**The Effectiveness of FeEDDHA Chelates in Mending and Preventing Iron Chlorosis in Soil-Grown Soybean Plants**

W. D. C. Schenkeveld and E. J. M. Temminghoff *Wageningen University The Netherlands* 

## **1. Introduction**

82 Soybean Physiology and Biochemistry

Urenã, R.; Rodríguez, F.; Berenguel, M.A. (2001). Machine vision system for seeds

Vale, F.X.R. (1985). *Aspectos epidemiológicos da ferrugem* (*Phakopsora pachyrhizi* Sydow) da soja

Vale, F.X.R.; Zambolim, L.; Chaves, G.M. (1990). Efeito do binômio temperatura-duração do

Vale, F.X.R.; Zambolim, L; Costa, L.C.; Liberato, J.R.; Dias, A.P.S. (2004). Influência do clima

Vargens, J.M.; Tanscheit, R.; Vellasco, M.M.B.R. (2003). Previsão de produção agrícola

Vianello, R.L.; Alves, A.R. (1991). *Meteorologia básica e aplicações*. Viçosa: Imprensa

Von Altrock, C. (1995). *Fuzzy Logic and NeuroFuzzy Applications Explained*. USA: Prentice

Yang, X.B.; Feng, F.(2001). Ranges and diversity of soybean fungal diseases in North

Yang, X.B.; Royer, M.H.; Tschanz, A.T.; Tsai, B.Y. (1990). Analysis and quantification of

Yang, X.B.; Tschanz, A.T.; Dowler, W.M.; Wang, T.C. (1991). Development of yield loss

Yorinori, J.T.; Lazzarotto, J.J. (2004). *Situação da ferrugem asiática da soja no Brasil e na América* 

Zambenedetti,E.B. *Preservação de Phakopsora pachyrhizi Sydow & Sydow e aspectos* 

Zadeh, L.A. (1965). Fuzzy sets. *Information and Control*, San Diego, v. 8, n. 3, p. 338-353, 1965.

soybean rust epidemics from 73 sequential planting experiments. *Phytopathology*, St.

models in relation to reductions of components of soybean infected with *Phakopsora* 

*do Sul*. Londrina: Embrapa Soja. 30 p. (Documentos, 236). Disponível em

*epidemiológicos e ultra-estruturais da sua interação com a soja* (*Glycine max* (L.). Merril). (2005). 92 p. Dissertação (Mestrado) - Universidade Federal de Lavras, Lavras, MG.

*in Agriculture,* Oxford, v. 32, n. 1, p. 1-20.

*Brasileira*, Brasília, v. 15, n. 3, p. 200-202.

*Automação*, Campinas, v. 2, n. 14, p. 114-120.

America. *Phytopathology*, St Paul, v. 91, n. 8, p. 769-775.

*pachyrhizi. Phytopathology*, St. Paul, v. 81, n. 11, p. 1420-1426.

<http://www. cnpso. embrapa. br> em:< dez. 2004.

Horizonte: Editora Perffil, p. 47-87.

Universitária/UFV. 449 p.

Paul, v. 80, n. 12, p. 1421-1427.

Hall. 384 p.

Federal de Viçosa, Viçosa, MG.

germination quality evaluation using fuzzy logic. (2001). *Computers and Electronics* 

(*Glycine max* L. Merrill). 104 p. Tese (Doutorado em Fitopatologia) - Universidade

molhamento foliar sobre a infecção por *Phakopsora pachyrhizi* em soja. *Fitopatologia* 

no desenvolvimento de doenças de plantas. In: VALE, F. X. R.; JESUS JUNIOR, W. C.; ZAMBOLIM, L. *Epidemiologia aplicada ao manejo de doenças de plantas*. Belo

baseada em regras lingüísticas e lógica fuzzy. *Revista Brasileira de Controle &* 

#### **1.1 Iron deficiency – The problem**

Iron (Fe) is an essential micronutrient for plants, humans and other animals. An adequate uptake of Fe is needed to ensure proper growth and development, as well as good health of organisms (Marschner, 1995; Vasconcelos and Grusak, 2007). When provided with insufficient quantities of Fe, organisms will suffer from Fe deficiency symptoms.

Fe deficiency is a worldwide problem in crop production, affecting yield both qualitatively and quantitatively (Mortvedt, 1991); plants do not reach their full growth potential, and the nutritional value is compromised, leading to economic losses and limitations in crop selection (Chaney, 1984). In extreme cases, Fe deficiency may result in complete crop failure (Chen and Barak, 1982). The list of plant species affected is vast and includes apple, citrus, grapevine, peanut, dryland rice, sorghum and soybean (Marschner, 1995).

