Synthesis of o,p-EDDHA and its detection as the main impurity in o,o-EDDHA commercial iron chelates

Ethylenediamine-N,N'bis(o-hydroxyphenyl)acetic acid (o,o-EDDHA) is one of the most efficient iron chelates employed to relieve iron chlorosis in plants. However, the presence of positional isomers of EDDHA in commercial iron chelates has been recently demonstrated, and among them, it has been claimed that ethylenediamine-N(o-hydroxyphenylacetic)-N'(p-hydroxyphenylacetic) acid (o,p-EDDHA) is the main impurity present in EDDHA fertilizers. Here we report the preparation of o,p-EDDHA, a compound whose synthesis had not been previously reported. The synthetic o,p-EDDHA is able to form ferric complexes, and it has been used as a standard in the analysis of the impurities of commercial iron fertilizers. The presence of o,p-EDDHA/Fe(3+) in commercial samples has been unambiguously demonstrated by HPLC.     

Theoretical speciation of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) in agronomic conditions

The presence of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) as the second largest component in commercial EDDHA iron chelates has recently been demonstrated. Here is reported the speciation of o,p-EDDHA by the application of a novel methodology through the determination of the complexing capacity, protonation, and Ca(2+), Mg(2+), Cu(2+), and Fe(3+) stability constants. The pM values and species distribution in solution, hydroponic, and soil conditions were obtained. Due to the para position of one phenol group in o,p-EDDHA, the protonation constants and Ca and Mg stability constants have different values from those of o,o-EDDHA and p,p-EDDHA regioisomers. o,p-EDDHA/Fe(3+) stability constants are higher than those of EDTA/Fe(3+) but lower than those of o,o-EDDHA/Fe(3+). The sequence obtained for pFe is o,o-EDDHA/Fe(3+) >/= o,p-EDDHA/Fe(3+) > EDTA/Fe(3+). o,p-EDDHA/Fe(3+) can be used as an iron chelate in hydroponic conditions. Also, it can be used in soils with limited Cu availability.     

Chromatographic determination of Fe chelated by ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid in commercial EDDHA/Fe3+ fertilizers

EDDHA/Fe3+ chelates are the most common fertilizers used to solve Fe chlorosis in established crops. Commercial products contain two regioisomers, ethylenediamine-N,N'-bis(o-hydroxyphenylacetic) acid (o,o-EDDHA)/Fe3+ and ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA)/Fe3+. Although several chromatographic methods exist for the determination of Fe3+ chelated by the o,o-EDDHA isomer, no method has been described for the quantification of Fe3+ chelated by o,p-EDDHA. In this work, factors that affect the behavior of o,p-EDDHA/Fe3+ in ion pair chromatography are reviewed: pH, ion pair reagent, and organic modifier. The best chromatographic performance was obtained with an aqueous mobile phase at pH 6.0 containing 35% acetonitrile and 5 mM tetrabutylammonium hydroxide under isocratic elution conditions. This method was applied to the quantification of commercial samples.     

Influence of the soil/solution ratio, interaction time, and extractant on the evaluation of iron chelate sorption/desorption by soils

Synthetic Fe chelates are the most efficient agricultural practice to control Fe deficiency in crops, EDTA/Fe3+ and o,o-EDDHA/Fe3+ being the most commonly used. Their efficacy as Fe sources and carriers in soils can be severely limited by their retention on it. The aim of this work is to evaluate the possible bias introduced in the studies of the iron chelate retention by soils. For that purpose, results obtained for EDTA and EDDHA iron chelates from two batch studies with different soil/solution ratios were compared with data obtained for a leaching column experiment. Moreover, different extractants were tested to study the o,o-EDDHA/Fe3+ and o,p-EDDHA/Fe3+ desorption from a calcareous soil, and also the effect of the interaction time in their retention process has been evaluated. In summary, the mobility through a calcareous soil of the studied iron chelates differs greatly depending on the type of iron chelate and also on the procedure used to evaluate the retention and the soil/solution ratio used. In general, the leaching column method is preferred because the achieved conclusions are more representative of the natural conditions, but batch methods are very useful as a preliminary experiment, especially one with a high soil/solution ratio. The iron chelate desorption could be quantified by using a sequential extraction with water, sodium sulfate, and DTPA as extractants. Under the experimental conditions used in this study, o,o-EDDHA/Fe3+ retention increased with interaction time.     

