A Critical Review of Methodologies for Evaluating Iron Fertilizers Based on Iron Reduction and Uptake by Strategy I Plants.

Under iron (Fe)-limited conditions, plants have developed strategies for acquiring this essential micronutrient. Several Fe sources have been studied as potential fertilizers, with Fe synthetic chelates being the most used to prevent and correct Fe chlorosis in crops. The determination of the activity of the Fe chelate reductase (FCR) enzyme has long been described in the literature to understand the efficiency of Strategy I plants in acquiring Fe from fertilizers under deficient conditions. Other experiments have focused on the translocation of Fe to the plant to define the effectiveness of Fe fertilizers. Yet, both assays are relevant in knowing the capacity of a novel Fe source and other compounds alleviating Fe chlorosis in Strategy I plants. This work reviews the methodologies that are used in FCR assays to evaluate novel Fe fertilizers, including the factors modulating the results obtained for FCR assay activity, such as the Fe substrate, the Fe level during the growing period and during the FCR assay, the pH, the choice of an in vivo or in vitro method, and the plant species. A discussion of the benefits of the concurrence of FCR and Fe uptake assays is then presented alongside a proposed methodology for assessing the effectiveness of Fe fertilizers, emphasizing the importance of understanding chemical and physiological plant interactions. This methodology unifies key factors that modify FCR activity and combines these with the use of the 57Fe tracer to enhance our comprehension of the efficacy of Fe-based fertilizers' effectiveness in alleviating Fe chlorosis. This comprehensive approach not only contributes to the fundamental understanding of Fe-deficient Strategy I plants but also establishes a robust method for determining the efficiency of novel sources for correcting Fe deficiency in plants.

Evaluation of Siderophores Generated by Pseudomonas Bacteria and Their Possible Application as Fe Biofertilizers.

The application of synthetic iron chelates to overcome iron deficiency in crops is leading to a high impact on the environment, making it necessary to find more friendly fertilizers. A promising alternative is the application of biodegradable iron chelates, such as those based on siderophores. In the present work, seven bacterial strains of the genus Pseudomonas were selected for their ability to secrete pyoverdine, a siderophore with a high affinity for iron, which could be used as a biofertilizer. The concentration of siderophores secreted by each bacterium expressed as desferrioxamine B equivalents, and the pyoverdine concentration was determined. Their potential as Fe biofertilizers was determined based on their capacity to complex Fe, determining the maximum iron complexation capacity at alkaline pH and selecting the RMC4 strain. The biostimulant capacity of the RMC4 strain was evaluated through the secretion of organic acids such as the hormone Indol-3-acetic acid or glutamic acid, among others, in a kinetic assay. Finally, the genome of RMC4 was determined, and the strain was identified as Pseudomonas monsensis. The annotated genome was screened for genes and gene clusters implicated in biofertilization and plant growth promotion. Besides iron mobilization, genes related to phosphorus solubilization, production of phytohormones and biological control, among others, were observed, indicating the suitability of RMC4 as an inoculant. In conclusion, RMC4 and its siderophores are promising sources for Fe biofertilization in agriculture.

[S,S]-EDDS Ligand as a Soil Solubilizer of Fe, Mn, Zn, and Cu to Improve Plant Nutrition in Deficient Soils.

The deficiencies of iron, manganese, zinc, and copper in calcareous soils are a worldwide problem affecting plant growth and fruit quality, usually minimized by the application of recalcitrant synthetic metal chelates. Biodegradable ligand [S,S]-EDDS is an eco-friendly substitute. This study investigates the capacity of [S,S]-EDDS to mobilize micronutrients from agronomic soils and improve plant nutrition. A batch and a plant experiment (Phaseolus vulgaris cv. Black Pole) with three agronomic soils was conducted to monitor the micronutrients solubilized by [S,S]-EDDS, the ligand degradation, and plant uptake. The results demonstrated the high capacity of [S,S]-EDDS to solubilize Fe and other micronutrients related to its chemical behavior and the enhancement of plant nutrition. The best results were shown in sandy-clay soil with low Fe, typically found in the Mediterranean areas. The results support the direct application of the ligand to soils and a possible biotechnological application of the ligand-producer bacteria.

