Ethylenediamine-di O-hydroxyphenylacetic Acid Research Articles

PvdN Enzyme Catalyzes a Periplasmic Pyoverdine Modification

Pyoverdines are high affinity siderophores produced by a broad range of pseudomonads to enhance growth under iron deficiency. They are especially relevant for pathogenic and mutualistic strains that inhabit iron-limited environments. Pyoverdines are generated from non-ribosomally synthesized highly modified peptides. They all contain an aromatic chromophore that is formed in the periplasm by intramolecular cyclization steps. Although the cytoplasmic peptide synthesis and side-chain modifications are well characterized, the periplasmic maturation steps are far from understood. Out of five periplasmic enzymes, PvdM, PvdN, PvdO, PvdP, and PvdQ, functions have been attributed only to PvdP and PvdQ. The other three enzymes are also regarded as essential for siderophore biosynthesis. The structure of PvdN has been solved recently, but no function could be assigned. Here we present the first in-frame deletion of the PvdN-encoding gene. Unexpectedly, PvdN turned out to be required for a specific modification of pyoverdine, whereas the overall amount of fluorescent pyoverdines was not altered by the mutation. The mutant strain grew normally under iron-limiting conditions. Mass spectrometry identified the PvdN-dependent modification as a transformation of the N-terminal glutamic acid to a succinamide. We postulate a pathway for this transformation catalyzed by the enzyme PvdN, which is most likely functional in the case of all pyoverdines.

Effect of Nano Fe-Chelate, Fe-Eddha and FeSO4 on Vegetative Growth, Physiological Parameters and Some Nutrient Elements Concentrations of Four Varieties of Lettuce (Lactuca sativa L.) In NFT System

In order to investigate the effects of different iron (Fe) sources (nano iron (Fe)-chelate, Fe- ethylenediamine-di(o-hydroxy phenyl acetic acid (EDDHA) and iron (II) sulfate (FeSO4)) on lettuce (Lactuca sativa) growth in alkaline solutions, an experiment was arranged in hydroponic system. This study showed that leaf Fe content and overall plant growth was significantly increased by Fe-chelate application, and the highest values of leaf Fe, plant pigments and vegetative growth were recorded in plants treated with nano Fe-chelate. The lowest Fe, chlorophyll, carotenoids and soluble sugars in leaves were observed in FeSO4 treatment. There were no difference in soluble sugars contents of plants between nano Fe-chelate and Fe-EDDHA treatments. Fertilization of lettuce plants with different Fe-chelate sources had a beneficial effect on the manganese (Mn) and zinc (Zn) uptake in plants. It is concluded that application of chelated form of Fe (especially nano Fe-chelate) must be performed in hydroponic system with alkaline water, to overcome Fe deficiencies and to make better nutritional status.

Iron Chelates Supplied Foliarly Improve the Iron Translocation Rate in Tempranillo Grapevine

Iron (Fe) chlorosis is a widespread nutritional disorder in grapevine, particularly in vineyards developed on calcareous soils. Despite the effective application of highly efficient Fe chelates such as Fe–ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) to the soil to solve the problem, the cost of the treatments and the loss of effectiveness of the chelate in the soil in rainfed crops limit their use. An alternative could be the utilization of Fe fertilizers directly supplied through foliar spray. Two Fe chelates [Fe–ethylenediaminetetraacetate (EDTA) and Fe–ethylenediamine disuccinic acid (EDDS)], an Fe complex (Fe-lignosulfonate), and an Fe salt (Fe-sulfate) were foliarly applied to mature Tempranillo tinto (Vitis vinifera L.), grown on a soil with a high active lime (most reactive calcium carbonate phase in soils) content (92.5 g kg−1 at 30–60 cm deep), and compared to an untreated control. Three shoots per vine and five vines per treatment were selected. One hundred mL of each Fe treatment (5 mM) were sprayed on mature leaves (treated), and young leaves (covered) were covered so Fe translocation could be studied. Treatments were repeated once after 2 weeks. Leaf blades were sampled before the first spray application and at the end of the assay, 4 weeks later, and 2 weeks after the second spray application. Micronutrient content [(Fe, manganese (Mn), copper (Cu), and zinc (Zn)] in leaf blades was determined and Fe/Mn ratio was calculated as a Fe nutrition index. Leaf chlorophyll content was measured weekly using a noninvasive Minolta SPAD chlorophyll meter. As expected, all Fe treatments resulted in greater leaf Fe concentration than in the untreated control; however, leaves sprayed with Fe-EDTA and Fe-sulfate showed the greatest Fe concentration. As consequence, the largest Fe/Mn ratio was found for Fe-EDTA and Fe-sulfate treatments, although no differences with Fe-EDDS treatment were found. Leaves sprayed with Fe-EDTA chelate showed not only the greatest SPAD readings at the end of the field experiment but also the greatest SPAD readings when measured on covered leaves from the 15th day after treatment application when compared to untreated control. Covered leaves from all vines sprayed with Fe treatments showed greater Fe concentrations than untreated control but only the Fe/Mn ratio for Fe-EDTA was significantly greater than that of untreated control. The inconsistency between total Fe concentration from the sprayed leaves and SPAD readings in both sprayed and covered leaves suggests that the proposed methodology is valid for assessing the Fe translocation rate from mature to young leaves when foliar fertilization was applied.

