**3. Results and discussion**

#### *Chlorosis*

92 Soybean Physiology and Biochemistry

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

> **o,p-FeEDDHA (mg l-1 Fe)**

blank - - - - - - 16%o,o 0.58 (8%) 0.61 (8%) 1.15 (16%) 5.02 (68%) 7.36 t = 0 34%o,o 1.07 (16%) 1.24 (18%) 1.26 (19%) 3.14 (47%) 6.71 t = 0 49%o,o 1.69 (22%) 2.03 (27%) 1.31 (18%) 2.50 (33%) 7.53 t = 0 99%o,o 3.44 (48%) 3.64 (51%) - 0.10 (1%) 7.18 t = 0

blank - - - - - - 30%o,o 0.60 (14%) 0.68 (16%) 0.79 (19%) 2.18 (51%) 4.25 t = 0 100%o,o 1.93 (48%) 2.00 (50%) - 0.05 (1%) 3.98 t = 0

blank - - - - - -

o,p \* \* 0.53 0.05 0,58 t = 3 and 6 weeks meso o,o - 0.56 - - 0.56 t = 0, 3 and 6

racemic o,o 0.58 - - - 0,58 t = 0, 3 and 6

o,o-mix low 0.29 0.31 - - 0.60 t = 3 weeks

o,o-mix high 0.87 0.93 - - 1.80 t = 0 and 3 weeks

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

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

0.29 0.31 1.06 0,10 1.76 t = 3 weeks

**rest-FeEDDHA (mg l-1 Fe)** 

**total Fe (mg l-1 Fe)** **Moment of application** 

weeks

weeks

used per pot.

**racemic o,o-FeEDDHA (mg l-1 Fe)** 

**meso o,o-FeEDDHA (mg l-1 Fe)** 

**Treatment** 

*Pot trial 1*

*Pot trial 2*

*Pot trial 3* 

o,o-mix low +

racemic and meso o,o-EDDHA

**2.4 Sampling and measurement** 

o,p

Inducing Fe deficiency chlorosis is a prerequisite for testing the effectiveness of the FeEDDHA components. In all three pot trials, the plants in the blank treatment became chlorotic, approximately a week after transfer of the seedlings to the pots. The development of chlorosis differed per pot trial; in pot trial 1 and 2, the degree of chlorosis reached a maximum after around three weeks, after which the difference in SPAD-index started to decrease. In pot trial 1, chlorosis in the youngest leaves of the blank treatment was actually entirely over-grown by the time the plants were harvested (Schenkeveld et al., 2008). Possibly, the decrease in degree of chlorosis is related to an increased root density in the pots as a result of an ongoing development of the root system. This high root density leads to increased rhizosphere effects and an enhanced ability of the plants to acquire Fe. In pot trial 3, the degree of chlorosis stabilized and remained more or less constant towards the end of the experiment.

#### **3.1 Effect of FeEDDHA treatment composition on Fe uptake**

#### *Pore water concentrations*

The Fe concentration in the pore water of the blank treatment was below detection limit, both in this and the other two pot trial experiments, indicating that FeEDDHA components were responsible for all Fe in solution in the FeEDDHA treatments. At harvest of pot trial 1, the total Fe concentration in the pore water proved linearly related to the o,o-FeEDDHA content of the FeEDDHA treatments (Figure 5). Racemic o,o-FeEDDHA accounted for approximately 80% of the Fe in solution, and meso o,o-FeEDDHA for the remaining 20%. o,p-FeEDDHA and rest-FeEDDHA had been removed from soil solution practically completely. These components have a tendency to adsorb due to a relatively high affinity for soil reactive surfaces. Moreover, upon interaction with soil, Cu may rapidly displace Fe from o,p-FeEDDHA resulting in solibilization of o,p-CuEDDHA (Garcia-Marco et al., 2006; Hernandez-Apaolaza et al., 2006; Schenkeveld et al., 2007). Hence, removal of FeEDDHA components from soil solution is to a large extent unrelated to plant processes. From the amount of Fe added with the FeEDDHA treatment only in between 4 and 20 % was retrieved at harvest. The recovery of racemic o,o-FeEDDHA and meso o,o-FeEDDHA was around 30% and 7%, respectively. The recovery of o,p-FeEDDHA and rest-FeEDDHA was below 1%.

Fig. 5. Fe and FeEDDHA component concentrations in soil solution of Santomera soil at harvest as a function of the o,o-FeEDDHA content of the FeEDDHA treatment. Error bars indicate standard deviations. (based on Schenkeveld et al., 2008)

#### *Fe uptake*

Fe uptake by soybean plants increased with increasing o,o-FeEDDHA content of the FeEDDHA treatment (Figure 6a). At low o,o-FeEDDHA content, the increase in Fe uptake is relatively strong, but the slope of the curve flattens with increasing o,o-FeEDDHA content, and eventually an optimum is reached (Schenkeveld et al., 2010a).