Fast Determination of a Novel Iron Chelate Prototype Used as a Fertilizer by Liquid Chromatography Coupled to a Diode Array Detector.

The environmental risk of the application of synthetic chelates has favored the implementation of new biodegradable ligands to correct Fe-deficient plants. This study developed and validated an analytical method for determination of a new prototype iron chelate─Fe(III)-benzeneacetate, 2-hydroxy-α-[(2-hydroxyethyl)amino]─(BHH/Fe3+) based on liquid chromatography with diode array detection, as a potential sustainable alternative. Chromatographic analysis was performed on a LiChrospher RP-18 in reverse-phase mode, with a mobile phase consisting of a mixture of acetonitrile (solvent A) and sodium borate buffer 0.20 mM at pH = 8 (solvent B) at a flow rate of 1.0 mL/min in isocratic elution mode. This method was fully validated and found to be linear from the limit of quantification (LOQ) to 50 mg/L and precise (standard deviation below 5%). The proposed method was demonstrated to be selective, precise, and robust. The developed methodology indicated that it is suitable for the quantification of iron chelate BHH/Fe3+.

Implications of the Mn:ligand ratio for Mn uptake by Glycine max L. plants fertilized with heptagluconate and gluconate complexes.

BACKGROUND: The environmental risk of the application of synthetic chelates has furthered the implementation of biodegradable complexes to correct manganese (Mn)-deficient plants. This study used the biodegradable ligands of heptagluconate (G7) and gluconate (G6) to test the influence of the Mn2+ :ligand ratio on their fertilizers' capacity to provide Mn to plants. The efficacy of these complexes to correct Mn-deficient soybean was evaluated in hydroponics and calcareous soil conditions and compared with the synthetic chelate EDTA (ethylenediaminetetraacetic acid). RESULTS: This study demonstrated that G7 was a biodegradable alternative to EDTA for supplying Mn, maintaining an adequate nutritional balance compared with G6, which reduced iron (Fe) uptake by the plants. The efficacy of the Mn complexes depended on both the ligand and the Mn:ligand ratio, with the 1:1 and 1:2 molar ratios of Mn2+ :G7 being the most effective complexes in the short term on the basis of their chemical structure and stability. CONCLUSION: The Mn2+ :G7 (1:1 and 1:2) complexes were found to be effective Mn sources for plant nutrition due to their chemical structures providing adequate stability in alkaline solution and their fast-action effect. © 2021 Society of Chemical Industry.

Assessing metal-lignosulfonates as fertilizers using gel filtration chromatography and high-performance size exclusion chromatography.

Lignosulfonates (LSs) are by-products from the paper industry used as biodegradable fertilizers. However, metal-LS ability to provide micronutrients to crops is related to the stability of the complex and the amount of metal complexed. This work evaluated these parameters using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FTIR), and 13C-nuclear magnetic resonance (NMR), along with gel filtration chromatography (GFC) and high-performance size exclusion chromatography (HPSEC), for different spruce, eucalyptus, and pine LSs. GFC and HPSEC pointed out that the amount and type of complexed metal in the LS depends on the molecular weight, pH, and sulphite pulping processes. Both techniques indicated that the low molecular weight LS enriched with phenolic groups has the highest Fe(III) complexing capacity. Also, Fe(III)/LS showed the formation of high molecular weight compounds, whereas Zn(II)/LS and Mn(II)/LS complexes did not form aggregates. Metal-LS fractionation provided considerable information to identify LSs with potential fertilizer capacity and to assess the effectiveness of their complexes.

Effect of Fe:ligand ratios on hydroponic conditions and calcareous soil in Solanum lycopersicum L. and Glycine max L. fertilized with heptagluconate and gluconate.

