Adamo Domenico Rombolà

Iron deficiency and chlorosis in orchard and vineyard ecosystems

Abstract Several perennial, deciduous, as well as evergreen fruit crops develop symptoms of iron deficiency—interveinal chlorosis of apical leaves—when cultivated in calcareous and alkaline soils. Under these conditions fruit yield and quality is depressed in the current year and fruit buds poorly develop for following year fruiting. This paper reviews the main fundamental and applied aspects of iron (Fe) nutrition of deciduous fruit crops and grapevine and discusses the possible development of sustainable Fe nutrition management in orchard and vineyard ecosystems. Cultivated grapevines and most deciduous fruit trees are made up of two separate genotypes the cultivar and the rootstock, providing the root system to the tree. The effect of the rootstock on scion tolerance of Fe chlorosis is discussed in terms of biochemical responses of the roots to acquire iron from the soil. Symptoms of iron chlorosis in orchards and vineyards are usually more frequent in spring when shoot growth is rapid and bicarbonate concentration in the soil solution buffers soil pH in the rhizosphere and root apoplast. Since the solubility of Fe-oxides is pH dependent, under alkaline and calcareous soils inorganic Fe availability is far below that required to satisfy plant demand, so major role on Fe nutrition of trees is likely played by the iron chelated by microbial siderophores, chelated by phytosiderophores (released into the soil by graminaceous species) and complexed by organic matter. As most fruit tree species belong to Strategy I-based plants (which do not produce phytosiderophores in their roots) Fe uptake is preceded by a reduction step from Fe3+ to Fe2+. The role of ferric chelate reductase and proton pump activities in Fe uptake and the possible adoption of these measurements for screening procedure in selecting Fe chlorosis tolerant rootstocks are discussed. In a chlorotic leaf the existence of Fe pools which are somehow inactivated has been demonstrated, suggesting that part of the Fe coming from the roots does not pass the leaf plasmamembrane and may be confined to the apoplast; the reasons and the importance for inactivation of Fe in the apoplast are discussed. The use of Fe chlorosis tolerant genotypes as rootstocks in orchards and vineyards represents a reliable solution to prevent iron chlorosis; in some species, however, available Fe chlorosis resistant rootstocks are not very attractive from an agronomic point of view since they often induce excessive growth of the scion and reduce fruit yields. As most fruit tree crops and grapes are high value commodities, in many countries growers are often willing to apply synthetic Fe chelates to cure or to prevent the occurrence of Fe deficiency. The application of iron chelates does not represent a sustainable way to prevent or cure iron chlorosis because of to their costs and of the environmental risks associated with their use. Since Fe chelates were introduced, little research on alternative means for controlling the chlorosis has been performed. Sustainable management of Fe nutrition in orchards and vineyards should include all genetical and agronomical means in order to naturally enhance Fe availability in the soil and in the plant. Special attention should be given to soil analysis and to prevention measures carried out before planting. Alternatives to iron chelates are being developed and in the future they should be included into the routine practices of managing fruit trees and grapevine under Integrated Production and Organic Farming.

Organic acids and Fe deficiency : a review

Organic acid concentrations often increase with iron deficiency in different plant parts such as roots, leaves and stem exudates. The review summarises data available on the changes in the concentrations of organic anions in plants with iron deficiency and the effects of these changes in plant metabolism. The paper reviews data available in the literature on the changes in xylem and apoplasmic fluid composition with iron deficiency, both in plants grown in controlled conditions and in the field, and discusses the possible ways of iron complexation and transport in these compartments. The characteristics of the iron reduction and uptake by the iron-deficient leaf mesophyll cells are also discussed, with especial emphasis in the possible roles of organic acids in these processes. Both the possible causes and functions of the organic acid concentration increases in iron-deficient plants are reviewed.

