Effectiveness of FeEDDHA, FeEDDHMA, and FeHBED in Preventing

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Effectiveness of FeEDDHA, FeEDDHMA and FeHBED in Preventing Iron Deficiency Chlorosis in Soybean Levi Marinus Bin, Liping Weng, and Marcel H.J. Bugter J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01382 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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Journal of Agricultural and Food Chemistry

Effectiveness of FeEDDHA, FeEDDHMA and FeHBED in Preventing Iron Deficiency Chlorosis in Soybean Levi M. Bina,b, Liping Wenga,*, Marcel H. J. Bugterb

Department of Soil Quality, Wageningen University, P.O. Box 47, 6700 AA, Wageningen, The Netherlands. Akzo Nobel Functional Chemicals, Dept. Micronutrients, P.O. Box 75730, 1070 AS, Amsterdam, The Netherlands * Corresponding author. Tel: +31 317 482332; email: [email protected].

1 ACS Paragon Plus Environment


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Abstract

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The performance of FeHBED in preventing Fe deficiency chlorosis in soybean (Glycine max

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(L.) Merr.) in comparison to FeEDDHA and FeEDDHMA was studied, as well as the

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importance of the ortho-ortho and ortho-para/rest isomers in defining the performance. To this

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end, chlorophyll production (SPAD), plant dry matter yield, and the mass fractions of

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important mineral elements in the plant were quantified in a greenhouse pot experiment. All

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three Fe chelates increased SPAD-index and dry matter yield compared to the control. The

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effect of FeHBED on chlorophyll production was visible over a longer time-span than that of

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FeEDDHA and FeEDDHMA. Additionally, FeHBED did not suppress Mn uptake as much as

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the other Fe chelates. Compared to the other Fe chelates, total Fe content in the young leaves

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was lower in the FeHBED treatment; however, total Fe content was not directly related to

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chlorophyll production and biomass yield. For each chelate, the ortho-ortho isomer was found

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to be more effective than the other isomers evaluated.

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Keywords

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EDDHA; EDDHMA; HBED; Iron Chelates; Iron Deficiency Chlorosis; (Glycine max (L.)

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Merr.)


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Introduction

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Iron is an essential mineral nutrient for plants. Sufficient iron uptake is vital for producing

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healthy crops, mainly because that the biosynthesis of chlorophyll requires relatively high

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amounts of iron 1. Deficient iron uptake leads to a reduction in leaf chlorophyll content known

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as iron deficiency chlorosis, which reduces yields and in extreme cases leads to necrosis of

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leaf tissue 2–4.

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Several factors affect the bioavailability of iron in soils, including pH, redox potential, CEC,

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and the presence of Fe complexing agents in the soil solution 5,6. Among these factors, pH is

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one of the predominant soil characteristics influencing Fe-solubility, as a result of the pH-

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dependent dissolution of soil Fe(III)-(hydr)oxides 3,7. High pH and high pH-buffering capacity

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result in a low concentration of dissolved Fe in calcareous soils 8,9. In addition to free Fe3+ ion

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species, hydrolyzed Fe(III) and Fe(III) complexed with soluble ligands also contribute to the

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soluble Fe in the soil solution

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organic matter (DOM) in soil solution, which will increase its solubility to a certain extent,

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but usually not enough to prevent iron deficiency

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graminaceous plant species (strategy II plants) such as grasses secrete phytosiderophores,

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which are natural chelating compounds that increase soluble Fe concentrations in the

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rhizosphere

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increase their iron uptake by three reactions: i) the excretion of protons to acidify the

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rhizosphere, thereby increasing the solubility of Fe3+; ii) the reduction of Fe3+ to Fe2+ by a

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Fe3+-chelate reductase; and iii) the release of organic acids and phenolic compounds, which

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act as chelates, into the rhizosphere 1,10,12,15–18.

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Various management strategies to prevent or cure iron deficiency chlorosis are implemented

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in agriculture to increase yields 2,19,20. Soil application of synthetic iron chelates is effective in

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counteracting iron deficiency chlorosis of plants grown on calcareous soils, and is the most

10,11

. Iron is able to form complexes with natural dissolved

10,11

. Under Fe deficient conditions,

1,10,12–14

. Most broadleaf plant species (strategy I plants), on the other hand,



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commonly applied technique in agriculture 13. Iron chelates with a relatively low affinity for

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Fe (FeEDTA, FeDTPA, etc.) are useful as foliar application, but are not effective when

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applied to calcareous soils 13. Only Fe chelates with a relatively high stability (e.g. FeEDDHA

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and analogues) are able to increase plant Fe uptake, reducing chlorosis in alkaline and

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calcareous soils 13.