BACKGROUND: The environmental risk from the application of synthetic chelates has led to the use of biodegradable complexes to correct Fe deficiency in plants. In this article, the Fe oxidation state, the Fe:ligand ratio, and the molecular weight distribution for heptagluconate (G7) and gluconate (G6) are considered as key factors for the efficacy of complexes as fertilizers. Complexes with different Fe:ligand ratios were prepared and analyzed by gel filtration chromatography (GFC). The ability of Fe:ligand ratios to provide Fe to tomato in hydroponics and soybean in calcareous soil was tested and compared with synthetic chelates (Fe3+ :HBED and Fe3+ :EDTA). RESULTS: G7 presented greater capacity to complex both Fe(II) and Fe(III) than G6, but the Fe(II) complexes exhibited poor stability at pH 9 and oxidation in solution. Gel filtration chromatography demonstrated the polynuclear nature of the Fe3+ :G7 at various ratios. The effectiveness of the Fe fertilizers depend on the Fe3+ :ligand ratio and the ligand type, the Fe3+ :G7 (1:1 and 1:2) being the most effective. Fe3+ :G7 (1:1) also presented a better response for the uptake of other micronutrients. CONCLUSION: Fe3+ :G7 molar ratios have been shown to be critical for plant Fe uptake under hydroponic conditions and with calcareous soil. Thus, the Fe3+ :G7 at equimolar ratio and 1:2 molar ratio can be an environmentally friendly alternative to less degradable synthetic chelates to correct Fe chlorosis in strategy I plants. © 2019 Society of Chemical Industry.

Evaluation of the Efficacy of Two New Biotechnological-Based Freeze-Dried Fertilizers for Sustainable Fe Deficiency Correction of Soybean Plants Grown in Calcareous Soils.

Currently, fertilization with synthetic chelates is the most effective agricultural practice to prevent iron (Fe) deficiencies in crops, especially in calcareous soils. Because these compounds are not biodegradable, they are persistent in the environment, and so, there is the risk of metal leaching from the soils. Thus, new, more environment-friendly efficient solutions are needed to solve iron-deficiency-induced chlorosis (IDIC) in crops grown in calcareous soils. Therefore, the central aim of this work was to prepare new freeze-dried Fe products, using a biotechnological-based process, from two siderophores bacterial (Azotobacter vinelandii and Bacillus subtilis) cultures (which previously evidenced high Fe complexation ability at pH 9) and test their capacity for amending IDIC of soybean grown in calcareous soils. Results have shown that A. vinelandii iron fertilizer was more stable and interacted less with calcareous soils and its components than B. subtilis one. This behavior was noticeable in pot experiments where chlorotic soybean plants were treated with both fertilizer products. Plants treated with A. vinelandii fertilizer responded more significantly than those treated with B. subtilis one, when evaluated by their growth (20% more dry mass than negative control) and chlorophyll development (30% higher chlorophyll index than negative control) and in most parameters similar to the positive control, ethylenediamine-di(o-hydroxyphenylacetic acid). On average, Fe content was also higher in A. vinelandii-treated plants than on B. subtilis-treated ones. Results suggest that this new siderophore-based formulation product, prepared from A. vinelandii culture, can be regarded as a possible viable alternative for replacing the current nongreen Fe-chelating fertilizers and may envisage a sustainable and environment-friendly mending IDIC of soybean plants grown in calcareous soils.

[S,S]-EDDS/Fe: A new chelate for the environmentally sustainable correction of iron chlorosis in calcareous soil.