The increase in Fe uptake with increasing o,o-FeEDDHA content suggests that Fe uptake is related to the Fe concentration in soil solution (Figure 5). This makes sense, since the limited solubility of Fe in calcareous soil is one of the primal causes for Fe chlorosis. The fact that Fe

were responsible for all Fe in solution in the FeEDDHA treatments. At harvest of pot trial 1, the total Fe concentration in the pore water proved linearly related to the o,o-FeEDDHA content of the FeEDDHA treatments (Figure 5). Racemic o,o-FeEDDHA accounted for approximately 80% of the Fe in solution, and meso o,o-FeEDDHA for the remaining 20%. o,p-FeEDDHA and rest-FeEDDHA had been removed from soil solution practically completely. These components have a tendency to adsorb due to a relatively high affinity for soil reactive surfaces. Moreover, upon interaction with soil, Cu may rapidly displace Fe from o,p-FeEDDHA resulting in solibilization of o,p-CuEDDHA (Garcia-Marco et al., 2006; Hernandez-Apaolaza et al., 2006; Schenkeveld et al., 2007). Hence, removal of FeEDDHA components from soil solution is to a large extent unrelated to plant processes. From the amount of Fe added with the FeEDDHA treatment only in between 4 and 20 % was retrieved at harvest. The recovery of racemic o,o-FeEDDHA and meso o,o-FeEDDHA was around 30% and 7%, respectively. The recovery of o,p-FeEDDHA and rest-FeEDDHA was

total Fe in solution

racemic o,o-FeEDDHA meso o,o-FeEDDHA

R2 = 0.98

R2 = 0.98

R2 = 0.99

0 0.5 1

**o,o-FeEDDHA fraction**

Fig. 5. Fe and FeEDDHA component concentrations in soil solution of Santomera soil at harvest as a function of the o,o-FeEDDHA content of the FeEDDHA treatment. Error bars

Fe uptake by soybean plants increased with increasing o,o-FeEDDHA content of the FeEDDHA treatment (Figure 6a). At low o,o-FeEDDHA content, the increase in Fe uptake is relatively strong, but the slope of the curve flattens with increasing o,o-FeEDDHA content,

The increase in Fe uptake with increasing o,o-FeEDDHA content suggests that Fe uptake is related to the Fe concentration in soil solution (Figure 5). This makes sense, since the limited solubility of Fe in calcareous soil is one of the primal causes for Fe chlorosis. The fact that Fe

below 1%.

*Fe uptake* 

0.0

indicate standard deviations. (based on Schenkeveld et al., 2008)

and eventually an optimum is reached (Schenkeveld et al., 2010a).

0.4

0.8

1.2

**[Fe] (mg l-1)**

1.6

2.0

uptake, unlike Fe concentration in soil solution, is not linearly related to the o,o-FeEDDHA content suggests a saturation effect, commonly observed with micronutrient uptake in relation to bioavailability (Marschner, 1995).

As a result of the FeEDDHA treatments, Fe uptake increased from 0.70 mg pot-1 in the blank to 1.75 mg pot-1 in the 99% o,o-FeEDDHA treatment; a 150% increase. The 16%o,o FeEDDHA treatment already increased Fe uptake by approximately 75%, to 1.22 mg pot-1. The additional Fe uptake in the FeEDDHA treatments in comparison to the blank only accounted for 7 to 15% of the Fe provided as FeEDDHA, and for 15 to 44% of the Fe added as o,o-FeEDDHA.

The increased Fe uptake manifested both in an increased Fe content of the shoot (Figure 6b), and in an increased dry weight yield (Figure 6c). The trends in Fe content and dry weight yield as a function of o,o-FeEDDHA content are similar as for Fe uptake. The relative effect on Fe content of the shoot: an increase from 31 to 60 mg kg(dw)-1 (≈ 100% increase), was larger than the relative effect on dry weight yield; an increase from 22.1 to 29.0 g(dw) pot-1 (≈ 30% increase). Comparable results were also obtained with soybean grown on another calcareous soil (Schenkveld et al., 2008; results not shown).

An important practical implication of these results for FeEDDHA application prior to the onset of chlorosis is, that for obtaining similar results in terms of crop yield and crop quality, a smaller dosage of FeEDDHA products with a higher o,o-FeEDDHA content is required in comparison to products with a lower o,o-FeEDDHA content.

Fig. 6. a) Fe uptake; b) Fe content of the shoot; and c) dry weight yield (shoot) of soybean plants grown on Santomera soil as a function of the o,o-FeEDDHA content of the FeEDDHA treatment. Error bars indicate standard deviations. (based on Schenkeveld et al., 2008)