Agronomic means for the control of iron deficiency chlorosis in deciduous fruit trees

Abstract Iron deficiency induced chlorosis represents the main nutritional disorder in fruit tree orchards grown on calcareous and/or alkaline soils. Until rootstocks tolerant to Fe deficiency chlorosis are available for most susceptible fruit species, the agronomic means of preventing or curing Fe deficiency chlorosis will be considered of utmost importance by fruit growers. Chlorosis of fruit trees has been successfully controlled through foliar or soil applications of Fe chelates, which are expensive and have to be applied annually. In this paper results of research carried out within an EU joint research project are reported, where the effectiveness of alternative, low‐input, environmentally friendly management techniques to control Fe deficiency chlorosis has been tested in established kiwifruit, peach and pear orchards located in the Po Valley (Italy), in the Ebro Valley (Spain) and in the area of Imathia (Greece). Iron sulphate supply to the soil proved to be effective only if applied together with high amounts of organic matter such as compost or manure. Promising results in preventing chlorosis were obtained by sowing a mixture of graminaceous species along the tree row and supplying them with Fe sulphate. Laboratory tests indicated that long lasting decreases of pH in calcareous soils are difficult to achieve. We have also followed two approaches using foliar sprays: 1) testing a variety of compounds which may activate the Fe pool likely present in chlorotic leaves (citric, sulphuric, ascorbic and indole‐3‐acetic acid) and 2) applying Fe sources alternative to synthetic Fe chelates. Sprays aiming to activate the Fe pools in a chlorotic leaf were generally effective, although rarely caused a full recovery. This suggests that inactivation of Fe occurs outside the mesophyll cells. Sprays of Fe sulphate in all the crops tested showed similar or even higher regreening effect than FeDTPA.

Iron source affects iron reduction and re‐greening of kiwifruit (Actinidia deliciosa) leaves

Abstract Among deciduous fruit plants, kiwifruit (Actinidia deliciosa) is one of the most susceptible to iron (Fe) chlorosis. To develop effective means for overcoming Fe chlorosis, it is of upmost importance to gain information about the reduction of Fe by leaf tissues, especially under conditions that lead to chlorosis. In the present study we have characterised the leaf Fe‐chelate reductase (FCR) in Fe sufficient and Fe deficient kiwifruit leaves and for the first time tested the hypothesis that FeIII‐malate is a suitable source of Fe for FCR, in addition to FeIII‐citrate. Under field conditions, we have also tested the re‐greening effects caused by the foliar application of different Fe sources, including FeIII‐malate, FeIII‐citrate, FeIII‐DTPA and an FeII source (FeSO4 + aminoacid‐polypeptide mixture) on chlorotic leaves. The results demonstrated that, similarly to other species, mesophyll tissues of A. deliciosa leaves are able to perform an enzymatic Fe reduction prior to Fe uptake. Plasma membrane enriched material extracted from Fe sufficient leaves reduced FeIII‐malate and FeIII‐citrate. The pH optimum was 6.0–6.2 for FeIII‐malate and 6.5 for FeIII‐citrate. The substrate‐dependence showed higher affinity for malate than for citrate. In contrast to the root level, the activity of the FCR of kiwifruit leaves was not enhanced under Fe deficiency. On the contrary, after two weeks of Fe depletion, the reduction of FeIII‐citrate was 4.5‐fold lower in the Fe deficient plants than in the Fe sufficient ones, while the reduction of FeIII‐malate was not significantly affected. Under field conditions, the Fe solutions caused regreening of chlorotic leaves, whose intensity and duration varied according to Fe source and Fe concentration. Among the treatments, the highest re‐greening effect was caused by FeIII‐DTPA and especially by the FeII source. FeIII‐citrate and FeIII‐malate were less effective in stimulating chlorophyll formation. All treatments increased leaf Fe concentration and content. Although less Fe from malate than from citrate penetrated into the leaves, the re‐greening effect from FeIII‐malate was intermediate between that of FeIII‐DTPA and the one caused by FeIII‐citrate. The results suggest that if FeIII‐malate can reach the plasmamembrane it provides a good source of Fe for leaf Fe uptake.