Iron deficiency in crops is usually prevented and cured by the application of synthetic Fe chelates such as EDTA/Fe and the o,o-EDDHA/Fe. However their persistence in soil calls for the implementation of new alternatives that present less of a risk to the environment. This study therefore evaluated the biodegradable chelating agent [S,S]-EDDS as a new source for Fe fertilisation in calcareous soils in relation to its chemical reactivity. The suitability of [S,S]-EDDS/Fe as an Fe fertiliser in a calcareous soil was investigated and compared to the traditional synthetic chelates EDTA/Fe and o,o-EDDHA/Fe. Plant experiments with soybean (Glycine max), 57Fe isotopic labelling, and batch incubations were conducted in a calcareous soil. The Fe concentration of plants treated with [S,S]-EDDS/Fe was similar to those treated with EDTA/Fe. A similar Fe concentration to the o,o-EDDHA/Fe treatment was achieved using a double dose of [S,S]-EDDS/Fe. Despite the degradation of [S,S]-EDDS limiting the durability of [S,S]-EDDS/Fe in soil, the Fe bound to the degradation products may be a determining factor in improving Fe uptake and translocation to leaves in plants treated with [S,S]-EDDS/Fe compared to other Fe sources. Speciation studies by modelling and batch experiments also supported the lower reactivity of [S,S]-EDDS/Fe with calcium compared to that of EDTA/Fe, possibly contributing to the permanence of [S,S]-EDDS/Fe in the calcareous soil. This study demonstrated for the first time, that [S,S]-EDDS may be an environmentally sustainable alternative to traditional synthetic chelating agents such as EDTA or o,o-EDDHA for curing Fe chlorosis in susceptible plants in calcareous soil.

Calcareous soil interactions of the iron(III) chelates of DPH and Azotochelin and its application on amending iron chlorosis in soybean (Glycine max).

In order to find new greener solutions for iron (Fe) induced chlorosis, two new chelating agents, N,N-dihydroxy-N,N'-diisopropylhexanediamide (DPH) and Azotochelin (AZO), were assessed for its effectiveness in mending induced chlorosis in soybean (Glycine max). DPH-Fe and AZO-Fe complexes were firstly tested for their soil interactions and capability to maintain Fe in a bioavailable form. Secondly, 57Fe-chelates of DPH and AZO were applied to the soil in a pot experiment with chlorotic soybean plants. Their growth, SPAD chlorophyll index, and the Fe concentration in plant tissues and the remaining soil were evaluated. An isotope deconvolution analysis by using the concentration of the Fe isotopes was used to distinguish the Fe coming from soil and from the 57Fe labelled fertilizer treatments. AZO and DPH have shown different interactions with soil and its components, with AZO showing less interaction than DPH. The application of AZO and DPH resulted in SPAD increase and Fe content. However, it was found that the Fe in plants had not come from the fertilizer application, but instead from natural sources. This is likely due to dissolution phenomena aided by the chelates added. Overall, AZO and DPH have shown good results in amending Fe induced chlorosis in calcareous soils and for this reason should be regarded as good green-candidates for Fe plant nutrition in calcareous soils.

Layered Double Hydroxides: Potential Release-on-Demand Fertilizers for Plant Zinc Nutrition.

A novel zinc (Zn) fertilizer concept based on Zn-doped layered double hydroxides (Zn-doped Mg-Fe-LDHs) has been investigated. Zn-doped Mg-Fe-LDHs were synthesized, their chemical composition was analyzed, and their nutrient release was studied in buffered solutions with different pH values. Uptake of Zn by barley (Hordeum vulgare cv. Antonia) was evaluated in short- (8 weeks), medium- (11 weeks), and long-term (28 weeks) experiments in quartz sand and in a calcareous soil enriched with Zn-doped Mg-Fe-LDHs. The Zn release rate of the Zn-doped Mg-Fe-LDHs was described by a first-order kinetics equation showing maximum release at pH 5.2, reaching approximately 45% of the total Zn content. The Zn concentrations in the plants receiving the LDHs were between 2- and 9.5-fold higher than those in plants without Zn addition. A positive effect of the LDHs was also found in soil. This work documents the long-term Zn release capacity of LDHs complying with a release-on-demand behavior and serves as proof-of-concept that Zn-doped Mg-Fe-LDHs can be used as Zn fertilizers.

Timing for a sustainable fertilisation of Glycine max by using HBED/Fe3+ and EDDHA/Fe3+ chelates.