Fe deficiency is typically found in crops grown on calcareous or alkaline soils, in arid and semi-arid regions of the world; these soils cover over 30% of the earths' land surface (Figure 1) (Alvarez-Fernandez, et al., 2006; Chen and Barak, 1982; Hansen, et al., 2006; Mortvedt, 1991). Fe is abundantly present in all soils including calcareous ones; in mineral soils the average Fe content is approximately 2% (20,000 μg/g) (Marschner, 1995; Mengel and Kirkby, 2001). Most agricultural crops require less than 0.5 μg/g in the plough layer (Lindsay, 1974). The occurrence of Fe deficiency in plants grown on calcareous soils, despite the excessive soil-Fe pool, is caused by a limited bioavailability of Fe in such soils.

#### **1.2 Symptoms of Fe deficiency**

Fe deficiency in plants typically causes chlorosis of leaf tissue because of inadequate chlorophyll synthesis; the leaves become pale green to yellow (Figure 2), often with darker coloured veins. In case of severe chlorosis, leaves can also become necrotic (Figure 2). Due to the reduction in photosynthetic capacity, carbon fixation by plants also becomes reduced, leading to slower growth rates and yield losses (Figure 2) (Alvarez-Fernandez, et al., 2006).

Fe chlorosis develops most strongly in young leaves, because growing plant parts (also fruits, buds and storage organs) have incomplete xylem structures. As a result, Fe is not directly transported from the roots to these sites with the highest demand, but remobilized from older plant parts and secondarily transported through the phloem (Grusak, et al., 1999;

Fig. 1. Global pH-map of the top soil (0-30 cm); red indicates pH < 5.5; yellow indicates 5.5 < pH < 7.3; green indicates pH > 7.3. Calcareous soils are to be found in the green areas. Source: ISRIC, 1995, derived from the WISE- database.

Zhang, et al., 1995). It has been observed that chlorotic leaves can have comparable or even higher Fe contents than green leaves (the "chlorosis paradox"). This phenomenon has been attributed to impaired expansion growth, leading to diminished dilution of the high Fe concentration in young leaves (Römheld, 2000). Fe deficiency also causes morphological changes in the roots: inhibition of root elongation, increase in diameter of apical rootzone, abundant root hair formation (Römheld and Marschner, 1981) and formation of rhizodermal transfer cells.

#### **1.3 Causes of Fe deficiency**

Two related soil characteristics are principally responsible for the low Fe availability in calcareous soils: 1) the relatively high pH (7 - 8.5) (Figure 1.1), and 2) the presence of a bicarbonate pH-buffer in soil solution (Boxma, 1972; Chaney, 1984; Lucena, 2000; Marschner, 1995; Mengel, et al., 1984; Mengel and Kirkby, 2001).

In order for soil-Fe to be taken up, it needs to be transported through the soil solution to the root surface. The solubility of soil Fe(hydr)oxides is a function of pH and the type of Fe(hydr)oxide. The concentration of inorganic Fe species in solution reaches a minimum around pH 7.5 - 8.5: in the order of 10-10 M (Figure 3); the free Fe3+ concentration is around 10-21 M (Lindsay and Schwab, 1982). For optimal growth, plants require an Fe concentration in soil solution in the order of 10-6 to 10-5 M (Marschner, 1995). Complexation by dissolved organic substances, like humic acids, fulvic acids and siderophores can increase the total Fe concentrations in soil solution by orders of magnitude in comparison to the inorganic Fe concentration (O'Conner, et al., 1971), but not always sufficiently to prevent Fe deficiency.

The bicarbonate pH-buffer prevents plants from adapting the rhizosphere pH and causes impairment of Fe deficiency stress response mechanisms (except in grasses). Although the pH-buffer capacity of calcareous soils is largely determined by the lime content, the dissolution of carbonate minerals is relatively slow in comparison to bicarbonate diffusion. Therefore, on the short term, the bicarbonate concentration in soil solution is more

Fig. 1. Global pH-map of the top soil (0-30 cm); red indicates pH < 5.5; yellow indicates 5.5 < pH < 7.3; green indicates pH > 7.3. Calcareous soils are to be found in the green areas.