Kinetic behavior of Fe(o,o-EDDHA)-humic substance mixtures in several soil components and in calcareous soils

Ferric ethylenediamine- N, N'-bis-(o-hydroxyphenylacetic)acid chelate (Fe(o, o-EDDHA)) is one of the most effective Fe fertilizers in calcareous soils. However, humic substances are occasionally combined with iron chelates in drip irrigation systems in order to lower costs. The reactivity of iron chelate-humic substance mixtures in several soil components and in calcareous soils was investigated through interaction tests, and their behavior was compared to the application of iron chelates and humic substances separately. Two commercial humic substances and two Fe(o, o-EDDHA) chelates (one synthesized in the laboratory and one commercial) were used to prepare iron chelate-humic substance mixtures at 50% (w/w). Various soil components (calcium carbonate, gibbsite, amorphous iron oxide, hematite, tenorite, zincite, amorphous Mn oxide, and peat) and three calcareous soils were shaken for 15 days with the mixtures and with iron chelate and humic substance solutions. The kinetic behavior of Fe(o, o-EDDHA) and Fe non-(o,o-EDDHA) (Fe bonded to (o,p-EDDHA) and other polycondensated ligands) and of the different nutrients solubilized after the interaction assay was determined. The results showed that the mixtures did not significantly reduce the retention of Fe(o, o-EDDHA) and Fe non-(o,o-EDDHA) in the soil components and the calcareous soils compared to the iron chelate solutions, but they did produce changes in the retention rate. Moreover, the competition between humic substances and synthetic chelating agents for complexing metal cations limited the effectiveness of the mixtures to mobilize nutrients from the substrates. The presence of Fe(o, p-EDDHA) and other byproducts in the commercial iron chelate had an important effect on the evolution of Fe(o, o-EDDHA) and the nutrient solubilization process.     

NMR analysis of the iron ligand ethylenediaminedi(o-hydroxyphenyl)acetic acid (EDDHA) employed in fertilizers

The exceptional efficiency of the iron chelate of ethylenediaminedi(o-hydroxyphenyl)acetic acid (o,o-EDDHA) in correcting iron chlorosis in plants and the medical applications of various metallic chelates of this compound have long been recognized. As commercial preparations of o,o-EDDHA usually contain impurities, a method for their detection is proposed. By using one- and two-dimensional nuclear magnetic resonance two impurities were identified. The structure of one of these compounds was assigned to an isomer of EDDHA containing at least one p-hydroxyphenyl moiety. The structure of the other impurity was tentatively assigned to a byproduct of the EDDHA synthesis: 2,6-di[CH(COOH)NHCH(2)CH(2)NHCH(COOH)Ar]phenol (Ar = hydroxyphenyl). Both compounds were also detected in the EDDHA extracted from a commercial iron fertilizer.     

Fe uptake from meso and D,L-racemic Fe(o,o-EDDHA) isomers by strategy I and II plants

One of the most efficient fertilizers to correct Fe deficiency in calcareous soils and waters with high bicarbonate content is based on ferric ethylenediamine-N,N'-bis(o-hydroxyphenylacetic) acid [Fe(o,o-EDDHA)]. Fe(o,o-EDDHA) forms two groups of geometric isomers known as meso and D,L-racemic. To determine the Fe uptake from meso and D,L-racemic Fe(o,o-EDDHA), four iron-efficient plants, two plants representative of strategy I (tomato and pepper) and two plants representative of strategy II (wheat and oats), were grown in hydroponic culture. Results indicated that strategy II plants took up iron from both Fe(o,o-EDDHA) isomers equally. However, strategy I plants took mainly the iron associated with the meso form (the lowest stability isomer).     