BACKGROUND: Efficient use of Fe chelates is crucial to avoid environmental risks and reduce economic losses. HBED/Fe3+ has been recently approved by the European Union for soil fertilisation, but studies delving into the best timing for its application are necessary. In this work, a batch incubation experiment and two biological experiments were developed to determine the optimal physiological stage for a sustainable application of HBED/Fe3+ in soil fertilisation compared with EDDHA/Fe3+ fertilisers using 57 Fe. RESULTS: HBED/Fe3+ demonstrated a high durability in soils and soil materials, maintaining more than 80% of Fe chelated after 70 days, and its application at an early physiological stage resulted in a high Fe accumulation in soybean and soil after 36 days. In contrast, the stability of EDDHA/Fe3+ decreased because of the retention of its lowest stable isomers. The best timing for chelates application was confirmed in a 52 day experiment. The application of HBED/Fe3+ at the early stage increased the Fe translocation to fruits; while o,o-EDDHA/Fe3+ accumulated more Fe in fruits when added at the fructification stage. CONCLUSION: The high HBED/Fe3+ stability in calcareous soil requires a differentiate application timing, and its addition at early physiological stages leads into the most efficient fertilisation. © 2016 Society of Chemical Industry.

Novel chelating agents for iron, manganese, zinc, and copper mixed fertilisation in high pH soil-less cultures.

BACKGROUND: Studies about simultaneous fertilisation with several micronutrients have increased in recent years, as Fe, Mn and Zn deficiencies may appear in the same culture conditions. In fertigation, the replacement of sulfates by synthetic chelates is essential in areas with high pH irrigation water and substrates. Ethylenediamine-N-(2-hydroxyphenylacetic acid)-N'-(4-hydroxyphenylacetic acid) (o,p-EDDHA) and ethylenediamine disuccinic acid (EDDS) are novel chelating agents whose efficacy in simultaneous fertilisation of Zn, Mn and Cu is unknown. This work evaluates the effectiveness of both ligands compared to traditional ligands (EDTA, HEEDTA and DTPA) applied as micronutrient chelate mixtures to soybean and navy bean plants grown in soil-less cultures at high pH by analysing the SPAD and micronutrient nutritional status, including the Composition Nutritional Diagnosis (CND) analysis tool. RESULTS: The application of micronutrients using o,p-EDDHA was more effective in providing Mn and Zn than traditional ligands or sulfates. The application using EDDS increased the Zn nutrition. The results are well correlated with the chemical stability of the formulations. CONCLUSION: The combined application of Mn and Zn as o,p-EDDHA chelates can represent a more effective source than traditional chelates in micronutrient fertiliser mixtures in soil-less cultures at a high pH.

Reactivity and effectiveness of traditional and novel ligands for multi-micronutrient fertilization in a calcareous soil.

This study compares the effectiveness of multi-micronutrient formulations containing iron (Fe), manganese (Mn), and zinc (Zn) with traditional (EDTA, DTPA, HEEDTA, and EDDHAm) or novel chelates (o,p-EDDHA, S,S-EDDS, and IDHA) and natural complexing agents (gluconate and lignosulfonate). The stability and reactivity of the formulations were studied on batch experiments with calcareous soil and by speciation modeling. Formulations containing traditional ligands maintained higher Mn but lower Zn concentration in soil solution than the novel ligands. The gluconate and lignosulfonate maintained low concentrations of both Mn and Zn in soil solution. Selected formulations were applied into calcareous soil and their efficacy was evaluated in a pot experiment with soybean. The formulation containing DTPA led to the highest Zn concentration in plants, as well as the formulation containing S,S-EDDS in the short-term, which correlated with its biodegradability. The application of traditional or novel ligands in formulations did not result in sufficient plant Mn concentrations, which was related to the low Mn stability observed for all formulations under moderate oxidation conditions. The results highlight the need to consider the effect of metals and ligands interactions in multi-nutrient fertilization and the potential of S,S-EDDS to be used for Zn fertilization. Furthermore, it is necessary to explore new sources of Mn fertilization for calcareous soils that have greater stability and efficiency, or instead to use foliar fertilization.