Response to iron‐deficiency stress of pear and quince genotypes 1

Abstract Roots of iron (Fe)‐efficient dicots react to Fe‐deficiency stress by strongly enhancing the ferric (Fe3+)‐reductase system and by lowering the rhizo‐sphere pH. In this study, we tested whether such adaptation mechanisms characterize pear and quince genotypes known to have differential tolerance to calcareous and alkaline soils. Two trials were performed using micropagated plants of three quince rootstocks (BA29, CTS212, and MC), three Pyrus communis rootstocks (OHxF51 and two selections obtained at the Bologna University: A28 and B21) and of two pear cultivars (Abbe Fetel and Bartlett, own‐rooted). In the first trial, plants were grown in a nutrient solution with [Fe(+)] and without [Fe(‐)] Fe for 50 days. Their root Fe‐reducing capacity was determined colorimetrically using ferrozine and FeEDTA, and Fe uptake of Fe(+) plants was estimated. In the second trial, the rhizosphere pH of plants grown in an alkaline soil was measured by a micro‐electrode. With the only exception of pears OHxF51 and A28...

Prevention of Iron‐Deficiency Induced Chlorosis in Kiwifruit (Actinidia deliciosa) Through Soil Application of Synthetic Vivianite in a Calcareous Soil

Abstract In this study we have tested the hypothesis that lime‐induced Fe deficiency chlorosis of kiwifruit may be prevented by the application of a synthetic iron(II)‐phosphate analogous to the mineral vivianite [(Fe3(PO4)2·8H2O)]. Two experiments, under greenhouse and field conditions, were performed. In the greenhouse, 1‐year old micropropagated plants (Actinidia deliciosa, cv. Hayward), grown in 3‐L pots on a calcareous soil, were treated in early autumn with soil‐applied: (1) synthetic vivianite (1.35 g plant−1) and (2) Fe‐EDDHA (24 mg Fe plant−1). The synthetic vivianite suspension, prepared by dissolving ferrous sulfate and mono‐ammonium phosphate, was injected into the soil as a sole application whereas the Fe‐EDDHA solution was applied four times at weekly intervals. The field experiment was conducted in a mature drip‐irrigated kiwifruit orchard located on a calcareous soil in the Eastern Po Valley (Italy). Treatments were performed in early autumn by injecting synthetic vivianite (1.8 kg tree−1) and Fe‐EDDHA (600 mg Fe tree−1) into four holes in the soil around each tree, at a depth of 25–30 cm. The Fe‐chelate application was repeated at the same rate in the following spring. Untreated (control) plants were used in both experiments. Autumn‐applied Fe fertilisers significantly prevented development of Fe chlorosis under greenhouse conditions whereas in the field only vivianite was effective. In conclusion, these 1‐year results show that vivianite represents an effective alternative to soil‐applied Fe chelates for preventing Fe chlorosis in kiwifruit orchards.

In vitro performance at high culture pH and in vivo responses to Fe-deficiency of leaf-derived quince BA 29 (Cydonia oblonga) somaclones regenerated at variable medium pH.