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The performance of an Fe chelate is largely determined by several characteristics: i) the

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ability of the ligand to maintain Fe in solution without being adsorbed to the soil solid phase

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or having the Fe exchanged by a competing cation; ii) the ability of the ligand to release Fe in

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the rhizosphere for plant uptake; and iii) the ability of the ligand to, once its original Fe has

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been delivered to the plant, pick up native Fe from the soil (shuttle-effect)

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pathways have been proposed by which Fe is released from chelates at the root surface, i.e.

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either by direct dissociation (releasing Fe(III)) or by enzymatic reduction (releasing Fe(II)) 24

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21–23

. Two

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. Since an excessively high stability and selectivity of a chelate would imply that the chelate

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cannot deliver Fe to plants, a fine balance between stability and reducibility (or speed of

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dissociation) will strongly define the ability of a chelate to efficiently deliver Fe to the plant.

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Among the high stability Fe chelates, FeEDDHA is currently the most widely used iron

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fertilizer

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(p,p-), and ortho-para (o,p-) positional isomers with different fractions of each isomer

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depending on the production conditions. In addition to the positional isomers, the mixture will

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also contain a fraction of diastereomers and polycondensates known as rest-FeEDDHA

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The position of the hydroxyl group on the phenolic ring of the positional isomers influences

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the complexation of EDDHA with Fe. If the hydroxyl group is in para position, it does not

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contribute to the binding of Fe. Therefore, o,o-FeEDDHA will bind iron more strongly than

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o,p-FeEDDHA, and o,p-FeEDDHA will bind iron more strongly than p,p-FeEDDHA 9. Since

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o,o-FeEDDHA is the most effective isomer in reducing iron deficiency chlorosis in calcareous

26

. The production of FeEDDHA yields a mixture of ortho-ortho (o,o-), para-para

30,31

.


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soils

, the %Fe as o,o-FeEDDHA is an obligatory label requirement for commercial

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FeEDDHA in the European Union.

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The chemistry of FeEDDHMA and FeHBED is largely comparable to that of FeEDDHA;

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however, FeEDDHMA has one methyl-group attached to each phenolic ring, instead of a

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hydrogen-atom in the case of FeEDDHA32. With HBED the hydroxybenzyl group is not

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directly attached to the carboxymethyl group, resulting in a lower rigidity of the molecule,

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which gives a better steric fit for Fe3+ and an approximately 4 log units higher complexation

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constant than that of FeEDDHA and FeEDDHMA (see Table 1).

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Several studies comparing FeEDDHA with other high stability Fe chelates, including

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FeEDDHMA and FeHBED, have been conducted in the past. These studies ranged from

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laboratory experiments

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because of its novelty, FeHBED has not been studied as extensively as FeEDDHA and

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FeEDDHMA. Even though in the past FeHBED was primarily applied in medical research, it

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was demonstrated in several experiments starting in as early as 1988 that FeHBED is as

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effective as FeEDDHA in counteracting iron deficiency chlorosis in plants8,22,25,29. However,

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the authors also found that FeEDDHA application resulted in a higher uptake of Fe on the

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short term, while FeHBED was suggested to give a better performance on the long term. This

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long term positive effect of FeHBED was attributed to its ability to maintain Fe in solution

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longer than FeEDDHA, and its higher stability 29.

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However, the number of comparative studies on FeHBED with other high stability Fechelates

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is still quite limited. Another issue, which has not been investigated, is the function of

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different isomers in defining the performance of FeHBED. Schenkeveld et al. (2008) found

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that for FeEDDHA the most important structural isomer determining the performance of this

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Fe chelate is o,o-Fe 23,30. Since the commercial production of FeHBED yields a product with a

21,27,28

to direct application of each Fe-chelate to crops

13

. However,



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high o,o-Fe fraction compared to FeEDDHA 29, knowledge of the relative importance of o,o-

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Fe in defining the performance of FeHBED may seem to be of little significance. However,

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obtaining knowledge on the relevance of structural isomers in the functioning of FeHBED is

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of paramount importance for our overall understanding of the ligand.