Effects of FeEDDS and EDDS on Peat-based Substrate pH and Cu, Fe, Mn, and Zn Solubility

Common chelating agents used in horticultural fertilizers like ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA) are not readily biodegradable and may persist in the environment, maintaining the capacity to solubilize heavy metals. For this reason, biodegradable chelating agents like ethylenediaminedisuccinic acid (EDDS) are being evaluated for use in horticultural crop production. Therefore, the objectives of the study were to determine the effects of FeEDDS and EDDS on substrate pH and copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) solubility in peat-based substrate compared with various Fe and chelate-ligand sources. Extractions were performed using the 1:2 by volume substrate analysis method with an incubation period of 24 hours. The control was distilled deionized water extractions. Iron-source (FS) extractants consisted of 1 mg·L−1 Fe solutions derived from FeEDDS, FeEDTA, FeDTPA, FeEDDHA, and FeSO4. Iron-source extractant solution pH ranged from 7.1 (FeEDDS) to 5.4 (FeSO4). The extract pH for all Fe-source treatments was similar at pH 6.7, demonstrating the buffering capacity of the peat-based substrate. Iron recovery rates for FS treatments were determined after subtracting Fe that was freely extracted with distilled-deionized water: FeSO4 (13%), FeEDDHA (68%), FeEDDS (73%), FeEDTA (102%), and FeDTPA (121%). Iron-source treatments were not different for Mn, averaging 0.03 mg·L−1, and Cu (0.04 mg·L−1) and Zn (0.24 mg·L−1) were greatest in the FeEDDS treatment. Chelate-ligand (CL) extractants consisted of 5 mm solutions of EDDS, EDTA, and DTPA. Chelate-ligand extractant solution pH ranged from 9.7 (EDDS) to 2.3 (DTPA), and extract solution pH ranged from 7.2 (EDDS) to 4.7 (DTPA). Extractant solutions of EDDS and DTPA resulted in the lowest and highest levels of Cu (0.06 and 0.14 mg·L−1, respectively) and Fe (4.3 and 13.1 mg·L−1, respectively) in extract solutions. Overall, these results suggest that there are no negative implications for the use of FeEDDS with peat-based substrate in terms of horticultural crop production based on substrate Fe solubility, which was not different from FeEDTA.

Global Gene Expression as a Function of the Iron Status of the Bacterial Cell: Influence of Differentially Expressed Genes in the Virulence of the Human Pathogen Vibrio vulnificus

Vibrio vulnificus multiplies rapidly in host tissues under iron-overloaded conditions. To understand the effects of iron in the physiology of this pathogen, we performed a genome-wide transcriptional analysis of V. vulnificus growing at three different iron concentrations, i.e., iron-limiting [Trypticase soy broth with 1.5% NaCl (TSBS) plus ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA)], low-iron (1 microg Fe/ml; TSBS), and iron-rich (38 microg Fe/ml; TSBS plus ferric ammonium citrate) concentrations. A few genes were upregulated under the last two conditions, while several genes were expressed differentially under only one of them. A gene upregulated under both conditions encodes the outer membrane porin, OmpH, while others are related to the biosynthesis of amino sugars. An ompH mutant showed sensitivity to sodium dodecyl sulfate (SDS) and polymyxin B and also had a reduced competitive index compared with the wild type in the iron-overloaded mice. Under iron-limiting conditions, two of the TonB systems involved in vulnibactin transport were induced. These genes were essential for virulence in the iron-overloaded mice inoculated subcutaneously, underscoring the importance of active iron transport in infection, even under the high-iron conditions of this animal model. Furthermore, we demonstrated that a RyhB homologue is also essential for virulence in the iron-overloaded mouse. This novel information on the role of genes induced under iron limitation in the iron-overloaded mouse model and the finding of new genes with putative roles in virulence that are expressed only under iron-rich conditions shed light on the many strategies used by this pathogen to multiply rapidly in the susceptible host.