Efficacy of Fe(o,o-EDDHA) and Fe(o,p-EDDHA) isomers in supplying Fe to strategy I plants differs in nutrient solution and calcareous soil

The FeEDDHA [iron(3+) ethylenediamine di(o-hydroxyphenylacetic) acid] is one of the most efficient iron chelates employed in the correction of iron clorosis in calcareous soils. FeEDDHA presents different positional isomers: the ortho-ortho (o,o), the ortho-para (o,p), and the para-para (p,p). Of these isomers, the p,p cannot chelate Fe in soil solution in a wide range of pH values, while both o,o and o,p can. The objective of this work was to compare the efficiency of both isomers (o,o and o,p) to provide Fe to two Strategy I plants (tomato and peach) in nutrient solution (pH approximately 6.0), as well as in calcareous soil (pH approximately 8.4; CALCIXEREPT). For this, chelates of both o,o-EDDHA and o,p-EDDHA with 57Fe (a nonradioactive isotope of Fe) were used, where the 57Fe acts as a tracer. The results obtained showed that the o,o isomer is capable of providing sufficient Fe to plants in both nutrient solution and calcareous soil. However, the o,p isomer is capable of providing sufficient Fe to plants in nutrient solution but not in calcareous soil.     

Fe Chelates for Remediation of Fe Chlorosis in Strategy I Plants

Abstract

Iron chlorosis is a mineral disorder due to low Fe in the soil solution and the impaired plant uptake mechanism. These effects increased with high pH and bicarbonate buffer. The solution to Fe chlorosis should be made by either improving the Fe uptake mechanism or increasing the amount of Fe in the soil solution. Among Fe fertilizers, only the most stable chelates (EDDHA and analogous) are able to maintain Fe in the soil solution and transport it to the plant root. In commercial products with the same chelating agent, the efficacy depends on the purity and the presence of subproducts with complexing activity, that can be determined by appropriate analytical methods such as HPLC. In commercial products declaring 6% as Fe‐EDDHA, purity varied from 0.5% to 3.5% before 1999, but in 2002 products ranging 3–5.4% chelated Fe are common in the Spanish market. Fe‐o,p‐EDDHA, as a synthesis by‐product with unknown efficacy, is present in all Fe‐EDDHA formulations. Commercial Fe‐EDDHMA products also contain methyl positional isomers. Fe‐EDDHSA synthesis produces condensation products with similar chelating capacity to the Fe‐EDDHSA monomer that can account for more than 50% of the chelated iron in the commercial products. Chelates with different molecules should be compared for their efficacy considering firstly their ability to maintain Fe in solution and secondly their capacity to release iron to the roots. Accepting the turnover hypothesis, their efficacy is also dependent thirdly on the ability of the chelating agent to form the chelate using native iron from the soil. The 1st and 3rd points are related to the chemical stability of the chelate, while plants make better use of iron from the less stable chelates. Plant response is the ultimate evaluation method to compare commercial products with the same chelating agent or different chelates.     

Effects of phosphate carriers,iron, and indoleacetic acid on iron nutrition and productivity of peanut on a calcareous soil

Results of a field experiment designed to assess the effects of phosphate carriers, iron (Fe), and indoleacetic acid (IAA) on the Fe nutrition of peanut grown on a calcareous soil showed that single superphosphate (SSP) was more effective than diammonium phosphate (DAP) in improving Fe nutrition and chlorophyll synthesis. Increased phosphorus (P) and Fe contents of chlorotic leaves showing symptoms of Fe deficiency suggested that Fe, despite absorption and uptake, was subjected to inactivation, and that the Fe content per se was not the cause of the observed chlorosis. Better amelioration of chlorosis with the SSP treatment as compared with DAP indicated a role of sulphur (S) in preventing inactivation of Fe, possibly caused by excessive P accumulation. A foliar spray of Fe‐EDDHA corrected the chlorosis, but a ferric citrate foliar treatment did not. This further suggested that the mobility of Fe was impaired in chlorotic plants. An IAA foliar spray only also tended to improve Fe nutrition. Significant increase in peanut productivity was observed following improvement in Fe nutrition both with soil and foliar treatments.     

