%o,o; 34%o,o; 49%o,o and 99%o,o) and a blank treatment were included in the experiment; the composition of the treatments is presented in Table 2. The treatments are named after the combined percentage of the Fe chelated by racemic and meso o,o-EDDHA and were given at t=0. The pot trial experiment had a run time of eight weeks. A more elaborate description of the experiment is provided in Schenkeveld et al., 2008.

#### *FeEDDHA-facilitated Fe uptake as a function of time - pot trial 2*

In pot trial 2, the relation between FeEDDHA component concentrations in the pore water and Fe uptake by plants was examined as a function of time. Soybean plants were offered two different FeEDDHA treatments, (30%o,o and 100%o,o) and a blank treatment. The treatments were equal in Fe dose (≈ 4 mg l-l Fe in the pore water; 0.07 mM), but differed in the percentage of Fe chelated by racemic and meso o,o-EDDHA. The composition of the treatments is presented in Table 2. FeEDDHA was applied once, at the start of the experiment. The pot trial with had a runtime of six weeks. Plants were harvested and soil solution was sampled every week. The experiment is described more elaborately in Schenkeveld et al., 2010a.

#### *Effect of moment of application on Fe uptake from FeEDDHA components- pot trial 3*

In pot trial 3, the influence of the moment of application on the effectiveness of individual FeEDDHA components in proving soybean plants with Fe was examined. The experiment involved a blank treatment and six FeEDDHA treatments: *o,p*; *meso o,o*; *racemic o,o*; *o,o-mix low*; *o,o mix-low + o,p*; and *o,o-mix high*. Two levels of FeEDDHA application were distinguished; a low level in the first four treatments, corresponding to a pore water concentration of around 0.6 mg l-1 Fe (i.e. 11 µM), and a high level in the latter two treatment, corresponding to a pore water concentration of around 1.8 mg l-1 Fe (i.e. 32 µM). The high level FeEDDHA application was included to ensure that Fe uptake had not yet reached a maximum in the low level application, which is prerequisite for comparing the effectiveness of the FeEDDHA components. The mixed treatments were included to examine potential synergetic effects. The composition of the treatments is presented in Table 2. With exception of the blank treatments, all pots received one FeEDDHA treatment, either at t=0 after transfer of the seedlings, after 3 weeks in the progressed vegetative stage, or after 6 weeks in the reproductive stage. Which FeEDDHA treatment was applied in which growth stage is indicated in Table 2. Treatments are named after the FeEDDHA treatment administered and the moment of application. The pot trial had a runtime of 8 weeks. Schenkeveld et al., 2010b describes the pot trial more elaborately.

All pot trial experiments were carried out in a greenhouse with 7 liter Mitscherlich pots containing either 6 kg (pot trial 1 and 2) or 5 kg (pot trial 3) of soil at 50% of the waterholding capacity. The experiments were done in triplicates. In pot trial 1 and 2, FeEDDHA solutions were mixed through the soil prior to filling the pots; in pot trial 3, the FeEDDHA treatments were applied through a sand column in the middle of the pot, which went up to a depth of approximately 10 cm into the soil. After FeEDDHA addition, the sand column was flushed with demineralized water.

Seeds of the Fe chlorosis susceptible soybean (*Glycine max* (L.) Merr.) cultivar Mycogen 5072 were germinated on quartz sand with demineralised water. After five days eight seedlings were transferred to each pot, which had been filled with soil one day prior to the transfer. Preparation of the pot trial, soil fertilization with macronutrients, foliar fertilization with micronutrients other than Fe, and plant care were performed as described in Schenkeveld et al. (2008). In pot trial 2, foliar fertilization was omitted. In pot trial 3 the amounts of macronutrients added to the soil were lowered, in proportion to the smaller quantity of soil used per pot.


Table 2. Treatment overview of the pot trials; \* o,p-EDDHA standard contains traces of racemic and meso o,o-EDDHA

#### **2.4 Sampling and measurement**

SPAD measurements were done, as described in Schenkeveld et al., 2008, on the youngest leaves throughout the pot trials, to monitor chlorosis. Chlorosis was established based on a significant difference (α = 0.05) in SPAD-indices between the blank and the treatment with the highest SPAD-index.

At harvest, the shoots were cut off directly above the soil surface. A 1 kg mixed subsample was taken from the soil. Roots were collected manually from the soil subsample, which was stored at 4 °C until further use. The shoots were washed with demineralized water and dried at 70 °C. After 48 hours, the shoots were weighed (dry weight). The mineral contents of the shoots were determined by microwave digestion with nitric acid, fluoric acid and hydrogen peroxide (Novozamsky, et al., 1996). Cu, Fe, Mn and Ni concentrations were measured by ICP-AES (Varian, Vista Pro). Fe uptake was calculated as the product of shoot dry weight yield and Fe content of the shoot. Roots were left out of consideration, due to contamination with soil material.

Pore water was collected from the soil subsample by centrifugation at 7,000 rpm for 15 minutes as described in Schenkeveld et al 2008. The pH of the pore water was measured directly after collection. Fe, Ca and Mg concentrations were measured by ICP-AES (Varian, Vista Pro); Cu, Al, Mn, Zn, Ni and Co concentrations were measured by ICP-MS (Perkin Elmer, ELAN 6000). The samples were acidified with nitric acid before ICP-analysis. FeEDDHA isomer concentrations were determined after separation by high-performance liquid chromatography (HPLC) as described in Schenkeveld et al. (2007). Preparation of experimental solutions and dilution of samples was done with analytical grade chemicals and ultra pure water.