Isotope pattern deconvolution as a tool to study iron metabolism in plants

Isotope pattern deconvolution is a mathematical technique for isolating distinct isotope signatures from mixtures of natural abundance and enriched tracers. In iron metabolism studies measurement of all four isotopes of the element by high-resolution multicollector or collision cell ICP-MS allows the determination of the tracer/tracee ratio with simultaneous internal mass bias correction and lower uncertainties. This technique was applied here for the first time to study iron uptake by cucumber plants using 57Fe-enriched iron chelates of the o,o and o,p isomers of ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA) and ethylenediamine tetraacetic acid (EDTA). Samples of root, stem, leaves, and xylem sap, after exposure of the cucumber plants to the mentioned 57Fe chelates, were collected, dried, and digested using nitric acid. The isotopic composition of iron in the samples was measured by ICP-MS using a high-resolution multicollector instrument. Mass bias correction was computed using both a natural abundance iron standard and by internal correction using isotope pattern deconvolution. It was observed that, for plants with low 57Fe enrichment, isotope pattern deconvolution provided lower tracer/tracee ratio uncertainties than the traditional method applying external mass bias correction. The total amount of the element in the plants was determined by isotope dilution analysis, using a collision cell quadrupole ICP-MS instrument, after addition of 57Fe or natural abundance Fe in a known amount which depended on the isotopic composition of the sample.

Experimental determination of the dissolution kinetics of zero-valent iron in the presence of organic complexants

Environmental context. Iron metal is being considered as a material to be used for the treatment of groundwater contaminated with toxic metals and organics. Although time-dependant information is available, predicting the long-term behaviour of this material has been complicated by the build-up of rust or other alteration phases on the surface of Fe metal. In addition to the build-up of rust, changes to important environmental factors also complicate these types of predictions. The research discussed in this paper uses a non-traditional experimental technique to isolate the impact of specific environmental factors (i.e. pH, temperature) and organic complexants on the dissolution of Fe metal. Abstract. The geochemical cycling of iron, the reactivity of iron minerals and, more recently, the reactivity of zero valent iron (α-Fe), have been the subject of numerous investigations for over more than three decades. These investigations provide a wealth of knowledge regarding the effect of pH, temperature, chelating agents etc. on the reactivity and mechanism(s) of dissolution for α-Fe and iron oxide/oxyhydroxide minerals. However, most investigations have been conducted under static conditions that promote the formation of a partially oxidised surface film (e.g. passivating layer). In the presence of a passivating layer, the proposed dissolution mechanisms are vastly different and are based on the composition of the partially oxidised surface film. The objective of this study was to quantify the dissolution of α-Fe under conditions that maintain the pO2 at a relatively constant level and minimise the formation of a passivating layer on the metal surface. Single-pass flow-through tests were conducted under conditions of relatively constant dissolved O2 [O2(aq)] over the pH(23°C) range from 7 to 12 and temperature range from 23 to 90°C in the presence of ethylenediamine tetraacetic acid (EDTA) and ethylenediamine di-O-hydroxyphenylacetic acid (EDDHA) to maintain dilute conditions and minimise the formation of a partially oxidised surface film and Fe-bearing secondary phase(s) during testing. Although more information is needed, these results suggest the adsorption of EDTA and EDDHA, or the diffusion of the oxidised Fe–organic complex from the surface of α-Fe, is the rate-limiting step in the dissolution reaction. Results also suggest that the rate of dissolution is independent of pH, has a non-linear dependence on the concentration of organic complexant, and the forward dissolution rate for α-Fe is as much as three orders of magnitude greater than when a passive film and corrosion products are present.

Possible role of jasmonic acid in the regulation of floral induction, evocation and floral differentiation in Lemna minor L.