Interaction of iron chelates with several soil materials and with a soil standard

Frequently the effectiveness of iron (Fe) chelates is low because they can be retained or destroyed by soil materials. The high cost of these Fe fertilizers makes it necessary to study soil material reaction with Fe chelates. Commercial Fe chelates with EDTA, EDDHA, and EDDHMA as ligands and their standards, prepared in the laboratory, were shaken for one hour with various soil materials [amorphous Fe(III) oxide, acid peat, calcium (Ca)‐montmorillonite and calcium carbonate (CaCO₃)] and with a soil standard made in the laboratory. After agitation, the chelate‐soil mixtures were filtered and the micronutrients and chelated Fe that remained in solution were determined. Among the soil materials used, amorphous Fe(III) oxide and acid peat had the greatest affect on the amount of chelated Fe remaining in solution. The type of chelating agent was the next major factor that affected the availability of soluble Fe following reaction with the soil materials. Another factor was the commercial formulation of the Fe chelates. The chelates comprised of EDDHA or EDDHMA maintained the highest percentages of chelated Fe in solution after interaction with the solid phases, except for the acid peat. The last soil material, acid peat, retained more chelated Fe for the Fe chelates with EDDHA or EDDHMA than with EDTA as the chelating agent. The commercial Fe‐EDDHA chelates had greater losses of chelated Fe than their standard after interaction with all the solid phases. The commercial Fe‐EDDHA chelate (Sequestrene) and the commercial Fe‐EDDHMA chelate (Hampirón) solubilized the highest amount of copper (Cu) from soil standard. This was attributed to the presence of by‐products in the commercial formulations since the Fe‐EDDHA standard did not have Cu in solution after the interaction. Therefore, the commercial Fe chelate by‐products are able to form Cu‐complexes which could affect chelated Fe and its availability to plants.     

Depressed growth of non‐chlorotic vine grown in calcareous soil is an iron deficiency symptom prior to leaf chlorosis

The development of iron deficiency symptoms (growth depression and yellowing of the youngest leaves) and the distribution of iron between roots and leaves were investigated in different vine cultivars (Silvaner, Riparia 1G and SO4) grown in calcareous soils. As a control treatment all cultivars were also grown in an acidic soil. Only the cultivars Silvaner and Riparia 1G showed yellowing of the youngest leaves under calcareous soil conditions at the end of the cultivation period. All cultivars including SO4 showed severe shoot growth depression, by 50 % and higher, before yellowing started or without leaf yellowing in the cultivar SO4. Depression of shoot growth occurred independently from that of root growth. In a further treatment the effect of Fe‐EDDHA spraying onto the shoot growth of the cultivar Silvaner after cultivation in calcareous soil was investigated. Prior to Fe application plants were non‐chlorotic, but showed pronounced shoot growth depression. Spraying led to a significant increase in shoot length, though leaf growth was not increased. Accordingly, depression of shoot growth of non‐chlorotic plants under calcareous soil conditions and with ample supply of nutrients and water has been evidenced to be at least partly an iron deficiency symptom. We suggest that plant growth only partially recovered because of dramatic apoplastic leaf Fe inactivation and/ or a high apoplastic pH which may directly impair growth. Since growth was impaired before the youngest leaves showed chlorosis we assume that meristematic growth is more sensitively affected by Fe deficiency than is chlorophyll synthesis and chloroplast development. In spite of high Fe concentrations in roots and leaves of the vines grown in calcareous soils plants suffered from Fe deficiency. The finding of high Fe concentrations also in young, but growth retarded green leaves is a further indication that iron deficiency chlorosis in calcareous soils is caused by primary leaf Fe inactivation. However, in future, only a rigorous study of the dynamic changes of iron and chlorophyll concentration, leaf growth and apoplastic pH at the cellular level during leaf development and yellowing will provide causal insights between leaf iron inactivation, growth depression, and leaf chlorosis.<?show $6#>     