Physiological responses in roots of the grapevine rootstock 140 Ruggeri subjected to Fe deficiency and Fe-heme nutrition.

Iron (Fe)-heme containing fertilizers can effectively prevent Fe deficiency. This paper aims to investigate root physiological responses after a short period of Fe-heme nutrition and Fe deficiency under two pH conditions (with or without HEPES) in the Fe chlorosis-tolerant grapevine rootstock 140 Ruggeri. Organic acids in root exudates, Fe reduction capacity, both roots and root exudates contributions, together with other physiological parameters associated to plant Fe status were evaluated in plants grown in hydroponics. Analyses of root tips by SEM, and Raman and IR spectra of the precipitates of Fe-heme fertilizers were performed. The physiological responses adopted by the tolerant 140 Ruggeri to the application of Fe-heme indicated an increased Fe reduction capacity of the roots. This is the first report showing oxalic, tartaric, malic and ascorbic as major organic acids in Vitis spp. root exudates. Plants reacted to Fe deficiency condition exuding a higher amount of ascorbic acid in the rhizosphere. The presence of HEPES in the medium favoured the malic acid exudation. The lowest concentration of oxalic acid was found in exudates of plants subjected to Fe-heme and could be associated to a higher accumulation in their root tips visualized by SEM analysis.

Blood meal-based compound. Good choice as iron fertilizer for organic farming.

Prevention of iron chlorosis with Fe synthetic chelates is a widespread agronomical practice but implies high costs and environmental risks. Blood meal is one of the main fertilizers allowed to be used in organic farming. Through this work a novel blood meal fertilizer was audited. Measurements such as FTIR, Raman, electron paramagnetic resonance, and Mössbauer spectroscopy, UV-visible properties, stability against pH, and batch experiments were performed to characterize and assess the reactivity on soil constituents and agronomic soils. The spectroscopy findings give clear indications that Fe is in the ferric oxidation state, is hexacoordinated, and has a low-spin form suggesting a similar structure to hemin and hematin. A spectrophotometric method at 400 nm was validated to quantify blood meal concentration at low electrolyte concentrations. Batch experiments demonstrated high reactivity of blood meal fertilizer with soil constituents, mainly in the presence of calcium, where aggregation processes are predominant, and its ability to take Fe from synthetic Fe (hydr)oxides. The beneficial profile of blood meal by a providing nitrogen source together with the capability to keep the Fe bound to porphyrin organic compounds makes it a good candidate to be used as Fe fertilizer in organic farming.

Biological activity of Fe(III) aquo-complexes towards ferric chelate reductase (FCR).

In this study we have obtained experimental evidence that confirms the high activity of aquo complexes III and IV towards the enzyme FCR, responsible for the reduction of Fe(III) to Fe(II) in the process of iron acquisition by plants. The in vivo FCR assays in roots of stressed cucumber plants have shown a higher efficiency of the family of complexes III and a striking structure-activity relationship with the nature of the substituent placed in a phenyl group far away from the metal center. The results obtained in this work demonstrate that all the aquo compounds tested interact efficiently with the enzyme FCR and hence constitute a new concept of iron chelates that could be of great use in agronomy.

Demetalation of Fe, Mn, and Cu chelates and complexes: application to the NMR analysis of micronutrient fertilizers.