Jasmonic acid (JA) is implicated in a wide variety of developmental and physiological processes in plants. Here, we studied the effects of JA and the combination of JA and ethylenediamine-dio-hydroxyphenyl-acetic acid (EDDHA) on flowering in Lemna minor in axenical cultures. JA (0.475–47.5 nmol l −1) enhanced floral induction in L. minor under long-day (LD) conditions. Under the same conditions, at a concentration of 237.5 nmol l −1, JA inhibited floral induction, and at a concentration of 475 nmol l –1 it prevented floral induction. Under LD conditions with LD preculture, a combination of EDDHA (20,500 nmol l −1) and JA (47.5 nmol l −1) had a synergistic effect on the promotion of floral induction. Floral induction was enhanced to the greatest extent in experiments with LD precultures. Microscopic examination of microphotographs of histological sections showed that JA and, to an even greater extent, JA + EDDHA at optimal concentrations promote apical floral induction (evocation). Furthermore, JA, and to an even greater extent JA in combination with EDDHA in an optimal concentration, also promote flower differentiation, especially the development of stamens, as is evident from the microphotographs. The experimental results show that JA promotes floral induction in other species of Lemnaceae from various groups according to their photoperiodic response. The results support our hypothesis that, in addition to previously ascribed functions, JA may regulate floral induction, evocation and floral differentiation. Our hypothesis is supported also by the results obtained by quantitative determination of endogenous JA levels in L. minor at three growth stages. The levels of endogenous JA decreased from 389 ng JA g –1 (fresh weight) of L. minor during the vegetative stage to 217 ng JA g –1 during the evocation stage, and to 37.5 ng JA g –1 during the flowering stage, which proves that JA is used for flowering.

Density functional complexation study of metal ions with amino polycarboxylic acid ligands: EDDHA and HBED in comparison to EDTA, EDDS, ODS, and ISA

Metal complexes of amino polycarboxylic acid ligands have been studied using density functional methods and a continuum solvation model (COSMO). Complexation energies for complexes of Mg 2+, Ca 2+, Mn 2+, Fe 3+, and Zn 2+ have been determined. The studied ligands include ethylenediaminedi-( o-hydroxyphenylacetic)acid (EDDHA) (including its ortho, ortho, ortho, para, and para, para-isomers), N, N′-bis(2-hydroxybenzyl)ethylenediamine- N, N′-diacetic acid (HBED), ethylenediamine tetraacetic acid (EDTA), ethylenediiminodibutanedioic acid (EDDS), meso-oxybis(butanedioic acid) (ODS), and 2,2′-bis(carboxymethyl)iminodiacetic acid (ISA). New, more accurate parameterisation is introduced to correct the neglected contributions in energetics. For o, o-EDDHA, the method is tested also for partly deprotonated forms of ligand. The results indicate that o, o-EDDHA 4− and HBED 4− are clearly the most effective ligands for all the metals.

IRON-REGULATED PROTEINS (IRPs) OF LEPTOSPIRA BIFLEXA SEROVAR PATOC STRAIN PATOC I

Background: Iron deficiency has been shown to induce the expression of siderophores and their receptors, the iron-regulated membrane proteins in a number of bacterial systems. In this study, the response of Leptospira biflexa serovar Patoc strain Patoc I to conditions of iron deprivation was assessed and the expression of siderophores and iron-regulated proteins is reported. Materials and Methods : Two methods were used for establishing conditions of iron deprivation. One method consisted of addition of the iron chelators ethylenediamine-N, N’-diacetic acid (EDDA) and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA) and the second method involved the addition of iron at 0.02 µg Fe/mL. Alternatively, iron sufficient conditions were achieved by omitting the chelators in the former method and adding 4 µg Fe/mL of the medium in the latter protocol. Triton X-114 extraction of the cells was done to isolate the proteins in the outer membrane (detergent phase), periplasmic space (aqueous phase) and the protoplasmic cylinder (cell pellet). The proteins were subjected to SDS-PAGE for analysis. Results: In the presence of the iron-chelators, four iron-regulated proteins (IRPs) of apparent molecular masses of 82, 64, 60 and 33 kDa were expressed. The 82-kDa protein was seen only in the aqueous phase, while the other three proteins were seen in both the aqueous and detergent fractions. These proteins were not identified in organisms grown in the absence of the iron chelators. The 64, 60 and the 33 kDa proteins were also demonstrated in organisms grown in media with 0.02 µg Fe/mL. In addition, a 24 kDa protein was found to be down-regulated at this concentration of iron as compared to the high level of expression in organisms grown with 4 µg Fe/mL. The blue CAS agar plates with top agar containing 0.02µg Fe/mL showed a colour change to orange-red. Conclusion: The expression of siderophores and iron-regulated proteins under conditions of iron deprivation was demonstrated in the non-pathogenic L.biflexa serovar Patoc.

Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sitB and sitD genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti.