Influence of micronutrient and phosphorous levels and chelator to iron ratio on growth,chlorosis, and nutrition of bluecrop highbush blueberries

‘Bluecrop’ blueberry plants were exposed to 2×2×2 factorial treatments of high and low phosphate and micronutrients (Cu, Zn, Mn, and B) and excess or stoichiometric concentrations of a chelator (EDDHA) added to Fe(NO₃), in pH 6.8 solution cultures. The effects on growth, Fe chlorosis and overall nutrition were accessed. Phosphorous was applied at 20 (low) or 400 (high) μM levels, micronutrients at low or high levels and either a 1:1 or 10:1 ratio of chelator to Fe concentration. Plants grown in excess chelator and low micronutrient supply grew normally. Plants grown in all high micronutrient solutions were chlorotic after 34 days. Plants in low micronutrient and stochiometric chelator to Fe ratio solutions were also chlorotic.

Foliar Fe was not a good indicator of Fe chlorosis development. High Cu levels in roots grown in high micronutrients without excess chelator may have been a causative factor in Fe chlorosis development, but not in reducing foliar Fe levels. There was no indication that excess chelator facilitated Fe transport as has been reported for other crops. However, this work demonstrates that highbush blueberry has an effective ability to absorb Fe from strong Fe chelates and remain non‐chlorotic even at high solution pH levels under an all nitrate‐nitrogen regime. Chemical name used: EDDHA‐ethylenediaminedi‐o‐hydroxyphenlyacetic acid.     

Uptake of Iron (59Fe) Complexed to Water‐Extractable Humic Substances by Sunflower Leaves

Abstract

A research was carried out to evaluate the leaves' ability to utilize Fe supplied as a complex with water‐extractable humic substances (WEHS) and the long‐distance transport of ⁵⁹Fe applied to sections of fully expanded leaves of intact sunflower (Helianthus annuus L.) plants. Plants were grown in a nutrient solution containing 10 µM Fe(III)‐EDDHA (Fe‐sufficient plants), with the addition of 10 mM NaHCO₃ to induce iron chlorosis (Fe‐deficient plants). Fe(III)‐WEHS could be reduced by sunflower leaf discs at levels comparable to those observed using Fe(III)‐EDTA, regardless of the Fe status. On the other hand, ⁵⁹Fe uptake rate by leaf discs of green and chlorotic plants was significantly lower in Fe‐WEHS‐treated plants, possibly suggesting the effect of light on photochemical reduction of Fe‐EDTA. In the experiments with intact plants, ⁵⁹Fe‐labeled Fe‐WEHS or Fe‐EDTA were applied onto a section of fully expanded leaves. Irrespective of Fe nutritional status, ⁵⁹Fe uptake was significantly higher when the treatment was carried out with Fe‐EDTA. A significant difference was found in the amount of ⁵⁹Fe translocated from treated leaf area between green and chlorotic plants. However, irrespective of the Fe nutritional status, no significant difference was observed in the absolute amount of ⁵⁹Fe translocated to other plant parts when the micronutrient was supplied either as Fe‐EDTA or Fe‐WEHS. Results show that the utilization of Fe complexed to WEHS by sunflower leaves involves an Fe(III) reduction step in the apoplast prior to its uptake by the symplast of leaf cells and that Fe taken up from the Fe‐WEHS complexes can be translocated from fully expanded leaves towards the roots and other parts of the shoot.     

Potential use of biodegradable chelate N-(1,2-dicarboxyethyl)-D,L-aspartic acid/Fe3+ as an Fe fertilizer

In the past several years, concern about the environmental fate of recalcitrant synthetic ligands (e.g., EDTA) has increased. The used of new biodegradable chelating agents such as imidodisuccinic acid (IDHA) has been proposed as an alternative. However, its application as an iron ligand to correct iron chlorosis in agriculture has not yet been studied. Then the objective of this work is to determine the fertilizer capacity of IDHA/Fe3+ using interaction assays with soils and soil materials and evaluating Fe nutrition of efficient and susceptible plants. Interaction of IDHA/Fe3+ with soil materials produces a reduction of the amount of soluble Fe. This is in good agreement with studies on the stability of the IDHA/Fe3+ chelate. In general, plant response to IDHA/Fe3+ in hydroponics is acceptable and better than that to EDTA/Fe3+. This good behavior seems to be related to the lower coordination of the iron in IDHA/Fe3+ with respect to EDTA/Fe3+.     