Summary The aims of the work were to obtain quince (Cydonia oblonga) BA 29 somaclones tolerant to high culture pH and lime soils, to test the effectiveness of in vitro selection methods based on the selection pressure exerted by pH during regeneration, and to understand the mechanisms for the improved performance under Fe-deficient conditions of eventually tolerant clones. Leaf-derived somaclones obtained at variable medium pH (5.7, 6.0, 6.5, 7.0 and 8.0) were compared with each other and with the control (i.e. shoots derived from BA 29 mother plants by standard micropropagation) for their in vitro growth, proliferation, rooting performance and pH-reducing ability at pH 7.0, 7.5 and 8.0, and for their Fe-chelate reductase (FCR) activity and soluble sugar and organic acid accumulation in the roots and leaf chlorophyll content when grown in soiless culture under Fe-efficient and -deficient conditions. Preliminary results on SPAD analysis on leaves of the same plants transferred to lime soil are also reported. The effect of pH selection pressure was not very clear; however, while all the clones hardly grew and proliferated in vitro at pH 8, the somaclones regenerated at pH 7 generally tended to have better growth and proliferation than control and other somaclones at pH 7.5, especially 7-A. In contrast, most of those regenerated at low pH, but 5.7-A, tended to have lower proliferation rates and shorter axillary shoots. Little differences were found in the rooting performances of the clones, although most somaclones seemed more able to reduce the pH than the control. After 10 d in hydroponic culture under Fe-deficient conditions, almost all the somaclones tended to have some FCR induction in the roots; however, it was detected only in 5.7-A and 7-A at day 30. Leaf chlorophyll determinations in soiless culture and lime soil indicated quite good tolerance to Fe-chlorosis for 6-A, 7-A and especially 5.7-A. When the in vitro and in vivo results are considered together, the somaclones 5.7-A, 7-A and 6-A seem the most interesting, their success in growing in Fe-deficient conditions being probably due partially to sorbitol accumulation in the roots, in particular for 6-A and 5.7-A, and also to inositol and citric acid, respectively for 5.7-A and 7-A. However, further plant evaluation on lime soil is needed to verify these preliminary results and to understand whether some of these somaclones can replace the widespread pear rootstock quince BA 29, as more tolerant to lime-induced Fe-chlorosis.

1)H NMR foodomics reveals that the biodynamic and the organic cultivation managements produce different grape berries (Vitis vinifera L. cv. Sangiovese).

The increasing demand for natural foods and beverages, i.e. prepared by excluding synthetic chemicals along the whole production chain, has boosted the adoption of organic and biodynamic cultivation methods which are based on protocols avoiding use of synthetic pesticides. This trend is striking in viticulture, since wine production is largely shaped by the varying drinking attitudes of environment-friendly consumers. Using (1)H NMR, the compositions of grape berries, collected at harvest in 2009 and 2011, in experimental plots cultivated either with biodynamic or organic methods, were compared. Although the analysis provides a comprehensive metabolic profile of berries, the resulting distinctive pattern consists of a few molecules. Lower content of sugars, coumaric and caffeic acids, as well as higher amount of γ-aminobutyric acid (GABA) were observed in biodynamic grapes. The (1)H NMR foodomics approach evidenced a diverse fruit metabolome that could be associated to a different physiological response of plants to the agronomic environment.

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.

Responses of in vitro cultured kiwifruit shoots to treatments with green amaranth aqueous extracts

Summary Because of environmental hazards related to the use of synthetic Fe-chelates in Fe-chlorosis control of plants, and with the purpose of using plant extract treatments as an alternative strategy, the effect of Amaranthus retroflexus L extracts on Actinidia deliciosa Liang et Ferguson shoot cultures was investigated, and the possibility of using in vitro cultures for rapid evaluation of the effect of such extracts on growth and Fe-chlorosis control in fruit plants is discussed. The extract was obtained by macerating for a week 100 g plant fresh weight in 1 litre of distilled water, filter-sterilizing and adjusting the pH to 5.65; the extract was added at 0 (control), 20, 2, 0.2 and 0.02 ml to 100 ml of previously autoclavesterilized modified Murashige and Skoog, MS, medium (PM), and salt-deficient PM/3 (third MS salt strength) and rooting RM (lacking growth regulators, half MS salt strength) media. The addition of 20 ml water extract to 100 ml PM/3 strongly reduced shoot and basal callus development, while 0.02 ml per 100 ml increased both with respect to the control. Rooting was hastened with 0.02 ml extract per 100 ml RM with respect to other concentrations. Moreover, roots produced with 0.02 and 0.2 ml extract per 100 ml RM were longer than with other treatments and in the control. The addition of 0.02 ml extract per 100 ml PM/3 medium, and of 0.02 to 2 ml extract per 100 ml RM improved the photosynthetic activity of cultures after five and four weeks in the proliferation and rooting phases, respectively.