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

Nature of impurities in fertilizers containing EDDHMA/Fe(3+), EDDHSA/Fe(3+), and EDDCHA/Fe(3+) chelates.

Iron chelates derived from ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA), ethylenediaminedi(o-hydroxy-p-methylphenylacetic) acid (EDDHMA), ethylenediaminedi(2-hydroxy-5-sulfophenylacetic) acid (EDDHSA), and ethylenediaminedi(5-carboxy-2-hydroxyphenylacetic) acid (EDDCHA) are remarkably efficient in correcting iron chlorosis in plants growing in alkaline soils. This work reports the determination of impurities in commercial samples of fertilizers containing EDDHMA/Fe(3+), EDDHSA/Fe(3+), and EDDCHA/Fe(3+). The active components (EDDHMA/Fe(3+), EDDHSA/Fe(3+), and EDDCHA/Fe(3+)) were separated easily from other compounds present in the fertilizers by HPLC. Comparison of the retention times and the UV-visible spectra of the peaks obtained from commercial EDDHSA/Fe(3+) and EDDCHA/Fe(3+) samples with those of standard solutions showed that unreacted starting materials (p-hydroxybenzenesulfonic acid and p-hydroxybenzoic acid, respectively) were always present in the commercial products. 1D and 2D NMR experiments showed that commercial fertilizers based on EDDHMA/Fe(3+) contained impurities having structures tentatively assigned to iron chelates of two isomers of EDDHMA. These findings suggest that current production processes of iron chelates used in agriculture need to be improved.

Involvement of the TonB system in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E.

Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5'phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with (14)C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon. Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.

Remedy of chlorosis induced by iron deficiency in plants with the fungal siderophore rhizoferrin

Although microbial siderophores are characterized by high affinity and selectivity for Fe3+, they are usually less efficient as Fe‐carriers to plants than synthetic chelates. An exception to this is rhizoferrin, a fungal siderophore produced by Rhizopus arrhizus isolated and purified by our group. A ferric complex of rhizoferrin was used in this study as an Fe source for tomato and cucumber ("strategy I") and barley and corn ("strategy II") plants grown in nutrient solutions. The Fe‐rhizoferrin complex was found to be an efficient carrier of Fe to these plants. The efficiency of this chelate is comparable with that of the commonly used ferric complexes of ethylenediaminetetraacetic acid (EDTA) and ethylenediamine‐di(o‐hydroxyphenyl)acetic acid (EDDHA). The application of Fe‐rhizoferrin resulted in enhanced plant weight in barley and corn and higher leaf chlorophyll concentration in tomato, barley and com. Iron uptake by Fe‐stressed cucumber plants from the 59Fe chelate of rhizoferrin and other chelators followed the order: EDDHA > rhizoferrin > EDTA > desferrioxamine B (DFOB); 59Fe translocation from roots to shoots followed the order: rhizoferrin = EDDHA > EDTA > DFOB. The high availability of Fe complexed by rhizoferrin for “strategy I” plants is related to its relatively high redox potential and low affinity to Fe2+. The high availability of Fe‐rhizoferrin to “strategy II” plants is related to its relatively low apparent stability constant with Fe3+, which results in the ability of this compound to transfer Fe3+ via ligand exchange to the phytosiderophores which is specifically taken up by the roots.

Isocratic ion-pair high-performance liquid chromatographic method for the determination of various iron(III) chelates

The micronutrient iron, an essential element for plant growth, is usually added as fertilizer in chelated form. An isocratic ion-pair chromatographic method was developed to identify and determine the total amount of chelate in fertilizers. Iron(III) chelates containing ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, trans-1,2-cyclohexanediaminetetraacetic acid, ethylenediaminedi( o-hydroxyphenylacetic) acid (EDDHA), also known as N,N′-ethylenebis-2-( o-hydroxyphenyl)glycine, ethylenediaminedi( o-hydroxy- p-methylphenylacetic) acid, N,N′-bis-(2-hydroxybenzyl)ethylenediamine-N,N′-dipropionic acid and N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid were well separated by this method. The mobile phase contained 0.03 M tetrabutylammonium chloride and 30% acetonitrile at pH 6.0. The stationary phase was a LiChrospher RP-18 column, the injection volume was 20 μl and the flow-rate was 1.5 ml/min. For the iron(III)-EDDHA chelate, linear range studies showed that the method is capable of determining Fe concentrations between 0.5 and 150 μg/ml, which permits the determination of the concentrations found in commercial fertilizers. With this method, separation and identification of the iron(III) complexes were obtained with good resolution and selectivity, including the separation of the geometric isomers of the complexes, in 15 min.