Iron fertilizers applied to calcareous soil on the growth of peanut in a pot experiment

A greenhouse pot experiment was conducted with peanuts (Arachis hypogaea L., Fabceae) to evaluate iron compound fertilizers for improving within-plant iron content and correcting chlorosis caused by iron deficiency. Peanuts were planted in containers with calcareous soil fertilized with three different granular iron nitrogen, phosphorus and potassium (NPK) fertilizers (ferrous sulphate (FeSO₄)–NPK, Fe–ethylendiamine di (o-hydroxyphenylacetic) (EDDHA)–NPK and Fe–citrate–NPK). Iron nutrition, plant biomass, seed yield and quality of peanuts were significantly affected by the application of Fe–citrate–NPK and Fe–EDDHA–NPK to the soil. Iron concentrations in tissues were significantly greater for plants grown with Fe–citrate–NPK and Fe–EDDHA–NPK. The active iron concentration in the youngest leaves of peanuts was linearly related to the leaf chlorophyll (via soil and plant analyzer development measurements) recorded 50 and 80 days after planting. However, no significant differences between Fe–citrate–NPK and Fe–EDDHA–NPK were observed. Despite the large amount of total iron bound and dry matter, FeSO₄–NPK was less effective than Fe–citrate–NPK and Fe–EDDHA–NPK to improve iron uptake. The results showed that application of Fe–citrate–NPK was as effective as application of Fe–EDDHA–NPK in remediating leaf iron chlorosis in peanut pot-grown in calcareous soil. The study suggested that Fe–citrate–NPK should be considered as a potential tool for correcting peanut iron deficiency in calcareous soil.     

Distribution and secondary effects of EDDHA in some vegetable species

Tomato (Lycopersicum esculentum), cucumber (Cucumis sativus), pepper (Capsicum annuum), and lettuce (Lactuca sativa) were grown on rockwool or perlite substrate with nutrient solution. Fe was administered as the Fe complex of the chelator ethylenediamine di-(o-hydroxyphenylacetic acid) (EDDHA) or Fe(NH₄)₂(SO₄)₂ in the nutrient solution or as inorganic iron in the substrate. Roots and leaves of plants grown on Fe-EDDHA contained EDDHA in quantities up to 0.27 × the amount of Fe, which is interpreted as an indication of the contribution of passive chelate absorption to Fe uptake. Fruits of tomato and pepper, and leaves of lettuce contained only traces of EDDHA. Breakdown of the chelator in leaves of pepper and tomato is estimated to have been between 0.5 and 2% per day. In tomato fruits, lycopene content was lowered in plants growing on Fe-EDDHA. Cucumber growing on Fe-EDDHA suffered from serious infection by the mildew Sphaerotheca fusca; the plants growing on an inorganic source of iron were resistant. These results exemplify physiological effects of EDDHA other than those directly associated with iron nutrition.     

Effect of different nitrogen‐forms and iron‐chelates on the development of stinging nettle

The development of stinging nettle (Urtica dioica L.) grown on culture solution containing with either ammonium or nitrate ions, or urea, was investigated under iron deficiency conditions, and with added FeEDTA or FeCto. Both seed‐cultured and vegetatively‐cultured stinging nettle plants produced normally developed green shoots when nitrate and 4 μM FeEDTA or FeCto were supplied. Stinging nettle plants were able to utilize Fe‐citrate, Fe‐ascorbate, and Fe‐malate effectively at the same concentration as well. When K3Fe(CN)6 was supplied, which is impermeable to the plasmalemma, and therefore is used to measure the reductive capacity of the roots, stinging nettle plants became chlorotic because the complex was stable at the pH of the culture solution. Urea did not induce chlorosis but inhibited growth. The plants died when ammonium was supplied as a sole N source. Applying bicarbonate and ammonium together prevented the plants from dying, but the plants became chlorotic. Total exclusion of iron from the culture solution resulted in iron‐deficiency stress reactions as has been described for other dicotyledonous plants (Strategy II).     