The application of nuclear magnetic resonance (NMR) for the quality control of fertilizers based on Fe(3+), Mn(2+), and Cu(2+) chelates and complexes is precluded by the strong paramagnetism of metals. Recently, a method based on the use of ferrocyanide has been described to remove iron from commercial iron chelates based on the o,o-EDDHA [ethylenediamine-N,N'bis(2-hydroxyphenylacetic)acid] chelating agent for their analysis and quantification by NMR. The present work extended that procedure to other paramagnetic ions, manganese and copper, and other chelating, EDTA (ethylenediaminetetraacetic acid), IDHA [N-(1,2-dicarboxyethyl)-d,l-aspartic acid], and complexing agents, gluconate and heptagluconate. Results showed that the removal of the paramagnetic ions was complete, allowing us to obtain (1)H NMR spectra characterized by narrow peaks. The quantification of the ligands by NMR and high-performance liquid chromatography showed that their complete recovery was granted. The NMR analysis enabled detection and quantification of unknown impurities without the need of pure compounds as internal standards.

Synthesis and chemical characterization of the novel agronomically relevant pentadentate chelate 2-(2-((2-hydroxybenzyl)amino)ethylamino)-2-(2-hydroxyphenyl)acetic acid (DCHA).

Iron chelates analogous to o,o-EDDHA/Fe(3+) are the fertilizers chosen to treat iron chlorosis in plants growing on calcareous soil. The isomer o,p-EDDHA/Fe(3+) presents less stability but faster assimilation by the plant than o,o-EDDHA/Fe(3+), because only five coordinating groups are able to complex Fe(3+). The new chelating agent 2-(2-((2-hydroxybenzyl)amino)ethylamino)-2-(2-hydroxyphenyl)acetic acid (DCHA) has been synthesized to obtain an iron fertilizer with intermediate stability between o,o-EDDHA/Fe(3+) and o,p-EDDHA/Fe(3+) and with fast assimilation. Its synthesis has been done starting from phenol, N-acetylethylendiamine, glyoxylic acid, and NaOH in a three-step sequence. The purity of the DCHA chelating agent, its protonation, and Ca(2+), Mg(2+), Fe(3+), and Cu(2+) stability constants, together with its ability to maintain iron in solution in different agronomic conditions, have been determined. The results indicate that the chelate DCHA/Fe(3+) has intermediate stability between those of o,o-EDDHA/Fe(3+) and o,p-EDDHA/Fe(3+) complexes and that it is capable of maintaining the Fe(3+) in agronomic conditions. This new chelating agent may be effective in correcting iron chlorosis in plants.

Chemical evaluation of HBED/Fe(3+) and the novel HJB/Fe(3+) chelates as fertilizers to alleviate iron chlorosis.

Iron chelates such as ethylenediamine-N,N'-bis(2-hydroxyphenylacetic) acid (o,o-EDDHA) and their analogues are the most efficient soil fertilizers to treat iron chlorosis in plants growing in calcareous soil. A new chelating agent, HJB (N,N'-bis(2-hydroxy-5-methylphenyl)ethylendiamine-N,N'-diacetic acid) may be an alternative to o,o-EDDHA since its synthesis yields a purer product, but its chemical behavior and efficiency as chlorosis corrector should be evaluated. In this research, a known analogous HBED (N,N'-bis(2-hydroxyphenyl)ethylendiamine-N,N'-diacetic acid) has also been considered. First, an ion-pair high performance liquid chromatography (HPLC) method has been tested for the HJB/Fe(3+) and HBED/Fe(3+) determination. The ability of HJB and HBED to maintain Fe in solution has been compared with respect to o,o-EDDHA. Theoretical modelization for HBED and HJB in agronomic conditions has been done after the determination of the protonation and Ca(II), Mg(II), Fe(III), and Cu(II) stability constants for HJB. Also, batch interaction experiments with soils and soil materials have been conducted. According to our results, HJB/Fe(3+) and HBED/Fe(3+) present high stability, even when competing cations (Cu(2+), Ca(2+)) are present, and have low reactivity with soils and soil components. The chelating agent HJB dissolves a higher amount of Fe than o,o-EDDHA, and it seems as effective as o,o-EDDHA in keeping Fe in solution. These results indicate that these chelates may be very efficient products to correct Fe chlorosis, and additional plant experiments should demonstrate plants' ability to assimilate Fe from HJB/Fe(3+) and HBED/Fe(3+).