Methods for the detection of Pseudomonas siderophores

The potentials of different testing methods for detecting the production of siderophores (universal chemical assay (CAS-agar assay), absorption spectrum, bioassay) were investigated. Twelve strains (5 species) of the genus Pseudomonas, including known siderophore producers and wild natural isolates, were included in the study. Combination of different methods to detect the production of bacterial siderophores provided more information than when using e.g. the CAS-agar assay alone. The bioassay was useful when the specificity of siderophores and their affinity for iron were investigated. Negative results in the CAS-agar assay and in absorption spectrum studies did not mean that competitive ligands against ethylenediamine-di-( o-hydroxyphenyl) acetic acid (EDDA) could not be produced, as was observed in the bioassay. The use of more than one test is essential when selecting bacterial strains for agroindustrial applications such as plant disease control.

Influence of nutrients on the development of gibbosity in fronds of the duckweedLemna gibbaL.

The duckweeds Lemna gibba L. and Lemna minor L. only grew well in undisturbed culture under axenic conditions in low light intensity when provided with a suitable energy source such as glucose. In media containing N03-N gibbosity (a convex ventral surface) was induced in the presence of the chelating agent ethylene-diamine-di-o-hydroxyphenylacetic acid (EDDHA). In nutrient solutions containing NO3-N as the only N source, but without EDDHA, L. gibba occasionally exhibited gibbosity in culture solutions of 40 cm3 volumes. More fronds were induced to exhibit gibbosity when the volume of the culture medium was increased from 40 cm3 to 200 cm3. Gibbosity was never induced in L. minor, neither was it induced in L. gibba in media containing NH4-N, even in the presence of NO3-N. There was no direct correlation between the occurrence of gibbosity and frond growth rate, but gibbosity occurred only when there was good frond growth. In the absence of a sugar, frond growth was enhanced by bubbling air through the culture solution in the light. Increasing the CO2 concentration in the air up to 1% enhanced growth and induced gibbosity. Carbon dioxide did not induce gibbosity in media containing NH4-N.

Chromatographic separations of metal chelates present in commercial fertilizers: I. Development of a gel permeation chromatographic separation method for the identification of metal chelates in commercial fertilizers

The separation of iron(III) chelates of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), hydroxy-2-ethylenediaminetriacetic acid (HEEDTA), ethylenediaminedi( o-hydroxyphenyl acetic acid (EDDHA) and ethylenediaminedi( o-hydroxy- p-methylphenyl)acetic acid (EDDHMA) and the Cu(II), Zn(II) and Mn(II) chelates of EDTA by gel permeation chromatography on Bio-Gel P2 and Fractogel HW-40(S) was investigated. The chelates FeDTPA, FeHEEDTA, FeEDDHA and FeEDDHMA were separated on a 35 × 1 cm I.D. Fractogel column using 0.05 M NaCl-0.005 M KH 2PO 4 (pH 8.0) with a total analysis time of 3 h. FeEDTA was not completely resolved from FeDTPA, and FeHEEDTA, CuEDTA, ZnEDTA and MnEDTA were found to co-elute with FeDTPA. The method is applicable to the qualitative analysis of fertilizers containing a single trace element in combination with a single chelating agent. Positive identification of a chelate is achieved by comparison of retention time data and identification of the metal present by atomic absorption spectrometry or inductively coupled plasma mass spectrometry. Quantification can be achieved by comparison of peak height or area with that of standards.

IRON HUMATE AS A SOURCE OF IRON FOR CITRUS IN HIGH-pH SOILS

Availability of iron (Fe) to plants is dependent on the stability of chelate to bind Fe. In soils with pH greater than 7.5, only EDDHA (Ethylenediamine di-o-hydroxyphenyl acetic acid) is the stable chelate to complex Fe. However, Fe EDDHA is an expensive material. Iron humate (IH) is a by-product of drinking water treatment, wherein, Fe is complexed with humic and fulvic fractions of the river water. Field experiments using Hamlin orange and Flame grapefruit on Swingle citrumelo rootstock conducted in two locations with high pH soils (> 7.5) have shown a significant increase in leaf Fe concentrations when IH was applied to the soil. Since the solubility of Fe from IH is lower than that of Fe EDDHA, the long-term effects of IH on growth and mineral nutrition of citrus are under investigation.