Zhang, et al., 1995). It has been observed that chlorotic leaves can have comparable or even higher Fe contents than green leaves (the "chlorosis paradox"). This phenomenon has been attributed to impaired expansion growth, leading to diminished dilution of the high Fe concentration in young leaves (Römheld, 2000). Fe deficiency also causes morphological changes in the roots: inhibition of root elongation, increase in diameter of apical rootzone, abundant root hair formation (Römheld and Marschner, 1981) and formation of rhizodermal

Two related soil characteristics are principally responsible for the low Fe availability in calcareous soils: 1) the relatively high pH (7 - 8.5) (Figure 1.1), and 2) the presence of a bicarbonate pH-buffer in soil solution (Boxma, 1972; Chaney, 1984; Lucena, 2000; Marschner,

In order for soil-Fe to be taken up, it needs to be transported through the soil solution to the root surface. The solubility of soil Fe(hydr)oxides is a function of pH and the type of Fe(hydr)oxide. The concentration of inorganic Fe species in solution reaches a minimum around pH 7.5 - 8.5: in the order of 10-10 M (Figure 3); the free Fe3+ concentration is around 10-21 M (Lindsay and Schwab, 1982). For optimal growth, plants require an Fe concentration in soil solution in the order of 10-6 to 10-5 M (Marschner, 1995). Complexation by dissolved organic substances, like humic acids, fulvic acids and siderophores can increase the total Fe concentrations in soil solution by orders of magnitude in comparison to the inorganic Fe concentration (O'Conner, et al., 1971), but

The bicarbonate pH-buffer prevents plants from adapting the rhizosphere pH and causes impairment of Fe deficiency stress response mechanisms (except in grasses). Although the pH-buffer capacity of calcareous soils is largely determined by the lime content, the dissolution of carbonate minerals is relatively slow in comparison to bicarbonate diffusion. Therefore, on the short term, the bicarbonate concentration in soil solution is more

Source: ISRIC, 1995, derived from the WISE- database.

1995; Mengel, et al., 1984; Mengel and Kirkby, 2001).

not always sufficiently to prevent Fe deficiency.

transfer cells.

**1.3 Causes of Fe deficiency**

Fig. 2. Examples of Fe deficiency symptoms in soybean plants. *Upper:* from left to right decreasing degree of chlorosis; *Lower left*: necrosis in the leaves; *Lower right:* reduced growth.

important for maintaining a high rhizosphere pH (Lucena, 2000). In addition to the role of bicarbonate as pH-buffer in soil solution, there has been much debate on bicarbonate uptake leading to Fe immobilization inside plants (Gruber and Kosegarten, 2002; Mengel, 1994; Nikolic and Romheld, 2002; Römheld, 2000).

### **1.4 Prevention and remediation of Fe deficiency**

When Fe stress response mechanisms of plants prove inadequate, techniques to prevent or remedy Fe deficiency need to be applied to avoid yield losses. Breeding and genetically modifying plants for a more efficient Fe uptake mechanism is a promising approach. Developing new cultivars should however be done carefully and requires much time. Once crops are in the field, application of Fe fertilizer is the most certain and efficient treatment to ensure that plants do not suffer from Fe deficiency.

Fig. 3. Hydrolysis species of Fe(3+) in equilibrium with soil-Fe (pKsol = 39.3; I = 0.03 M), after Lindsay (1979).

Fe fertilizers can be administered through trunk injection, foliar application, and soil application. Trunk injection is expensive and only suitable for trees. Foliar application does not provide full control of Fe chlorosis, but can be useful as complementary technique next to soil application (Alvarez-Fernandez, et al., 2004). Soil application is the most common technique to manage Fe deficiency in soil grown crops (Lucena, 2006). The technique is based on increasing the Fe concentration in soil solution. On calcareous soils, soil application of Fe fertilizers based on organic Fe salts, Fe complexes of lignosulfonates, citrates, gluconates, and synthetic Fe chelates of limited stability (e.g. FeEDTA, FeDTPA and FeHEDTA) has limited or no result, because these fertilizers are not able to maintain Fe in soil solution. Only Fe chelates of higher stability (FeEDDHA and derivatives, with phenolic functional groups) are effective and provide the most efficient treatment to control Fe deficiency (Lucena, 2006).

#### **1.5 Fe deficiency in soybean**

Fe deficiency chlorosis is a persistent, yield-limiting condition for soybean (*Glycine max* (L.) Merr.) production in regions with calcareous soils (Inskeep and Bloom, 1986). In the North Central U.S., Fe deficiency is responsible for an estimated loss in soybean grain production of \$120 million per year (Hansen et al., 2004). Foliar Fe treatments and soil application of Fe chelates can be efficient in alleviating Fe deficiency chlorosis in soybean. However, in agricultural practice, these methods are only economically feasible for high-value crops and not for soybean (Fairbanks 2000).

Although soybean is not a target species for application of synthetic Fe chelates, it is an attractive test species due to the availability of soybean cultivars with a high susceptibility to Fe deficiency, the ease in handling of the plants, and the relatively short growth cycle in comparison to many of the target species (e.g citrus trees and grape vines). There is much experience with soybean in Fe chlorosis research; in nutrient solutions, in pot cultures and in the field (e.g. Garcia-Marco et al. 2006; Goos et al. 2004; Goos and Johnson 2000; Heitholt et al. 2003; Wallace and Cha 1986).