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The aim of this study is to find an answer to these two questions: i) Is the ability of

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FeEDDHA, FeEDDHMA, and FeHBED in preventing iron deficiency chlorosis of plants (in

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this case soybean) comparable? ii) Does the o,o-isomer define the performance of all three

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chelates? Answering these questions will improve our understanding regarding the

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relationship between the iron chelate properties and its effectiveness in correcting iron

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deficiency related symptoms in crops.

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Materials and Methods

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Soil Sample

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In order to test the efficacy of each chelate in preventing Fe deficiency in soybean, a pot

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experiment was conducted. The soil used in the pot experiment was collected from

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Santomera, Spain, and was expected to cause iron deficiency chlorosis in soybean plants in

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concurrence with previous experiments conducted with soil taken from the same location 30.

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The soil was obtained from the top layer (0 - 20 cm). Pre-treatment consisted of drying (40

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°C) and sieving to pass a 2 mm screen. The soil was classified as an Entisol (Table 2). The

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content of available Fe in the soil was found to be low in terms of DTPA-extractable Fe (1.3

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mg kg-1) and relatively low in terms of oxalate-extractable Fe (1.47 g kg-1) (Table 2).

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Fe chelate solutions


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The FeEDDHA, FeEDDHMA and FeHBED used for this experiment were prepared by Akzo

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Nobel Functional Chemicals B.V. (Arnhem, the Netherlands). For each chelate both a

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formulation with 100% o,o-Fe and a formulation with approximately 30% o,o-Fe (31%; 26%;

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39%, for FeEDDHA, FeEDDHMA and FeHBED, respectively) were prepared.

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Experimental Setup

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Because of its susceptibility to Fe deficiency chlorosis, soybean (Glycine max (L.), cultivar

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Mycogen 5072, was chosen as crop plant for this experiment. Prior to planting, the soybean

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seeds were germinated on quartz sand using demineralized water. After 7 days the soybean

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seedlings were transplanted to pre-cleaned, 7 liter porcelain enameled steel plate pots

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(Mitscherlich pots) filled with 6 kg soil, at a plant density of 9 seedlings per pot. The pot

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experiment consisted of 10 treatments, including one control treatment (no Fe chelate), and

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nine Fe chelate treatments (3 iron chelates x 3 dosages and isomer compositions, i.e. 17.7 mg

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L-1 Fe with 100% o,o-Fe, 17.7 mg L-1 Fe with ± 30% o,o-Fe, and 6.9 mg L-1 Fe with 100%

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o,o-Fe). The Fe dosage is expressed as mg Fe L-1 of soil water, which is 0.135 L kg-1 soil (at

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50% water holding capacity). Three replicates per treatment were used. Since one of the

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objectives of the pot experiment was to test the importance of o,o-Fe in supplying Fe to

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plants, a treatment with a low fraction (about 30%) of o,o-Fe was compared to two 100% o,o-

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Fe treatments (a low and a high dosage). The concentrations of o,p-/rest-Fe and o,o-Fe in the

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mixed isomer treatment cumulated to a total Fe concentration similar to that of the high

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dosage 100% o,o-Fe treatment, while the concentration of o,o-Fe in this mixed isomer

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treatment is equal to the Fe concentration in the low dosage 100% o,o-Fe treatment.

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For each pot, 6 kg of soil was mixed with the iron chelate solutions, and put into the pots

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several days before the transplanting of the seedlings. At this stage the following nutrients 7 ACS Paragon Plus Environment


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were also added to the soil in each pot: 40 mmol NH4NO3, 25 mmol K2HPO4, 20 mmol

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CaCl2, 10 mmol MgSO4, 0.5 mmol H3BO3, and 3.75 µmol (NH4)6Mo7O24. These nutrients

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were given in a single application before the plants were transferred, whereas Zn, Cu and Mn

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were applied as foliar application (see below). Prior to adding the soil to the pots, the soil

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moisture content was adjusted with demineralized water to 50% of the water holding capacity

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of the soil (135 L kg-1). The pots were placed in the greenhouse using a randomized block

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design, consisting of three blocks. Each block contained one treatment replicate, and the pots

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within the block were shifted around in a random pattern on a weekly basis.