Iron‐Enriched Azolla as a slow‐release biofertilizer for cucumber plants grown in a hydroponic system

The unique ability of dried plant residues Azolla to adsorb iron (Fe) was employed to formulate and test an organic Fe biofertilizer. A simplified experimental system was established to examine the effectiveness of Fe‐enriched Azolla as a source of Fe for the remedy of Fe‐deficient plants. The optimal Fe‐enrichment level needed to achieve a complete recovery of starved plant by the Fe‐Azolla complex was tested using a bioassay system of hydroponically grown cucumbers. Dried Azolla plants were mixed a with a solution of ferrous sulfate (FeSO₄) at pH 2.0, rinsed, and dried to form organic, compact material containing 4% (w/w) Fe bound to Azolla. The Fe‐Azolla complex was applied to the nutrient solutions of Fe‐deficient cucumber seedlings. Growth rates and development measurements as well as chlorophyll and the Fe‐containing catalase activity tests have been performed. The effect of the slowly released Fe in correcting Fe deficiency were followed for three weeks and compared with the efficiency of additions of several synthetic Fe chelates. Iron‐starved plants exhibited fast regreening of the chlorotic interveinal tissues after the addition of Fe‐Azolla complex to the nutrient solutions. Iron starvation decreased the activity of catalase. Iron‐treated‐starved plants exhibited recovery of catalase activity compared to the low level activity measured untreated Fe‐starved plants. Iron‐enriched Azolla treatment was found equivalent to Fe‐EDTA and Fe‐EDDHA. This study is the first step in our research program aimed to establish the application of Fe‐enriched Azolla as a bioagent for the benefit of Fe‐deficient crops.     

Micronutrient deficiencies in rainfed calcareous soils of Pakistan. I. Iron chlorosis in the peanut plant

Abstract

Peanut (Arachis hypogaea L.) is susceptible to iron (Fe) chlorosis, however, plant analysis diagnostic criteria are lacking for determining the intensity of chlorosis in this crop. As total Fe content is a misleading index of Fe nutritional status of plants, determination of physiologically active Fe fraction (Fe²⁺) is suggested for the purpose. In a nutrient indexing survey of the chlorosis‐affected peanut crop grown in the rainfed Potohar plateau of Pakistan, o‐phenanthroline extractable Fe²⁺ concentration in plants decreased with increasing severity of chlorosis and thus proved an effective technique for determining the intensity of Fe chlorosis. Green plants contained 40.1 to 67.3 mg Fe²⁺/kg, mildly chlorotic 32.1 to 40.0 mg Fe²⁺/kg, moderately chlorotic 28.0 to 32.0 mg Fe²⁺/kg, and severely chlorotic <28.0 mg Fe²⁺/kg. The minimum Fe²⁺ requirement in green plants was estimated to be 40 mg/kg on dry weight basis. In rainfed field experiments on a calcareous Typic Hapludalfs soil, foliar sprays of 1% solution of sequestrene (NaFeEDDHA) proved superior to the foliar sprays of 0.5% FeSO₄.7H₂O in correcting Fe chlorosis in two cultivars of peanut. Maximum increase in pod yield with sequestrene was 42% in cv. BARD‐92 and 27% in cv. BARD‐699 over the respective control yields. Ferrous concentration in plants increased with both the Fe sources, however, a substantial increase was recorded only with sequestrene. As peanut is a low‐input high‐risk rainfed crop, correction of Fe chlorosis by using sequestrene may not be economically feasible. Thus, development and/or screening of peanut varieties tolerant to Fe chlorosis is suggested by employing Fe²⁺ analysis technique.