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Evapotranspiration of the plants and soil was quantified by weighing the pots, and the water

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lost was replenished with demineralized water on a daily basis. To ensure that no nutrient

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other than iron would limit plant growth, a micronutrient solution in the form of a foliar spray

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was applied two times during the experiment, with the first application 20 days and the

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second application 32 days after transplanting the seedlings. The fertilizer solution on the first

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application date consisted of 0.2 mM Cu, 0.82 mM Mn and 0.27 mM Zn in the form of

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dissolved EDTA salts. On the second application date, half of this dosage was applied. The

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soybean plants were grown in a greenhouse at a 20 °C (± 3 °C), and with additional lighting

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supplied for 16 h d-1 with 400 Watt HPI lamps.

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Measurements

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Twice per week, SPAD measurements from the leaves were obtained to compare leaf

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chlorophyll content between treatments. The relation between SPAD-index and chlorophyll

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content is widely accepted, and has been proven by Schenkeveld et al. (2008). The

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measurements were obtained using a Minolta-502 SPAD meter. Per plant, two of the youngest

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fully opened trifoliate leaves, two leaves from the second youngest trifoliate, and two leaves


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from the next trifoliate were measured for 4 plants per pot. SPAD was measured midway

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between the leaf edge and the central vein.

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After growing for six weeks, the shoots were harvested by cutting them directly above the soil

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surface. The shoots were cleaned using demineralized water and split into young leaves (the

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youngest fully expanded trifoliate including the top meristem) and the rest of the plant. The

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two plant components were dried at 70 °C, and subsequently weighed to compare the dry

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weight yields between each treatment. In addition, the mineral nutrient content (Al, Ca, Cu,

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Fe, Mg, Mn and Zn) of the two individual plant parts was obtained by measuring on ICP-AES

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(Varian, Vista Pro) after microwave digestion at 175 °C (Discover, CEM, USA) using 16 M

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nitric acid.

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Statistical Analyses

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Data analyses were performed using the statistical programs GenStat (15th edition) and IBM

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SPSS Statistics (version 22). The obtained data were first tested for normality using Q-Q

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plots, or a Shapiro-Wilk test (α ≤ 0.05). Additionally, the data were tested for homogeneity of

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variance using Bartlett’s homogeneity of variance test. Subsequently, the data for the dry

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weights and the mineral concentrations of the plant components were analyzed using

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ANOVA, with Duncan’s multiple range as post-hoc test (α ≤ 0.05). If, even after

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transformation, no homogeneity of variance could be achieved, a Welch’s One-way ANOVA

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with Games-Howell post-hoc analysis was performed. The SPAD-index measurements over

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time were analyzed using a Restricted Maximum Likelihood (REML) model. For the specific

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time points, the SPAD-index measurements were compared using a split-plot ANOVA

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(n=240), with Duncan’s multiple range as post-hoc test (α ≤ 0.05). The split-plot ANOVA



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was applied to correct for the pseudo-replication caused by using the measurements of each

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plant instead the average of the whole pot 33.

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Results and Discussion

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SPAD-Index Measurements over Time

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The SPAD-index results of the youngest fully opened trifoliate are discussed here, since the

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youngest trifoliate is most susceptible to Fe chlorosis 32, whereas a similar trend was found in

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the results of the other leaves (results not shown). Figures 1, 2 and 3 illustrate the changes in

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SPAD-index over time for each Fe chelate. Chlorosis was strongest in the control treatment

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on day 14 (2 weeks), while chlorosis was weakest on day 41 (6 weeks), at the termination of

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the experiment. For all the treatments, the SPAD-index generally increased over time, but

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with different rates. The fluctuations in the curves are mainly caused by shifting from one set

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of trifoliate leaves to the next fully opened set. The results indicate that all three Fe chelates

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have significantly increased the SPAD-index in the first 4 weeks of the pot experiment

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compared to the control treatment (Figure 1, 2, 3). For the 17.7 mg Fe L-1 (100% o,o-Fe)

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treatment, the increase in SPAD-index from day 10 over time is strongest for FeHBED among

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the three Fe chelates (the slope of the trend line is 0.16), whereas the increase is least strong

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for FeEDDHA (trend line has a slope of 0.069).

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As the experiment progresses, there is a shift in how each Fe chelate influences leaf

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chlorophyll production (Figure 4). At the start of the experiment (day 14) the only significant

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(F prob.

Effectiveness of FeEDDHA, FeEDDHMA, and FeHBED in Preventing

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